prostate carcinogenesis [35–38]. In the past two decades, research studies have shown that GTCs influence multiple biochemical and molecular cascades that inhibit several hallmarks of carcinogenesis relevant to prostate carcinogenesis. With an acceptable safety profile, GTCs are ideal candidates for PCa chemoprevention. Laboratory studies demonstrate that EGCG can affect several cancer-related proteins, including p27, Bcl-2 or Bcr-Abl oncoproteins, Bax, matrix metalloproteinases (MMP-2 and MMP-9), the androgen receptor (particularly important in PCa development and progression), epidermal growth factor receptor, activator protein 1, and some cell cycle regulators [29,35]. Using cell culture systems, Adhami et al. [39] were able to show that EGCG induces apoptosis, cyclin kinase inhibitor WAF-1/p21-mediated cell cycle-dysregulation, and cell growth inhibition. In cDNA microarrays, EGCG treatment of LNCaP cells induced genes that exhibit growthinhibitory effects and repressed genes belonging to the G-protein signaling network [40]. The ubiquitin/proteasome pathway plays a critical role in activation of the cellular apoptotic program and the regulation of apoptosis [41]. Our work demonstrated that GTC specifically inhibits the chymotrypsin-like activity of the proteasome in several tumor and transformed cell lines, including prostate cell lines, resulting in the accumulation of two natural proteasome substrates–p27 (Kip1) and nuclear factor kappa B (NF-kB) inhibitor alpha, which inhibit transcription factor NF-kB, leading to growth arrest in the G(1) phase of the cell cycle. Synthetic analogs of EGCG were observed to be more potent as proteasome inhibitors compared to EGCG. Polyphenon E® (Poly E) and Sunphenon® 90D are standardized formulations of green tea containing 50% of the catechins from EGCG. We observed that Poly E® (>50% EGCG, 80% total catechins) preferentially inhibits the proteasomal chymotrypsin-like activities over other activities, with an IC50 value of 0.88 μM [41–44]. Standardized GTC formulations of Poly E® and Sunphenon® 90D in equal concentrations were evaluated in vitro. Pre-treatment with Sunphenon® 90D downregulated NF-kB in H2O2-treated C2C12 cells, while activating caspase-3 (Figure 1) [45]. Incubation of human primary osteoblasts with Sunphenon® 90D significantly reduced oxidative stress and improved cell viability [46]. EGCG has been shown to have both anti-inflammatory properties, such as through the influence of T-cell proliferation and inhibition of NF-kB, and neuroprotective properties by acting as a free radical scavenger [47,48]. More specifically, EGCG's antioxidant properties deplete reactive oxygen species, thus preventing DNA damage and inhibiting NF-kB-induced inflammation, angiogenesis, and cell survival that could otherwise propel cancer development and progression [49]. In summary, we and others have reported convincing evidence suggesting that GTCs inhibit proliferation and cell cycle events and induce apoptosis through multiple mechanisms.

The association of green tea intake with PCa risk has been investigated in several epidemiological studies. In a meta-analysis of 9 case-control studies, there was a statistically significant 57% lower risk of PCa, comparing subjects with the highest relative to lowest green tea consumption, whereas there was a null association in a meta-analysis of 4 cohort studies [50]. Similar results were observed in a more recent meta-analysis of 3 case-control and 4 cohort studies: no statistically significant associations were observed across cohort studies, while a statistically significant 55% lower odds of PCa was observed for highest versus lowest green tea intake in the case-control studies [51]. This inconsistency could be due, in part, to differences in study design, residual confounding factors such as by diet/lifestyle and biological factors, and varying formulations and subtypes of green tea studied. These studies were mostly limited to men in Asian countries, where approximately 20% of green tea is consumed globally and where mortality from PCa is the lowest compared to Western populations [36], where green tea consumption is a more recent phenomenon. Asian men who migrate to the US have a relatively increased risk of PCa compared to their counterparts in their countries of origin, potentially as a result of acculturation and adoption of Western diets [38]. Although the above study findings have been mixed potentially due to confounding by variation in geographical location, tobacco and alcohol use, and other lifestyle factors (mainly diets) [37,52]—taken together, studies among Asian populations demonstrate a protective effect of GTCs as related to PCa [37,38,53]. Another

highly plausible confounder of GTC-PCa associations is the gut microbiome, which has increasingly been implicated in the modulation of carcinogenesis. The gut microbiome comprises densely populated commensal and symbiotic microbes [54] whose composition is highly influenced by the host's dietary intake. The gut microbiome also produces metabolically active metabolites that interact with host-signaling pathways and gene expression, impacting cancer initiation and progression [55,56]. Multiple studies have observed differences in the gut microbiome between various racial and ethnic groups, even amongst those living in the same community. These differences are potentially attributed to lifestyle, dietary, social, and other uncharacterized exposures that result in variations across racial and ethnic groups [57,58]. Using fecal shotgun metagenomic data analyzed amongst 106 Japanese individuals compared with those of 11 other nations, the composition of the Japanese gut microbiome was more abundant in the phylum Actinobacteria, in particular, genus *Bifidobacterium*, compared to others [59]. In line with increased PCa rates in Asian populations living in the US, studies have shown that the gut microbiome of Southeast Asian immigrants changes after migration to the US [60], potentially indicative of an incompatibility between the incorporation of Western lifestyles with the traditionally harbored microbiome of this population [61]. These studies have provided the basis for understanding that the gut microbiome can act as an important mediating factor in investigations of diet and lifestyle differences that potentially promote cancer risk.

**Figure 1.** Mechanistic pathway by which GTCs prevent PCa progression. In vitro studies [41–44] demonstrate that GTCs block proteasomal activity in PCa cells, leading to build-up of proteasomal substrates Kip1 and Ik-b α that subsequently downregulate the activity of NF-κB. This inhibits the cell cycle and elicits apoptosis in these PCa cells. GTCs, green tea catechins; Ik-b α, NF-κB inhibitor alpha; NF-κB, nuclear factor kappa B; PCa, prostate cancer. Created with Biorender.com (accessed on 1 July 2022).

#### **3. Pre-Clinical Evidence of the Safety and Effectiveness of GTCs in PCa Carcinogenesis**

Several promising pre-clinical studies of GTC effects on prostate carcinogenesis were completed that were highly clinically relevant [39,62–66]. In studies evaluating oral GTCs (vs. pure EGCG) administered to transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, greater bioavailability [29,35,62–66] of GTC was observed compared to administering EGCG alone [64]. Oral infusion of a polyphenolic fraction isolated from green tea extract at a human achievable dose (i.e., six cups of green tea per day) in a TRAMP mouse model, compared to water-fed mice [62], demonstrated significant delays in primary prostate tumor incidence and burden. Overall, they observed a decrease in prostate (64%) and genitourinary (72%) weight from baseline weight, inhibition of serum insulin-like growth factor-1 (IGF-1), and restoration of insulin-like growth factor binding protein-3 levels (IGFBP-3). Additionally, a significant reduction in the protein expression of proliferating cell nuclear antigen and apoptosis in the prostate was observed in GTC-fed mice compared to water-fed mice, resulting in reduced dissemination of cancer cells, thereby causing inhibition of development, progression, and metastasis to distant organ sites. Our team evaluated [65] the safety and efficacy of GTCs at various doses (200, 500, and 1000 mg EGCG in GTC/kg/day) in reducing the progression of PCa in a TRAMP mouse model. Significant decreases in the number and size of tumors in treated TRAMP mice were observed compared with untreated animals. We observed a dose-dependent inhibition of metastasis in GTC-treated mice (*p* = 0.0003). After 32 weeks of treatment with standardized formulation of GTC, it was found to be well-tolerated with no evidence of toxicity in C57BL/6J mice [65]. Apart from significant reductions in tumor size and multiplicity, GTCs also prevented metastatic progression of PCa in the TRAMP and other relevant mouse models. Collectively, these findings from pre-clinical studies, using doses relevant for translation to human clinical trials, provide evidence for safety and chemopreventive effects of GTCs.

#### **4. Clinical Evidence of Bioavailability, Safety, and Effectiveness of GTCs in Modulating Prostate Carcinogenesis**

Early phase I/II studies [67–76] conducted over the past decade found that doses of GTCs containing 200–1200 mg of EGCG per day (Poly E®) were tolerated by subjects, including men with precancerous lesions such as high-grade prostatic intraepithelial neoplasia, atypical small acinar proliferation, or early stage PCas.

Additionally, early phase I trials assessing standardized formulations of GTCs (Sunphenon 90D®) demonstrated increasing doses of plasma EGCG with increasing doses of the supplement [77–79]. Oral intake of GTCs in healthy subjects containing 225, 375, and 525 mg EGCG (Sunphenon® 90D) demonstrated a significant dose-dependent increase in plasma concentrations of EGCG to 657, 4300, and 4410 pmol EGCG/mL, respectively [80]. Consumption of Sunphenon® 90D containing 246 mg EGCG significantly increased plasma EGCG, which was highly correlated with attenuation of plasma phosphatidylcholine hydroperoxide levels, a marker of antioxidant capacity. Although increased bioavailability (as indicated by higher concentrations of EGCG in plasma) [81–83] occurs when GTCs are consumed in a fasting state [69] as opposed to a fed state, increased toxicity has also been reported when GTCs are taken in a fasting state. Similarly, increased bioavailability and tolerance to a multiple dosing schedule compared to a single daily dose of EGCG has been reported in phase II trials [67,75,84]. A summary of the concentration of GTCs in plasma with intervention trials targeting men at high risk for PCa is presented in Table 1. Mean plasma concentrations of EGCG varied among all these trials, potentially due to varying duration of intervention, doses, methods used in analyzing plasma EGCG, ethnicity of the target population, and nutritional and lifestyle habits. Another potential explanation may be differences in gut microbial capacity to process GTCs, as described below.

**Table 1.** Concentration of GTCs in plasma in interventional trials targeting men with PCa.

BID, twice a day; EGCG, (−)-epigallocatechin-3-gallate; GTC, green tea catechins; HGPIN, high-grade prostatic intraepithelial neoplasia; PCa, prostate cancer; Poly E, Polyphenon E; PSA, prostate specific antigen; RP, radical prostatectomy; SD, standard deviation; TID, three times a day.

Overall, prior studies support that GTCs are generally safe for consumption in human populations. A phase II/III trial (NCT00799890) was recently completed to evaluate the effect of 200–800 mg Sunphenon® 90D on attenuating brain atrophy, targeting patients with primary or secondary chronic-progressive multiple sclerosis treated for 36 months. An additional study (NCT00951834), using a maximum dose of 800 mg EGCG for 18 months to target early Alzheimer's, has been completed with results pending. No toxicities have been reported in either of these trials. In a more recently reported phase II clinical trial that evaluated the effects of 1315 mg of total catechins, containing 843 mg of EGCG, vs. placebo in modulating mammographic density, 1075 women were evaluated in a 12-month intervention. Overall, 26 women (5.1%) in the green tea extract arm developed moderate to severe abnormalities in liver function tests during the intervention period [86,87]. In three randomized trials [84,88–90] of the effects of GTCs (800 mg EGCG), or green tea as a beverage, in men diagnosed with localized PCa prior to prostatectomy, no toxicities were observed. These trials did not collect and analyze samples to assess interactions of the gut microbiome with GTC safety and toxicity.

A summary of the changes observed in intermediate endpoint biomarkers of PCa among Phase II GTC clinical trials is presented in Table 2. The findings from our study [75] and those of Bettuzzi et al. [67,68] suggest that a daily intake of the standardized GTC formulation administered non-fasting for 12 months in divided doses: (a) accumulates in plasma; (b) reduces serum PSA; and (c) reduces the cumulative rate of progression to PCa with no toxicities [67,68,75]. In the study of the effects of green tea beverages [88], nuclear staining of NF-κB was significantly decreased in radical prostatectomy (RP) tissue of men consuming GTC (*p* = 0.013), but not black tea (*p* = 0.931), compared to water control. Further, GTCs were detected in prostate tissue from 32 of 34 men consuming green tea but not in the other groups; evidence of a systemic antioxidant effect was observed (i.e., reduced urinary 8-hyroxydeoxy-guanosine) only with green tea consumption (*p* = 0.03) [88]. In randomized trials of the effects of GTCs (800 mg EGCG) or green tea as a beverage among men diagnosed with localized PCa, prior to prostatectomy, a reduction in serum PSA was observed [88,89]. Nguyen et al. [84] observed that the proportion of subjects who had a decrease in Gleason score between biopsy and surgical specimens was greater among those randomized to GTCs, but this finding was not statistically significant for the full duration of the intervention. In an open-label, single-arm, two-stage phase II clinical trial, 26 men with positive prostate biopsies received 800 mg EGCG/day (Poly E®) for 3–6 weeks until undergoing RP. EGCG administration lowered serum concentrations of hepatocyte growth factor (HGF), vascular endothelial growth factor, IGFBP-3, IGF-1, and PSA in these patients, with no elevation of liver enzymes [89]. More recently, Lane et al. [85] completed a 6-month randomized controlled trial of green tea and lycopene among men with elevated serum PSA but negative prostate biopsies. They randomized men to consume food sources of

each of these agents, to standardized formulations, or to placebo. Plasma levels of both lycopene and EGCG were higher in the treatment arms compared to the placebo arm with concentrations among the dietary source arm of these formulations being greater than the capsule group and the placebo arm. All interventions were tolerated well by the participants; however, men preferred the capsules to using food sources of lycopene and green tea. No biomarkers of disease progression were assessed in this study.

**Table 2.** Changes in intermediate endpoint biomarkers of PCa observed in Phase II clinical trials using GTCs.

Abbreviations: 8OHdG, 8-hydroxydeoxy-guanosine; ASAP, atypical small acinar proliferation; BID, twice a day; CI, confidence interval; GTC, green tea catechins; HGF, hepatocyte growth factor; HGPIN, high-grade prostatic intraepithelial neoplasia; EGCG, epigallocatechin-3-gallate; PCa, prostate cancer; Poly E, polyphenon E; PSA, prostate specific antigen; RP, radical prostatectomy; TID, three times a day; VEGF, vascular endothelial growth factor.

