**Figure 1.** Cisplatin or natural compounds effects on cells viability. (**A**) HUVEC and (**B**) PE/CA-PJ49 cells were either left untreated or treated 24 h with different concentrations of CisPt, CRM. Data shown are representative of three independent experiments and are expressed as mean of three replicates ± SD (*n* = 3). Untreated cells were considered to have 100% viability. Viability% = (T − B)/(U − B) × 100, (where T, absorbance of treated cells; U, absorbance of untreated cells; and B, absorbance of blank). (**C**) Tumor cell viability compared to normal HUVEC cell viability after treatment with CisPt and / or RSV,CRM; (\* *p* < 0.05, \*\* *p* < 0.005; \*\*\* *p* < 0.0005).

#### *4.2. E*ff*ects of CisPt and*/*or Natural Compounds RSVor CRM on the Cell Proliferation Process*

PE/CA-PJ49 and HUVEC cells were treated with 5 μM and 20 μM CisPt and/or 40 μM RSV, 15 μM CRM for 24 h. Analysis of the effect induced by the individual treatment with CisPt, RSV or CRM on the proliferation process of the PE/CA-PJ49 tumor cells and of the normal HUVEC cells are shown in Figure 2A,B. Treatment with 20 μM CisPt for 24 h determined a significant decrease of the proliferation process compared to the 5 μM CisPt treatment, both in the case of tumor cells PE/CA-PJ49 (\*\* *p* = 0.0065) and that of normal cells HUVEC (\*\* *p* = 0.0011). Treatment with 40 μM RSV for 24 h affected the

proliferation of tumor cells PE/CA-PJ49 much more than the one of normal cells HUVEC (\*\* *p* = 0.0063) (Figure 2A).

**A. CisPt and/or RSV B. CisPt and/or CRM**

**Figure 2.** The index proliferation (IP) of HUVEC and PE/CA-PJ49 cells treated with CisPt and/or (**A**) RSV (**B**) CRM was calculated as IP = absorbance of treated cells/absorbance of untreated cells. Results are expressed as DO mean values of three determinations ± standard deviation (SD). Untreated cells were considered to have IP equal 1. (\*\* *p* < 0.005; \*\*\* *p* < 0.0005).

Analysis of the effect induced by the combined treatment of 5 μM CisPt + 40 μM RSV on PE/CA-PJ49 tumor cells showed that RSV can amplify the effect induced by lower CisPt concentration by recording a decrease of the IP (5 μM CisPt + 40 μM RSV vs. 5 μM CisPt; \* *p* = 0.0145). In case of applying the same treatment on the normal HUVEC cells no significant changes of IP were registered (5 μM CisPt + 40 μM RSV vs. 5 μM CisPt; non-significant, *p* = 0.105). Using a high concentration of CisPt, the proliferative response of tumor cells to the combined treatment of 20 μM CisPt + 40 μM RSV recorded a significant decrease in IP (20 μM CisPt + 40 μM RSV vs. 40 μM RSV; \*\* *p* = 0.0093). Treatment of normal cells with 20 μM CisPt + 40 μM RSV led to a response similar to that induced by treatment with 20 μM CisPt (non-significant, *p* = 0.53), but there was a significant decrease in IP compared to the response induced by treatment with 40 μM RSV alone (\*\* *p* = 0.005) (Figure 2A). These results show that the effect of treatment with 20 μM CisPt is dominant in the treatment of normal or tumor cells. Treatment with 15 μM CRM for 24 h on tumor cells PE/CA-PJ49 led to a much more intense decrease of the proliferative process compared to the effect induced on normal HUVEC cell proliferation (\*\* *p* = 0.0065) (Figure 2B). Applying the combined 5 μM CisPt + 15 μM CRM treatment to normal cells did not alter the proliferative process compared to the effect induced by individual treatment with 5 μM CisPt (non-significant, *p* = 0.784) or 15 μM CRM (non-significant, *p* = 0.209). IP of tumor cells PE/CA-PJ49 treated with 5 μM CisPt + 15 μM CRM suggested a significant inhibition of the proliferative process compared to the effect induced by treatment with 5 μM CisPt (\*\* *p* = 0.008) or 15 μM CRM (\*\* *p* = 0.0042). The combined treatment of 20 μM CisPt + 15 μM CRM very strongly inhibited the proliferation of tumor cells compared to the effect induced by treatment only with CRM (\*\*\* *p* = 0.0006) (Figure 2B). The combined treatment applied to normal cells HUVEC led to a decrease in IP, but the dominant effect on cell proliferation appears to be due to the high concentration of CisPt (20 μM CisPt + 15 μM CRM vs. 15 μM CRM; \* *p* = 0.0424). Comparative analysis of the proliferative response of PE/CA-PJ49 tumor cells versus the normal HUVEC cells to the combined CisPt + CRM treatment shown a significantly different cellular behavior (\*\* *p* = 0.004 –5 μM CisPt + 15 μM CRM; \*\* *p* = 0.0051 – 20 μM CisPt + 15 μM CRM) (Figure 2B).

#### *4.3. Modulation of p21 Protein Expression by Natural Compounds and*/*or Cisplatin Treatment*

In cancer development and evolution p21 protein might acts as an oncogene or tumor suppressor and for this reason it could be an important player in processes such as the cancer aggressiveness or the response to chemotherapy [63,64]. In this study, using ELISA assay we analyzed the expression level of p21 in the tumor cells (PE-CA/PJ49) compared to a normal cells (HUVEC). In addition, we looked at how treatment with CisPt, RSV or CRM applied individually or in combination could affect cell p21 protein expression. The data showed that the p21 protein is expressed more (3.4×) in the untreated tumor cells of the PE-CA/PJ49 vs. normal cells of the HUVEC line (625/184 pg/mL). Individually applied treatment with CisPt, 40 μM RSV or 15 μM CRM did not significantly affect the expression of p21 protein (*p* = non-significant) in normal HUVEC cells. Treatment of normal cells with 20 μM CisPt + 40 μM RSV induced an increase of p21 protein expression compared to untreated cells (*p* < 0.029) or to cells treated only with RSV (\*\* *p* < 0.0085). A significant increase in p21 protein expression of HUVEC cells was recorded in the case of the combined treatment 5 μM CisPt + 15 μM CRM compared to untreated cells (\*\* *p* < 0.0048) (Figure 3 and Table 4).

**Figure 3.** The total p21 protein expression (pg/mL) in normal cell line-HUVEC and in tumor cell line-PE/CA-PJ49 cells treated 24 h with CisPt and/or RSV, CRM. The experiments were performed in triplicates. Results are expressed as mean values of three determinations ± standard deviation (SD). (\*\* *p* < 0.005, \*\*\* *p* < 0.0005; \*\*\*\* *p* < 0.00005).

PE/CA-PJ49 tumour cells were treated 24 h with 5 μM CisPt (\*\*\* *p* = 0.0009) or 20 μM CisPt (\*\* *p* = 0.0039) showed a significant increase of total p21 protein expression compared to untreated cells (control). 40 μM RSV (\*\*\*\* *p* < 0.0001) applied alone, determined a significant increase of the total p21 protein expression in PE/CA-PJ49 cells versus control (Figure 3). On the contrary, after combined treatment with 5 μM CisPt + 40 μM RSV (\* *p* < 0.014) versus treatment with 5 μM CisPt alone the results showed a slight increase in p21 expression of PE/CA-PJ49 cells. As shown in Figure 3 and Table 4 the simultaneous treatment with 20μMCisPt+40μMRSV induced in tumor cells a significant decrease of p21 protein expression comparatively with the effect induced by RSV alone (\*\* *p* = 0.0058). These results support the notion that the dominant effect is attributed to the high concentration of CisPt. Thus we can say that RSV can potentiate the induced effect of 5 μM CisPt but cannot counteract

the induced effects of treatment with a much higher concentration of CisPT on p21 protein expression in tumor cells.

**Table 4.** The effects of CisPt, RSV, CRM treatment applied alone or in combination for 24 h on the level of p21 protein expression (n-fold p21protein expression) in normal cell line-Huvec and in tumor cell line-PE/CA-PJ49. The n-fold p21expression was calculated using the formula: n-fold p21expression = p21 pg/mL treatment/p21 pg/mL control.

PE/CA-PJ49 tumor cells treatment with 15 μM CRM led to a significant increase of the p21 protein expression compared to control (\*\* *p* = 0,0022). Combined treatment 15 μM CRM and 5 μM CisPt (\*\*\*\* *p* = 0.0001) or 20 μM CisPt (\*\*\*\* *p* = 0.0001) led to a much higher amplification of p21 protein expression compared to CisPt only-treated cells (Figure 3 and Table 4). Our data showed that in PE/CA-PJ49 tumor cells the effect induced by the CisPt and CRM was cumulative. In addition, the analysis reveal the potential of CRM to sustain and amplify the effect induced by CisPt treatment, regardless of the CisPt concentration used.

#### *4.4. Modulation of P21 Gene Expression by Natural Compounds and*/*or Cisplatin Treatment*

Changes of the P21 gene expression have been found in various types of malignancy including head and neck [65–67] but the impact of the P21 level on the disease progression and prognosis remains controversial. Therefore, we performed in vitro experiments to assess how natural compounds (RSV or CRM) modulated the P21 expression in head and neck tumor cells PE/CA-PJ49 versus normal cells HUVEC. Analysis of the expression level of P21 was performed by real-time qPCR assay in order to identify their role in response to chemotherapeutic agent-CisPt. Obtained data from this study may help define how P21 acts in modulating the DNA repair processes of tumor cells, in hope of finding new effective strategies in the treatment of head and neck cancer. Using real-time qPCR assay, we evaluated the P21 gene expression induced by CisPt and/or RSV, CRM treatment of HUVEC and PE/CA-PJ49 tumor cells. The data showed that RSV treatment acts differently on the two cell lines analyzed. RSV induces a slight decrease in P21 gene expression in HUVEC cells and an increase in the PE/CA-PJ49 tumor cells. Treatment with 5 μM CisPt (\* *p* = 0.027) or 20 μM CisPt (\*\* *p* = 0.016) induces an antagonist response compared to the effect induced by RSV treatment alone in P21 gene expression of normal HUVEC cells. The results obtained from the combined CisPt + RSV treatment show that RSV did not significantly modify the P21 gene expression generated by CisPt treatment in normal cells. CRM treatment applied alone did not affected P21 expression in HUVEC cells. The combined 5 μM CisPt + 15 μM CRM treatment showed a significant increase in P21 expression in normal cells (\*\* *p* = 0.019 versus 15 μM CRM). On the contrary, 20 μM CisPt + 15 μM CRM treatment of HUVEC cells induced a decrease of P21 gene expression compared with the effect induced by CRM alone (\* *p* = 0.021) or 20 μM CisPt (\* *p* = 0.013) (Figure 4 and Table 5).

**Figure 4.** The effect of treatment with CisPt, RSV, CRM applied independently or in combination on P21 gene expression in tumor cells PE/CA-PJ49 compared to normal cells HUVEC. Each sample was performed in duplicate. The samples were analyzed using the formula 2-ΔΔCt = gene expression. (\* *p* < 0.05, \*\* *p* < 0.005; \*\*\* *p* < 0.0005).

**Table 5.** n-fold P21gene expression in PE/CA-PJ49 tumor cells versus normal cells HUVEC treated 24 h with CisPt and/or RSV, CisPt and/or CRM. The results were obtained using gene reference HPRT.

RSV alone induced a significant increase of P21 expression (\*\* *p* = 0.006) while 5 μM CisPt (*p* = non-significant) applied individually induced a slight increase of the P21 gene expression compared to untreated PE/CA-PJ49 tumor cells. The application of the combined treatment 5 μM CisPt + 40 μM RSV in tumor cells led to a significant increase in P21 gene expression compared to untreated cells (\*\* *p* = 0.002). When using a higher concentration of CisPt together with RSV, a significant increase in P21 gene expression was observed in tumor cells versus control (\*\* *p* = 0.009) or versus RSV (\* *p* = 0.046) (Figure 4 and Table 5). These data support the modulatory role of RSV in tumor cells on the effect induced by 20 μM CisPt on the gene expression of P21 without influencing the effect induced by 5 μM CisPt.

Although CRM appears to induce a slight increase in P21 gene on the PE/CA-PJ49 tumor cells, this is not significantly different from P21 expression in untreated cells. The use of combination treatment with 5 μM CisPt + 15 μM CRM showed a significant increase in P21 in tumor cells (\*\* *p* = 0.0014) compared to the effect induced by individual treatment with CRM. Analysis of the induced effect of 20 μM CisPt +15 μM CRM in PE/CA-PJ49 tumor cells also showed a significant increase in P21 gene expression compared to the induced effect of CRM alone (\*\*\* *p* = 0.0009) or compared to the 20 μM CisPt effect (\*\* *p* = 0.0018) (Figure 4 and Table 5). These data demonstrate the ability of CRM to support the effect induced by CisPt treatment on P21 gene expression levels.

In conclusion, CisPt treatment led to an increase of the P21 gene expression level of in both lines. RSV treatment for 24 h had a different effect in PE-CA/PJ49 tumor cells (inducing an increase) versus the effect induced in normal HUVEC cells (induced a decrease) in P21 gene expression. CRM

alone amplified slightly P21 gene expression in both lines. Applied in combination with CisPt, CRM amplified the effect induced by 5 μM CisPt in both lines, the effect being twice as high in tumor cells. When the high concentration of 20 μM CisPt was used, the effects induced by CRM in tumor cells are the same as those recorded at CRM + 5 μM CisPt. However, in the case of normal cells, 20 μM CisPt + CRM treatment caused a drastic decrease in P21 gene expression, probably due to the toxic effect of CisPt, which killed a large number of normal HUVEC cells.

#### *4.5. E*ff*ects of the Natural Compounds (RSV,CRM) on Cell Cycle Phases Distribution in Cisplatin Treated Cells*

Cell cycle phases distribution was evaluated to further characterise the cytotoxic effect of CisPt and/or RSV, CRM on tumor cells PE-CA/PJ49 compared with HUVEC normal cells.

