**Meet the editor**

Professor K.L. Narasimhan completed his training in Pediatric Surgery in 1987 and has been interested in Pediatric Urology. He has been working on spina bifida from 1990. He has over 110 scientific publications in peer reviewed journals. He has special interest in prevention of neural tube defects. He also has several publications related to myelomeningoceles, neurogenic bladder,

posterior urethral valves and obstructive uropathy.

Contents

**Preface IX**

**Part 1 The Role of Folic Acid**

Lourdes García-Fragoso,

Prinz-Langenohl Reinhild,

Chapter 1 **The Role of Folic Acid**

Chapter 6 **Selective Abortion**

Chapter 8 **Advances in Genetics**

Patrizia De Marco

John A. A. Nichols

Ramya Iyer and S. K. Tomar

**Part 2 Genetics of Neural Tube Defects 139**

**and Prevention of Neural Tube Disease 1**

**in the Prevention of Neural Tube Defects 3**

Inés García-García and Carmen L. Cadilla

Chapter 3 **Primary Prevention of Neural Tube Defects 47**  Claudine Nasr Hage and Grace Abi Rizk

Pietrzik Klaus and Holzgreve Wolfgang

Chapter 5 **Antenatal Prevention of Neural Tube Defects 91**  Naomi Burke, Tom Walsh and Michael Geary

Chapter 7 **Folate and Prevention of Neural Tube Disease 117**

**of Non Syndromic Neural Tube Defects 141**

**and Folic Acid Fortification as Contrasting Strategies for Prevention of Congenital Neural Tube Defect 98**

Chapter 2 **Strategies for Prevention of Neural Tube Defects 31**  Hiroko Watanabe and Tomoyuki Takano

Chapter 4 **Prevention of NTDs – Proposal of a New Concept 65**

### Contents

#### **Preface** XI

Patrizia De Marco

X Contents

#### Chapter 9 **Neural Tube Defects in Algeria 163**  Bakhouche Houcher, Samia Begag, Yonca Egin and Nejat Akar

Chapter 10 **Association of** *A80G* **Polymorphism in the** *RFC1* **Gene with the Risk for Having Spina Bifida-Affected Offspring in Southeast Mexico and Interaction with** *C677T-MTHFR* **179**  Lizbeth González-Herrera, Orlando Vargas-Sierra, Silvina Contreras-Capetillo, Gerardo Pérez-Mendoza, Ileana Castillo-Zapata, Doris Pinto-Escalante, Thelma Canto de Cetina and Betzabet Quintanilla-Vega

#### **Part 3 Surgical Treatment of Large Thoraco-Lumbar Neural Tube Defects 191**

#### Chapter 11 **Treatment of Large Thoraco-Lumbar Neural Tube Defects 179**  Mehrdad Hosseinpour

### Preface

The book Neural Tube Defects - Role of Folate, Prevention Strategies and Genetics has several eminent international authors and the book is a resource for anybody who is interested in this very important subject. The book has been divided into 3 sections; The role of Folic acid and prevention of Neural Tube disease which has seven chapters. Genetics of Neural Tube Defects having 3 chapters and a Surgical Treatment of Large Thoraco-lumbar Neural Tube Defects which includes a chapter on surgical treatment of large lumbo - sacral defects. The authors are distinguished and the chapters are a product of their extensive research.

I am glad that Intech took the initiative to publish this book for online free access.

**Dr. Kannan Laksmi Narasimhan**  Consultant in Ped Surgery, KK women's and children's hospital, Bukit Timah Road, Singapore

**Part 1**

**The Role of Folic Acid**

**and Prevention of Neural Tube Disease**


*3.3. Aryne-Enolether Substrates*

In 2017, a strategy of synthesizing indeno[1,2-*c*]quinoline derivatives was developed by the Liu group through gold(I)-catalyzed cascade cyclization with 18 examples and up to 99% yield (Scheme 19) [29]. The coordination of gold species to the *β* position of the triple bond initiated an attack of the enolether to generate indene intermediates (**86**). Intermediates (**87**) were produced by the activation of double bonds in the conjugated enolether with [Au]+, which were converted to aromatic intermediates (**88**) via intramolecular condensation with the release of MeOH after protodemetalation. In oxygen, the intermediates (**88**) were further oxidized to a more stable indeno[1,2-*c*]quinoline product (**89**). Notably, the electrondonating effect of the amine on the right benzene ring played a crucial role in the initiation of the above transformation.