In summary, evidence from epidemiological, in vitro, pre-clinical, and early phase trials completed by our team and others have shown that the standard GTC formulations (a) accumulate in plasma and tissue; (b) reduce serum PSA and cumulative rate of progression to PCa; and (c) are potent inhibitors of PCa carcinogenesis through multiple mechanisms without toxicities at these doses, establishing the evidence needed for further development of GTCs in phase II clinical trials targeting men at exceptional risk or those diagnosed with low risk PCas. Self-reported patient race/ethnicity, medical history, family history of cancer, lifestyle factors such as smoking and alcohol use, and dietary intake have been accounted for in many of these studies; however, to date, the role of the gut microbiome in the absorption, safety, and modulation of prostate carcinogenesis has not been evaluated. Although the data on the safety, effectiveness, and potential mechanism of GTCs in prostate carcinogenesis appears promising, there are gaps in knowledge pertaining the role of the gut microbiome in modulating the bioavailability and toxicity of GTCs and in prostate carcinogenesis. With significant variations observed in GTC bioavailability (Table 1) as well as in modulation of intermediate endpoint biomarkers with GTCs (Table 2) in prior clinical trials, it is imperative to evaluate the contribution of the gut microbiome to modulating the interrelationships among GTC chemoprevention and PCa progression.

#### **5. The Gut Microbiome, PCa, and GTCs**

Predictive biomarkers of responses to secondary chemoprevention are presently lacking. Identification of biomarkers, such as the gut microbiome, predictive of favorable clinical responses to secondary chemoprevention has the potential to substantially facilitate clinical decision-making. Numerous studies found that the gut microbiome directly effects drug metabolism, efficacy, and toxicity, potentially affecting disease development and progression [91]. For example, in oncology, there exists convincing evidence to support that the antitumor effects of immunotherapies can be enhanced or inhibited by the gut microbiome [92,93].

The gut microbiome likely has critical roles in regulating the bioavailability of GTCs and absorption of bioactive phenolic GTC metabolites, as demonstrated in laboratory and pre-clinical models (Figure 2). Although dietary polyphenols are absorbed by the small intestine, accumulating evidence suggests that they are metabolized to a greater extent in the colon by bacterial enzymes [94,95]. EGCG is hydrolyzed by bacteria to gallic acid or EGC and further converted to multiple metabolites, such as 5-(3,5-dihydroxyphenyl)-4hydroxyvaleric acid and 5-(3 ,5 -dihydroxyphenyl)-g-valerolactone [95]. These metabolites are then either taken up via the portal vein and transported to the liver or excreted in the feces.

**Figure 2.** Examples of chemopreventive effects of GTCs in the context of PCa via gut microbiome modulation. (**A**) GTCs like EGCG have been evidenced to alter microbial composition, such as increasing abundance of Bifidobacterium [95]. This genus, for example, is known to increase production of SCFAs [95,96] which inhibit inflammatory pathways initiated by NF-κB that would otherwise propel carcinogenesis [97]. (**B**) The gut microbiome can enzymatically alter GTCs like EGCG to produce metabolites including gallic acid, EGC, valeric acid, and valerolactone, that subsequently travel to the bloodstream to exert potential chemopreventive benefits (e.g., regulating HDAC 1 and 2 and suppressing cell-cycle-related genes) [95,98–100]. EGC, epigallocatechin; EGCG, epigallocatechin gallate; GTCs, green tea catechins; HDAC, histone deacetylase; IL-6, interleukin-6; NF-κB, nuclear factor kappa B; SCFA, short chain fatty acid; TNF-α, tumor necrosis factor alpha. Created with Biorender.com (accessed on 1 July 2022).

On the other hand, several pharmacologic agents, including GTCs, were shown to influence gut microbiome composition and function. For example, in a study of 10 volunteers who drank 1000 mL of green tea daily for 10 days, Bifidobacteria abundance was increased [101]. In multiple animal studies, green tea polyphenols had similar effects on Bifidobacteria and other effects, including decreasing the Firmicutes/Bacteroidetes ratio [102,103]. In turn, gut microbiome composition and function may directly and indirectly influence PCa progression, such as through production of metabolically active metabolites or regulation of hormones and inflammation, as described below [104–106]. Given the substantial preliminary evidence for interrelationships among GTCs, the gut

microbiome, and prostate carcinogenesis, it is highly likely that the gut microbiome may mediate etiological effects of GTCs, including effects on PCa progression and development of adverse events; however, little is known regarding these interrelationships among humans.

The gut microbiome has biologically plausible roles in PCa such as via its influence on hormone and inflammation regulation and production of metabolically active metabolites [104–106]. For example, gut microbes produce sex hormones, such as androgen, and, in a study by Pernigoni et al., multiple species among mice and humans produced androgens from androgen precursors, in turn promoting progression of castrate resistant PCa [107]. In a study of both mice and PCa patients, Proteobacteria was increased after antibiotic exposure, and was in turn associated with development of PCa in mice and with metastasis of PCa among humans [108]. In a study of mice on a high-fat diet, the resultant alterations to the mice fecal microbiome promoted histamine biosynthesis and increased inflammatory cancer cell growth [109]. Previous human studies of the gut microbiome and PCa included a case-control study comparing 16S rRNA sequenced fecal bacteria among 64 men with PCa and 41 without PCa, finding differences in beta diversity, higher abundances of Bacteroides and Streptococcus species, and differences in folate and arginine pathways [110]. Another case-control study compared the gut metagenome among 8 men with benign prostatic conditions and 12 men with intermediate or high risk clinically localized PCa, finding higher relative abundance of Bacteriodes massiliensis and lower relative abundances of Faecalibacterium prausnitzii and Eubacterium rectalie amongst men with intermediate/high-risk PCa [111]. In a comparison of men with and without prostate enlargement, the ratio of Firmicutes to Bacteroidetes was higher among men with enlarged prostates, potentially related to prostate inflammation [112]. Finally, evidence supports the study of the gut microbiome across the disease continuum of PCa, with evidence demonstrating that the gut microbiome may be modified by PCa treatment, including androgen deprivation therapy, among more advanced PCa patients [113].

#### **6. Challenges and Future Directions**

There is currently sufficient evidence that establishes the need to evaluate the role of the gut microbiome in modifying the response to GTCs among men diagnosed with PCa. However, there are several challenges and pitfalls pertaining to studying GTCs in the context of modification by the gut microbiome. At present, biospecimens and data available to study the role of the gut microbiome in the effects of GTCs are sparse and further research is clearly needed among diverse populations. Studying the gut microbiome itself presents several challenges, as it is a complex, dynamic ecosystem that is driven by numerous known and unknown factors, such as dietary intake, requiring comprehensive measurement of potential confounding factors. It has been well documented that methods for stool collection, DNA extraction, and sequencing can influence downstream gut microbiome metrics, potentially resulting in inconsistent study findings that hinder progress in the field. To ensure high-quality, reproducible results, it is critical to establish contemporary and validated methodologies and to optimize protocols and procedures for fecal sampling, handling, processing, and microbiome analyses [114,115].

To fill existing gaps in knowledge, detailed characterizations of the gut microbiome and its metabolites among extensively phenotyped human subjects are needed. Although sequencing of the 16S rRNA gene classifies bacteria based on conserved single marker genes, there is a lack of detailed resolution. Shotgun metagenomic sequencing, which comprises the untargeted sequencing of all DNA present in a sample, provides more detailed taxonomic information than 16S rRNA sequencing. Characterizing microbial genes, strains, and functions may provide deeper insight into GTC-gut microbiome interactions. In addition, the gut microbiome and metabolome have moderate-to-high intraindividual variability and are 'high-dimensional' in that there are typically large numbers of microbes/metabolites relative to the numbers of subjects. As a result, collecting repeat samples is particularly useful for reducing bias in estimating effects/associations and increasing statistical power [116]. Other challenges include addressing limitations of previous human

studies of microbiome-PCa associations, including small sample sizes, inclusion of more advanced PCas, and cross-sectional design leading to concerns with reverse causality. Studies among large populations of men with serial microbiome and intermediate biomarker endpoint assessments are critically needed.

Future studies evaluating GTCs in prostate carcinogenesis may also include a metabolomics approach to assess EGCG- and microbiome-related metabolites from stool samples preand post-treatment with GTCs. In addition, these studies must include the evaluation of the correlation among specific microbial species/strains with (a) plasma levels of EGCG; (b) multiple markers of toxicity and safety; and (c) surrogate endpoint biomarkers, such as serum PSA, as an indicator for PCa progression, to provide timely evidence for a role of the gut microbiome in mediating the effects of GTCs on PCa progression. To our knowledge, studies collecting serial stool samples longitudinally to measure the microbiome in relation to these intermediate biomarkers of prostate carcinogenesis are currently unprecedented. Further, as in all biomedical research, there should be an emphasis in recruiting and studying disproportionately affected populations. In PCa, African American (AA) men are known to have the highest PCa risk. There is accumulating evidence that exposures associated with race may collectively and individually influence gut microbiome composition [57,58]. Therefore, with the inclusion of AA men in these clinical trials, we may be able to provide data to inform secondary chemoprevention efforts among this particularly at-risk population by studying a comprehensive biomarker (the gut microbiome). Finally, this review is focused and specific to the current data on the safety, effectiveness, and molecular mechanisms of GTCs in prostate carcinogenesis. Other cancers, like breast and colon cancers, may similarly be impacted by both GTCs and the gut microbiome. Collectively, these studies are critical in understanding the dynamics of the gut microbiome as we develop and evaluate promising agents such as GTCs for cancer chemoprevention.

**Author Contributions:** Conceptualization and writing, N.B.K. and D.A.B.; writing—original draft preparation and writing—review and editing, S.H.; Conceptualization and writing—original draft preparation, J.P.-S., M.P., B.J.M., R.L., J.D. and A.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** Financial support for this project was provided by funds from National Cancer Institute R01CA235032.

**Conflicts of Interest:** The authors declare that they have no competing financial or other interests in the results obtained within this manuscript.

 Breast cancer stem cells (BCSCs) are a rare population of cells present therein that cause cancer aggressiveness, relapse, drug-resistance, poor therapeutic outcome and a decrease in overall survival of these patients. The published literature indicates that dietary phytochemicals have the potential to target stemness and self-renewal properties in luminal A-derived BCSCs. The aim of this review is to highlight the anticancer potential of dietary phytochemicals against luminal A-derived BCSCs and their underlying mechanism(s). These findings necessitate in-depth preclinical and clinical studies on phytochemicals to explore their role in breast cancer prevention and treatment.

**Abstract:** Breast cancer is heterogeneous disease with variable prognosis and therapeutic response. Approximately, 70% of diagnosed breast cancer represents the luminal A subtype. This subpopulation has a fair prognosis with a lower rate of relapse than the other clinical subtypes. Acquisition of stemness in luminal A subtype modifies the phenotype plasticity to accomplish increased aggressiveness and therapeutic resistance. Therefore, targeting luminal A-derived breast cancer stem cells (BCSCs) could be a promising strategy for its prevention and treatment. Extensive studies reveal that dietary phytochemicals have the potential to target BCSCs by modulating the molecular and signal transduction pathways. Dietary phytochemicals alone or in combination with standard therapeutic modalities exert higher efficacy in targeting BCSCs through changes in stemness, self-renewal properties and hypoxia-related factors. These combinations offer achieving higher radio- and chemo- sensitization through alteration in the key signaling pathways such as AMPK, STAT3, NF-kB, Hedgehog, PI3K/Akt/mTOR, Notch, GSK3β, and Wnt related to cancer stemness and drug resistance. In this review, we highlight the concept of targeting luminal A-derived BCSCs with dietary phytochemicals by summarizing the pathways and underlying mechanism(s) involved during therapeutic resistance.

 Introduction**

Breast cancer is a heterogeneous disease that generally initiates in the milk glands or in ducts, which is referred to as lobular/ductal carcinoma in situ. These neoplastic breast cancer cells migrate through the gland/duct walls and proliferate in the surrounding tissue, which ultimately transform into an invasive phenotype. Breast cancer is frequently characterized by four major molecular subtypes that includes the luminal A (HR+/HER2−), luminal B (HR+/HER2+), basal-like (HR−/HER2−), and HER2-enriched (HR−/HER2+) subpopulations. The molecular subtypes (luminal A, luminal B, basal-like, and HER2

**Citation:** Prajapati, K.S.; Gupta, S.; Kumar, S. Targeting Breast Cancer-Derived Stem Cells by Dietary Phytochemicals: A Strategy for Cancer Prevention and Treatment. *Cancers* **2022**, *14*, 2864. https:// doi.org/10.3390/cancers14122864

Academic Editor: Andrea Manni

Received: 27 May 2022 Accepted: 8 June 2022 Published: 10 June 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

enriched) constitute approximately 73%, 11%, 12%, and 4% of breast cancer [1]. The other less common molecular subtypes include claudin-low and molecular apocrine forms. These molecular subtypes assist patient categorization, allowing them for better management of the disease as well as therapy-type decisions. Of the four major subtypes, luminal A tumors grow at a slower rate and are less aggressive with fairly high survival and low recurrence [1]. Conventional therapies target rapidly growing malignant cells that result in extensive elimination of tumors. Nonetheless, the surviving fraction constituting the minimal residual disease expands and undergoes multi-lineage differentiation to reconstitute the tumor. The reemerged tumor is highly aggressive, drug resistant, and comprises a new phenotype exhibiting increased aggressiveness and stemness. These cells are often referred as "cancer stem-like cells".  These cells have self-renewal properties and express stemness markers. BCSCs play an important role in the causation of drug resistance and results in a poor clinical outcome. Researchers are finding ways to target and remove the bulk of the tumor mass along with BCSCs for effective prevention and/or treatment of breast cancers. BCSCs play an important role in cancer metastasis due to the aberrant expression of some stemness-related factors, such as CD44, SOX2, OCT4, c-MYC, KLF4, Nanog, and SALL4 [2,3]. Besides the high expression of stemness and self-renewal markers, the aberrant expression of molecular signaling pathways including Wnt/β-catenin, Notch, Hedgehog, JAK-STAT, and PI3K/Akt/mTOR in BCSCs are shown to be involved in the pathophysiology of the disease [4,5]. Overall, the present literature reveals that the occurrence of BCSCs in the tumor microenvironment positively correlates with disease recurrence, low survival rate, chemo/radio therapy resistance, and low therapeutic output [6].

#### **2. Clinical Characteristics of Breast Cancer Subtypes**

Advanced molecular techniques provide a clear-cut characterization of breast cancer subtypes with better prediction and prognosis. Gene-based molecular assays, such as 70 gene and 80-gene signatures, 21-gene Recurrence Score, PAM50-ROR (50-gene Prediction Analysis of Microarrays and Risk of Recurrence), Endo-Predict, and the Breast Cancer Index (BCI) provide a precise characterization of breast cancer subtypes with better prediction and prognosis. The 21-gene recurrence score quantifies the probability of distant recurrence in patients with node-negative, ER+ breast cancer treated with tamoxifen into low, intermediate, and high-risk categories. The PAM50 classifies the intrinsic subtypes (luminal A, luminal B, HER2 enriched, basal, and normal breast) with risk of recurrence (low, intermediate, and high). The PAM50-ROR determines the probability of distant recurrence over 10 years. The 70-gene signature has the ability to divide patients into lowor high-risk corresponding to 10-year distant-metastasis-free survival (DMFS). The 80-gene signature divide patients into luminal, basal, and HER2 intrinsic subtypes. The combined 70- and 80-gene signatures are also able to classify breast cancer patients into luminal A-like (luminal subtype and low-risk), luminal B-like (luminal and high-risk), HER2, and basal subtypes [7,8].