PE/CA-PJ49 tumor cells and HUVEC normal cells were treated with 5 μM or 20 μM CisPt in the presence or absence of 40 μM RSV or 15 μM CRM for 24 h. The progression through the cell cycle phases induced by treatment were evaluated by flow cytometry using a FACSCanto II flow cytometer. Data analysis were performed using the ModFit 3.2 program which offers the possibility of evaluating the cell cycle phases distribution (G0/G1, S, G2 + M).

The results obtained in the case of the tumor cells PE-CA/PJ49 revealed that single 5 μM or 20 μM CisPt applied treatment decreased of the phase G0/G1 (24.2% or 22.84% versus 41.24 in untreated cells) associated with a slight increase of the synthesis phase (50.5% or 49.6% versus 43.2% in control cells) and followed by the increase of G2+M phase (25.3% or 27.5% versus 15.5% in untreated cells) (Figure 5A,B). The effects induced by the 40 μM RSV treatment in PE/CA-PJ49 tumor cells shown a small decrease of G0/G1 phase (41.24 to 39.05%) associated with an increase in phase S (38.1 to 41.52%) and phase G2 + M (17.56 to 19.41%) compared to untreated cells (Control). Combined CisPt treatment with RSV applied to PE/CA-PJ49 induced an increase of G0/G1 phase (48.4% compared with the untreated cells 41.2%) and a decrease of the synthesis phase (34.1% versus 43.2% in control). Cell cycle phases distribution did not differ significantly when use different concentration of CisPt (5 or 20 μM) and RSV 40 μM are combined. Treatment of PE/CA-PJ49 tumor cells with 15 μM CRM alone or in combination with CisPt induced a decrease in synthesis phase (CRM = 38.6%; CisPt 5 μM + CRM = 33.6%; CisPt 20 μM + CRM = 35.14%; versus control = 43.2%) accompanied by an increase of the G2 +M phase (CRM=33.6%; CisPt 5uM + CRM = 32.2%; CisPt 20uM + CRM = 34.02%; versus control = 15.5%) (Figure 5A,B). These results suggested that the combined CisPt treatment with CRM induced a tendency to block the cell cycle in G2+M phase in tumor cells.

The HUVEC cells used as the normal line showed a distribution in the synthesis phase of less than 20% (untreated cells). The treatments applied individually 5 μM or 20 μM CisPt caused an increase of the synthesis phase to 29.2% and 39.65%, respectively, compared to 19.1% recorded in untreated cells (Control). These data show that treatment with CisPt affects normal HUVEC cells especially at higher concentrations. 40 μM RSV and 15 μM CRM did not appear to significantly affect the cell cycle when applied individually, and cannot influence the effect induced by CisPt treatment when used in combination (Figure 6A,B).

#### *4.6. E*ff*ects of the Natural Compounds (RSV,CRM) on the Apoptotic Process in Cisplatin-Treated Cells*

Apoptosis can be seen as an efficient method of preventing malignant transformation, as it ensures the removal of cells which present with genetic alterations. Deficient apoptosis can promote the development of cancer, both through the accumulation of cells found during division and the blocking of the removal of cells with high malignant potential genetic variations. The factors which determine the cells to follow one of three possible pathways are not yet known – the start of apoptosis after cellular injury, the repair of the lesion or the continuation of the cellular cycle. Many therapeutical agents have anti-tumoral effects generated by their capacity to activate the apoptotic process [68]. Tumoral cells PE/CA-PJ49 and normal cells HUVEC were treated for 24 h with CisPt, RSV, CRM in order to detect the effect induced by treatment with CisPt on tumor cells in the presence or absence of natural compounds (RSV or CRM). The flow cytometric analysis was performed using DIVA 6.2

software and allowed to discriminate viable cells (FITC−PI− = Q3) from necrotic cells (FITC−PI+ = Q1) and early apoptosis (FITC+PI− = Q4) from late apoptosis (FITC+PI+ = Q2).

**Figure 5.** The effect of treatments with CisPt, RSV, CRM applied independently or in combination on tumor cells PE/CA-PJ49 on cell cycle phases distribution. (**A**) flow cytometry histograms; (**B**) cell cycle phases distribution (%).

Analysis the RSV or CRM effects on the apoptotic process of tumor cells PE/CA-PJ49 is shown in the Table 6 and Figure 7. Our results show that the untreated tumor cells PE/CA-PJ49 (Control) had low total apoptosis (Q2 + Q4 = 2%). Treatment with 40 μM RSV induced a raise of total apoptosis to 27.2%, much higher than the total apoptosis induced by 5 μM CisPt (12.5%) or 20 μM CisPt (17.4%). In the case of simultaneous treatment with CisPt and RSV a raise of the total apoptosis was registered (5 μM CisPt + RSV = 31.1%; 20 μM CisPt + RSV = 34.9%) and it seems to have been generated by the presence of RSV.

**Figure 6.** The effect of treatments with CisPt, RSV, CRM applied independently or in combination on normal cells HUVEC on the cell cycle phases distribution. (**A**) flow cytometry histograms; (**B**) cell cycle phases distribution (%).

**Figure 7.** Natural compounds (RSV or CRM) effects on the apoptotic process of PE/CA-PJ49 tumor cells treated 24 h with CisPt and/or RSV or CRM (Dot-plot analysis).

Treatment for 24 h with 15μMCRM (29.6%) has determined an increase of the total apoptosis in the tumor cells PE/CA-PJ49. Combined treatment CRM with CisPt applied to the PE/CA-PJ49 cells for 24 h has determined a raise of total apoptosis to 28,7% when using 5 μM CisPt or to 40,3% when using 20 μM CisPt (Table 6 and Figure 7).

Effects of the natural compounds RSV or CRM on apoptosis process in normal HUVEC cells were analyzed after applying the same treatment scheme used in the case of PE/CA-PJ49 tumor cells. As shown in Table 7 and Figure 8, treatment with 5 μM or 20 μM CisPt, RSV or CRM did not significantly modify the apoptosis, compared to untreated HUVEC cells (Control). However, combined treatment led to an increase of the apoptotic process. Thus, 5 μM and 20 μM CisPt combined with RSV have determined apoptosis in 14% and 21.6% of the analysed cellular population, a value that was double the one obtained when using individual stimuli (Table 7 and Figure 8). Combined treatment CRM with 5 μM or 20 μM CisPt has induced a raise of apoptosis in 19,7% or 23.9% of the analysed cellular

population (Table 7 and Figure 8). Comparative analysis of the obtained data shows that both RSV and CRM have the capacity to stimulate the apoptosis of PE/CA-PJ49 tumor cells without significantly affecting the apoptotic process of normal HUVEC cells (RSV-PE/CA-PJ49 versus RSV HUVEC = 27.2% versus 5.1%; CRM-PE/CA-PJ49 versus CRM-HUVEC = 28.2% versus 7.2%). In conclusion, the two analyzed natural compounds (RSV and CRM) have the ability to amplify the apoptotic process induced by CisPt treatment in PE/CA-PJ49 tumor cells.

**Table 7.** Apoptosis of HUVEC cells induced by 24 h treatment with CisPt and/or RSV, CRM.

**Figure 8.** Natural compounds (RSV or CRM) effects on the apoptotic process on normal HUVEC cells treated 24 h with CisPt (Dot-plot analysis.).

#### **5. Discussion**

The toxic effects of CisPt are dose-dependent and affect the kidneys and bone marrow, which are accompanied by an increase in transaminases and serum creatinine. In addition, CisPt therapy alone has not been found to be effective in treating patients with HNSCC. Cisplatin efficacy appear to be more increased in combination with other chemotherapeutic agents and/or radiation therapy, but side effects or resistance to these drugs are often mentioned [69,70]. This observation suggests that the combination of cisplatin with other anti-tumor agents could be much more effective in the treatment and evolution of head and neck cancer. Some natural compounds due to their chemosensitizing potential, antitumoral activity and ability to reduce the side effects of drugs used in conventional cancer therapy protocols promise to be one of the important components of combinatorial therapy [71,72]. Natural compounds must be well tolerated and have long-lasting benefits. They can also act at the level of different signaling pathways responsible for the tumorigenesis process in HNSCC, which recommends them as agents with multiple molecular targets [73,74]. The experimental approch of this study focused on the enhancing chemotherapy response while lowering side effects and the incidence of drug resistance. To design novel combinatorial therapeutic strategy for improving the patients' prognosis and response to chemotherapy is needed. To streamline the response to chemotherapy, we analyzed how the use of CisPt in combination with CRM and RSV may influence some cellular processes such as proliferation, P21 gene expression, apoptotic process, and cell cycle development in HNSCC cell line (PE/CA-PJ49) compared to a normal cell line (HUVEC). The results showed that the viability of the tumor and normal cells was affected by CisPt treatment in the same way in both cell lines in a concentration-dependent manner. RSV or CRM treatment affected the viability of tumor cells PE/CA-PJ49 more than of normal HUVEC cells. RSV and CRM have the ability to amplify the inhibitory effect of 5μM CisPt induced on PE-CA/PJ49 tumor cells without significantly affecting the 5 μM CisPt-induced effect in normal cells. The use of a high concentration of CisPt (20 μM) together with a natural compound (CRM or RSV) led to the observation that the effect induced by CisPt on cell proliferation is dominant in both tumor cells and normal cells. Because p21 can act as a tumor suppressor or a tumor-promoting protein [75], and this makes more difficult to establish its function in the evolution of cancer we analysed the role of P21 in response to chemotherapy. Using ELISA and RT-PCR were evaluated protein and gene P21 expression in tumor PE/CA-PJ49 cells and normal HUVEC cells treated with CisPt in presence of RSV or CRM. A significant increase of total p21 protein expression was recorded in PE/CA-PJ49 tumor cells treated with 5 μM CisPt and 20 μM CisPt compared with the effect induced by RSV applied alone. However, it was observed that RSV can potentiate the induced effect of 5 μM CisPt but cannot influence the effects induced by 20μMCisPt treatment on p21 protein expression in tumor cells. In PE/CA-PJ49 the CRM treatment amplified the increase of p21 protein expression induced by CisPt treatment, regardless the CisPt concentration. Increased p21 expression was correlated with a descreased of proliferative activity of tumor cells and a good response to CisPt therapy.

Analysis of P21 gene expression level showed that CisPt treatment induced an increase of P21 expression in both lines. RSV treatment influenced in a different manner P21 expression, inducing an increase in PE/CA-PJ49 and a decrease in normal HUVEC cells. In combination with CisPt, RSV did not influence the CisPt-induced effect in HUVEC cells. On the contrary in PE/CA-PJ49 tumor cells, using the combination CisPt+RSV induced an increase in P21 gene expression and this way, RSV can modulate the effect induced by 20 μM CisPt. CRM alone did not modify the P21 gene expression in both lines. In combination with CisPt, CRM amplified the effect induced by 5 μM CisPt in both lines, the effect being higher in tumor cells. Not significant differences were observed in P21 expression in tumor cells when used the high concentrations of CisPt (20 μM). In normal cells was recorded a drastic decrease in P21 gene expression upon treatment with 20 μM CisPt + CRM. These data support the toxic effect of CisPt used in high concentration on normal cells. The treatment of tumor cells with RSV or CRM in the presence of CisPt induced an increase in protein and gene expression levels of p21. These results may be associated with a more effective response of tumor cells to treatment with lower concentrations of CisPt using RSV or CRM as therapeutic adjuvants.

The cell cycle ensures the cell can proliferate and grow by passing through the G1, S, G2 and M phases. Cancer cells reveal disorders in the cell cycle progression which contributes to exacerbate cell proliferation and to loss of genomic integrity. Treatment with 5 μM or 20 μM CisPt applied alone on tumor cells induced a slight transition from G1 to S phases accompanied by the increase of the G2+M phase (25.3% or 27.5% versus 15.5% in untreated cells). Cell cycle phases distribution did not differ significantly when different concentrations of CisPt (5 or 20 μM) were used. Treatment of PE/CA-PJ49 tumor cells with 40 μM RSV did not affect significantly the cell cycle phases compared to untreated cells. PE/CA-PJ49 cells treated with CisPt + RSV showed an increase of the G0/G1 phase and a decrease in the S phase. Treatment of PE/CA-PJ49 tumor cells with 15 μM CRM alone or in combination with CisPt induced a decrease the number of cells in S phase which was accompanied the massive blockage of cells in G2/M phase. In addition, the results provide evidence that RSV and CRM induce an increase in the apoptotic process of tumor cells. Interaction of RSV or CRM with CisPt in triggering tumor cells apoptosis indicated the amplifier role of the used natural compounds with respect to cisplatin effect. Therefore, associated chemotherapy of CisPt with natural compounds (RSV or CRM) as adjuvants might have a beneficial effect in decreasing the CisPt doses and in reducing its adverse reactions.

#### **6. Conclusions**

In conclusion, the results suggest that RSV and CRM act against proliferation and sustain the effects of cisplatin by the induction of cell cycle arrest, amplification of DNA damage and contributing to cancer cell destruction by increasing the apoptotic process. In addition, RSV and CRM amplify the expression of P21 in tumor cells and might contribute to increasing the sensitivity to cisplatin.

**Author Contributions:** G.G.P.-M. had equal contribution with M.B. in designed the research, data acquisition, analysis and interpretation of data, and wrote the manuscript. V.R., R.H. and C.D.S. performed the experiments, data acquisition, analysis and interpretation of data and manuscript drafting. N.R. and C.C.D. contributed to statistical analysis, critical revision of the manuscript for important intellectual content. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Grant PN-III-P1-1.2-PCCDI-2017-0341/2018 and Competitiveness Operational Programme (COP) A1.1.4. Grant ID: P\_37\_798 MyeloAL-EDiaProT, Contract 149/26.10.2016, (SMIS: 106774) and ERAPerMed\_Joint Transnational Call for Proposal (2019) for Personalised Medicine: Multidisciplinary Research Towards Implementation, BronchoBOC, Contract 140/2020.