**Scheme 19.** Gold(I)-catalyzed syntheses of indeno[1,2-*c*]quinoline derivatives.

In 2018, Liu et al., used 1,5-enyne substrates to synthesize a series of 2-aryl indenone derivatives in the catalysis of a gold catalyst (Scheme 20) [53]. Intermediates (**92**) were formed via a gold-catalyzed cycloisomerization. An O2-mediated radical addition to intermediates **92** afforded intermediates (**93**) that underwent aromatization to yield peroxy intermediates (**94**), which were subsequently transformed into oxonium intermediates (**95**) through the cleavage of the peroxide bond with the coordination of [Au]+. Finally, indenone products (**96**) were achieved by the hydrolysis of oxonium with the release of MeOH. The above free radical reaction process was verified via control experiments and heavy atom labeling.

**Scheme 20.** Gold(I)-catalyzed syntheses of 2-aryl indenone derivatives.


**Ivalin Induces Mitochondria-Mediated Apoptosis Associated with the NF-**κ**B Activation in Human Hepatocellular Carcinoma SMMC-7721 Cells**

**Zhuo Han 1,**†**, Fang-yuan Liu 1,**†**, Shi-qi Lin 1,**†**, Cai-yun Zhang 1, Jia-hui Ma 1, Chao Guo 1, Fu-juan Jia 1, Qian Zhang 1, Wei-dong Xie 1 and Xia Li 1,2,\***

Academic Editor: Kyoko Nakagawa-Goto Received: 6 September 2019; Accepted: 22 October 2019; Published: 22 October 2019

**Abstract:** Ivalin, a natural compound isolated from *Carpesium divaricatum*, showed excellent microtubule depolymerization activities among human hepatocellular carcinoma in our previous work. Here, we investigated its functions on mitochondria-mediated apoptosis in hepatocellular carcinoma SMMC-7721 cells. DAPI (4 ,6-diamidino-2-phenylindole) staining, annexin V-fluorexcein isothiocyanate (FITC) apoptosis detection, and western blotting were applied to explore the apoptotic effect of Ivalin. Next, the induction effect of Ivalin on the mitochondrial pathway was also confirmed via a series of phenomena including the damage of mitochondria membrane potential, mitochondria cytochrome c escape, cleaved caspase-3 induction, and the reactive oxygen species generation. In this connection, we understood that Ivalin induced apoptosis through the mitochondrial pathway and the overload of reactive oxygen species. Furthermore, we found that the activation of nuclear factor-κB (NF-κB) and subsequent p53 induction were associated with the apoptotic effect of Ivalin. These data confirmed that Ivalin might be a promising pro-apoptotic compound that can be utilized as a potential drug for clinical treatment.

**Keywords:** Ivalin; *Carpesium divaricatum*; hepatocellular carcinoma; mitochondria-mediated apoptosis; NF-κB

### **1. Introduction**

Clinical data have shown that hepatocellular carcinoma (HCC) is the most common and dominating primary tumor with a high incidence rate in adults worldwide [1–3]. Since many advanced treatments have been used in recent years, chemotherapy is still widely applied to treat HCC in the research of clinical trials [4]. However, both the occurrence of drug resistance and recurrences are the key hurdles associated with chemotherapy [5]. Therefore, the exploration and evolution of novel therapeutic agents for the treatment of HCC are greatly of need.

Abstracted compounds from plants utilized into an effective anticancer drug is one of the fastest developing therapeutics for chemotherapy [6,7]. The large family Asteraceae contains over 25,000 species (such as *Aster* and *Carpesium*) and many species have shown that it is best utilized as sources of edible oils, vegetables, pesticides, medicines and so on. In addition, it is considered to be an ideal source of effective natural compounds with the structure of sesquiterpenoids, particularly, an eudesmane framework [8]. Eudesmane-type sesquiterpenoids and their biological functions including antifungal,

antitumor, and antibacterial have been the fastest growing field of pharmacological and synthetic studies during the last two decades [9]. Telekin and 1-oxoeudesm-11(13)-eno-12,8a-lactone (OEL), examples of compounds with an eudesmane framework, have been reported to strongly restrain cell proliferation via the induction of mitochondria-mediated apoptosis [10,11]. Ivalin (Figure 1), another compound with an eudesmane framework, was abstracted from the traditional herb *Carpesium divaricatum* [12]. Our previous works demonstrated that Ivalin can serve as a novel microtubule inhibitor by depolymerizing microtubules and resulted in cell proliferation inhibition in hepatocellular carcinoma SMMC-7721 cells [13]. Here, we report that Ivalin enforced the procedure of apoptosis in the same cells by triggering reactive oxygen species (ROS) generation and the participation of the mitochondria pathway.