Clinical reports suggest that at least half of the newly diagnosed breast cancer belongs to luminal A subtype. Initially, in 2011–2013, the oncologist proposed the molecular basis for the treatment of early breast cancer, which defined luminal A breast cancer patients as having a estrogen receptor (ER) positive, progesterone receptor (PR) positive (20%), HER2 negative, and Ki67 positivity of <14% [9,10]. This definition was based upon the gene profile (gene-based assay) and immunohistochemistry (IHC-based markers) of the tumor. The patients with IHC-based luminal A tumors were demonstrated to have better disease-free survival if PR expression was >20%. Especially for the luminal A breast cancer subtype, the patients and clinicians prefer surrogate IHC-based markers over gene-based biomarkers to establish the subtype. Overall, both the gene- and IHC-based biomarkers have been utilized for subtyping and treatment preference for breast cancer patients [11].

#### **3. Relevance of Targeting Luminal A-Derived Breast Cancer Stem Cells**

It has been widely accepted that the luminal cells of the mammary gland are unipotent, i.e., produce one cell type and have a self-renewal potential after evolution. Song et al. (2019) reported that during pregnancy or hormonal stimulation, the luminal cells give rise to luminal-derived basal cells (LdBCs) expressing the basal markers and ERα receptors. These cells respond to hormones and possess stem-cell renewal capability in the mammary gland [12]. Previously, it was reported that the Wnt and Notch signaling are determinants of the basal fate and luminal lineage. Recent findings align with the previous report that LdBCs demonstrated increased Wnt signaling (Figure 1). The plasticity of mammary luminal cells are associated with tumor progression in breast cancer patients [12]. The molecular subtype of breast cancer shows differential therapeutic response. Mei et al. (2020) studied the cancer stemness relationship with the molecular subtype of breast cancer in a comparative manner. The group developed MCF-7 cells (referred to as OKMS), which concomitantly overexpressed stemness-related genes (OCT4, KLF4, MYC, and SOX2) at the mRNA and protein level. These OKMS cells exhibited relatively low ER and higher HER2 expression. The cell growth and migration potential was increased up to 1.5 fold and the cancer stem cell population was increased up to 16% in OKMS cells, compared to parental MCF-7 cells. The OKMS cells demonstrated a drug response similar to HER2 positive cells after treatment with tamoxifen and trastuzumab. These results suggest a shift towards more aggressive and malignant tumors. However, luminal A cells exhibit slow growth and are less malignant but increase in stemness, transforming them to a more aggressive phenotype [13]. In another study, Yousefnia et al. (2019) reported that after a few passages, luminal A cells demonstrate a greater number of mammosphere formation in comparison to triple negative breast cancer cells [14]. Kim et al. (2012) demonstrated that tumor-derived pure luminal-like cells were capable of initiating invasive tumors, and generate larger tumors in comparison to basal-like cells in the in vivo model [15]. In addition, the stemness of cancer cells has the ability to change the tumor microenvironment in favor of supporting their aggressive natures [16]. Overall, these studies suggest that the stemness in the luminal A subtype transforms these cells to a more aggressive breast cancer phenotype. Since the luminal subtype forms the majority of breast cancer cases, targeting luminal A-derived BCSCs could provide a better therapeutic efficacy.

**Figure 1.** Sequential molecular events during self-renewal property acquisition in luminal A cells. LdBCs—Luminal-derived basal cells.

Epidemiologic and preclinical studies suggest that dietary phytochemicals possess chemopreventive properties against various cancer types. These phytochemicals possess

anticancer, antioxidant, antiviral, antibacterial, and various other pharmacological activities. Dietary phytochemicals, such as curcumin, quercetin, resveratrol, silibinin, lycopene, and emodin have been found to be useful in decreasing cancer incidence. A number of studies summarize the anticancer potential of phytochemicals and their importance in drug development. Dietary phytochemicals have the ability to reduce cancer promotion by inhibiting cancer cell proliferation, survival, invasion/metastasis, angiogenesis, and eliminating toxic carcinogens from the body through targeting different cancer-related signaling pathways. Dietary compounds also regulate cell cycle progression, inflammatory cytokines, and oxidative stress response in cancer cells, and have the potential to modulate small non-coding RNAs' expression alone or in a synergistic manner. More importantly, dietary phytochemicals have a greater ability to prevent/inhibit the formation of a small population of cancer cells often referred to as cancer stem-like cells by targeting various signaling pathways [17–27]. Therefore, targeting the luminal A-derived BCSCs by dietary phytochemicals could be a better strategy in the prevention and clinical management of breast cancer. The involvement of dietary phytochemicals in the management of BCSCs may provide significant contribution in this direction due to their minimal toxicity, low-cost, and bioavailability. In this article, we have reviewed the putative role of dietary phytochemicals in targeting luminal A-derived BCSCs in order to provide a strategy to increase the therapeutic efficacy in breast cancer patients. As most studies have focused on the role of dietary and non-dietary phytochemicals in cancer stem cell pathophysiology, to date, there is no comprehensive analysis to explore the role of dietary phytochemicals related to luminal A-cell-derived BCSCs. Here, we discuss the molecular mechanism(s) and pathways through which these phytochemicals target luminal A-cell-derived BCSCs. In addition, we also provide information related to pharmacokinetics, metabolism, bioavailability, dosage, and changes in the microbiome.

#### **4. Source of Data**

The literature published (in the English language) and indexed in the PubMed database was utilized for the present review. The relevant studies were retrieved through the use of "breast cancer stem cell, luminal A, mammosphere" as keywords in searches of the database. The literature that contained the luminal A breast cell-derived cancer stem cells and phytochemicals was filtered. Various in vitro luminal A breast cancer cells (BT483, CAMA1, EFM19, HCC1428, HCC712, IBEP2, KPL1, LY2, MCF-7, MDAMB134, MDAMB134VI, MDAMB175, MDAMB175VII, MDAMB415, T47D, ZR751, and ZR75B) were taken into consideration; however, most of the studies were focused on the MCF-7 cell-derived mammosphere, which demonstrated increased stemness/self-renewal markers. Further, the literature was filtered on the basis of dietary and non-dietary phytochemicals by finding the associated published information (Table 1). Data published on the role of dietary phytochemicals on luminal A-derived breast cancer stem cells were only considered in the study. Dietary phytochemicals viz. curcumin, naringenin, resveratrol, genistein, quercetin, silibinin, and thymoquinone, comparatively cited in the literature, were used in the review. Additional phytochemicals such as ginsenoside Rg3, hisperidin, pristimerin, pterostilbene, 3-O-(E)-p-Coumaroyl betulinic acid and withaferin A, which are less studied, are discussed under a common heading, namely "other dietary phytochemicals".

**Table 1.** List of dietary phytochemicals and associated secondary metabolite group targeting luminal A breast cancer cells.

#### **5. Curcumin and Stemness in BCSCs**

Curcumin belongs to the polyphenolic group of phytochemicals and is an important component of the spice turmeric all around the globe, especially in India. It is used for its coloring ability and taste. Moreover, curcumin has been used in various medicinal preparations of Ayurveda and Chinese Medicinal systems. Numerous studies report potent biological activity, and applications in the food, biotechnology, and cosmetics industry. Curcumin is well-known for its health promoting and disease preventive properties [41]. Yang et al. (2020) studied the effect of curcumin (alone and in combination with nanoparticles) in the radiation-treated (4Gy) MCF-7 cell-derived mammosphere. The group found that curcumin significantly increased the radio-sensitivity in the breast sphere cells alone and in combination with the gold nanoparticles. More than 60% of the sphere population was significantly damaged by curcumin treatment. Curcumin pretreatment in the radiationexposed breast cancer MCF-7 and MDA-MB-231 cell-derived mammosphere demonstrated increased apoptosis and reactive oxygen species formation, G0/G1 phase cell cycle arrest, and decreased HIF-1α and HSP90 protein expression [42]. Sarighieh et al. (2020) tested the efficacy of curcumin in cancer stem cells isolated from MCF-7 cells. The cells were sorted in the presence of CD44+/CD24− surface markers, and the curcumin treatment was rendered both in hypoxic and normoxic conditions. The curcumin-treated MCF-7 derived cancer stem-like cells demonstrated early apoptosis and G2/M phase arrest under hypoxic conditions. Under normoxic conditions, the curcumin induced S and G2/M phase arrest in the cells [43]. The curcumin inhibits HIF1 nuclear translocation by degrading ARNT (aryl hydrocarbon receptor nuclear translocator), which is required for the transcription of hypoxia-related genes in breast cancer stem cells. The treatment also decreases HIF1 and HIF-2α expression in MCF-7 cell-derived mammospheres. In another study, Borah et al. (2020) demonstrated that curcumin in combination with GANT61 (Gli1 and 2 inhibitor of hedgehog signaling pathway) loaded PLGA (poly(lactic-co-glycolic acid) nanoparticles demonstrated self-renewal inhibitory potential in MCF-7 cell-derived mammospheres [44]. Attia et al. (2020) reported the effect of curcumin in combination with paclitaxel and vitamin D on MCF-7 cells. The study demonstrated that it improved anticancer activity and anti-drug resistance efficacy in MCF-7 cells by a decrease in proliferation and increased apoptosis. Further treatment significantly decreased cancer stemness markers (multidrug resistance complex and aldehyde dehrogenase-1) at the protein level [45]. Liu et al. (2019) studied the effect of a curcumin coating on a synthetic polymer (oligomeric hyaluronic acid-hydrazone bond-folic acid-biotin), which forms curcumin nano-actiniaes (Cu-NA). The Cu-NA demonstrated increased toxicity in MCF-7 cells, breast cancer stem cells, and in the in vivo anticancer experiment in comparison to free curcumin and/or other control groups [46]. Hu et al. (2019) demonstrated that curcumin has the potential to inhibit the cancer stemness property (Oct4, Nanog, and Sox2) and the epithelial to mesenchymal

(EMT) transition in luminal A cells. The group isolated the CD44+CD24−/low subpopulation of MCF-7 cells, which shows breast cancer stem-like properties. The study reported that curcumin treatment resulted in a decreased cell proliferation and colony formation potential in these cells [47]. Hashemzehi et al. (2018) demonstrated the efficacy of a novel curcumin formulation (phytosomal encapsulated curcumin) on thrombin-induced cellular proliferation and metastasis in MCF-7 cells. The study demonstrated that curcumin formulation exerts its anticancer, anti-metastatic, and anti-stemness potential by activating AMPK signaling [48]. Li et al. (2018) demonstrated that curcumin treatment decreases the expression of stemness markers (ALDH1A1, CD44, Nanog, and Oct4) and inhibit Sonic hedgehog and Wnt signaling pathways in MCF-7 cell-derived mammospheres [49]. In another study, the folate decorated curcumin-loaded nanostructured lipid carriers (FA-CUR-NLCs) formulation demonstrated enhanced antitumor activity (in comparison to standard curcumin) in MCF-7 cell-inoculated animal models [50]. Yuan et al. (2018) studied the effect of the curcumin and doxorubicin combined nano-formulation (CURDOX-NPs) against drug resistance MCF-7 cell (MCF-7/ADR)-derived mammospheres. The study reported that the CURDOX-NPs significantly reduced the mammosphere formation potential in vitro and demonstrated tumor growth regression (~33%) in a mouse xenograft model [51]. Zhou et al. (2017) showed that curcumin has the potential to inhibit the drug resistance potential in breast cancer stem-like cells by improving Bcl-2-mediated apoptosis. The apoptosis inducing efficacy of curcumin was more pronounced during the combined treatment with Wnt and PI3K inhibitors. The treatment significantly decreased the antiapoptotic proteins and increased the expression of pro-apoptotic proteins in targeted cells [52]. In a different study, Zhou et al. (2015) reported that curcumin has the potential to inhibit the cancer stem cell self-renewal potential in MCF-7 cell-derived mammospheres by lowering the expression of drug efflux transporters (ABCG2). Curcumin treatment also improved the anti-cancer efficacy of mitomycin C in the in vitro breast cancer stem cell model [53]. Further, Zhou et al. (2011) d that the co-treatment of curcumin and mitomycin C significantly reduced the mitomycin C-associated side-effects and improved its efficacy in a breast cancer xenograft model. Curcumin co-treatment altered the creatinine/blood urea nitrogen level and glutamic oxalacetic transaminase/glutamic pyruvic transaminase activity towards normal levels. The study indicated that curcumin mitigates the kidney reacted toxicity due to mitomycin C treatment in MCF-7 xenograft models. Moreover, curcumin and mitomycin C synergistically induced cell cycle arrest via the p38 MAPK pathway in the in vivo model compared to respective alone treatment(s) [54].

Other studies indicate that STAT3 (Signal Transducer and Activator of Transcription 3) and NF-κB (Nuclear factor-κB) signaling pathways play an important role in the maintenance of cancer stem-like properties in cancer cells. Chung and Vadgama, (2015) studied the effect of curcumin and EGCG co-treatment in CD44+ MCF-7-derived BCSCs. The study demonstrated a significant decrease in stem cell population, and decreased STAT3 phosphorylation and STAT3-NFkB interaction in the curcumin-treated cells [55]. Furthermore, curcumin co-treatment with interferon-β/retinoic acid (IFN-β/RA) in the MCF-7 athymic nude mouse model demonstrated synergistic anticancer potential. Curcumin increases the IFN-β/RA level with a simultaneous decrease in cyclooxygenase-2 (COX-2) activity and increases the DNA damage-inducible gene 153 (GADD153) expression, resulting in reduced tumor growth [56]. A summarization about the curcumin potential against luminal A-derived breast cancer stem cells includes (i) the decrease in self-renewal/stemness marker(s) expression, (ii) the possessed radio-sensitization and chemo-sensitization potential, (iii) targets AMPK, STAT3, and NF-kB signaling pathways and hypoxia-related markers, (iv) decreases the expression of multi-drug resistance transporters (ABCG2), and (v) the possessed synergetic anti-breast cancer stem cell potential. The overall mechanism(s) of the curcumin regulation of breast cancer stemness and associated signaling pathways is shown in Figure 2.