**Acknowledgments:** Competitiveness Operational Programme (COP) A1.1.4. ID: P\_37\_798 MyeloAL-EDiaProT, Contract 149/26.10.2016, (SMIS: 106774), MyeloAL

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Dietary Supplements for Male Infertility: A Critical Evaluation of Their Composition**

**Andrea Garolla 1,\*, Gabriel Cosmin Petre 1,**†**, Francesco Francini-Pesenti 2,**†**, Luca De Toni 1, Amerigo Vitagliano 3,4, Andrea Di Nisio 1 and Carlo Foresta 1**

Received: 17 April 2020; Accepted: 18 May 2020; Published: 19 May 2020

**Abstract:** Dietary supplements (DS) represent a possible approach to improve sperm parameters and male fertility. A wide range of DS containing different nutrients is now available. Although many authors demonstrated benefits from some nutrients in the improvement of sperm parameters, their real effectiveness is still under debate. The aim of this study was to critically review the composition of DS using the Italian market as a sample. Active ingredients and their minimal effective daily dose (mED) on sperm parameters were identified through a literature search. Thereafter, we created a formula to classify the expected efficacy of each DS. Considering active ingredients, their concentration and the recommended daily dose, DS were scored into three classes of expected efficacy: higher, lower and none. Twenty-one DS were identified. Most of them had a large number of ingredients, frequently at doses below mED or with undemonstrated efficacy. Zinc was the most common ingredient of DS (70% of products), followed by selenium, arginine, coenzyme Q and folic acid. By applying our scoring system, 9.5% of DS fell in a higher class, 71.4% in a lower class and 19.1% in the class with no expected efficacy. DS marketed in Italy for male infertility frequently includes effective ingredients but also a large number of substances at insufficient doses or with no reported efficacy. Manufacturers and physicians should better consider the scientific evidence on effective ingredients and their doses before formulating and prescribing these products.

**Keywords:** fertility; ingredients; male reproduction; semen parameters; supplements

#### **1. Introduction**

Infertility is a pathological condition defined as the inability of a sexually active, non-contracepting couple to achieve pregnancy in one year [1]. Both male and female factors can lead to infertility. In particular, according to the causes, it has been reported that 29.3% is due to a male factor, 37.1% to a female factor, 17.6% to both male and female factors, with the remaining percentage considered as idiopathic [2].

It is estimated that around 10%–15% of all couples are affected by infertility, thus representing a global concern in most developed countries [3].

Among male infertility causes, many recent studies have emphasized the role of genital tract inflammation, incorrect lifestyles and malnutrition [4]. On this regard, weight excess and other conditions such as metabolic syndrome, alcohol abuse, cigarette smoking, exposure to environmental pollutants etc. have been strongly related to a decrease in sperm quality and fertility. A major driving hypothesis is that these conditions, by inducing an elevation of reactive oxygen species (ROS) and nitrogen species (RONS), are able to alter the balance of the redox status of both the steroidogenic cell population and the germ line cell populations, leading to the impairment of the hypothalamic–pituitary–testicular axis and the reduction of sperm quality [5].

A large number of recent studies have focused on the ability of many substances, generally termed as *nutraceuticals*, to improve the hormonal status and sperm parameters by different mechanisms [6]. Nutraceuticals are used as ingredients of dietary supplements (DS), widely marketed for the prevention or treatment of the most disparate pathological conditions. From a legislative point of view, the European Food Safety Agency (EFSA) defines that DS are not intended for the treatment or prevention of disease in humans, but only to support specific physiological function [7]. Currently, DS are widely prescribed to improve physiological aspects related to male fertility.

Many DS are available on the market with various formulations, containing both nutrients and botanicals at different doses. Despite many authors demonstrating positive effects of some ingredients on semen parameters and fertility outcomes [8], many others have also shown a lack of efficacy and even potentially harmful side effects [9]. In a recent position statement, the Italian Society of Andrology and Sexual Medicine (SIAMS) summarized the state of the art on each single ingredient currently used in the andrological field. In this paper, authors concluded that there was still limited scientific evidence on the possible role of any nutraceutical in andrology and the use of antioxidants could be suggested in patients with idiopathic infertility in the presence of documented abnormal sperm parameters only after a specific diagnostic workup. However, to date, no regulation or guidelines are available for the use of these products, generating confusion for both prescribers and patients [10]. Moreover, several factors make it difficult to empirically address the right ingredient for the right patient. In particular, it is difficult to identify the correct DS since each product contains different ingredients at different doses.

The purpose of this study was to critically evaluate the composition of DS employed in male infertility, using the Italian market as a sample.

#### **2. Materials and Methods**

In order to evaluate the potential efficacy of DS, a systematic literature review on substances used to improve sperm parameters was preliminarily performed. The literature search was conducted in MEDLINE, Scopus, EMBASE, and Cochrane Library registers until 31 March 2020. Only randomized clinical trials (RCTs) and systematic reviews or meta-analysis of RCTs were considered eligible. With the aim to rule out possible interactions between ingredients, only studies that used active substances alone or in combination with at most the other three ingredients were considered. The key terms used for the search were: fertility or male reproduction or semen parameters and supplements or ingredients. Figure 1 displays the flow diagram of the selection of eligible papers.

To establish the efficacy of each ingredient we considered only those having at least one RCT or systematic review or meta-analysis of RCTs, demonstrating a significant effect on any sperm parameters involved in male fertility. Significance was set at *p*-value < 0.05. When evaluating the findings of meta-analyses, we verified whether statistical methods incorporated substantial heterogeneity (Higgins I2 > 30%) into a random-effects model, as appropriate. Regarding the daily dose of each active ingredient with nutrient characteristics, we referred to the tolerable Upper intake Levels (UL) as reported in Dietary Reference Intake (DRI) [11].

**Figure 1.** Flow diagram of the selection of eligible papers.

Based on the results of available articles, we were able to identify, for each active ingredient, the minimal effective daily dose (mED) able to improve sperm parameters. To define mED we used the lowest effective dose reported in RCTs, systematic review or meta-analysis of RCTs. Therefore, we classified the ingredients contained in each supplement and suggested daily dose into three categories: reported efficacy with a dose achieving the mED (A), reported efficacy but with a dose below mED (B) and unreported data of efficacy (C). To classify DS, we created a formula taking into consideration the three classes of ingredients and their number:

$$Score = \frac{(2A + B - C)}{2N} \times (A + B/2)^2$$

In particular, the above formula was conceived based on the following sequential steps:

efficacy) the number of moderate efficacy ingredients (*A* + *B*/*2*), finally obtaining a corrected score for each supplement.

(5) Given the distribution of the scores resulted in three main clusters, we classified DS into three categories, resembling the efficacy of the ingredients: higher expected efficacy (corrected score ≥ 4), lower expected efficacy (4 < corrected score > 1) and no expected efficacy (corrected score ≤ 1).

We collected the names and formulations of the DS registered in Italy by referring to the register of the Italian Ministry of Health [12].

#### **3. Results**

The literature search on active ingredients allowed us to identify 41 studies (RCTs or meta-analyses) reporting their efficacy on sperm parameters (Figure 1). By this analysis we found that 18 of these ingredients had a reported efficacy. The complete list of ingredients with clinical evidence of efficacy, the respective references, evaluated sperm parameters and employed daily doses, are summarized in Table 1. In the right column, the mED of each ingredient is reported. In some studies, marked with an asterisk, the employed dose exceeded the reported UL. In particular, all the studies involving zinc evaluated the effect of this ingredient at a dose exceeding UL. For each active ingredient, the evidence of efficacy was supported by at least two RCTs or meta-analysis, excluding astaxanthin, D-aspartic acid and L-citrulline, which had only one reference.

**Table 1.** Active ingredients with evidence of efficacy, references, evaluated sperm parameters, employed daily doses and minimal effective dose (mED).

**Table 1.** *Cont.*

\* The employed dose exceeded/reach UL. LC: L-Carnitine; LAC: Acetyl L-Carnitine; EPA: Eicosapentaenoic acid; DE: Dry Extract; DHA: docosahexaenoic acid. Rev: Review; Met: Meta-analysis.

Ingredients without clinical evidence in the improvement of sperm parameters (no RCT or meta-analyses) are listed in Table 2.

> Astragalus DE Damiana DE Nettle DE Catuba DE Ecklonia bicyclis DE L-Taurine Glutathione Glucosamine SOD Vitamin D3 Vitamin B1 Riboflavin Niacin Vitamin B5 Vitamin B6 Biotin Manganese

**Table 2.** Ingredients without clinical evidence of efficacy.

DE: Dry Extract; SOD: super oxide dismutase.

We found 21 DS marketed in Italy for male infertility. Their composition and the daily doses of their active ingredients are summarized in Table 3. Moreover, for each supplement, the scores of expected efficacy and the symbols summarizing the efficacy of their ingredients are reported.

#### *Nutrients* **2020** , *12*, 1472

**Table 3.** *Cont.*

**Table3.***Cont.*

score of supplement's potential efficacy; EV: efficacy value of active ingredients; evidence of ingredients and dose efficacy: (A) reported efficacy and achievement of mED, (B) reportedefficacy but below mED and (C) unreported efficacy. DE: Dry Extract; SOD: super oxide dismutase; DHA: docosahexaenoic acid.

#### *Nutrients* **2020** , *12*, 1472

A detailed analysis of this table raised the following considerations: (i) all supplements were mixtures of active ingredients; (ii) in each supplement the number of ingredients ranged from 2 up to 17, with a mean number higher than 7; (iii) 13 of 21 supplements contained at least one ingredient without reported efficacy; (iv) 19 supplements had ingredients below mED; (v) indeed, 1 supplement contained seven ingredients dosed below mED; (vi) 1 supplement contained only active ingredients satisfying mED; (vii) the product number 9 had a nutrient reaching UL (zinc 40 mg/day); (viii) zinc was the most used ingredient, followed by selenium, arginine, coenzyme Q, folic acid and carnitine. These substances were present in more than 50% of DS, whereas all the remaining ingredients were represented in 10% or less of products.

The distribution of DS into the three classes of efficacy is reported in Figure 2. Two DS out of 21 (9.5%) were included in the higher expected efficacy group. The majority of remaining products (71.4%) fell in the lower expected efficacy group, and four (19.1%) in the group with no efficacy.

**Figure 2.** Distribution of supplements in classes of expected efficacy. \* This supplement has a content of Zinc reaching the UL. Numbers refer to a specific supplement.

#### **4. Discussion**

This critical review aimed to evaluate the formulation of supplements for male infertility using the Italian market as a sample. In general, there is still poor evidence in terms of large well-designed randomized and placebo-controlled trials availability, supporting the efficacy of nutraceutical products in the field of male reproductive health [54,55]. Nevertheless, these products are commonly administered to infertile patients [8,56]. Since a medical prescription is not necessary to purchase dietary supplements, subjects seeking fertility may have easy access to these products [10,57]. As a proof of concept, the Italian market of supplements generated 3.3 billion euros in 2019, with an increase of 4.3% compared to 2018 [58].

Whilst a rational use of supplements may be potentially beneficial for the improvement of sperm parameters, we need to stress that their uncontrolled use is potentially harmful for patients' health due to direct toxic effects and interaction with drugs or nutrients [59]. In this respect, we were surprised to point out that all RCTs and meta-analyses on zinc for male infertility relied on doses always exceeding the UL. Over this background, in the near future it would be desirable to better define thoughtful criteria for each supplement in use.

Our analysis found that beside the gap of literature, the market of food supplements is still supported by poor scientific evidence. The majority of DS contained a huge number of ingredients, up to 17. The mixture of such a high number of ingredients may generate different issues, including a low concentration of each substance (i.e., necessitating of two or more administrations to reach the daily effective dose), a large volume of pills and a high risk of interactions. What is more, we found that some ingredients included in many DS had no scientific evidence of efficacy (i.e., astragalus, vitamin D3, taurine and riboflavin). The formulation of pills with a large number of ingredients, some of which cause uncertain benefits, denotes a gap of knowledge of potential biologic targets by manufacturers. Moreover, it has been reported that some plant extracts, present in many of these supplements, are likely to interact with drug metabolism [60,61]. This aspect raises further concerns on the safety of these products.

Very frequently, nutrients were present in DS at a dosage below mED. This situation was more common among products with a high number of ingredients. The administration of any active substance with a dose below mED appears as scientifically unjustified due to uncertainties in the therapeutic results. Differently, when the number of ingredients was small, the dose often satisfied mED. Another major aspect in the evaluation of supplements concerns safety. Some ingredients, particularly when administrated in high doses, are not free from risks when used as dietary supplements. For example, folates can mask the B12 deficiency favoring the progression of neurological damage [62]. The combination of these two vitamins could have a synergic effect in improving homocysteine metabolism hence the sperm quality. It should be noted that vitamin B12, when present, was rarely associated to folic acid [63,64]. Furthermore, zinc reduces the copper intestinal absorption interfering with its carrier [65]. With respect to this, we want to stress that one supplement on the market contained a dose of zinc reaching the UL.

On a positive note, our analysis revealed that some active ingredients with reported efficacy are frequently present in analyzed supplements. Previous studies demonstrated that some ingredients are particularly effective in specific patients' conditions. Substances with antioxidant properties are indicated in inflammation of the male accessory glands, both related to microbial and non-microbial origin. Several studies performed in asthenozoospermic infertile patients, showed that the positive effect of selenium supplementation is dependent on the correct structure of the mitochondrial capsule [66,67]. Carnitine supplementation induced a significant increase in sperm motility in cases of asthenozoospermia with preserved mitochondrial function [68,69]. Due to the key role of zinc in the processes of DNA compaction, administration of this micronutrient was successful in improving sperm morphology and DNA integrity in patients with prostate abnormalities [70,71].

Based on active ingredients reaching mED we created a grading scale of supplements distinguishing three classes of expected efficacy. Three products were present in the higher class, some of which contained ineffective or underdosed ingredients. Most of the supplements fell in the lower group of expected efficacy. In this class, a large number of ineffective or underdosed products were also present. For an adequate evaluation of these classes, we considered the number of the effective ingredients as the most important criterion of efficacy. A relevant aspect was the use of ineffective or underdosed ingredients that should be absent or less than possible. Another parameter to evaluate a product was the presence of a lower number of ingredients.

We acknowledge the application of a non-validated statistical method to calculate scores for each DS may represent a point of weakness in this study. Very recently, a validated formula to score supplements was suggested by Kuchakulla et al. [72], based on the Budoff's score, previously conceived by cardiologists to evaluate their procedures [73]. However, this scoring system when applied to DS, does not take into account the effective dose of ingredients, a crucial point in the evaluation of their efficacy. For example, using this approach, a DS containing ingredients at ineffective or toxic doses would be considered useful. As a point of strength, our scoring system relied on high quality evidence coming from RCTs or a systematic review and meta-analyses of RCTs, which represents a reliable approach to critically weighing the expected efficacy of dietary supplements. The same approach could be applied to evaluate products used in other clinical conditions.

In conclusion, this study showed that most DS marketed in Italy for male infertility contain ingredients with reported efficacy in the improvement of sperm parameters. Nevertheless, a non-negligible number of DS are mixtures of substances with uncertain or unreported benefits, whose administration may be unhelpful or even harmful for infertile patients. On that basis, we believe manufacturers should carefully scrutinize scientific evidence before delivering each supplements' formulation. Accordingly, physicians should evaluate the composition of DS and the dose of each single constituent before considering their clinical use. Finally, the choice for DS should be tailored to the specific patient's fertility problem.

**Author Contributions:** A.G. and G.C.P. contributed to the conception/design of the research and acquisition/analysis of the literature data; A.G., G.C.P. and F.F.-P. equally contributed and drafted the manuscript; A.D.N. concepted and performed data analyses; L.D.T., A.V. and C.F. critically revised the paper for important intellectual content. All authors revised and approved the final manuscript, and agreed to be fully accountable for ensuring the integrity and accuracy of the work. All authors had full access to all the data in the study and are able to take responsibility for the integrity of them and the accuracy of the analysis.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors thank Marco Ghezzi for helpful discussion.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* ω**-3 and** ω**-6 Polyunsaturated Fatty Acids, Obesity and Cancer**

#### **Stefania D'Angelo, Maria Letizia Motti and Rosaria Meccariello \***

Dipartimento di Scienze Motorie e del Benessere, Università di Napoli Parthenope, via Medina 40, 80133 Napoli, Italy<EMAIL_ADDRESS>(S.D<EMAIL_ADDRESS>(M.L.M.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +39-081-5474668

Received: 5 August 2020; Accepted: 7 September 2020; Published: 10 September 2020

**Abstract:** Recently, nutraceutical bioactive compounds in foods have been discovered for their potential health benefits regarding the prevention of chronic disorders, such as cancer, and inflammatory, cardiovascular, and metabolic diseases. Dietary omega-3 polyunsaturated fatty acids (ω-3PUFAs), including alpha-linolenic acid, docosapentaenoic acid, and eicosapentaenoic acid, are mostly attractive. They are available for the customers worldwide from commonly used foods and/or as components of commercial food supplements. The anti-inflammatory and hypotriglyceridemic effects of these fatty acids are well known, whereas pro-inflammatory properties have been recognized in their dietary counterparts, the ω-6PUFAs. Both ω-3 and ω-6PUFAs contribute to the production of lipid mediators such as endocannabinoids that are notably involved in control of food intake, energy sensing, and food–related disorders. In this review, we present ω-3 and ω-6PUFAs and their derivatives, endocannabinoids; discuss the anti-obesity effects of ω-3PUFAs; their roles in inflammation and colorectal cancer development; and how their action can be co-preventative and co-therapeutic.

**Keywords:** obesity; ω-3PUFAs; ω-6PUFAs; endocannabinoids; CRC; fatty acids

#### **1. Introduction**

The prevalence of obesity has increased worldwide. Obesity represents a major health challenge because it substantially increases the risk of comorbidities, including cardiovascular disease, hypertension, type 2 diabetes, dyslipidemia, nonalcoholic fatty liver disease, obstructive sleep apnea, and some cancers, thereby contributing to a decline in both quality of life and life expectancy [1]. This syndrome is a complex condition involving social, biological, and psychosocial factors. The genesis of obesity is multifactorial: it is characterized by chronic low-grade inflammation, primarily due to an imbalance between the production/secretion of pro-inflammatory cytokines vs. anti-inflammatory cytokines [2]; deregulated lipid and glucose metabolism in metabolic organs is thought to be a critical factor. High-calorie diets and sedentary lifestyles are the most important factors in the development of obesity. As a consequence, global anti-obesity strategies focus on dietary and lifestyle modifications. In fact, weight loss, energy restriction, and nutrient dense diets can restore this imbalance, at least in part [3]. Therefore, the most used approaches aim at suppressing appetite, normalizing lipid metabolism, and increasing energy expenditure [4], through limitation of sugar and fat consumption, promotion of physical activity, consumption of fruits and vegetables, and pharmacological approaches.

Dietary interventions using natural bioactive food compounds have emerged as promising therapeutic tools for metabolic diseases, with limited deleterious side effects. Composition of the diet may affect metabolic and endocrine functions and overall energy balance [5]. Studies conducted in both animal models and humans support the assertion that dietary bioactive compounds can increase energy expenditure and thermogenesis, providing benefits in preventing/limiting obesity. Most health recommendations emphasize diets rich in fruits and vegetables, which have lower caloric density and higher nutrient density [6]. Such diets would provide significant amounts of phytochemicals, bioactive components with nutraceutical effects due in part to their anti-oxidant and anti-inflammatory properties [7]. Natural bioactive compounds, for example, the polyphenols (epigallocatechin, resveratrol, curcumin, quercetin, oleuropein, anthocyanins, ellagic acid, and others), have been studied as factors with possible indirect or direct impacts on specific molecular pathways, associated with the pathophysiologies of different syndromes [8,9] due to their well-documented anti-oxidant [10–12], anti-proliferative [13–16], anti-inflammatory, anti-cancer, anti-aging, and anti-obesity effects [17–21]. In addition to polyphenols, other nutraceuticals with an anti-obesity effects are the dietary ω-3 polyunsaturated fatty acids (PUFAs), which can act on adipose tissue inflammation, in contrast to omega-6 (ω-6) PUFAs, which exhibit pro-inflammatory properties. Both ω-3 and ω-6PUFAs contribute to the production of lipid mediators such as endocannabinoids, which are notably involved in control of food intake, energy sensing, and food–related disorders; reproduction; inflammation; the stress response; and cancer, among other things [22–28].

Therefore, in this review article we present ω-3 and ω-6PUFAs and their derivatives, endocannabinoids; discuss the anti-obesity effects of ω-3PUFAs; their role in inflammation and colorectal cancer (CRC) development; and co-preventative and co-therapeutic applications.

#### **2.** ω**-3. and** ω**-6PUFAs**

The amount and type of dietary fat in the diet are important factors influencing adipose tissue function and whole-body metabolism, with important health ramifications. Fatty acids (FA) are hydrocarbon chains with a carboxyl group at one end and a methyl group at the other. FA species are classified by their varying degrees of saturation into three main classes: saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) [29].

SFAs have a simple carbon chain containing no double bonds; MUFAs contain one double bond; and PUFAs are classified as carbon chains containing two or more double bonds. The variations in the chemical structures of these diverse classes can lead to dissimilar physiological activities. For example, SFA has been linked to the development of metabolic dysfunction; contrariwise, MUFAs and PUFAs have helpful activities in metabolism [30].

PUFAs are additionally classified into ω-3 and ω-6 groups, based on the position of the first double bond from the methyl end of the fatty acid. The structural dissimilarities of these FAs also give rise to functional differences, in terms of their actions on inflammation and metabolism [5].

ω-3 FAs are PUFAs with more than one carbon-carbon double bond in their backbones. They are polyunsaturated because their chains have numerous double bonds. One way in which a FA is named derives from the position of the first double bond, counted from the tail, that is, the omega (ω-) or the n-end. Thus, in ω-3 FAs, the first double bond is between the third and fourth carbon atoms from the tail end; then, they have a double bond at the third carbon from the methyl end of the carbon chain.

Humans do not own the essential ω-3 desaturase to add a double bond at the 15th carbon of a long chain FAs, and are, therefore, unable to endogenously synthesize α-linoleic acid (ALA 18:3n-3) and linoleic acid (LA 18:2n-6), making them vital FAs.

In addition to ALA, ω-3 PUFAs can be defined as a heterogeneous mix of FAs, among which eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DHA, 22:6n-3) are presently thought to be the most bioactive of the ω-3 species; however, docosapentaenoic acid (DPA, 22:5), an intermediate of EPA and DHA, may also have positive health properties [30,31].

ω-6PUFAs are also essential fatty acids and normally have metabolically distinct properties to ω-3PUFAs. While the human body cannot synthesize ω-3 and ω-6PUFAs, it does have the capability to further metabolize these FAs through stages of elongation and desaturation. ALA can be metabolized to EPA and DHA by Δ6 desaturase and Δ5 desaturase correspondingly, while LA is transformed to arachidonic acid (AA 22:4n-6). However, the change of ALA to DHA is very inefficient with <10% transformation in females and <3% in males [30,32]. While ALA is the favorite substrate for

Δ6 desaturase, plenty of dietary linoleic acid has been observed to suppress the change of ALA to DHA, which may be a confounding factor in these data. Evidence suggests that supplementing with stearidonic acid (18:3n-3) may increase the efficiency of transformation to DHA, demonstrating Δ6 desaturase as a rate limiting step. There is also a degree of individual change in the lipidome following ω-3 integration in humans, which may be a factor in the equivocal metabolic conversion measured in many human integration trials [30,33].

The structural differences of the FAs give rise to functional dissimilarities, in terms of their actions on inflammation and metabolism. For example, due to the pro-inflammatory actions of saturated FAs, intake of these molecules is associated with an increase in cardiovascular disease risk. In contrast, ω-3PUFAs have anti-inflammatory capability, and their intake is connected to a decrease of in cardiovascular disease risk. Dietary FAs are involved in glucose-insulin homeostasis and modulating adipose tissue properties [5]. High saturated-fat intake causes adipose tissue inflammation and obesity in mice; those effects can be partially reduced when these high-fat diets are energy-restricted [29]. Instead, dietary EPA supplementation ameliorates adipose tissue inflammation, regardless of adipose tissue mass. These data, taken as a whole, demonstrate the importance of AF in modulating the adipose tissue properties [34].

Saturated FA, generally, contributes to adipose tissue inflammation, probably due to TLR-2 and TLR-4 activation, and switching of downstream pro-inflammatory signaling pathways comprising the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway [29]. In contrast, ω-3 FA, primarily DHA and EPA, mitigate adipose tissue inflammation in diverse animal models of obesity [34]. Compared to ω-6PUFAs such as AA, ω-3PUFAs generate less eicosanoids with inflammatory properties. Additionally, ω-3PUFAs competitively decrease AA-mediated inflammatory eicosanoid prostaglandin E2 (PGE2) synthesis. As a whole, food sources rich in healthy fats can provide health benefits in both the long and short term.

Figure 1 summarizes the essential FAs and their dietary sources. In general, the primary source of ω-3PUFAs in the human diet is marine products, in particular phytoplankton that enters at multiple levels in the food chain [34], fatty fish, and cod liver oil, food rich in DHA and EPA [35,36].

**Figure 1.** Essential fatty acids and dietary source (AA: arachidonic acid; ALA: α-linolenic acid; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; LA: linoleic acid; PUFA: polyunsaturated fatty acid).

In plants, ALA can be extracted from seeds such as flaxseed (linseed), green leafy vegetables, legumes, and nuts. Vegetable oils such as sunflower, corn, perilla, canola, and soybean are the principal sources of LA, and they provide a small amount of ALA [31].

#### *2.1. Endocannabinoids: PUFAs Derivatives Involved in the Central and Peripheral Control of Food Intake*

The endocannabinoid system (ECS) comprises lipid mediators capable of binding to and activating cannabinoid receptors—traditionally, the central and the peripheral cannabinoid receptor, CB1 and CB2 respectively, membrane G-coupled receptors originally found to be mainly expressed in the brain and peripheral tissues. The system also includes a large set of biosynthetic and metabolizing enzymes and transporters [37]. Since the discovery in the 1990s of the first endocannabinoids, anandamide (AEA) and 2-arachinonoylglycerol (2-AG), the relevance of ECS has been progressively widening due to the inclusion in the system of non-canonical cannabinoid receptors (i.e., the transient receptor potential cation channel subfamily V member 1 (TRPV1); the orphan G-coupled receptors GPR18, GPR119, and GPR55; and also peroxisome proliferator-activated receptors (PPARs) such as PPARα and γ), the discovery of several "endocannabinoid-like" compounds, and the large spectrum of biological functions [38]. Currently ECS represents a conserved, widely-expressed signaling system involved in the control of most biological activities within the brain and peripheral tissues, from food intake to reproduction, the immune response, and cancer, among others [22–28,39], and it is susceptible to epigenetic modulation by diet [40].

Traditionally, AEA and 2-AG are the N-ethanolamide and the glyceryl ester of ω-6PUFA AA respectively, and represent the main endogenous ligands of CB1 and CB2, with AEA having low CB2 affinity, and 2-AG capable of binding both receptors [41]. The endocannabinoids N-docosahexaenoyl ethanolamine (DHEA) and N-eicosapentanoyl ethanolamine (EPEA) are ω-3 DHA and ω-3 EPA derivatives respectively; docosahexaenoylglycerol (DHG) and eicosapentaenoyl glycerol (EPG) are the glycerol esters of ω-3 DHA and EPA derivatives and have been discovered following the investigations on 2-AG analogues. While ω-6 AA derivatives AEA and 2-AG bind the canonical receptors [41], ω-3 EPA/DHA derived endocannabinoids, and endocannabinoid-like compounds exhibit lower affinity binding to CB1/CB2 than AEA/2-AG, and in some cases bind the aforementioned non canonical cannabinoid receptors [42]. Currently, ω-3 derived endocannabinoids are known at lesser extent than canonical ones, and their basic characterizations support possible involvement in inflammation, neuroprotection, and cancer [42]. Details concerning the metabolic/hydrolyzing pathways linking ω-3 and ω-6, endocannabinoids, and inflammatory mediators are summarized in Figure 2.