**Figure 2.** Effect of curcumin on luminal A-derived mammosphere. Yellow-colored circle represents curcumin. HIF-1α—Hypoxia inducing factor-1α, AMPK—AMP-activated protein kinase, EMT— Epithelial mesenchymal transition, Shh—Sonic hedgehog, ALDHA1—Aldehyde dehydrogenase A1, SRY (sex determining region Y)-box 2, and MDR—Multi-drug resistance.

#### **6. Phytoestrogen and BCSCs**

Phytoestrogens are polyphenolic naturally occurring secondary metabolites of plants. These molecules are structurally and functionally similar to the mammalian major sex hormone, 17-β-oestradiol (E2). Coumestans and isoflavones are widely researched phytoestrogens. These compounds are found in a variety of foods and possess a protective effect against hormone-related cancers and other diseases. The literature search related to the present review demonstrated that naringenin, resveratrol, genistein, apigenin, and quercetin possess the potential to target luminal A-derived BCSCs.

#### *6.1. Genistein*

Genistein is a naturally occurring isoflavone present in various dietary sources, including legumes and soybean products [57]. A hydroxyl group at carbon 7 forms a glycosidic linkage with sugar molecules and generates its dietary carbohydrate conjugates [58]. Genistein elicits several pharmacological activities, such as protein tyrosine kinase and DNA topoisomerase II inhibition, and the induction of antioxidant enzymes. The report demonstrates the role of genistein in the induction of apoptosis, cell cycle arrest, cell proliferation and metastasis inhibition in breast cancer experimental models. Genistein alters the expression of cancer-associated signaling pathways alone or in combination with standard chemotherapeutic drugs [43]. The Krüppel-like factor 4 (KLF4), an evolutionary conserved zinc finger-containing transcription factor, regulates cellular proliferation. In cancer, it functions in an organ-specific manner and displays both an oncogenic and tumor suppressive role.  A high KLF4 expression was positively associated with the increased self-renewal/stemness markers and side-population in experimental settings [59]. A later report demonstrates that genistein in combination with sulforaphane significantly reduces KLF4 expression at mRNA and protein levels in MCF-7 cells [60]. BCSCs undergo increased endocytosis (clathrin and caveolin independent)

than analogous non-stem cancer cells. Genistein was unable to inhibit the endocytosis in the MCF-7-derived mammosphere but demonstrated a mammosphere reduction potential [61]. Genistein inhibit mammosphere formation in MCF-7 cells by downregulating the Hedgehog and PI3K/Akt pathway and inhibiting abiogenesis in mammary glands [62–64]. Further, it has been reported that genistein target adipogenesis and stem cell formation in an interleukin-6 (IL-6) independent manner [64]. In a similar study, genistein at a 25 μM dose increases the sphere formation potential in MCF-7 cell-derived tertiary spheroids. The genistein increased protease inhibitor 9 (PI-9, granzyme b inhibitor) expression and decreased estrogen receptor isoform (ERα66) [65]. It also reduced the mammosphere formation potential in MDA-MB-231 cells both in co-cultured with MCF-7 cells and in solo culture at micro and nanomolar concentrations. Although the size of the spheroids was reduced in both cultures, the morphological changes were observed only in the cocultured experimental setup. Another study revealed that genistein inhibits the stem cell formation in MDA-MB-231 cells via induction PI3K/Akt and MEK/ERK signaling pathways in a paracrine manner through the increased expression of amphiregulin in MCF-7 cells [66]. Genistein alone or in combination with other phytoestrogens has been reported to inhibit/reduce tumor growth both in in vitro and in vivo systems. The combination of genistein (GEN) and lignan enterolactone (ENL) (100 mg/kg ENL + 100 mg/kg GEN) demonstrated that the inhibition of estradiol (E2) induced in MCF-7 cells established tumor growth and angiogenesis in mice [67]. The published literature thus far suggests the effect of genistein against luminal A-derived BCSCs through the (i) downregulation of the Hedgehog and PI3K/Akt pathway, (ii) suppression of the oncogenic transcription factors and (iii) inhibition of the mammosphere formation in ER− breast cancer cells in a paracrine manner.

#### *6.2. Naringenin and Resveratrol*

Naringenin belongs to flavanones, a subclass of flavonoid. The compound is widely distributed in tomatoes, citrus, and other fruits. It is also found in glycoside form, known as naringin in various dietary sources. Naringenin is insoluble in water and soluble in organic solvents. The compound possesses antioxidant, anti-inflammatory, anti-aging, anticancer, anti-asthma, and anti-viral properties. Moreover, it has been utilized in infertility, immuno-depression, constipation, hepatic damage, pregnancy, and obesity as a therapeutic molecule [68]. Naringenin has been reported to possess anticancer activity, induce apoptosis, and cell cycle arrest at different concentrations in various human breast cancer cell lines. Naringenin has been well documented in managing the cancer cell growth and proliferation, migration, and multi-drug resistance both in in vitro and in vivo by targeting signaling pathways such as Jak/Stat3, Notch1, p38/MAPK, NF- B, PI3K/Akt, and COX2. Nanoparticle formulations of naringenin improves chemosensitization and anticancer potential [68]. Curcumin-naringenin loaded dextran-coated magnetic nanoparticles (CUR-NAR-D-MNPs) in combination with radiotherapy has been studied in the MCF-7 cell-inoculated mouse model. CUR-NAR-D-MNPs, in combination with radiotherapy, reduced tumor volume and induced cell cycle arrest and apoptosis by modulating the expression of p21, p53, TNF-α, CD44, and ROS signaling in experimental animals [69]. The group also studied the effect of phytoestrogens (naringenin, resveratrol, and quercetin) in the MCF-7 cell-derived xenograft model. The findings revealed that the phytoestrogen treatment inhibit the survival of breast tumor initiating cells, and restrict tumor growth rates and tumor initiation by increasing DAXX protein levels. Phytoestrogens demonstrated DAXX-mediated anti-breast cancer activity in the order of naringenin > resveratrol > quercetin at 20 mg/kg dose [70].

Resveratrol is a lipid soluble polyphenolic compound. The phytochemical is found in cis and trans conformations in dietary sources. The dietary source of resveratrol includes cranberry, red/white grapes, strawberry, peanuts, etc. Resveratrol has been well documented for its cardio-protective and cancer preventive properties [71]. After hormonal therapy, the tumor initiating cells in ER+ breast cancer typically demonstrate increased Notch signaling activity and resistance to therapy. These cells do not express the death

domain-associated protein 6 (DAXX), resulting in the activation of notch signaling and formation of therapy-resistant tumor initiating cells (TICs) [70]. It is quite interesting that the treatment of MCF-7 cell-derived TICs with phytoestrogens (such as naringenin and resveratrol) demonstrated increased DAXX expression, which results in the inhibition of the Notch signaling-mediated TIC-cell enrichment. It should be noted that naringenin and resveratrol demonstrate this effect by selectively targeting both ERα and ERβ forms of the estrogen receptor. The findings of Peiffer et al. (2020) indicate that the selective inhibition of ER receptor isoforms by phytochemicals could be a novel therapeutic approach to target cancer stemness and tumor initiation in therapy resistant ER+ positive cells [70].

#### *6.3. Quercetin*

Quercetin (3,5,7,3 ,4 -pentahydroxylflavone) belongs to a flavonol subclass of flavonoid. It is found in various fruits and vegetables, but at a larger quantity in apple and onion. Quercetin protects body tissues alone or in combination with other dietary antioxidants, such as vitamin C/E and carotenoids. The phytochemical is well known for its beneficial role in human health, as it is a powerful antioxidant, lowers cholesterol and erectile dysfunction, improves blood circulation, reduces inflammation, lowers the risk of neurodegenerative disease, and possesses anticancer properties [72]. Quercetin-3-methyl ether (Q3ME) is a natural analog of quercetin found in various plants. 7-O-geranylquercetin is an alkylated derivative of quercetin synthesized to overcome the poor solubility of quercetin. The compound possesses potent anti-tumor activity. It has been demonstrated that the derivative treatment in MCF-7/ADR cells inoculated in BALB/c nude mice significantly reversed drug resistance by down-regulating the expression of P-gp protein and its encoding gene MDR1 [73]. Cao et al. (2018) reported that quercetin has the potential to target MCF-7 cell-derived BCSCs alone or in combination with DAPT, a γ-secretase inhibitor. Q3ME inhibits the mammosphere formation by regulating the expression of genes involved in cancer stemness and inhibited the Notch and PI3-AKT signaling pathways [74]. The increased expression of P-glycoprotein (membrane transporter) mediated multidrug resistance (MDR) in cancer cells play a major role in decreasing the therapeutic outcome in breast cancer patients [75–77]. In this context, nuclear translocation of YB-1, a oncogenic transcription factor, is in association with the P-gp overexpressed in breast cancer cells and is associated with the stemness property [78]. Li et al. (2018) demonstrated that quercetin decreased YB-1 translocation, P-gp expression in luminal A breast cancer cells, and in the drug-resistant MCF-7 cell-derived stem-like cell population [79]. Similarly, Li et al. (2018) reported that quercetin, in combination with doxorubicin, significantly decreased the MCF-7-derived breast cancer stem-like cells [80]. Further, Li et al. (2018) demonstrated that quercetin significantly decreased the clone and sphere formation potential in MCF-7 cell-derived BCSCs [81]. Quercetin also modulated the PI3K/AkT/mTOR (phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin) signaling pathway in these cells [82]. Earlier, Imai et al. (2012) reported that the quercetin derivative (LY294002) has the capability to reduce the drug efflux in BCSCs by inhibiting the PI3K/Akt signaling pathway [83]. A summarization of the literature on the anticancer effects of quercetin against luminal A-derived breast cancer stem cells includes (i) targeting the ERα receptor, Notch and PI3/AKT/mTOR signaling pathways, (ii) lowering the expression of the multidrug resistance transporter, and (iii) inhibits the nuclear translocation of proteins involved in the breast cancer stemness and self-renewal process.

#### *6.4. Silibinin*

Silibinin is a natural flavonolignan compound. It is a major constituent of Silymarin, extracted from *Silybum marianum* (milk thistle), which is used for the treatment of liver diseases. Recently, silibinin has been reported to exert significant anti-neoplastic effects against breast, prostate, lung, colon, and skin cancer in the in vitro and in vivo model. It has been reported that metabolic heterogeneity plays an important role in cancer stem cell maintenance and self-renewal. Bonuccelli et al. (2017) found that MCF-7 cells possess high

PGC1α activity and reactive oxygen species production, and NADH levels in the mitochondria possess increased stem cell formation potential. Silibinin significantly reduced the mammosphere formation in this metabolic form of MCF-7 cells [84]. Studies have indicated that silibinin is a glycolytic inhibitor and also inhibits glucose uptake. Dadras et al. (2016) studied the MCF-7 cells stemness inhibition potential of silibinin-entrapped nanoparticles. The study demonstrated a significant reduction in the potential of free silibinin in MCF-7 cancer stem cell viability in the in vitro assay [85]. Published reports on the efficacy of silibinin in luminal A-derived breast cancer cells are at the preliminary levels. There is a need to explore the underlying mechanism of stemness and the self-renewal property inhibition potential of silibinin in both the in vitro and in vivo models. The literature so far has demonstrated that silibinin exerts its pharmacological effect on luminal A-derived breast cancer stem cells by targeting metabolic pathways, and reducing the viability of MCF-7 cell-derived mammospheres.

#### *6.5. Thymoquinone*

*Nigella sativa* L. (Ranunculaceae), commonly known as black cumin, is an important spice in Europe, South West Asia, and North Africa. Thymoquinone (TQ) belongs to the quinone group of phytochemicals and is the most abundant constituent of *Nigella sativa* seeds in volatile oil. Several pharmacological activities of TQ have been reported, including anti-histaminic, anti-inflammatory, anti-microbial, antioxidant, immunomodulatory, and anti-tumor effects. Various studies demonstrate that TQ possesses less toxicity and has low adverse effects on normal cells [86]. TQ demonstrated reduced self-renewal properties in the MCF-derived mammosphere. The TQ treatment decreased the number and size of spheroids in comparison to the control group. TQ, alone or in combination with the emodin, a natural anthraquinone derivative, lowered the cancer stemness-related markers viz. OCT-4, SOX-2, NANOG, and ALDH1/2 in MCF-7-derived BCSCs [87]. In another study, the TQ decreased the stem cell population by 12%; in combination with paclitaxel, the efficacy was increased up to 32%, as compared with the non-treated cells. Whereas, the paclitaxel treatment alone demonstrated an 8% reduction in stem cell population. In another study, the gemcitabine was unable to decrease the stem cell population; however, in combination with TQ, a 12% reduction was observed [88]. These studies indicate that TQ may be used in combination with standard chemotherapeutic drugs to target BCSCs effectively. However, detailed mechanisms are warranted to establish the BCSCs' reduction potential of TQ, in combination with the standard therapeutic drugs in pre-clinical models, including a reduction in mammosphere size and number alone or in combination with a phytochemical, as well as the chemosensitization potential of BCSCs. The luminal Aderived mammosphere reduction and breast cancer stemness and self-renewal regulating pathways' inhibiting potential of phytoestrogens are summarized in Figure 3.

**Figure 3.** Effect of phytoestrogen(s) on the luminal A-derived mammosphere. ALDH1—Aldehyde dehydrogenase 1, Krüppel-like factor 4, and BCSCs—Breast cancer stem cells.

#### **7. Other Dietary Phytochemicals**

Hisperidin is a natural polyphenolic compound generally known as a citrus flavonoid. The compound is pharmacologically active and possesses significant anticancer activity. Hesperidin and its aglycone derivative (hesperitin) inhibit metastasis and tumor growth in MCF-7-inoculated experimental mice. Hesperitin (at a 1000 and 5000 ppm concentration) prevent tumor growth by reducing the plasma estrogen level and pS2 gene expression in

the ovariectomized, and the aromatase overexpressing in the MCF-7 athymic xenograft mouse model. The hesperitin treatment inhibited aromatase activity and increased the cell cycle arrest and apoptosis in experimental animals [89]. Recently, Hermawan et al. (2021) reported the anticancer activity of hesperidin in luminal A-derived breast cancer spheroids using bioinformatics and an in vitro approach. A decreased sphere and colony formation potential was observed in hesperidin-treated MCF-7 cells [90].

Pristimerin is a natural occurring triterpenoid compound mainly isolated from the *Celastraceae* and *Hippocrateaceae* family. It exerts anticancer activity against different types of cancer both in in vitro and in vivo experimental models. Cevatemre et al. (2018) demonstrated that pristimerin inhibited breast tumor growth and induced apoptosis by cleaving PARP and activating caspase-3 in MCF-7-derived spheroids and in the mouse xenograft model [91]. Pristimerin demonstrated increased apoptosis, cytoplasmic vacuolation, endoplasmic reticulum stress, unfolded protein response, and autophagy flux blockagemediated death in breast cancer spheroids. The phytochemical inhibited the Wnt signaling pathway in MCF-7-derived mammospheres by degrading the low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt co-receptor [91].