Endocannabinoids, via CB1, have a recognized role as orexigenic factors, in that they stimulate food intake and body fat deposition [26,43]; at the periphery ECS activity is largely reported in the gastrointestinal tract, and liver and adipose tissue, along with possible involvement in the microbiota–gut–brain axis and obesity onset, as excellently reviewed in [23]. Obese subjects display high endocannabinoid tone in the plasma and brain; furthermore, altered expression of CB1 and higher endocannabinoid levels in the muscle, adipose tissue, pancreas, and liver have been reported ([23] for a recent review). Consistently, CB1 activation increases food intake [44], whereas its pharmacological and genetic impairment reduces food assumption, protecting against the development of obesity, liver steatosis, and related inflammation [45,46]. Traditionally endocannabinoid signaling via CB1 is involved in the central control of food intake exerting its activity within the hypothalamus. This brain region catches and integrates the environmental cues, including fuel availability, in order to maintain energy homeostasis [47], projects neuronal networks towards different nuclei within the hypothalamus or in the brain stem controlling both the homeostatic regulation of energy balance, and biological functions deeply related to energy homeostasis and availability, such as reproduction [26]. Appetite inhibiting neuropeptides, such as proopiomelanocortin (POMC) precursor and cocaine-amphetamine regulated transcript (CART), and appetite stimulating neuropeptides such as neuropeptide Y (NPY), melanin-concentrating hormone (MCH), and agouti related protein (AgRP), are produced within the hypothalamic arcuate nucleus (ARC) to mediate the adaptive changes in food intake and energy expenditure in response to nutrient availability and peripherally produced "metabolic sensors" (i.e., glucose from liver, interleukin-6 (IL-6) from muscle, leptin from white adipose tissue, insulin, amylin and pancreatic polypeptide from pancreas, glucagon-like peptide-1, gherlin, cholecystokinin and peptide YY from the gastro-intestinal tract, and lastly gut microbiome-derived signals) [23,26,47]. ECS activity may be affected by metabolic sensors and may modulate neuronal population producing orexigenic/anorexigenic-peptides [26]. Among "metabolic sensors," leptin is the major peripheral indicators of body metabolic reserves acting as satiety signal [48]. In the natural mutant mice for *Leptin* gene, the Ob/Ob mice, over activation of ECS has been reported in the hypothalamus whereas leptin inhibits the hypothalamic activity of ECS [49]. Additionally leptin resistance, a condition causing food intake alteration and consequent obesity development, has been linked to the over-activation of the ECS [49], with a sex-specific epigenetic modulation of *cnr1*, the gene encoding CB1, following maternal high fat diet (HFD) [50]. Therefore, CB1 antagonist-based therapy has been used for the treatment of obesity, but, in spite the promising anti-obesity effects, the treatment caused severe psychiatric side effects and has been discontinued in patients [51].

**Figure 2.** A schematic representation of the metabolic/hydrolyzing pathways linking ω-3 and ω-6, endocannabinoids, and inflammatory mediators. The syntheses of DHEA and EPEA, and AEA from ω-3 and ω-6 PUFAs respectively, requires the activity of N-acetyltransferase (NAT) followed by N-acyl phosphatidylethanolamine-specific phospholipase D (NAPEPLD). The synthesis of 2-AG from ω-6 PUFAs requires the subsequent activity of phospholipase Cβ (PLCâ) and diacylglycerol lipase (DAGL). The hydrolysis of the endocannabinoids requires the activity of the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). The activity of lipoxygenases (LOX), cyclooxygenase (COX), and cytochrome P450 enzymes (CyP450) drives the production of inflammation mediators. AA: arachidonic acid; AEA: anandamide; 2-AG: 2-arachinonoylglycerol; DHA: docosahexaenoic acid; DHEA: N-docosahexaenoyl ethanolamine; EPA: eicosapentaenoic acid; EPEA: N-eicosapentanoyl ethanolamine; Mar1: maresin 1; NPD1: neuroprotectin D1; PUFA: polyunsaturated fatty acid; RvD1: resolvin D1.

A deep interplay between the synthesis and actions of the AA, DHA, and EPA-derived endocannabinoids and ECS occurred, as recently reviewed [52]. In this respect, many authors suggest that dietary PUFAs and in particular the ω-6/ω-3 ratio may affect the endogenous tone of endocannabinoids with consequences on health [26,53]. For example, in animal models fed for three or four months with a ω-3 deficient diet, low DHA levels in the brain with effects on ECS and synaptic plasticity have been discovered [54,55]; similarly, long-term EPA and DHA supplementation reduces AEA and 2-AG levels, with reciprocal increases in levels of the corresponding endocannabinoid-like EPA- and DHA-derived molecules [52].

Lastly, dietary intervention may epigenetically affect ECS in animal models, and cell lines [40]; and at present, a recurrent process potentially influencing the development of eating disorders such as binge-eating has been related to the epigenetic modulation of FAAH [56], the gene encoding for the main endocannabinoid hydrolyzing enzyme.

#### **3. The Antiobesity E**ff**ects of** ω**-PUFAs**

DHA and EPA exert numerous beneficial effects, and they act as natural hypolipidemics, decrease risk of cardiovascular syndromes and could prevent the progress of insulin resistance and obesity [57].

Human dietary intervention trials suggest that fish oil (EPA and DHA) supplementation might decrease waist circumference. ω-3PUFAs decrease adiposity by numerous effects. For example, DHA and EPA start AMP-activated protein kinase (AMPK), which in turn actives FA β-oxidation in adipose tissue [5]. DHA and EPA are also promoting mitochondrial biogenesis, which can increase energy metabolism [58].

In rodents, DHA and EPA also raise FA oxidation in the small intestine and liver in in vivo experiments. DHA and EPA prevent hepatic lipogenesis in an AMPK and PPARα-dependent manner [5]. PPARs are transcription factors that form heterodimers with retinoid X receptors in the promoter regions of different genes implicated in glucose and lipid e metabolism [59]. PPARγ works as a main regulator of adipogenesis and checks numerous genes and adipokines in glucose and lipid metabolism. ω-3PUFAs work as ligands for PPARγ, and it has been observed that PPARγ plays a evident role in the capability of ω-3PUFAs, particularly DHA, to prompt M2 macrophage polarization and thereby decrease inflammation since these data are not detected in PPARγ-knockdown RAW264.7 cells [60]. The EPA- and DHA-mediated increment in FA oxidation and decrease in lipogenesis could be accountable for their anti-obesity actions [61].

In Figure 3, some signaling mechanism-mediating effects of ω-3PUFAs are summarized.

DHA and EPA may act as anti-inflammatory agents directly. EPA improves adipose tissue inflammation and decreases insulin resistance. The ameliorations in adipokines' profiles are characterized by rises in anti-inflammatory adipokines, such as adiponectin, and reductions in pro-inflammatory cytokines, such as IL-6, tumor necrosis factor-alpha (TNF-α), monocyte chemo attractant protein 1 (MCP-1), and plasminogen activator inhibitor 1 (PAI-1). EPA and DHA's action of normalizing plasma adiponectin concentrations appears to be largely responsible for their insulin sensitizing action. This favorable activity on adiponectin secretion seems to be PPARγ-dependent, because fish oil fails to raise plasma adiponectin in PPARγ-null mice [62].

Prostaglandins, eicosanoids with pro-inflammatory action, are secreted by adipocytes. AA-originated eicosanoids such as thromboxane A2 and PGE2 possess stronger inflammatory action than EPA-originated ones. Since EPA contends with AA for incorporation into cell membranes, it is possible that enhancement dietary EPA intake decreases synthesis of AA-originated eicosanoids. Indeed, EPA hinders AA-induced secretion of PGE2 from 3T3-L1 adipocytes in vitro [63].

**Figure 3.** Some signaling mechanisms mediating effects of ω-3PUFAs (AMPK: AMP-activated protein kinase; FFAR: free fatty acid receptor; IL-: Interleukin-; MCP-1: Monocyte chemo attractant protein 1; NF-kB: nuclear factor-κB; PGE2: Prostaglandin E2; PPAR: Peroxisome proliferator-activated receptor; PUFA: polyunsaturated fatty acid; TNF-α: Tumor necrosis factor-alpha).

A close link was observed between inflammatory markers, BMI, and body fat percentage. NF-κB, a key transcription factor in gene expression and cytokine inflammation is inhibited by ω-3 PUFAs. Studies in humans and in vitro have shown that ω-3PUFAs are involved in the reduction of cytokines, such as IL-1, IL-6, and TNF-α, whose concentrations are high in cases of obesity [64]. The ω-3PUFAs behave as agonists for numerous free fatty acid receptors (FFARs) typical of different cell types, involved in both the inflammatory response and energy homeostasis. Some unsaturated and saturated long-chain fatty acids can activate FFAR1 and FFAR4 [65]. For example, FFAR4 stimulation prevents lipopolysaccharide (LPS)-mediated release of inflammatory cytokines, such as TNF-α and IL-6 in the macrophage-type RAW264.7 cell line [64].

Current data show that reduction of inflammation is an active process. EPA and DHA-derived resolvins and protectins are key examples of inflammation resolution agonists. Experiments involving treatment with resolvins or transgenic restoration of protectins have shown a slowdown of the adipose tissue macrophage infiltration, and enhanced insulin resistance in rodents. Secretion of these mediators could be another mechanism by which DHA and EPA ameliorate the inflammation in adipose tissue [5].

Human clinic trials have been organized to evaluate the effects of the intake of ω-3PUFAs (using as food different types of fish with different contents of DHA and EPA) on the variation of composition and body weight, and also on the evaluation of the caloric content of food intake. Fish oil and fishes have also been used in dietetic interventions of different duration and with or without associated physical activity, to evaluate a possible weight loss.

Participant-reported diet diaries show evident decreases in fat, carbohydrate, and total caloric intake with ω-3PUFAs integration [66], but others reported no variation in energy intake [29,67]. Since most trials only indicated total caloric intake, the action of ω-3PUFAs integration on macronutrient and energy intake should be repeated in larger trials to conclusively establish the action of these PUFAs on weight reduction in humans. Weight reduction data appear more encouraging when ω-3PUFA

integration and calorie restriction are combined, but it is problematic to draw deductions due to the diversity of calorie limit programs in dissimilar trials. Combined ω-3PUFAs supplementation and calorie restriction compared to calorie restriction alone or replacement of saturated fatty acids determined a major amelioration in insulin resistance and reduction of TGs [68,69]. ω-3PUFAs could decrease body weight, thereby improving the metabolic profile through various mechanisms: alteration in adipokines release; modification of gene expression in adipose tissue; adipokine-mediated or adipokine-connected pathways; variation in carbohydrate metabolism; appetite suppression; rise in fat oxidation; intensification in energy expenditure (probably by thermogenesis); initiation of mechanisms related to muscle anabolism; and, lastly, epigenetic actions.

The adipose tissue increase in obesity happens via hyperplasia (augmentation in adipocyte number due to adipogenesis) and adipocyte hypertrophy (growth of adipocytes). Both ω-3 and ω-6 PUFAs can bind and/or control transcriptional factors that regulate genes implicated in pre-adipocyte differentiation. Principally, AA and its derivatives act as ligands for PPARγ and PPARδ to cause fat cell differentiation and quicken maturation by increasing lipoprotein lipase expression in vitro [70]. Importantly, concentrations of ω-6 and ω-3PUFAs in human subcutaneous tissue are associated with less adipocyte size; improved saturated FA concentrations lead to amplified fat cell size. Considering all these data, it is possible to propose that ω-3PUFAs stimulate adipogenesis and a healthy expansion of adipose tissue during positive energy balance, stimulating a metabolically healthy phenotype [70].

Numerous trials have observed that ω-3PUFAs modulate adipokine secretion. Obese individuals have high plasma leptin values indicative of leptin resistance. Weight-loss-connected to decrease in leptin could act on hunger and a lower metabolic rate and ultimately lead to weight regaining [71]. EPA integration reduces the decrease in blood leptin values, which happens during weight loss in obese women, proposing a potentially prominent role of EPA in weight loss conservation [72]. ω-3PUFA-mediated consequences on leptin are related to a various factors, such as energy balance and kind of diet, which could determine incompatible data anyway [29]. It has been suggested that the anti-inflammatory capacities of ω-3PUFAs integration cause a rise in adipocyte adiponectin synthesis and get better leptin sensitivity. This interaction could have a substantial influence on body weight control.

A study described evident sensations of fullness in the attendees who took higher ω-3 PUFA content food compared to those who took lower ω-3PUFA content meals both immediately and 2 h after eating the meal. [73]. Consequently, it is possible that an increase in the feeling of satiety after a meal rich in ω-3PUFA content can help weight loss by reducing the next food intake. Additionally, FFAR4 could mediate appetite reduction. ω-3PUFAs are agonists for FFAR4, which provokes the secretion of cholecystokinin, a hormone that is synthesized, is freed from the small intestine, and is related to appetite suppression [74].

Brown adipose tissue (BAT) is a particular fat that disperses excess energy into heat (non- shivering thermogenesis) through mitochondrial uncoupling protein 1 (UCP1). Current studies confirm the metabolic activity of BAT by revealing BAT as a crucial regulator in ensuring energy balance by rising thermogenic energy consumption. An important quantity of BAT is dose in healthy adults and most children and adolescents, but not in the obese adults, indicating that loss of operative BAT depots is a contributing obesity factor. Cold- and diet-induced thermogenesis mediated by UCPs in the presence of ω-3PUFA have been analyzed in some studies [75]. UCPs are inner mitochondrial proteins moving hydrogen ions across the mitochondrial inner membrane [76]. ω-3PUFAs enhance mitochondrial oxidative capability in skeletal muscle and WAT, probably through UCP-3 up-regulation, but not in liver or BAT. Nevertheless, because most trials were carried out at 20 ◦C, it is uncertain whether increment in mitochondrial oxidative capability is ω-3PUFA-mediated or cold caused. Mechanisms relating the role of ω-3PUFAs in probable induction of energy expense and reduction of body fat should be investigated further at different temperatures since thermogenic markers act even at 22 ◦C [76].

Control of lipid metabolism may change by ω-3PUFA type, and by fat store. For example, EPA is preferably aimed to β-oxidation, while DHA and DPA avoid catabolism and are stored

in tissues. Furthermore, hormone-sensitive lipase, and gene expressions of fatty acid synthase, phosphoenol-pyruvate carboxykinase, and lipoprotein lipase, in retroperitoneal fat, are reduced with DHA and mixed EPA/DHA integration but not with EPA integration alone [77]. Additionally, ultimately, ω-3 PUFAs control lipid metabolism, encouraging fatty acid oxidation and repression of lipogenesis, causing a positive lipid profile and adipocyte metabolism.