Pterostilbene is a natural dimethylated analogue of resveratrol. The compound is present in blueberries in larger quantity. Pterostilbene inhibits cancer cell proliferation, invasion, and metastasis, and induced apoptosis in several experimental models. Mak et al. (2013) studied the effect of pterostilbene in M2 tumor-associated macrophages (M2TAMs) inducing stem cell generation potential in MCF-7 cells. The pterostilbene treatment significantly reduced the BCSC generation in MCF-7 cells co-cultured with the M2TAMs by decreasing the CD44+/CD24− cell population, and reduced the migratory and invasive capabilities of BCSCs. Moreover, the pterostilbene treatment reduced NF-κB, vimentin and Twist1, and elevated the E-cadherin expression in MCF-7-derived mammospheres [92]. Later, Wu et al. (2015) reported that pterostilbene produced more toxicity in MCF-7-derived cancer stem cells in comparison to MCF-7 cells. Pterostilbene significantly induced necrosis-mediated cellular membrane damage, reduced stemness markers CD44 and c-Myc, and inhibited hedgehog, Akt and GSK3β signaling pathways in MCF-7-derived spheroids [93].

Ginsenoside Rg3 (GRg3), which belongs to the *Araliaceae* family, is an important pharmacological active constituent of *Panax ginseng*. GRg3 has been known to possess potent anticancer activity by modulating several oncogenic pathways. Oh et al. (2015) comparatively studied the efficacy of low and high GRg3 containing red ginseng extract in the MCF-7 cell-derived mammosphere [94]. The high GRg3 content extract demonstrated more pronounced effect on the mammosphere by decreasing their self-renewal potential. In a different study, GRg3 decreased the stemness and self-renewal potential in MCF-7 cells' spheroids by targeting the PI3K/Akt signaling pathway, modulating Sox-2 and Bmi-1 self-renewal markers, and inhibiting the nuclear translocation of the HIFα factor [95].

Withaferin A (WA) is a major pharmacological active ingredient of Indian Ginseng (*Withania somnifera*). It belongs to the steroidal lactone group of compounds. WA inhibits luminal A-derived BCSCs by reducing the urokinase type plasminogen activator receptor (uPAR) and polycomb group protein Bmi-1 expression, which are well known factors to drive stemness and self-renewal properties in BCSCs. Inhibition of Notch4 activity reduces stem cell activity and tumor formation both in in vitro and in vivo breast cancer models [96]. Kim and Singh (2014) reported that WA has the potential to inhibit Notch4 activation and reduce KLF4 expression and ALDH1 activity in MCF-7 and SUM159 cancer stem cells [97]. The modulation of miRNAs by phytochemicals is an important strategy to modulate the expression of target mRNAs at a transcriptional level in cancer cells [13,98]. Higher expression of miR-6844 has been demonstrated in clinical specimens of invasive breast cancer patients in comparison to normal subjects. Recently, our group reported that miR-6844 is highly expressed in the luminal A-cell-derived mammosphere, compared to normal non-sphere breast cancer cells. The WA treatment significantly reversed the expression of miR-6844 and inhibited the mammosphere formation potential [99]. The

aberrant expression of Notch signaling target genes has been positively correlated with the stemness and self-renewal property of BCSCs. Recently, we demonstrated that natural phytochemical 3-O-(E)-p-Coumaroylbetulinic acid possesses stemness and self-renewal inhibition potential in the MCF-7 cell-derived mammosphere by downregulating the Notch signaling pathway. The phytochemical altered the Notch target genes such as E-cadherin, Hey1, and Hes1, and breast cancer stemness markers viz. c-Myc, COX2, CD44, OCT4, NANOG, CD44, and EpCAM in MCF-7 cell-derived mammospheres [100].

Overall, the phytochemicals discussed above possess the potential to target luminal Aderived BCSCs by the selective elimination of these cells through the inhibition of Akt, Wnt, hedgehog/GSK3β signaling pathway, and nuclear translocation of the HIFα factor. These underlying mechanisms of the luminal A-derived breast cancer stemness and self-renewal inhibition potential of dietary phytochemicals is summarized in Figure 4 and Table 2.

**Table 2.** Stemness/self-renewal signaling regulations by dietary phytochemicals in luminal A-derived BCaSCs.

BCa—Breast cancer, BCaSCs—Breast cancer stem cells, Akt—Protein kinase B, SOX-2—(Sex determining region Y) box-2, Bmi-1—Polycomb protein complex, HIF-1α—Hypoxia inducing factor-1α, Wnt—Wingless/Integrated, LRP6—Low-density lipoprotein receptor-related protein 6, LC3-II—Light chain 3, NF-κB—Nuclear factor-kappa B, uPAR—Urokinase type plasminogen activator receptor, KLF4—Kruppel-like factor-4, ALDH1—Aldehyde dehydrogenase 1, AMPK-AMP—activated protein kinase, STAT3—Signal transducer and activator of transcription 3, HSP90—Heat shock protein 90, ARNT—Aryl hydrocarbon receptor nuclear transporter, Oct4—Octamer binding transcription factor, EMT—Epithelial mesenchymal transition, ABCG2—ATP-binding cassette super family G member 2, PI3K—Phosphoinositide-3-kinase, DAXX—Death-associated protein 6, IL-6—Interleukin-6, mTOR—Mammalian target of rapamycin, P-gp—P-glycoprotein, YB-1—Y-binding protein 1, and MDR—Multidrug resistance.

**Figure 4.** Luminal A-derived breast cancer stemness/self-renewal inhibiting potential of various dietary phytochemicals including *Withaferin A*, Ginsenoside Rg3 (GRg3), Pterostilbene, Hisperidin, and Pristimerin. The phytochemicals target Notch signaling, Wnt, hypoxia, NF-B, and Akt pathways involved in the maintenance of stemness and self-renewal. Withaferin A reduces the mammosphere formation in luminal A breast cancer cells by modulating the expression of Sox2, c-MYC, and CD44 via targeting Notch signaling pathway. Pterostilbene inhibits NF-κB, vimentin, and Twist1, and elevated E-cadherin expression. Ginsenoside Rg3 inhibits the nuclear translocation of HIF-1α, Sox-2, and Bmi-1 marker expression via targeting hypoxia and PI3K/Akt signaling pathway. Pristimerin mediated modulation of autophagy and degradation of LRP6, a Wnt co-receptor of Wnt signaling pathway and the expression of stemness markers. Hesperidin arrest cell progression at G0/G1 phase by targeting cyclin D1. Hesperidin modulates the expression of p21 and p53 in luminal A-derived mammosphere. [KLF4—Kruppel-like factor 4, HIF-1α—Hypoxia inducing factor-1α, VEGFA—Vascular endothelial growth factor A, Hh—Hedgehog, PTCH1—Patched1, Smo—Smoothened, GLI1—Gli family zinc finger 1, Kinesin family member 7, FZ—Frizzled, TCF/LEF—T-Cell factor/Lymphoid enhance factor, IKB—Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, mTOR—Mammalian target of rapamycin, PIP3—Phosphatidylinositol (3,4,5)-trisphosphate, PIP2—Phosphatidylinositol-4, 5-bisphosphate, PTEN—Phosphatase and tensin homolog, PI3K—Phosphoinositide 3-kinases, Akt—Protein kinase B, GREB—cAMP-response element binding protein, NICD—notch intracellular domain, HAT— Histone acetyltransferases; APH-1—anterior pharynx—defective 1, *PSEN*-Presenilin-1, NCSTN— Nicastrin, MDM2-*Mouse double minute* 2 homolog, Akt—Protein kinase B, SOX-2-(Sex determining region Y) box-2, Bmi-1—Polycomb protein complex, HIF-1α—Hypoxia inducing factor-1α, Wnt— Wingless/Integrated, LRP6—Low-density lipoprotein receptor-related protein 6, LC3-II—Light chain 3, NF-κB—Nuclear factor-kappa B, KLF4—Kruppel-like factor-4, AMPK—AMP-activated protein kinase, PI3K—Phosphoinositide-3-kinase, IL-6—Interleukin-6, mTOR—Mammalian target of rapamycin, P-gp—P-glycoprotein, YB-1—Y-binding protein 1, MDR—Multidrug resistance, AMP— Adenosine monophosphate, ADP—Adenosine diphosphate, PIP3—Phosphatidylinositol (3,4,5) trisphosphate, PIP2—Phosphatidylinositol-4, 5-bisphosphate, PTEN—Phosphatase and tensin homolog, PI3K—Phosphoinositide 3-kinases, and Akt—Protein kinase B].

#### **8. Pharmacokinetics and Bioavailability**

Liver is the primary site of metabolism for phytochemicals, together with the intestine and gut microbiota. After several metabolic reactions, phytochemical structures are modified in the hepatocytes and enterocytes, and form subsequent inactive/bioactive metabolites. Following oral ingestion, phytochemicals undergo extensive metabolism that includes chemical reactions including reduction, sulfation, and glucuronidation in the liver, kidneys, and intestinal mucosa. These metabolized products generated in the liver and intestine, as a result of their biotransformation, exhibit enhancement in their biological activity. Studies have demonstrated that the human microbiota and brush border enzymes are involved in phytoestrogen metabolism and active metabolite synthesis. Gut microbiota composition influences phytochemical bioavailability and inter-individual effects. Gut bacteria, for example, convert genistein, soy's most abundant isoflavone, into several metabolites that target and alter estrogen-dependent and non-estrogenic pathways with a variety of biological activities. As such, individual differences in the microbiome, therapeutic possibilities, and anticancer effects may be unique to each person [101–103]. Overall, these variables hinder the clinical development of dietary phytochemicals. Preclinical studies suggest that the intestinal P-glycoprotein efflux pump is responsible for the limited bioavailability of the phytochemicals. Blocking ABC transporters with quercetin increases the bioavailability and decreases their efflux. Other studies demonstrate that the combination of dietary phytochemicals such as curcumin and piperine exhibits greater bioavailability. Reports on humans suggest that the intake of nonsteroidal anti-inflammatory agents, reserpine, and blood thinners exhibit adverse effects taken together with phytochemicals [102]. Efforts are also directed to enhance the bioavailability of phytochemicals to exhibit better preventive and/or therapeutic responses. In this direction, the high-bioavailability formulation of phytochemicals is under development [104,105]. Food processing such as heating, drying, and grinding, and factors such as climate change and plant stress could impact the bioavailability of dietary phytochemicals, having an effect on their biological activity. Technological strategies such as nanoparticle synthesis, phytochemical(s) in lecithin, the phosphatidylcholine carrier, and solid lipid nanoparticles demonstrate an increase in the bioavailability of dietary phytochemicals [105–107].

In preclinical studies, phytochemicals exhibit an antitumor potential at the doses 1–200 μM and 2–100 mg/kg body weight without any apparent toxicity utilized in in vivo models [54,56,73,89]. In recent studies, it is noted that dietary phytochemicals/phytoestrogen exhibit a significant reduction in tumor volume in the MCF-7-inoculated mouse xenograft model at 2–100 mg/kg body weight [54,56,73,89]. These doses can be extrapolated in studies on humans as well. Moreover, curcumin (500 mg BID; NCT01740323), genistein (100 mg; NCT00244933), resveratrol (474 mg phenolics/day; NCT03482401), ginsenoside Rg3 (20 mg BID; NCT01717066), and thymoquinone (500 mg; NCT04852510) have been studied in various clinical trials in the range of previously reported in vivo dosages or at higher dosages. These clinical trials on dietary phytochemicals either alone or in combination with standard chemotherapy demonstrated encouraging results in breast cancer patients with less toxicity and lower side effects (NCT03072992).

#### **9. Study Strengths and Limitations**

Few studies have summarized the role of dietary phytochemicals in targeting BC-SCs. Some other available reviews related to this subject are discussed under cancer stem cell physiology and related hallmarks, and their modulation by phytochemicals. The present review summarizes the role of dietary phytochemicals and their effect on luminal A-derived BCSCs' pathophysiology at the molecular level. The review emphasizes that dietary phytochemicals possess increasing potential to target breast cancer subtype (luminal A) cell-derived stem cells. This review also highlights the efficacy of dietary phytochemical-derived nanoparticles, and co-treatment with other drugs against luminal A-derived BCSCs. However, there are some limitations in the study, such as the lack of the extensive information on the effect of dietary phytochemicals in luminal A cells derived in

in vivo experimental models. Because the luminal A-derived stem cell formation is an important event in the breast tumor microenvironment, further detailed studies are required to assess the stemness initiation, self-renewal, and chemotherapy resistance potential of dietary phytochemicals.

#### **10. Conclusion and Future Prospects**

Breast cancer luminal A subtype possess estrogen receptor and thus are responsive to hormone therapy. The luminal A subtype constitutes approximately 80% of the total breast cancer cases and demonstrates better prognosis and therapeutic susceptibility. Luminal A-derived T47D and MCF-7 cell culture models form tight three-dimensional cell–cell adhesion structures in comparison to luminal B breast cancer cells. As the breast tumor microenvironment constitutes nearly all subtypes of cancer cells, out of these, luminal A cells are slow growing and have the capability to transform into other breast cancer subtypes in response to changes in hormone levels and physiological states, including pregnancy status and various therapeutic modalities. Targeting luminal A cells at the initiation of breast cancer with phytochemicals could inhibit cancer progression and minimizes the casual switching to various subtypes. The dietary intake of these phytochemicals could offer prevention towards initiation and development of breast cancer, whereas cotreatment with standard chemotherapeutic drugs has the potential to increase the efficacy and therapeutic response. The present review highlights that dietary phytochemicals have the potential to target luminal A-derived BCSCs by lowering their stemness and self-renewal properties. These dietary phytochemicals demonstrate their anticancer effects by the modulation of signaling pathways, including AMPK, STAT3, NF-kB, Hedgehog, PI3K/Akt/mTOR, Notch, GSK3β, and Wnt, and other, as well as via the regulation of the mechanism(s) involved in the process of proliferation or drug resistance. These phytochemicals have the ability to target puttive molecular and biochemical events in luminal cell-derived mammospheres. The present review necessitates in-depth preclinical and clinical studies on dietary phytochemicals alone or in combination with the standard treatment to explore their cancer prevention and treatment potential.

**Author Contributions:** S.K. conceived and designed the study; S.K. wrote original draft of the manuscript; K.S.P. prepared figures and tables; S.G. wrote and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by [Department of Science and Technology, India] grant number [EEQ/2016/000350].