The determination of ω-6 and ω-3PUFAs composition in the cell membrane of red blood cells (RBC) represents a biomarker of dietary intake and endogenous metabolism; in addition, it is a precise way to perform estimated studies and clinical trials in order to value their effects in weight increase and obesity. Harris et al. led a prospective study to observe the link between baseline RBC membrane phospholipids of ω-3PUFAs, ω-6PUFAs, ω-6/ω-3 ratio, and *trans* FA with the variations in body weight and the risk of becoming obese or overweight during a mean of 10.5 years follow up in the NIH Women's Health Initiative Study. This prospective analysis provided a strongly suggestive sign that ω-3PUFAs in RBC membrane phospholipids are reversely connected, while *cis* ω-6, ω-6/ω-3 ratio and *trans* fatty acids are favorably related with weight gain [78].

#### *Recommendations for PUFAs Intake*

The recommended intake for ω-3PUFAs is based on governing body. The Dietary Guidelines for Americans recommend consuming about 230 g/week of fish, corresponding to approximately 250 mg/day of EPA and DHA [29]. This recommended intake corresponds to consuming fish twice weekly, including one serving of oily fish. The U.S. Food and Drug Administration claimed that levels up to 3 g/day are considered as safe, while other authorities suggested at up to 5–6 g/day [79].

However, in intervention studies reporting a favorable health effect, the intake of fish oils or their derivatives resulted in long chain ω-3PUFAs daily intakes well above those "suggested" 200 mg/day and ranged from 0.5 to 9 g/day. Consequently, this justifies readjustments of nutritional guidelines to an upper level. Governments (UK, Belgium, The Netherlands, France, New Zeeland, and Australia) and health organizations (American Heart Association, FAO/WHO, American Dietetic Association,) now advise dietary consumption for total ω-3 PUFA of 1.4 to 2.5 g/day, with EPA and DHA ranging from 140 to 600 mg/day depending on the authority issuing guidelines, FOA/WHO making a relatively low recommendation of 250 mg/day, the average being around 500 mg/day [80]. This means minimum of 2 intake of fish/week (30–40 g/day), including one of oily fish (tuna salmon, sardine, and mackerel). In the light of the literature and inter individual changeability in PUFA metabolism and requirement, perhaps the minimal EPA+DHA supplies for healthy adults should reach 0.5–1 g/day (2–4 servings per week of fish, half of oily fish); that is minimal intake proved to reduce obesity and, in general, metabolic syndrome [81], with a total serving of ω-3 PUFA of 5–6 g/day as found in ancestral nutrition to which our metabolism is best fit [82]. Such levels are met in the traditional Japanese diet as it contains 80–100 g fish and shellfish/d/capita [81,83].

Numerous studies have discussed the actions of ω-3 PUFAs integration on obesity, in both animal and human models, highlighting possible mechanisms for ω-3PUFAs in decreasing body weight, improving body composition and counteracting the contrary metabolic effects of obesity [28,84]. However, clearly, findings of prospective studies concerning the favorable actions of ω-3 intake on obesity are far from agreement. The manifest discrepancies may have arisen due to differing or inadequate methods of data collection on food intake (food frequency questionnaires), changes in cooking procedures and other unaccounted for lifestyle behaviors (exercise, etc.) from study to study and among diverse study populations [29]. Therefore it is not possible at present to decide the ω3 dose to hinder obesity, neither through food, nor through integration. The advice is to act through nutritional interventions. In general, it is recommendable to replace the intake of SFAs with PUFAs. The World Health Organization (WHO) prompts eating at least two servings of oily fish per week, which is rich in the ω-3PUFAs (DHA and EPA) [85]. International and national guidelines on healthy eating agree in advising the intake of ω-3, both marine and vegetable, with a diverse and balanced diet, containing foods in which they are naturally present. In subjects at cardiovascular risk and on a

diet low in these fatty acids, or in patients in secondary prevention, integration at diverse levels should be estimated with the specialized doctor [81]. Human intervention trials indicate potential benefits of ω-3PUFAs supplementation, especially when combined with energy-restricted diets or exercise, but more well-controlled and long-term trials are needed to confirm these effects and identify doses for antiobesity-action.

ω-6PUFAs are pro-inflammatory and commonly occur in poultry, eggs, corn, and most vegetable oils, and also in processed and fast foods. ω-6PUFAs have pro-inflammatory capability and are considered to be the counterpart of ω-3PUFAs, which are anti-inflammatory [18]. A high-fat diet and the Western dietary pattern feature high quantities of ω-6PUFAs and low amounts of ω-3PUFAs. Instead, a low-fat diet (e.g., traditional Japanese diet) is low in ω-6PUFAs and high in ω-3PUFAs [86]. Data show that a higher ratio of ω-6/ω-3PUFAs increases inflammation and the probability of chronic inflammatory syndromes, including cardiovascular disease, obesity, and nonalcoholic fatty liver syndrome [18]. Preclinical studies show that ω-6 PUFAs have a tumor-enhancing effect. In a recent Japanese cohort study, incorporating 38,200 women, ω-6PUFA intake was positively associated with breast cancer risk [81,87].

Many data discussed the importance of preserving a low omega–6/omega–3 ratio for decreasing inflammation. Decreasing the ω-6/ω-3 ratio seems to reduce the inflammatory response to a high-fat meal [88]. A stable ω-6/ω-3 is one of the most significant dietary factors in the inhibition of obesity: a lower ω-6/ω-3 ratio should be reputed in the management of obesity [89]. A high omega-6 fatty acid intake and a high ω-6/ω-3 ratio are connected with weight gain in both animal and human investigations, whereas a high ω-3 FAs intake reductions the risk for weight gain [89].

Several sources of information recommend that human beings evolved on a diet that had a ratio of ω-6 to ω-3PUFA of about 1/1; whereas today, Western diets have a ratio of 10/1 to 20–25/1, demonstrating that Western diets are lacking in ω-3PUFA related with the diet on which humans evolved and their genetic patterns were established [90,91].

Due to agribusiness and modern agriculture western diets enclose unnecessary levels of omega-6 PUFAs but very low levels of ω-3PUFAs, leading to an unhealthy ω-6/ω-3 proportion, instead of 1:1 that was during evolution [90]. It is thought that hominids' foods during the Paleolithic era were high in seafood and low in seeds and vegetable oils, which led to a ω-6/ω-3 proportion of about 1:1 [30,92,93]. ω-6PUFAs are related to the synthesis of pro-inflammatory mediators while omega-3 PUFAs produce less powerful inflammatory mediators and inflammatory resolving proteins, so manipulating this proportion may bring about helpful health outcomes.

A balanced ω-6/ω-3 FA ratio (1:1 to 2:1 is optimal) is vital for homeostasis and regular development throughout the lifespan [92,94,95]. However, there are significant genetic variables in fatty acid biosynthesis including desaturase 1 and desaturase 2, which encode rate-limiting enzymes for FA metabolism. Data connected to genotyping of the desaturase region analyzed in human populations show that present-day humans vary dramatically in their capability to produce long-chain PUFAs [89,96].

In clinical investigations and intervention trials it is indispensable that the background diet is precisely determined in terms of the ω-6 and ω-3 FAs content. Because the concluding concentrations of ω-6 and ω-3PUFAs are defined by both dietary intake and endogenous metabolism, it is important that in all clinical investigations and intervention trials the ω-6 and ω-3 FAs are precisely defined in the red blood cell membrane phospholipids [89].

Mice fed the lowest ω-6/ω-3 ratio had the lowest non-HDL (i.e., atherogenic lipoporteins) and inflammation (IL-6). Mice fed lower ω-6/ω-3 ratio diets also had less macrophage cholesterol increase and less aortic atherosclerotic lesions. The lowest ω-6/ω-3 ratio (1:1) diet led to the least atherosclerotic formation and the severity of atherosclerosis augmented as the ω-6/ω-3 proportion increased [97].

Using long-chain ω-3PUFAs to suppress low-grade inflammation may advantage numerous chronic syndromes such as atherosclerosis, rheumatoid arthritis, diabetes, dyslipidaemia, obesity and heart failure. The ingesting of ω-6 seed oils may have the contrary action [98].

The consequences of extreme ω-6PUFAs remain controversial: ω-6PUFAs have intrinsic cardiovascular protective actions, justifying the latest FAO/WHO recommendations on maintaining high ω-6PUFAs consumptions if ω-3PUFA ones are fulfilled [99]. However, ω-6PUFAs compete with ω-3PUFAs for processing to eicosanoids, thereby limiting synthesis of anti-inflammatory ω-3PUFA derived mediators [100]. Moreover, there are convincing proofs that a low ω-6/ω-3PUFA ratio is determinant for the inhibition of pathologies connected to the metabolic syndrome, as colorectal cancer [101].

Deduced from ancestral nutrition, in an ideal balanced diet, fat should represent no more than 20–30% of total energy intake amongst which 5–6 g/day of ω-3PUFAs with a great percentage of EPA+DHA and the ω-6-to-ω-3 proportion should average 1 [102,103]. To keep in with a developmental approach and with the epigenetic consequences of the diet, a proportion of ω-6/ω-3 around 1 in breast milk should serve as a bench mark to decide the correct dietary requirements during pregnancy, lactation, and infant feeding [81,104].

#### **4. Role of** ω**-3PUFAs in Inflammation and Colorectal Cancer Development**

A large body of literature highlights the importance of dietary intake for the risk and progression of chronic disorders including inflammatory and neoplastic disease [105]. Nowadays it is well known that inflammation is a predisposing factor for cancer capable to promote the insurgence of several types of tumors [106], and that an inflammatory microenvironment is an essential component of all tumors. During metastasis development it has been shown that microenvironment modulates the capability of tumor cells and cancer stem cells to evade the innate immune response and survive. The metastatic niche is a complex system including several cell types, as vascular, stromal, and above all inflammatory and immune cells, in addition to many other molecules which provide survival, immune surveillance protection and metabolic requirements. The interaction among all these factors determines metastatic dissemination [107].

Only a few of all cancers depend on germline mutations, while the majority is determined by somatic mutations and environmental factors. Most often cancer is caused by chronic inflammation such as chronic infections, tobacco smoke, inhalation pollutants (such as silica and asbestos), and dietary factors (some forms of cancer are linked to obesity) [108]. Furthermore, in some cases exogenous diet-derived miRNAs might substantially contribute to the pool of circulating miRNAs, regulating tissue homeostasis and interfering with human health [109].

CRC is a multifactorial disease caused by multiple genetic and environmental factors. These include the type of diet, the lifestyle, the intake of alcoholic beverages, smoking, obesity, genomic abnormalities, alterations in the signaling pathways, chronic activation of the inflammatory response, oxidative stress, dysbiosis, etc. These factors work by altering intestinal homeostasis [110]. In fact, despite still lacking extensive epidemiological studies to date, most cases of early-onset colorectal cancer (EO-CRC) arise sporadically and are attributable to environmental factors [111]. In recent years, due to the activation of preventive screening activated in the population aged ≥50 years, the incidence of CRC in Western countries has stabilized and even decreased. In contrast, the incidence of CRC among people under the age of 50 has increased in both Europe and the United States, thereby representing a major public health problem [112,113]. However, CRC remains the fourth leading cause of cancer death in the world dependent on a close relationship between inflammation and environmental factors [114].

The gastrointestinal tract is not only responsible for digestion and absorption of nutrients but also represents a powerful barrier against pathogens and toxins harmful to the individual. It also has an endocrine function responsible for maintaining the metabolic homeostasis of the whole organism. Since the intestine comes into direct contact with food, it is very sensitive to dietary factors which directly influence both its structure and function [115]. In fact, the crypts and intestinal villi are structurally influenced by external factors by changing their size in response to changes in the diet [116].

Since chronic inflammation has been shown to promote the onset of CRC in humans, ω-3PUFAs, due to their strong anti-inflammatory function, have been shown to be protective against colon cancer [117,118].

Some studies have been conducted on the efficacy of ω-3PUFAs in the prevention of CRC through the integration of purified EPA with DHA or fish oil (FO). These studies have shown the importance of ω-3PUFAs in inhibiting the uncontrolled proliferation of CRC cells both when administered in large quantities for short times (8–9 g of EPA+DHA/day for 2 weeks) and in smaller quantities for longer times (2.5–4 g of EPA+DHA/day for 3–6 months). However, the effect on the control of intestinal cell proliferation was not seen in patients with the same supplementation but with a high-fat basal diet and a low ω-3/ω-6PUFAs ratio. For this reason, the effectiveness of ω-3PUFAs depends on both the total lipid content and the ω-3/ω-6PUFAs ratio [119].

Many studies have been conducted in CRC models to explain the molecular mechanisms to the base of the anti-inflammatory and anti-neoplastic activity of ω-3PUFAs. First of all, ω-3PUFAs are incorporated in phospholipid membrane inducing an alteration in structure, fluidity and function of lipid rafts. These membrane changes influence the activity of membrane receptors leading at the inhibition of signaling pathways involved in the activation of pro-inflammatory molecules of cell survival and apoptosis [120–122]. Moreover, in CRC, ω-3PUFAs modulate inflammatory pathways, generating lipid mediators implicated in the resolution of inflammation including resolvine, protectin, and maresins [123].

The ω-3PUFAs exert their antitumor actions through different mechanisms, involving proliferation, apoptosis, and migration. Their affects involve COX-dependent or COX-independent mechanisms, and they act on different pathways such as Wnt/β-catenin and Hippo or by regulation of oxidative stress and the expression of Granzyme B. To date, there are numerous papers in the literature that describe different mechanisms of action of ω-3PUFAs in CRC, as summarized in Table 1.

**Table 1.** ω-3PUFAs target different molecular pathways acting on classical hallmarks of cancer, i.e., proliferation, apoptosis, and migration.

Abbreviations: COX: cyclooxygenase, GPR: G-coupled receptor, Gαs: G-protein alpha subunit, PKA: protein kinase A, EMT: epithelial mesenchymal transition.