**Acknowledgments:** S.K. acknowledges the Department of Science and Technology, India for providing financial support in the form of the DST-SERB Grant [EEQ/2016/000350]. S.K. also acknowledges DST-India for providing a Departmental grant to the Department of Biochemistry, Central University of Punjab, Bathinda, India in the form of the DST-FIST grant. K.S.P. acknowledges DBT, India for providing the Senior Research Fellowship.

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

#### **References**

## *Correction* **Correction: Bishayee et al. Lotus (***Nelumbo nucifera* **Gaertn.) and Its Bioactive Phytocompounds: A Tribute to Cancer Prevention and Intervention.** *Cancers* **2022,** *14***, 529**

**Anupam Bishayee 1,\* , Palak A. Patel 1, Priya Sharma <sup>1</sup> , Shivani Thoutireddy <sup>1</sup> and Niranjan Das <sup>2</sup>**

#### **Table Legend**

In the original article, there was a mistake in the legend for Table 2 [1]. In the table legend (page 24), "in vivo" appears incorrectly which should be replaced by "in vitro". The correct legend appears below.

**Table 2.** Potential anticancer effects and mechanisms of action of *N. nucifera*-derived constituents based on in vitro studies.

#### **Error in Table**

In the original article, there were mistakes in Table 3 as published. In the table legend (page 32), "in vitro" appears incorrectly which should be replaced by "in vivo". Additionally, the content of the table is same as Table 2, which should be replaced by a correct table. The corrected Table 3 along with the title appear below.

**Table 3.** Potential anticancer effects and mechanisms of action of *N. nucifera*-derived constituents based on in vivo studies.

**Citation:** Bishayee, A.; Patel, P.A.; Sharma, P.; Thoutireddy, S.; Das, N. Correction: Bishayee et al. Lotus (*Nelumbo nucifera* Gaertn.) and Its Bioactive Phytocompounds: A Tribute to Cancer Prevention and Intervention. *Cancers* 2022, *14*, 529. *Cancers* **2022**, *14*, 2116. https:// doi.org/10.3390/cancers14092116

Received: 6 April 2022 Accepted: 6 April 2022 Published: 24 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original article has been updated.

#### **Reference**

1. Bishayee, A.; Patel, P.A.; Sharma, P.; Thoutireddy, S.; Das, N. Lotus (*Nelumbo nucifera* Gaertn.) and Its Bioactive Phytocompounds: A Tribute to Cancer Prevention and Intervention. *Cancers* **2022**, *14*, 529. [CrossRef] [PubMed]

## *Review* **Lotus (***Nelumbo nucifera* **Gaertn.) and Its Bioactive Phytocompounds: A Tribute to Cancer Prevention and Intervention**

**Anupam Bishayee 1,\* , Palak A. Patel 1, Priya Sharma <sup>1</sup> , Shivani Thoutireddy <sup>1</sup> and Niranjan Das <sup>2</sup>**

**Simple Summary:** The plant *Nelumbo nucifera* (Gaertn.), commonly known as lotus, sacred lotus, Indian lotus, water lily, or Chinese water lily, is an aquatic perennial crop belonging to the family of Nelumbonaceae. *N. nucifera* has traditionally been used as an herbal medicine and functional food in many parts of Asia. It has been found that different parts of this plant consist of various bioactive phytocompounds. Within the past few decades, *N. nucifera* and its phytochemicals have been subjected to intense cancer research. In this review, we critically evaluate the potential of *N. nucifera* phytoconstituents in cancer prevention and therapy with related mechanisms of action.

**Abstract:** Cancer is one of the major leading causes of death worldwide. Accumulating evidence suggests a strong relationship between specific dietary habits and cancer development. In recent years, a food-based approach for cancer prevention and intervention has been gaining tremendous attention. Among diverse dietary and medicinal plants, lotus (*Nelumbo nucifera* Gaertn., family Nymphaeaceae), also known as Indian lotus, sacred lotus or Chinese water lily, has the ability to effectively combat this disease. Various parts of *N. nucifera* have been utilized as a vegetable as well as an herbal medicine for more than 2000 years in the Asian continent. The rhizome and seeds of *N. nucifera* represent the main edible parts. Different parts of *N. nucifera* have been traditionally used to manage different disorders, such as fever, inflammation, insomnia, nervous disorders, epilepsy, hypertension, cardiovascular diseases, obesity, and hyperlipidemia. It is believed that numerous bioactive components, including alkaloids, polyphenols, terpenoids, steroids, and glycosides, are responsible for its various biological and pharmacological activities, such as antioxidant, anti-inflammatory, immune-modulatory, antiviral, hepatoprotective, cardioprotective, and hypoglycemic activities. Nevertheless, there is no comprehensive review with an exclusive focus on the anticancer attributes of diverse phytochemicals from different parts of *N. nucifera*. In this review, we have analyzed the effects of *N. nucifera* extracts, fractions and pure compounds on various organ-specific cancer cells and tumor models to understand the cancer-preventive and therapeutic potential and underlying cellular and molecular mechanisms of action of this interesting medicinal and dietary plant. In addition, the bioavailability, pharmacokinetics, and possible toxicity of *N. nucifera*-derived phytochemicals, as well as current limitations, challenges and future research directions, are also presented.

**Keywords:** *Nelumbo nucifera*; phytochemicals; cancer; prevention; therapeutic benefits; molecular mechanisms

#### **1. Introduction**

Cancer is the second leading cause of both morbidity and mortality throughout the world, with an estimated 19.3 million new cancer cases and almost 10 million cancer

**Citation:** Bishayee, A.; Patel, P.A.; Sharma, P.; Thoutireddy, S.; Das, N. Lotus (*Nelumbo nucifera* Gaertn.) and Its Bioactive Phytocompounds: A Tribute to Cancer Prevention and Intervention. *Cancers* **2022**, *14*, 529. https://doi.org/10.3390/cancers 14030529

Academic Editor: Raffaele Capasso

Received: 24 December 2021 Accepted: 17 January 2022 Published: 21 January 2022 Corrected: 24 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

deaths in the year 2020 [1]. Interestingly, it has been suggested that more than 40% of all cancer deaths could be prevented via lifestyle changes, including diet [2]. High dietary consumption of fruits and vegetables (more than 400 g/day) may prevent at least 20% of all cancers [3]. Dietary intervention may also improve the efficacy of cancer chemotherapy and lower the risk of long-term complications in cancer patients [4]. The cancer-preventive potential of various fruits, vegetables, spices, whole grains, and herbs is attributed to the presence of secondary plant metabolites, also known as phytochemicals. These naturallyoccurring phytochemicals are also utilized in the discovery and development of anticancer drugs [5,6]. Emerging preclinical and clinical data shows that bioactive food components contain enormous cancer-preventive and anticancer therapeutic potential due to their unique ability to impact various cancer hallmarks, namely sustained proliferation, cell death resistance, energy metabolism, immune surveillance evasion, inflammation, invasion, angiogenesis, and metastasis, by modulating a plethora of oncogenic and oncosuppressive signaling pathways [7–17].

*Nelumbo nucifera* Gaertn., commonly known as lotus, sacred lotus, Indian lotus or Chinese water lily, is a well-known dietary and medicinal plant. It is a large, perennial aquatic plant that belongs to the family *Nelumbonaceae* and consists of a sole genus Nelumbo with two species, *N. nucifera* and *N. lutea*, which are called Asian lotus and American lotus, respectively [18,19]. In general, lotus refers to the Asian lotus, which is mainly distributed in India, China, Nepal, Sri Lanka, Thailand, Japan, New Guinea and Australia [20–22]. In contrast, the American lotus is predominantly found in the eastern and southern regions of North America and north of South America [18,23,24]. Since ancient times, the lotus flower (Figure 1) has been recognized as a spiritual object for Hindus, Buddhists and Egyptians, and is considered a symbol of longevity in Chinese traditional culture [25,26]. Lotus is the national flower of India and Vietnam. As a vegetable as well as a medicinal and ornamental plant, lotus has been cultivated for more than the last 7000 years [19]. Almost all parts of the lotus plant have been used as food and medicine for more than 2000 years in Asia [27]. Lotus has a significant economic value in various Asian countries, where it is emerging as a horticultural model plant [26]. China is the largest producer and consumer of lotus in the world [27]. The rhizome (modified stem), perianth (non-reproductive part of the flower) and seeds of lotus are popular food ingredients and are used in various food products due to their delicious taste and nutritional value [28,29].

**Figure 1.** Photographs of lotus (*N. nucifera*). (**A**) Flower; (**B**) leaves; (**C**) rhizomes and (**D**) natural habitat.

Various parts of the lotus plant (Figure 1), from root to shoot, have documented use in different traditional systems of medicines, such as Indian traditional medicine (Ayurveda) and Chinese traditional medicine [30–32]. The whole plant, as well as crude extracts, fractions and constituents, have been found to possess numerous biological and pharmacological activities, including antioxidant, anti-inflammatory, immunomodulatory, antipyretic, antibacterial, antiviral, antifungal, antidiarrheal, diuretic, antiamnestic, antithrombotic, antiarrythmic, antidiabetic, hypocholesterolemic, antiobesity, antiaging, antiatherosclerotic, antifibrotic, sedative, antineurodegenerative, memory-improving, antifertility, hepatoprotective, skin-protective, cardiovascular-protective, and anticancer properties [24,25,29,31,33–36]. The remarkable health-promoting and disease-mitigating activities of the lotus plant have been correlated with the presence of numerous bioactive phytocompounds, including polyphenols, flavonoids, phenolic acids, alkaloids, terpenoids, steroids, fatty acids, and glycosides [25,31,33–36].

During the last several decades, *N. nucifera* has been subjected to intense research that evaluated the antineoplastic effects of various parts and active constituents of this dietary and medicinal plant. However, to the best of our knowledge, a comprehensive state-of-the-art review of all available anticancer studies has not been performed. Most of the previous reviews mainly focused on traditional and ethnopharmacological uses, biosynthesis, phytochemical analysis, industrial applications, and broad-spectrum health benefits of *N. nucifera* in which an overview of anticancer potential represents a minor representation [29,31–34,37]. Several prior publications highlighted pharmacological activities of selected phytochemicals of *N. nucifera,* and these reports did not have a sole focus on cancer [25,35,36,38,39]. Hence, the aim of this work has been to perform a systematic and critical analysis of fragmentary studies to provide an up-to-date and complete assessment of cancer-preventive and anticancer therapeutic attributes of *N. nucifera* and its bioactive phytocomponents with an understanding of the cellular and molecular mechanisms of action. Moreover, the bioavailability, pharmacokinetics and possible adverse effects of *N. nucifera*-derived phytochemicals, as well as current limitations, challenges and future research directions, are also discussed.

#### **2. Chemical Constituents of** *N. nucifera*

Several parts of *N. nucifera* are known to contain various pharmacologically active constituents and the most prominent phytochemical classes include alkaloids, flavonoids, terpenoids polysaccharides, steroids, essential oils, tannins, glycosides, proteins, fatty acids, minerals, and vitamins [29,31,34,40–43]. The main bioactive constituents of *N. nucifera* are alkaloids and flavonoids [33]. Different parts of *N. nucifera* contain several types of phytochemicals. For example, the leaves are rich in flavonoids and alkaloids, the flowers and plumules are rich in flavonoids, the seeds are rich in alkaloids, and the rhizome is rich in starch [29,40,42,44]. Procyanidins are the chief active components of *N. nucifera* receptacles [29]. A list of diverse groups of major phytochemicals present in different parts of *N. nucifera* is provided in Table 1.

Various bioactive alkaloids in different parts of *N. nucifera* include *N*-nornuciferine, nuciferine, roemerin, 2-hydroxy-1-methoxyaporphine, (6*R*,6a*R*)-roemerine-*N*β-oxide, (*S*) armepavine, (+)-1(*R*)-coclaurine, (−)-1(*S*)-norcoclaurine, lotusine, isoliensinine, liensinine, neferine, liriodenine, asimilobine, pronuciferine, oleracein E, demethyl-coclaurine, dauricine, *cis*-*N*-coumaroyltyramine, *cis*-*N*-feruloyltyramine, *trans*-*N*-coumaroyltyramine, and *trans*-*N*-feruloyltyramine (Figure 2) [31,33,34,40,44–47].

**Figure 2.** Chemical structures of major bioactive alkaloids isolated from different parts of *N. nucifera*.

The different parts of the *N. nucifera* plant, such as leaves, roots, seeds, and flowers, contain several bioactive flavonoid molecules, including flavonols, flavons, flavan-3 ols, flavanons, and anthocyanins [36]. The flavonols found in *N. nucifera* are myricetin, quercetin, kaempferol, and isorhamnetin, whereas the flavon molecules are diosmetin, syringetin, apigenin, luteolin, and chrysoeriol [36]. The flavan-3-ol molecules include catechin, epicatechin, catechin rhamnoside, gallocatechin gallate, and epigallocatechin gallate, along with its dimer and polymers, such as procyanidin dimer B1 and elephantorrhizol. The flavanones include naringenin and taxifolin, and the anthocyanins are

cyanidin, delphinidin, malvidin, petunidin, and peonidin [36]. The major bioactive flavonoids derived from different parts of *N. nucifera* are quercetin, kaempferol, apigenin, isorhamnetin, luteolin, myricetin, syringetin, and diosmetin (Figure 3) [33,36]. A total of 16 flavonoid *C*-glycosides and 56 flavonoid *O*-glycosides have been isolated from different parts of *N. nucifera* [36]. The main flavonoid *O*-glycosides in the different parts of *N. nucifera* include myrycetin 3-*O*-galactoside, myrycetin 3-*O*-glucoside, quercetin 3- *O*-arabinopyranosyl-(1→2)-galactopyranoside, myrycetin 3-*O*-glucuronide, quercetin 3- *O*-rhamnopyranosyl-(1→6)-glucopyranoside (rutine), quercetin 3-*O*-galactoside (hyperoside), quercetin 3-*O*-glucoside (isoquercitrin), kaempferol 3-*O*-robinobioside, quercetin 3-*O*-glucuronide, kaempferol 3-*O*-galactoside, isorhamnetin 3-*O*-rutinoside, kaempferol 3- *O*-glucoside (astragalin), syringetin 3-*O*-glucoside, isorhamnetin 3-*O*-glucoside, kaempferol 3-*O*-glucuronide, kaempferol 7-*O*-glucoside, diosmetin 7-*O*-hexose, and isorhamnetin 3-*O*glucuronide [25,42,48]. The key flavonoid *C*-glycosides in the different parts of *N. nucifera* are luteolin 8-*C*-*β*-D-glucopyranoside (orientin), luteolin 6-*C*-*β*-D-glucopyranoside (isoorientin), apigenin 8-*C*-*β*-D-glucopyranoside (vitexin), apigenin 6-*C*-*β*-D-glucopyranoside (isovitexin), apigenin 6-*C*-*β*-D-glucopyranosyl-8-*C*-*β*-D-glucopyranoside, luteolin 6-*C*-*β*-Dglucopyranosyl-8-*C*-*β*-D-pentoside, luteolin 6-*C*-*β*-D-pentosyl-8-*C*-*β*-D-glucopyranoside, apigenin 6-*C*-*β*-D-glucopyranosyl-8-*C*-*β*-D-xylopyranoside, apigenin 6-*C*-*β*-D-xylopyranosyl-8-*C*-*β*-D-glucopyranoside, apigenin 6-*C*-*β*-D-glucopyranosyl-8-*C*-*β*-D-arabionoside, apigenin 6-*C*-*β*-D-arabionosyl-8-*C*-*β*-D-glucopyranoside, apigenin 6-*C*-*β*-D-glucopyranosyl-8- *C*-*β*-D-rhamnoside, and apigenin 6-*C*-*β*-D-rhamnosyl-8-*C*-*β*-D-glucopyranoside [25]. The *N. nucifera* leaves, roots, and seed kernels contain several phenolic acids that can be classified into two categories: hydroxybenzoic acids and hydroxycinnamic acids [36]. The phenolic acids under the group of hydroxycinnamic acids include caffeic acid, protocetechuic acid, chlorogenic acid, cinnamic acid, ferulic acid, *p*-coumaric acid, and sinapic acid. The hydroxybenzoic acids include gallic acid, hydroxybenzoic acid, *p*-hydroxybenzoic acid, syringic acid, and vanillic acid [36].

**Figure 3.** Chemical structures of major bioactive flavonoids isolated from different parts of *N. nucifera*.

#### *2.1. Leaves*

The leaves of *N. nucifera* are rich in alkaloids and flavonoids [29,40,44,45,47]. The phytochemical analysis of EtOAc-soluble fraction of 80% MeOH extract of leaves of *N. nucifera* revealed the presence of 33 significant bioactive constituents, including 13 megastigmanes, 1 sesquiterpene, 8 alkaloids, and 11 flavonoids [45]. The various bioactive alkaloids in *N. nucifera* have raised significant interest due to their versatile chemical and biological activities [46]. The leaves of *N. nucifera* are a rich source of various bioactive alkaloids [44–47]. The 95% EtOH extract of leaves of *N. nucifera* yielded six potent bioactive alkaloids, namely (+)-1(*R*)-coclaurine, (−)-1(*S*)-norcoclaurine, nuciferine, liensinine, neferine, and isoliensinine [40]. The high-performance liquid chromatography (HPLC) coupled with ion trap/time-of-flight mass spectrometry (LC/MS-ITTOF) analysis of *N. nucifera* leaf extract reported the existence of five isoquinoline alkaloids: dehydronuciferine, *N*-nornuciferine, *O*-nornuciferine, nuciferine, and roemerine [49]. The non-aqueous capillary electrophoresis coupled with ultraviolet and mass spectroscopy (NACE-UV-MS) analysis is a method developed by Do et al. [50] to determine the major alkaloids, such as (−)-caaverine, (+) isoliensinine, (+)-norarmepavine, (−)-armepavine, (−)-nuciferine, (−)-nornuciferine, and (+)-pronuciferine, present in *N. nucifera* leaves. One alkaloid, *N*-methylasimilobine, has also been reported from the MeOH extract of *N. nucifera* leaves [51].

The potential bioactive flavonoids in the leaves of *N. nucifera* are quercetin, kaempferol, and luteolin [45,51]. The methanol extracts of the leaves of *N. nucifera* revealed the presence of several flavonoid compounds, including catechin, quercetin, quercetin-3-*O*-glucopyranoside, quercetin-3-*O*-glucuronide, quercetin-3-*O*-galactopyranoside, kaempferol-3-*O*-glucopyranoside, and myricetin-3-*O*-glucopyranoside [52]. The bioactive quercetin-based flavonoids and glycosides in *N. nucifera* leaves are (+)-catechin, hyperoside, isoquercitrin, and astragalin [29,40]. The in vitro lipolysis assay of the leaf extract of *N. nucifera* identified the presence of five bioactive flavonoid molecules, namely quercetin 3-*O*-*α*-arabinopyranosyl-(1→2)-*β*galactopyranoside, (+)-catechin, hyperoside, isoquercitrin, and astragalin [53].

The gas chromatography-mass spectrometry (GC-MS) analysis of hexane extract of the leaves of *N. nucifera* revealed the presence of 38 compounds, in which 15 compounds are the essential oil composition with 9,12,15-octadecatrienoic acid, linoleic acid ethyl ester, *n*-hexadecanoic acid, hexadecanoic acid, ethyl ester, and methyl (*Z*)-5,11,14,17 eicosatetraenoate [54]. The GC-MS analysis of essential oils of *N. nucifera* leaves detected the presence of 95 constituents, in which the major constituents are *l*-(+)-ascorbic acid 2,6 dihexadecanoate, *trans*-phytol, hexahydrofarnesyl acetone, pentadecyl acrylate, *β*-ionone, geranyl acetone, propionic acid decyl ester, farnesyl acetone, and heneicosane [41].

The analysis of a MeOH extract of the leaves of *N. nucifera* showed the presence of five norsesquiterpenes/megastigmanes, such as (*E*)-3-hydroxymegastigm-7-en-9-one, (3*S*,5*R*,6*S*,7*E*)-megastigma-7-ene-3,5,6,9-tetrol, dendranthemoside B, icariside B2, and sedumoside F1 [51]. Two bioactive triterpenes, namely alphitolic acid and maslinic acid, have also been extracted from the MeOH extract of *N. nucifera* leaves [51].

The leaves of *N. nucifera* also contain several nonvolatile organic acids, such as alphitolic, maslinic, gallic, tartaric, malic, and anisic acids [29,51,55].

#### *2.2. Plumules*

The plumule of *N. nucifera* contains 7.8% moisture, 4.2% ash, 12.5% crude oil and 26.3% protein [56]. The *N. nucifera* plumule oil is a rich source of several fatty acids. The major fatty acids in *N. nucifera* plumule oil are linoleic acid (50.4%) and palmitic acid (18.0%), followed by oleic acid (13.5%), behenic acid (6.8%), arachidic acid (3.30%), linolenic acid (3.0%), and stearic acid (2.90%) [56]. The major triglyceride components in *N. nucifera* plumule oil include linoleic acid-linoleic acid (12.80%), *β*-palmitic acid-linoleic acid (11.27%), *β*-oleic acid-linoleic acid (9.43%), *β*-palmitic acid-linoleic acid-oleic acid (8.58%), *β*-behenic acid-linoleic acid (8.32%), *β*-palmitic acid-oleic acid-linoleic acid (8.28%), *β*-palmitic acid-linoleic acid-arachidic acid (7.99%), *β*-palmitic acid-linoleic acid-palmitic acid (7.71%), and *β*-linoleic acid-oleic acid-linoleic acid (7.13%) [56].

The unsaponifiable components in *N. nucifera* plumule oil are very high (up to 14–19%) due to the presence of several sterol compounds [56]. The major sterols present in *N. nucifera* plumule oil are *β*-sitosterol (31.75%), Δ5-avenasterol (19.66%), and campesterol (6.28%), followed by stigmasta-7,25-dien-3-ol (3.49%), 24-methylene-9,19-cyclolanostan-3*β*-ol (3.79%), *α*-sitosterol (3.41%), stigmasterol (2.67%) ergosta-8,24(28)-dien-3-ol (2.5%), ergosta-5,24 dien-3-ol (1.71%), lanost-7-en-3-one (1.51%), and stigmasta-7-en-3-ol (0.96%) [56].

The ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry analysis of *N. nucifera* plumule extracts revealed the presence of several bioactive constituents which are mainly of the alkaloid and flavonoid group of compounds [57]. The bioactive alkaloid components in *N. nucifera* plumule extracts include higenamine, lotusine, 4 -methylcoclaurine, isoliensinine, liensinine, neferine, and nuciferine [57]. Duan and Jiang [58] reported a new benzylisoquinoline alkaloid, namely nelumstemine [1-(4 -hydroxybenzoyl)-6,7-dimethoxy-3,4-dihydroisoquinoline], which was isolated from the stems of *N. nucifera*. The bioactive flavonoid constituents in *N. nucifera* plumule extracts include apigenin-6-*C*-*α*-L-glucopyanosyl-8-*C*-*β*-D-glucopyranoside and apigenin-6-*C*-*α*-L-arabofuranosyl-8-*C*-*β*-D-glucopyranoside [57].

#### *2.3. Seeds and Rhizomes*

Starch is the major ingredient (up to 70% of the dry weight) of both the rhizome and seeds of *N. nucifera* [28]. The raw and dried rhizome and seeds of *N. nucifera* are good sources of niacin and vitamin B6 [28]. The raw and dried portions of the rhizome and seeds of *N. nucifera* contain various minerals, vitamins and fatty acids [28]. The major minerals present in the seeds and dried rhizome of *N. nucifera* include potassium (16,300 μg/g), phosphorus (1715 μg/g), and magnesium (1650 μg/g), followed by aluminum (470 μg/g), calcium (445 μg/g), manganese (57 μg/g), sodium (33 μg/g), cobalt (16 μg/g), strontium (15), iron (13 μg/g), zinc (13 μg/g), copper (10 μg/g) and vanadium (7 μg/g) [24,28]. Other important nutritional agents are fat (72.17%), crude fiber (10.60%), moisture (10.50%), total ash (4.5%), proteins (2.7%), and crude carbohydrates (1.93%) [59]. The seed possesses higher energy with 348.45 cal per 100 g [59]. Vitamin C and folate are the main vitamins present in both the raw rhizome and seeds of *N. nucifera* [28]. *N. nucifera* seeds also contain several vitamins in large amounts, such as vitamin B1 (2.24 mg/kg), vitamin B2 (0.13 mg/kg), vitamin B6 (3.03 mg/kg), vitamin C (39.4 mg/kg), and vitamin E (4.6 mg/kg) [24].

Alkaloids are the key secondary metabolites in the seeds of *N. nucifera* [31]. The major alkaloids present in the seeds of *N. nucifera* are lotusine, isoliensinine, liensinine, dauricine, pronuciferine, nuciferine, procyanidin, neferine, roemerine, and armepavine [31]. The chloroform fraction of hot MeOH extract of the embryos of seeds of *N. nucifera* led to the isolation of three novel bisbenzylisoquinoline alkaloids, such as nelumboferine, nelumborines A and B, along with four previously reported alkaloids [55].

The protein, minerals, amino acids, and unsaturated fatty acids are the rich sources of the seeds of *N. nucifera* [31]. The seeds of *N. nucifera* also contain mainly four types of monosaccharide, namely D-galactose, L-arabinose, D-mannose and D-glucose [59]. The main nutrients in *N. nucifera* seeds are proteins and carbohydrates [24]. The level of polysaccharides in the rhizome of *N. nucifera* is very high [29]. Two antioxidant micromolecular constituents, such as (±)-gallocatechin and (−)-catechin, along with antioxidant macromolecular constituents as well as a polysaccharide–protein complex having *α*/*β*-pyranose and *α*-furanose ring of molecular mass 18.8 kDa, have been reported from the rhizome of *N. nucifera* [60]. The polysaccharide–protein complex is composed of mannose, rhamnose, glucose, galactose and xylose in the molar ratio of 2:8:7:8:1 [61]. The abundance of essential amino acids in *N. nucifera* seeds is very high [24]. The major essential amino acids present in the seeds of *N. nucifera* are methionine (1.64%), valine (1.10%), isoleucine (0.99%), lysine (0.97%), phenylalanine (0.86%), histidine (0.50%), threonine (0.45%), and leucine (0.15%) [24]). The key carbohydrates of *N. nucifera* seeds are starch, polysaccharides and oligosaccharides [24]. The main nutrients of *N. nucifera* seeds represent starch (55.77%), crude protein (16.2%), water (14.0%), sugar (8.13%), ash (4.05%) and fat (2.05%) with a total calorific value of 1432 kJ/100 g [24].

*N. nucifera* seeds are also rich sources of various fatty acids, such as 14-methylpentadecanoic acid, 8,11-octadecadienoic acid, anti-9-octadecenoic acid, 18-carbonate, behenic acid, 20 carbonate, 9,12,15-octadecatrienoic acid, 14-carbonate, 23-carbonate, pentadecanoate, 17 carbonate, maleic-7-hexadecene acid, anti-8-octadecenoic, and maleic-9-octadecenoic acid [24].

From the MeOH extract of *N. nucifera* rhizome, a new ursane triterpenoid ester, urs-12 *en*-3*β*-*O*-9*E*,12*E*-octadecadienoate, was isolated [62]. The fresh seed epicarps of *N. nucifera* are rich in flavonols and their glycosides. The most important flavonols in *N. nucifera* epicarps are myricetin, quercetin, kaempferol, and isorhamnetin [63]. The CC along with RP-HPLC and HPLC–ESI-MS analysis of *N. nucifera* seed epicarp at three different ripening stages, the green ripening stage, half ripening stage, and full ripening stage, revealed the presence of four bioactive polyphenolic compounds: catechin, epicatechin, hyperoside, and isoquercitrin [64]. It is reported that the levels of these four bioactive polyphenolic compounds are different during seed ripening. The levels of catechin and epicatechin decrease in the ripening stage, whereas the levels of hyperoside and isoquercitrin increase during the ripening stage [64]. The rhizome of *N. nucifera* contains considerable amounts of various phytochemicals, such as tannins, saponins, and phenolic acids [65]. The major phytochemicals present in *N. nucifera* embryo include liensinine, isoliensinine, neferine, nuciferine, lotusine, pronuciferine, rutin, hyperin, and demethylcoclaurine [33].

#### *2.4. Flowers*

Several classes of bioactive phytochemicals, such as polyphenols, flavonoids, tannins and terpenoids, have been isolated from the ethanolic extracts of *N. nucifera* pink flower stamens and petals [43]. Nakamura et al. [66] reported the presence of a new alkaloid, namely *N*-methylasimilobine *N*-oxide, along with 11 bioactive benzylisoquinoline alkaloids, in the methanolic extracts of flower buds and leaves of *N. nucifera*. The major phytochemicals present in the flowers of *N. nucifera* are quercetin, luteolin, luteolin glucoside, kaempferol, kaempferol-3-*O*-glucoside, and isoquercitrin [33]. The ethanolic extract of *N. nucifera* petals revealed the presence of nine potent bioactive benzylisoquinoline alkaloids: (+)-juziphine, (+)-isococlaurine, (−)-*N*-methylisococlaurine, (−)-*N*-methylcoclaurine, (+)-nor-roefractine, (+)-armepavine, (−)-caaverine, (−)-lirinidine, and (+)-glaziovine [67]. The colors of the petals of *N. nucifera* are different only due to the presence of two flavonoids, namely isorhamnetin and kaempferol [68].