Multiple molecular mechanisms causing an increased apoptosis of CRC cells depend on the action of ω-3PUFAs. First of all, ω-3PUFAs influence the redox state of the cells: indeed, there is a link between anti-tumor effects of ω-3PUFAs and oxidative stress. PUFAs may induce an increase in apoptotic potential of CRC cells by enhancing the concentration of intracellular reactive oxygen species (ROS), inducing an elevated cancer cells apoptosis by the loss of mitochondrial membrane potential, ROS generation, activation of caspase 3 and 9, and by an increase of Bax/Bcl2 ratio [124].

Another important anti-inflammatory mechanism involves COX, a major player in inflammation. COX hyperactivation in the CRC, induces in turn the production of PGE2, a powerful pro-inflammatory and pro-carcinogenic agent [125]. ω-3PUFAs exert their anti-inflammatory role by modulating COX activity. In this respect, EPA, acting as an alternative substrate for COX-2, induces a switch in production from pro-tumorigenic PGE2 to three series PGs (PGE3) that abrogate the antiapoptotic activity of PGE2 in CRC cells [126]. However, the anti-cancer mechanism of ω-3PUFAs in CRC could also be explained

by a COX2-indipendent mechanism. Indeed, DHA and EPA inhibit the proliferation and induce the apoptosis of CRC cells in vitro and in animal models. At molecular level, involvement of the Hippo pathway, cytoplasmic retention of phosphorylated YAP by GPRs (GPR40 and GPR120)-Gαs-PKA cascade has been reported [127]. Moreover, the GPR120 is expressed on macrophages and regulates their polarization reducing inflammation [128].

In addition to apoptosis, ω-3PUFAs can also influence proliferation and migration capability of CRC cells. In colorectal cancer, the Wnt-β-catenin signaling pathway is the key regulator of tumor development, and alterations in this cellular signaling pathway can be found in most patients [129]. It has been shown that dietary ω-3PUFAs are able to inhibit significantly intestinal polyp growth in mice, correlating with the ECS described in Section 2.1. In fact, CB1 up-regulation reduces β-catenin and its transcriptional target c-myc, both involved in regulation of cell proliferation. In CRC patients, cancer tissue shows a significant inhibition of CB1 expression levels, compared to adjacent normal tissue, demonstrating that the "protective" action of endocannabinoids via CB1 is lost in the tumor [130]. Moreover, D'Eliseo and colleagues have studied the effect of DHA on migration of CRC cells and demonstrated that DHA inhibits Granzyme B expression, reducing CRC cells capacity to undergo epithelial mesenchimal transition (EMT) and invade matrigel [131].

Finally, ω-3PUFAs regulate the expressions of genes involved in inflammation and colon cancer development also through epigenetic modifications [132–134]. In fact, more recently, ω-3PUFAs have been attributed the ability to influence the epigenetic regulation of genes involved in the polarization of macrophages, negatively regulating the colorectal carcinogenesis; however, the interesting topic is not fully understood [135].

Taken together, although the many different mechanisms, ω-3PUFAs play anti-inflammatory and anticancer effects acting on the classic hallmarks of cancer, i.e., cell proliferation, apoptosis and migration.

To date, one of the most important problems in the treatment of tumors, including sporadic colorectal cancer, is the development of resistance to anti-tumor treatments and tumor relapse that can be related to self-renewing of cancer stem/stem-like cells (CSC/CSLC) within a tumor mass. Many groups have studied the effects exerted by ω-3PUFAs on cancer stem-like cells.

With the immunophenotyping of CSLC, the anti-CD133 antibody was found to be effective for isolating a population of colon cancer cells that retained the properties of stem cells (CSLC), while anti-cytokeratin 20 (CK20) and anti-Mucin-2 (MUC2) were specific epithelium colonic differenziation markers. EPA treatment induces an increase in of CK2 and MUC2 and an inhibition of CD133 expression. This means that the EPA could induce a more differentiated state of most cancer cells and could trigger the reduction of the stem state of the CSLC, as demonstrated by the reduction of the expression of the CD133 marker [136].

Moreover, Yang and colleagues showed an antiproliferative and proapoptotic effect on the dedifferentiated SW620 colon cell line, treated with DHA and EPA [137].

Additionally Sam et al., evaluating the effects of DHA and EPA treatments on LS174T cells, a model for colorectal cancer initiating cells with stem cell-like properties, demonstrated that ω-3PUFAs induce cell growth inhibition and promote cell death by down-regulating survivin expression and activating caspase-3 [138].

The effect of ω-3PUFAs on CSLC may be an important goal for cancer therapy and will constitute an interesting challenge for future studies. Anyway, the anti-tumor activity of ω-3PUFAs, shown through multiple mechanisms, suggests that they could have an important therapeutic role in the management of CRC.

#### **5. Conclusions**

Obesity is a preventable disease that can be treated through proper diet and exercise. A balanced ω-6/ω-3 ratio 1–2/1 is an important dietary factor in the prevention of obesity, along with physical activity. Different pro- and anti-inflammatory properties are exerted by ω-6 and ω-3PUFAs themselves and by their derivatives, such as endocannabinoids, lipid mediators deeply involved in the control of many biological functions, including the inflammatory response and the central and local control of food intake and energy homeostasis. Therefore, appropriate dietary intervention has primarily relevance in the prevention and the treatment of obesity in that it maintains the efficiency of key signaling pathways and avoids long term/chronic inflammatory states.

Inflammation is a predisposing factor for cancer, CRC included, with ω-3PUFAs exhibiting anti-cancer properties, once again confirming the need for a balanced ω-6/ω-3 ratio for health preservation.

The discovery of cancer stem cells offers a new perspective in cancer therapy. Since CSCs contribute to cancer onset and relapse after conventional therapy, they can represent a unique fundamental therapeutic target to completely cure cancer. Thus, the effect of ω-3s on CSLC may be an important goal for cancer therapy and will constitute an interesting challenge for future studies. Anyway, the anti-tumor activity of ω-3s, performed through multiple mechanisms, suggests that they could have an important therapeutic role in the management of CRC.

**Author Contributions:** Conceptualization, M.L.M., S.D., and R.M.; writing—original draft preparation, M.L.M., S.D., and R.M; supervision, R.M.; funding acquisition, R.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Prin-Miur 2017 to R.M. project code 20175MT5EM.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Review*

### **The Crosstalk between Prostate Cancer and Microbiota Inflammation: Nutraceutical Products Are Useful to Balance This Interplay?**

**Felice Crocetto 1,**†**, Mariarosaria Boccellino 2,**†**, Biagio Barone 1, Erika Di Zazzo 3,\*, Antonella Sciarra 4, Giovanni Galasso 2, Giuliana Settembre 2, Lucio Quagliuolo 2, Ciro Imbimbo 1, Silvia Bo**ff**o 5, Italo Francesco Angelillo 6 and Marina Di Domenico 2,5**

Received: 31 July 2020; Accepted: 26 August 2020; Published: 31 August 2020

**Abstract:** The human microbiota shows pivotal roles in urologic health and disease. Emerging studies indicate that gut and urinary microbiomes can impact several urological diseases, both benignant and malignant, acting particularly on prostate inflammation and prostate cancer. Indeed, the microbiota exerts its influence on prostate cancer initiation and/or progression mechanisms through the regulation of chronic inflammation, apoptotic processes, cytokines, and hormonal production in response to different pathogenic noxae. Additionally, therapies' and drugs' responses are influenced in their efficacy and tolerability by microbiota composition. Due to this complex potential interconnection between prostate cancer and microbiota, exploration and understanding of the involved relationships is pivotal to evaluate a potential therapeutic application in clinical practice. Several natural compounds, moreover, seem to have relevant effects, directly or mediated by microbiota, on urologic health, posing the human microbiota at the crossroad between prostatic inflammation and prostate cancer development. Here, we aim to analyze the most recent evidence regarding the possible crosstalk between prostate, microbiome, and inflammation.

**Keywords:** prostate cancer; microbiota; nutraceutical compounds

#### **1. Introduction**

Prostate cancer (PCa) is the second most commonly diagnosed malignancy in men and the fifth leading cause of tumor-associated death worldwide [1].

Global estimations are approximating 800,000 new PCa cases and 300,000 deaths per year [2], and this condition poses a significant health concern in the future due to the gradual aging of the

population. Genetics, family history, African descent, advanced age, diet, and environment are well-established risk factors for PCa development. However, the relevant pathways accounting for PCa development are not fully clarified [3–5]. The role of androgenic stimulation and the deficit of apoptosis of prostate cells are well-known explanations regarding the incidence and progression of PCa. Recent studies have also hypothesized a crucial role of microenvironment, infections, inflammation, and cytoskeletal changes induced by steroid integrating signals [6,7], influencing patients' outcomes and the rationale for the immunological treatment of PCa [8–12].

Chronic inflammation is a prominent contributing factor to the benign and malignant prostatic growth; however, the potential stimulus that induces or maintains this chronic inflammation remains poorly characterized [13]. Inflammation, sex hormones, and many other factors (e.g., infections, diet, physical activity, drugs), are known to affect the microbiota. The microbiota is a complex community composed of fungi, parasites, bacteria and viruses living within the human body. Microbiota components interact with each other and with the host, impacting, eventually, the overall human health. The purpose of this study is to summarize and analyze the most recent evidence regarding the possible crosstalk among prostate, microbiota, and inflammation.

#### **2. Prostate and Chronic Inflammation**

The role of inflammation in the carcinogenesis of a solid tumor is an accustomed aspect [13]. In fact, two key inflammatory cytokines, IL-6 and IL-2, have been convincingly implicated in prostate cancer pathogenesis. Inflammation may also contribute to impairing immune surveillance mechanisms, which are partially mediated by NK cells [14,15]. Repeated tissue damage and regeneration produce highly reactive nitrogen species (RNS) and oxygen species (ROS), which are responsible of cancer development and progression [16–19]. The underlying biological mechanism relies on DNA modifications of cells caused by this continuous process of damage and repair [20–22]. However, if there is a strong and proven connection between solid tumors and inflammation, the role of this condition in PCa development is still debatable and under revision. Different studies have suggested how chronic prostatitis could induce proliferation of stromal and glandular cells in response to ROS production, eliciting general tissue damage, and vascular injury [23,24]. ROS, moreover stimulate NF-kβ and TNF-α pathways by activating their proper kinases [25]. The morphological modification in the prostate tissue, associated with chronic and acute inflammation, is a glandular atrophy with hyperplasia called proliferative inflammatory atrophy (PIA) [26]. Up to 40% of PIA lead to the transition to a high-grade prostatic intraepithelial neoplasia (PIN), a precursor of PCa. Although some evidence of molecular changes has been observed in PIA, no certain clonal genetic alterations have been found in this condition [27]. However, genes such as NKX3.1 and CDKN1B have been shown to be downregulated in PIA, as in PIN and PCa, while the increased transcription of Hsp27 and PRDX6 could promote processes leading to tumorigenesis [28]. Several PCa susceptibility genes, such as MIC1, RNASEL, MSR1, PON1, TLR4, OGG, BRCA2 and CHEK2, are involved in prostate carcinogenesis and in other critical processes, as a host response to steroids, infection, inflammation and oxidative stress [29,30]. Furthermore, despite significant changes in inflammatory cellular infiltration between prostatitis, benign prostatic hyperplasia (BPH) and PCa have been found; the role of innate and adaptive immunity has not been completely cleared [13] (Figure 1). Chronic inflammation could have, moreover, a significant effect on cancer progression and metastatic invasion due to neo-angiogenesis and activation of epithelial–mesenchymal transitions (EMTs) [31,32]. These biologic and pathogenic processes are correlated to various molecules defined as biomarkers/indicators of normal, or pharmacologic, responses to a therapeutic intervention [33]. The control of these phenomena triggers pathways, as migration, proliferation, cell growth, apoptosis, and adhesion through various downstream effectors. The first key element that regulates cell proliferation, migration, and invasion in PCa is p85αPI 3Kinase [34–38]. Evidence from the literature supports the role of angiogenesis in human cancer progression, including PCa. The vascular endothelial growth factor (VEGF) is a potent angiogenic factor [39,40]. Several miRNAs, functioning as tumor suppressors or oncogenes

are deregulated in prostate tumorigenesis. miRNA dysregulation progress has a key role in prostate cancer [41]. Anti-VEGF therapy and combined chemotherapy treatments trigger apoptosis in cancer and, in particular, in prostate cancer [42–50].

**Figure 1.** Chronic prostatitis and immune cell infiltration. Outlined in blue are aggregates of lymphocytes, plasmacells and istiocites, which surround damaged glands (black arrows). In red circles, multinucleated giant cells are outlined.

Different cancer types (i.e., lung cancer, pancreatic cancer, glioblastoma, meningioma, myeloma, and myeloma) are characterized by distinct patterns revealed by corona composition, constituting a "fingerprint" for each cancer type [51–54].

Classically, the peripheral zone of the prostate gland is a common site of PCa development, while the transitional zone is mostly affected by benign prostatic hyperplasia [55]. However, in about 20% of cases, the two conditions subsist in the same zone and, despite different pathogenic pathways, several well-established epidemiologic studies confirm that both conditions are hormone-dependent and could be associated with a previous chronic prostatic inflammation [56]. A certain degree of inflammation is almost always present when prostate specimens are sampled: the REDUCE trial demonstrated on 8224 men that, indeed, 77.6% of biopsies are positive for some grade of inflammation, with the majority (>80%) showing a mild chronic inflammation [57]. To further support these findings, men diagnosed with prostatitis have an increased risk of developing, in the future, PCa compared to those without any grade of prostate inflammation. Specifically, 18% of those patients will develop PCa [58]. Chronic and acute inflammation is also frequently found in prostate tumor specimens obtained from prostatectomies and transurethral resections [59]. A study conducted by Daniels et al. on 5821 men >65 years old reported a positive association between a previous history of prostatitis and PCa (OR 5.4, 95% CI = 4.4–6.6) [60]. Similarly, Cheng et al. showed that protracted prostatitis symptoms could significantly increase the odds of PCa in 68,675 men (RR 1.3, 95% CI = 1.10–1.54) [61]. In addition, Dennis et al. reported, in a meta-analysis of 11 case-control studies, the evidence of a statistically significant risk of developing PCa in patients with a previous history of prostatitis (OR 1.6, 95% CI = 1–2.4) [62] and analogous results were found by a similar meta-analysis on 20 case-control studies (OR 1.50, 95% CI 1.39–1.62) [63]. Finally, a recent and wide meta-analysis by Perletti et al. reported, in 422,943 patients, a significant association between PCa and previous prostatitis (OR 1.83, 95% CI = 1.43–2.35) [4]. However, despite those data, the real impact of chronic inflammation on prostate carcinogenesis has been challenging to define. In particular, it is not easy to estimate the real

incidence of prostatitis due to the asymptomatic majority of cases (5–10%) [64]. Moreover, evidence that seems to show an increased risk for acute prostatitis rather than for chronic prostatitis, is influenced by the same potential detection bias.

#### *Etiology of Prostate Chronic Inflammation*

The etiology of chronic inflammation preceding PCa development remains unknown, however, infections and chemical trauma are often correlated to chronic inflammation.

Several putative etiological agents have been identified, from the xenotropic murine leukemia virus related-virus XMRV to different strains of bacteria [58,65–67]. Several studies support the potential role of infectious agents in PCa etiology with evidence that up to 87% of PCa patients show microbial DNA in their prostate [68,69]. However, if no clear association had been shown with HPV or other sexually transmitted viruses, men with previous gonorrhea or syphilis infections had a 60% increased risk of developing PCa [70]. A study based on animal models reported a mutagenic activity of inflammation caused by *Escherichia coli* in the prostatic gland, with the induction of epithelial hyperplasia, an increased tendency to apoptosis, and somatic mutations [71]. Moreover, the presence of an induced prostatic infection with *Escherichia coli*, in addition to the consumption of a diet enriched with a cyclic amine, the 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) (a well-known prostatic carcinogen in rodents), further increased the risk of PCa development in mice with a marked drop in survival rate compared with PhIP-alone-treated animals, thus suggesting chronic inflammation as an enabling characteristic of PCa [72]. Cai et al., reported a significant increase in Gram-positive strains in patients with chronic prostatitis and a successively diagnosed PCa [73], while other significant associations between cancer development and infection were shown also for *Mycoplasma hominis* [74] and *Trichomonas vaginalis* [75,76]. In particular, previous *Trichomonas vaginalis* infection could create a favorable microenvironment, promoting PCa cell proliferation and invasiveness (activating the epithelial–mesenchymal transition), in addition to an increased overall inflammatory state of the gland [77]. Twu et al. reported, in fact, how *Trichomonas vaginalis* secretes a protein (TvMIF), which is 47% similar to the human macrophage migration inhibitory factor (HuMIF), which is reported to be elevated in PCa [78]. *Propionibacterium acnes*, which is frequently isolated in prostate tissue, has also been thought to have an influence on the development of PCa due to the association with reported histological inflammation in prostate-derived tissue models and prostatectomy specimens [79]. To further outline the role of *Propionibacterium acnes* in prostate carcinogenesis, Ugge et al. retrospectively analyzed the association between the presence of acne vulgaris during adolescence and the occurrence of PCa in 243,187 men for a median follow up of 36.7 years; 1633 of those patients developed PCa, reporting an adjusted OR of 1.43 (95% CI = 1.06–1.92) [80]. However, a recent meta-analysis by Zhang et al. did not find a significant association between acne and PCa, questioning this relation [81]. An EPICAP study reported instead the association between sexually transmitted and urinary tract infections and PCa, with an increased risk of developing this malignancy in patients with a previous history of prostatitis (OR 2.95, 95% CI = 1.26–6.92) and in patients who did not assume non-steroidal anti-inflammatory drugs (OR 2.00, 95% CI = 1.37–2.91) [82]. To further support the role of chronic inflammation in increased PCa risk, St. Hill et al. showed how EBV, HIV, HBV, HCV, or HSV chronic infections were associated with an increased risk of occurrence of PCA. Similarly, the risk was also increased in men with other chronic inflammatory diseases or conditions such as osteoporosis, diabetes mellitus, arthritis, or cardiovascular disease; however, currently, no inflammation marker could be associated with a higher risk of PCa development [83].

#### **3. Microbiota in Urological Disease**

#### *3.1. Urinary and Prostate Microbiota*

Commensal microorganisms colonize barrier surfaces of all multicellular organisms, coevolving and adapting with the host for more than 500 million years. As result, the commensal microbiota affects many processes of their hosts via biologically active molecules, playing critical roles in human diseases, in particular cancers and autoimmune conditions, influencing the innate and adaptive immune response [84]. The discovery of communities of bacteria in the genitourinary tract and their role in urologic diseases has introduced novel factors and implications in the pathophysiology of these conditions. The advent of such molecular-based methods as the quantitative real-time PCR and amplification of 16S rRNA for the identification and characterization of microbial populations has permitted the discovery of previously unrevealed microbial populations. Historically, the bladder, and generally the urinary tract, has always been considered sterile, however, recent studies have revealed important evidence of the presence of microbes in bladders of patients without clinical infection [85,86]. Human urinary microbiota characteristics depend on the age, gender, and disease status of individuals [87–92], and understanding its role in urological diseases is of particular interest. Moreover, novel molecular methods have made it possible to characterize the bladder microbiota formed by *Burkholderia cenocepacia* and different strains of *Lactobacilli* in urologic chronic pelvic pain syndrome (UCPSS), which was considered to be defined as "the absence of identifiable bacterial infection" [93]. Particularly interesting is that, in the same condition, an increased rate of *Lactobacilli*, compared to the remaining flora, was instead revealed in the urine of patients with Interstitial Cystitis (IC) [94]. Furthermore, *Lactobacillus casei* and *Lactobacillus rhamnosus* could also have interesting applications in the treatment of bladder cancer, demonstrating a decreasing effect on rates of metastasis and recurrence due to an enhanced recruiting of natural killer cells, both in vitro and in vivo [95]. Accordingly, different studies have hypothesized a link between prostate microbiota and pro-inflammatory bacterial species. In 2016, Mandar et al. reported a lower rate of *Lactobacilli* in patients with chronic prostatitis, while Shoskes et al. reported, for the same condition, higher rates of *Clostridia* and *Bacteroides* compared with controls [96,97]. In 2015, Yu et al. described how bacterial strains present in prostatic secretions, seminal fluid and voided urine are different among patients with BPH and PCa. Specifically, there are lower rates of *Eubacterium* and *Defluviicoccus* and higher rates of *Bacteroidetes* in patients with PCa, hypothesizing the role of certain bacteria in the induction of chronic inflammatory states, with enhanced production of factors favoring tumorigenesis [98]. An analogous study conducted on 135 PCa patients by Shrestha et al. in 2018, reported an increased presence of *Anaerococcus lactolyticus* and *obesiensis*, *Streptococcus anginosus*, *Varibaculum cambriense*, *Actinobaculum schaalii*, and *Propionimicrobium lymphophilum*. All patients were previously diagnosed with urinary tract infection caused by *Enterobacteriaceae*, which were instead more abundant in patients with BPH [99]. Similar conclusions were reported by Alanee et al., confirming the possible association between urinary and fecal microbiota with PCa after examination of prostate biopsies, which were characterized by higher rates of *Streptococcus anginosus*, *Anaerococcus lactolyticus*, and *Varibaculum cambriense* [100]. Analogously, Bhudia et al., reported increased rates of *Staphylococcus epidermidis*, *Streptococci*, *Corynebacterium amycolatum*, *Peptoniphilus harei*, and *Fusobacterium nucleatum* in prostate secretions of PCa patients [101]. Cavarretta et al. reported an abundance of *Propionibacterium* spp. and *Staphylococcus* spp. in 16 tumoral and peritumoral prostatectomy specimens [102]. Similarly, Feng et al. examined 65 radical prostatectomy specimens, reporting an increased rate of *Escherichia coli*, *Propionibacterium acnes*, *Pseudomonas* spp. and *Acinetobacter*; in particular, *Pseudomonas* has a gene expression profile that strongly correlates with human small RNA's profile, and that could be also related to metastasis [68]. Moreover, the same authors identified increased bacterial content (especially *Escherichia* spp. and *Acidovorax* spp.) in prostate specimens of African men, which were also associated with elevated tumor hypermutation, suggesting the possibility of a bacterially driven oncogenic transformation [69].

#### *3.2. Gut Microbiota*

The role of microbiota in urological diseases and PCa is, however, not limited to bacteria related to the urinary tract. Modification of the gut microbiota could modify the risk of incurring PCa and be influenced by the tumorigenesis process itself [103] (Table 1). Liss et al. reported significant differences in bacteria obtained via rectal swab between PCa and healthy patients, with an increase in certain genera such as *Bacteroides* and *Streptococci* and impoverishment of bacteria related to folate and biotin production [104]. Golombos et al., similarly, confirmed the abundance of *Bacteroides* in PCa patients and reported an increased presence of *Faecalibacterium prausnitzii* and *Eubacterium rectalie* in BPH patients [105]. Potential alterations of gut microbiota could influence, both directly and indirectly, prostate health via bacterial metabolites, and influence the enteric endocrine system [106]. Multiple studies have shown that gut microbiota also modulates the response to chemotherapy acting on the translocation, immunomodulation, metabolism, and enzymatic degradation of drugs [107]. This consideration, moreover, is valid also for androgen axis-targeted therapy in PCa treatment, which is influenced in its clinical response and antitumoral efficiency by gut microbiota. Conversely, androgen axis-targeted therapy enhances *Bacteroides* and *Streptococci* rates in the gastrointestinal tract while lowering overall bacterial diversity [108]. Besides, an analysis of the fecal microbiota of healthy volunteers and PCa patients by 16S rDNAsequencing, showed a greater abundance of *Akkermansia muciniphila* and *Ruminococcaceae* spp. in the microbioma of patients treated with oral androgen receptor axis-targeted therapies such as enzalutamide, bicalutamide and abiraterone acetate [109]. Finally, there are suggestions that butyrate, an anti-inflammatory micronutrient produced by *Faecalibacterium prausnitzii* and *Eubacterium rectale*, could be implicated in one of the pathways for the prevention of PCa, although further studies are required [105].

#### **4. Nutraceutical Aspects in the Interplay between Prostate and Microbiota**

#### *4.1. Unsaturated Fatty Acids*

Olive oil and unsaturated fats, high vegetable consumption, fruit intake, and allium vegetables, typical aspects of the Mediterranean diet, were related to a decreased risk of several cancer types. In particular, countries following the Mediterranean Diet have lower PCa incidence and mortality compared to other European regions. However, there are few studies that have assessed the effect of the Mediterranean diet on PCa incidence. Further large-scale studies are required to clarify the effect of the Mediterranean diet in order to establish the role of this diet in the PCa prevention [110,111]. PCa has a well-known association with food and, in particular, with fat intake; moreover, there is a relationship between PCa and gut microbiota that changes based on the diet [112]. A low-fat diet and/or intensive exercise involves changes in serum hormones and growth factors in vivo, which could reduce growth and induce apoptosis of LNCaP prostate tumor cells in vitro [113]. Low-fat diet-fed mice show significantly lower levels of prostate-specific serum antigen (PSA), insulin and Igf1 mRNA levels compared to mice with a high-fat diet, as well as a delayed tumor-growth rate in LAPC4 xenografts [114]. A high-fat diet induces, in fact, lipid accumulation in PCa and promotes metastasis via abnormal sterol regulatory element-binding protein (SREBP)-dependent lipid metabolism [115]. Several epidemiological studies suggest that an increased intake of saturated fatty acids and a sedentary lifestyle decreases the survival rate of PCa patients, whilst unsaturated fatty acids and physical activity reduce the risk of PCa [116,117]. In recent years, *n*-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), present in fish oil, have been found to influence cancer cell proliferation. EPA and DHA were, moreover, effective in decreasing the proliferation, invasion, and migration of prostate PC3 cancer cells as well [118]. As known, sex hormones also play an important role in the development and progression of PCa. In prostate-specific Pten-/-mice, the reduction in serum cholesterol lowers intraprostatic androgens and suppresses tumor progression, although it does not change the incidence of PCa [119]. In transgenic mice, the consumption of high amounts of unsaturated fatty acid ω-3, produces a significant slow-down of prostate tumorigenesis by affecting estradiol, testosterone, and androgen receptor levels, suggesting a specific role of unsaturated fatty acids in the regulation of sex hormones, which may be the basis of fat-induced PCa progression [120].

#### *4.2. Carnitine*

Carnitine, and in particular its acetylated derivative, Acetyl-l-Carnitine (ALCAR) is involved in mitochondrial membrane trafficking in catabolic and anabolic pathways. Several studies have documented the antioxidant and scavenger activity of this compound, utilized in clinical settings related to disorders where the oxidative stress acts as a promoting factor (e.g., diabetes, Alzheimer's disease, and other neurometabolic disorders) [121,122]. ALCAR reduces PCa cell viability and induces apoptosis; moreover, ALCAR impairs the adhesion, invasion and migration of PC3, DU145, LNCaP, and BPH cells, eliciting a decreasing effect on TNF-α and other proinflammatory cytokines, such as

IL-6, CCL2 and CXCL12 [123]. Besides, ALCAR was able to limit inflammatory angiogenesis, in vitro and in vivo, downregulating the VEGF/VEGFR2, CXCL12/CXCR4, and FAK pathways [124].

#### *4.3. N-acetylcysteine (NAC)*

*N*-acetylcysteine (NAC) is an exogenous antioxidant primarily used as a mucolytic agent and as an antidote of acetaminophen toxicity. Its effects on increasing glutathione levels and scavenging free radicals pose NAC as a powerful antioxidant. The association of NAC with phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), two compounds present in cruciferous vegetables (cauliflower, cabbage, and broccoli) inhibit LNCaP and DU145 cell growth in a dose-dependent manner, increasing p21, a potent inhibitor of cyclin-dependent kinases mediating cell replication, up to apoptosis. Besides, SFN-NAC reduces PSA and the expression of the androgen receptor [125,126]. NAC alone inhibits the growth of PC3 cells suppressing the transcription of nuclear factor (NF)-κB, while increasing Cyr61 levels and activating the Erk pathway [127]. Finally, NAC shows a significant anti-migration and anti-invasion activity on DU145 and PC3 cells, limiting the metastatic ability of those cells [128].

#### *4.4. Monoterpenes*