**Table 1.** List of phytochemicals reported from the different parts of *N. nucifera*.

#### **3.** *N. nucifera* **Extracts, Fractions and Pure Compounds in Cancer Research**

#### *3.1. Literature Search Methodology*

The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) criteria [76], which is recommended for reporting systematic reviews, was followed for this work. The literature search was conducted using various databases, such as PubMed, ScienceDirect, Google Scholar, and Scopus. Publication data was not a criterion when filtering research articles. The last search was performed in December 2021. Various combinations of keywords that were used included: *Nelumbo nucifera*; lotus; cancer; in vivo, in vitro; tumor; prevention; treatment, proliferation, apoptosis, and clinical studies. Reviews, systemic reviews, meta-analyses, letters to editors, book chapters, conference abstracts, and unpublished results were not included, and only articles written in the English language were considered. Additionally, available clinical studies pertaining to *N. nucifera* and cancer were searched on clinicaltrials.gov. The quality of each animal study was evaluated according to SYRCLE's RnB tool that investigates sources of bias [77]. Reference lists from reviews and collected articles were checked for relevant publications. Articles not related to cancer or *N. nucifera* have been excluded. Upon applying these exclusion criteria, eligible full-length articles were obtained and reviewed, and a collaborative decision was made for their incorporation for further analysis. Figure 4 illustrates the overview of the literature search and study selection.

**Figure 4.** A PRISMA flowchart illustrating the literature search and study selection process relevant to the anticancer potential of *N. nucifera*. The total number of in vitro and in vivo studies 95 is greater than the number of studies included in this work (64) because many publications contained results based on more than one organ-specific cancer or study type (in vitro and in vivo).

#### *3.2. Preclinical Studies (In Vitro and In Vivo)*

Potential anticancer effects and mechanisms of action of *N. nucifera*-derived extracts, fractions and pure compounds have been investigated using various cancer cell lines and animal tumor models. Tables 2 and 3 summarize relevant in vitro and in vivo results, respectively.

#### 3.2.1. Breast Cancer

The anticancer properties of various parts of the *N. nucifera* plant have been extensively investigated using different breast cancer cell lines. Karki et al. [78] explored the anticancer properties of the aqueous rhizome extract of *N. nucifera* on the MDA-MB-231 human breast cancer cell line. It was found that the extract inhibited the proliferation and migration of those cells by decreasing the levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 (Table 2). Yang et al. [79] treated MCF-7 human breast cancer cells with flavonoid-rich aqueous *N. nucifera* extract (NLE) and found that it inhibited the proliferation of MCF-7 cells by arresting them in the G0/G1 phase of the cell cycle. Mechanistically, NLE induced p53 phosphorylation (p-p53), increased levels of cyclin-dependent kinase inhibitory proteins, such as p21, p27, and p16, downregulated cyclin expressions, and decreased levels of cyclin D1/cyclin-dependent kinase 4 (CDK4) and cyclin E/CDK2 complexes which led to the upregulation of Rb/E2F pathway and ultimately resulted in a G1 phase arrest. Another important observation was the NLE-mediated inactivation of fatty acid synthase (Fas). Quantitative HPLC analysis revealed gallic acid as the most abundant flavonoid within the

extracts, followed by rutin. In a follow-up study, the same research group [80] treated MDA-MB-231 human breast cancer cells with the aqueous leaf extract used in the previous study (NLE) to determine its anticancer effect. NLE treatment reduced cell viability, migratory and invasive properties by decreasing the levels of phosphorylated forms of extracellular signal-regulated kinase (p-ERK), phosphoinositide-3-kinase (p-PI3K), protein kinase B (p-Akt) as well as rat sarcoma gene (RAS) and mitogen-activated extracellular signalregulated kinase (MEK). In addition, NLE inhibited angiogenesis by downregulating the expression of connective tissue growth factor (CTGF), MMP-2, vascular endothelial growth factor (VEGF) and nuclear factor-κB (NF-κB) p65, while increasing levels of tissue inhibitor of metalloproteinase-2 (TIMP-2). In an extension of previous studies, Wu et al. [81] observed that NLE and *N. nucifera* leaf polyphenol extract (NLPE) reduced cell viability and suppressed cell proliferation in 4T-1 breast cancer cells and exhibited anti-invasive and antimigratory effects in MDA-MB-231 and 4T-1 cells. These effects occurred mechanistically through increasing apoptosis, decreasing levels of Ras homolog family member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1), cyclin-dependent kinase 42 (Cdc42) and suppressing activation of ERK1/2, p38 MAPK. Additionally, NLE and NLPE reduced the expression of protein kinase Cα (PKCα) in 4T-1 cells. Arjun et al. [82] used methanol and acetone leaf extracts of *N. nucifera* to investigate their anticancer effects on MCF-7 human breast cancer cells. Although both leaf extracts inhibited proliferation and reduced viability of human breast cancer cells, the methanol extract produced the maximum inhibitory effect. However, the underlying mechanism of action of these effects has not been reported.

In addition to the leaf, other parts of the *N. nucifera* plant have also been studied for their anticancer activities. For example, *N. nucifera* flower receptacles have been investigated by Krubha and Vasan [83]. A methanolic extract from *N. nucifera* floral receptacles has shown antiproliferative and cytotoxic activities against MCF-7 human breast cancer cells by increasing antioxidant activity as demonstrated by 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay.

Various researchers used pure compounds isolated from the *N. nucifera* plant to investigate their anticancer properties against breast cancer in vitro. In one study, MDA-MB-231 human breast cancer cells were treated with isoliensinine, liensinine and neferine. Although all three compounds exhibited antiproliferative effects, isoliensinine was found to be the most potent phytochemical. Isoliensinine induced cell cycle arrest at the G1 phase, which possibly occurred by downregulating cyclins and upregulating a cyclin-dependent kinase inhibitor, p21. In addition, isoliensinine also induced cell death by inducing apoptosis mediated by induction of reactive oxygen species (ROS) and alterations of Bcl-associated X protein (Bax), B cell lymphoma 2 (Bcl-2), caspase-3, and poly (ADP-ribose) polymerase-1 (PARP-1). Finally, isoliensinine activated both p38 MAPK and c-Jun N-terminal kinase (JNK) signaling, contributing to apoptosis induction [84].

The anticancer effects of neferine, another bisbenzylisoquinline alkaloid found abundantly in the *N. nucifera* plant, have been investigated by many researchers. Yang et al. [85] reported that neferine decreased MCF-7 cell viability; however, a combination treatment of neferine and dehydroepiandrosterone (DHEA), an endogenous steroid hormone, exerted a greater growth-inhibitory effect. Neferine enhanced the anticancer effect of DHEA to induce apoptosis mediated by increased levels of pro-apoptotic factors, such as caspase-3, caspase-8, caspase-9, Bax, p53, p21, transcription factor E2F1, Fas, Fas ligand (FasL), and decreased levels of antiapoptotic factors, such as Bcl-2, B-cell lymphoma-extra-large (Bcl-xL), human inhibitor of apoptosis protein-1 (HIAP-1), HIAP-2, and survivin. From the green seed embryos of *N. nucifera*, Kadioglu et al. [86] isolated neferine and compared its anticancer effect against taxol- and doxorubicin-resistant as well as sensitive MCF-7 cells. Neferine induced cytotoxicity of multidrug-resistant MCF-7 cells to a greater extent compared to the effect on sensitive cell lines by increasing rhodamine 123 (R123) uptake and inhibiting P-glycoprotein (P-gp). In a follow-up study, Law et al. [87] also reported that neferine induced cytotoxicity in drug-resistant and -sensitive MCF-7 cells. Interestingly, autophagic cell death occurred in apoptosis-resistant cells via green fluorescent protein

(GFP)-light-chain 3 (LC3) puncta formation and ryanodine receptor (Ryr) activation, which subsequently activated the calcium-dependent kinase (caMKKβ), leading to the activation of the 5 -adenosine monophosphate-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling pathway. In another study, Liu et al. [88] investigated the anticancer effects of neferine on MDA-MB-231 human breast cancer cells. Neferine significantly reduced cell proliferation, migration and invasion as well as increased apoptosis via regulation of the expression of miR-374a and fibroblast growth factor receptor 2. Neferine also suppressed the activation of PI3K, Akt, MEK, and ERK signaling pathways.

Similar to neferine, liensinine and nuciferine are two other active bisbenzylisoquinoline alkaloids found in the *N. nucifera* plant. Kang et al. [89] treated MCF-7 and MDA-MB-231 breast cancer cells with liensinine and nuciferine to investigate their anticancer properties. Liensinine and nuciferine both decreased cell viability and inhibited cell proliferation via apoptosis induction and cell cycle arrest in a concentration-dependent manner. Apoptosis was mediated by a mitochondrial-dependent pathway by decreasing Bcl-2/Bax ratio, activating caspase-3, and activating caspase-mediated PARP cleavage. Liensinine was found to be more potent in inhibiting proliferation, invasion and migration in both cell lines compared to nuciferine. In addition, both compounds inhibited osteoclast differentiation and bone resorption by blocking the receptor activator of NF-κB ligand and decreasing the secretion of cathepsin K and MMP-9. Additionally, the anticancer effects of liensinine have been studied by Zhou et al. [90], and the mechanistic results are rather unique. It was determined that treatment of MDA-MB-231 and MCF-7 human breast cancer cells with liensinine reduced the viability of these cells. Mechanistically, liensinine inhibited autophagy and induced the accumulation of autophagosomes and lysosomes by inhibiting their fusion via the recruitment of Ras-related protein Rab-7a (RAB7A) to lysosomes. This inhibition of autophagy/mitophagy by liensinine led to the sensitization of doxorubicininduced cell death of breast cancer cells via increased dynamin-1-like protein (DNM1L) dephosphorylation and translocation to mitochondria and increased mitochondrial fission.

Very recently, Huang et al. [91] isolated three new pairs of benzyltetrahydroisoquinoline alkaloid epimers, seco-neferine A-F, from an ethanolic extract of plumula nelumbinis, a commonly used health food and traditional Chinese medicine. All six compounds were tested against MDA-MB-231 human breast cancer cells, and seco-neferine F displayed a maximum but moderate cytotoxic effect. The mechanisms behind the observed anti-breast cancer effect have not been explored.

There are at least three studies that investigated the anticancer potential of *N. nucifera* using in vivo breast cancer models. In addition to the in vitro studies presented earlier, Yang et al. [79] evaluated the effects of NLE on nude mice with an MCF-7 tumor xenograft and found that it effectively reduced tumor volume and tumor weight. Mechanistically, it was demonstrated that NLE can suppress intratumor expression of human epidermal growth factor receptor 2 (HER2), phospho-HER2 (p-HER2), and Fas (Table 3). Chang et al. [80] studied the effects of NLE in female C57BL/6 nude mice xenografted with MDA-MB-231 cells treated with various concentrations of NLE (0.5, 1, and 2%). There was a significant reduction of tumor size in animals receiving NLE-treated cells compared to control. Immunohistochemical staining of tumor tissue with CD31 antibody revealed NLEmediated inhibition of angiogenesis as demonstrated by CD31. However, mechanisms of action of the observed antitumor and antiangiogenic effects were not studied in vivo. In addition to the in vitro experiment presented earlier, Zhou et al. [90] further performed in vivo studies to replicate the effects of liensinine on the autophagy of breast cancer cells. In this study, 5–7-week-old female nude mice were inoculated with MDA-MB-231 cells and treated with either liensinine (60 mg/kg) or doxorubicin (2 mg/kg) alone or a combination of both. It was determined that the combination treatment of liensinine and doxorubicin significantly reduced tumor growth. Mechanistically, liensinine suppressed autophagy/mitophagy, increased mitochondrial translocation of DNM1L, and increased the colocalization of DNM1L and translocase of outer mitochondrial membrane 20 (TOMM20). All of these mechanisms led to the sensitization of doxorubicin-induced cell death via the promotion of DNM1L-mediated mitochondrial fission.

#### 3.2.2. Cervical Cancer

*N. nucifera*'s anticancer effect on cervical cancer has gained recent research interest. Currently, there are at least three studies that have explored this area. Maneenet et al. [67] reported a considerable cytotoxic activity of an ethanolic extract of *N. nucifera* petals against HeLa human cervical cancer cells with a preferential cytotoxicity (PC50) of 10.9 μM. Phytochemical characterization of the extract revealed the presence of nine benzylisoquinoline alkaloids which exhibited significant antiausterity activity against HeLa cells. One compound, namely (−)-lirinindine, showed the greatest cytotoxicity as evidenced by cell shrinkage, plasma blebbing, and ultimately total cell death within 10 h. Mechanistically, the cytotoxic effect was achieved via induction of apoptosis, activation of caspase-3, decreased levels of Bcl-2, and downregulation of the Akt/mTOR pathway. Li et al. [92] studied the anticancer effects of liensinine on Caski, C33A, HeLa, and SiHa human cervical cancer cells. The alkaloid inhibited the proliferation of all cervical cancer cells and suppressed the colony formation of C33A and HeLa cells. A mechanistic study indicated that liensinine registered cell cycle arrest at the G0/G1 phase, increased apoptosis, elevated the levels of caspase-9 and p21 and decreased the expression of Mcl-1, CDK2, p-Akt, and GSK3α. Dasari et al. [93] focused on researching the effects of neferine on HeLa and SiHa cervical cancer cells. Treatment with various concentrations of neferine suppressed viability, induced cytotoxicity, and reduced migration in a concentration-dependent manner. Neferine induced apoptosis by promoting excess ROS formation, increasing oxidative damage, activating the expression of a DNA damage response marker (pH2AX) and upregulating the levels of pro-apoptotic markers, such as cytochrome c, Bax, cleaved caspase-3, caspase-9 and cleaved PARP1, while downregulating anti-apoptotic factors, such as Bcl-2, translational controlled tumor protein (TCTP), procaspase-3, and procaspase-9. Neferine was also shown to induce autophagy by promoting the conversion of LC3 protein from LC3-I to LC3-II and by increasing the formation of autophagosomes which was determined by an increase in autophagy factors, such as Beclin1, atg-4, atg-5, and atg-12. Migration of the cancer cells was also reduced due to cell cycle arrest at the G1/G0 phase of the cell cycle.

#### 3.2.3. Colon Cancer