Nuclear factor-κB (NF-κB), a conventional transcription factor, is important for the execution and control of apoptosis [14]. NF-κB, in different given stimulations such as in ROS accumulation and cancer therapeutic agents, complexly regulates the program of apoptosis via its downstream target genes, which include the p53 protein and so on [15,16]. Detecting both the protein and mRNA levels of NF-κB and p53 will help us to further perceive and recognize the influence of pro-apoptosis and underlying theory of Ivalin. The results from these experiments showed that the initiate of NF-κB and subsequent p53 induction were associated with the apoptotic effect of Ivalin in SMMC-7721 cells.

### **2. Results**

### *2.1. Apoptotic E*ff*ect of Ivalin*

Our previous studies confirmed that Ivalin (Figure 1) was significantly cytotoxic to SMMC-7721 cells (IC50: 4.34 ± 0.10) with a lower effect toward the normal cell line HL7702 (IC50: 25.86 ± 0.87) [13]. In response to characterizing the cell growth inhibition effect of Ivalin, we monitored morphological changes in SMMC-7721 cells after 24 h of treatment. Compared to the untreated cells, Ivalin treatment increased the apoptotic body formation as well as nuclear condensation, which were the significant morphologic alterations related to apoptosis (Figure 2A).

**Figure 1.** Structure of Ivalin.

When cells were undergoing apoptosis, the phosphatidylserine in the inter surface of the plasma membrane transforms to the outer surface, which can be stained with Annexin V. In this connection, we performed flow cytometry to further quantify the apoptotic effect of Ivalin via dual stained cells with Annexin V-fluorexcein isothiocyanate and propidium iodide. The results shown in Figure 2B revealed that the proportion of Annexin V-stained cells increased with the percentages increased from 4.57%, 9.28%, 16.6%, to 47.32% after treating with 0 to 8 μmol/L Ivalin, respectively. Therefore, we believe that Ivalin may strongly increase the ratio of apoptotic cells in SMMC-7721 cells.

The Bcl-2 family consists of members with a pro-apoptotic or the opposite effect and the balance between them may regulate the fate of cells [17,18]. Bcl-2 and Bax, the most common proteins with vital roles in the Bcl-2 family, were analyzed by western blot after Ivalin treatment. Results revealed that Ivalin-treatmen triggered the altered expression of Bcl-2 and Bax in SMMC-7721 cells (Figure 2C). The increase in the Bax protein and decrease in the Bcl-2 protein expression levels further confirmed the pro-apoptotic effect of Ivalin as suggested above.

**Figure 2.** SMMC-7721 cells treated with Ivalin causing apoptosis. (**A**) Fluorescence micrographs of untreated and Ivalin treated SMMC-7721 cells with 4 ,6-diamidino-2-phenylindole (DAPI). Magnification: 100×. (**B**) Results from the flow cytometry analysis, the quantification of the apoptotic cells after indicate treatment. (**C**) Western blot showed that Ivalin induced apoptosis by enhancing the Bax and declining the Bcl-2 expression. \* *p* < 0.05; \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control group.

### *2.2. Ivalin Triggered the Loss of Mitochondrial Membrane Potential (MMP) in SMMC-7721 Cells*

We next stained the cells with JC-1 to measure the cellular MMP in response to Ivalin treatment. Cells treated with Ivalin led to the loss of MMP in a concentration-dependent manner (Figure 3). Meanwhile, the increased mitochondrial membrane permeability in treated cells may result in the translocation of mitochondria cytochrome c to cytosol. Figure 4A illustrates an apparent release of cytochrome c from the mitochondria to cytosol in the experimental groups. Furthermore, the treatment with Ivalin concentration-dependent increased the level of cleaved caspase-3 in the experimental groups (Figure 4B). The above findings indicate that the mitochondria-mediated pathway was associated with Ivalin-induced apoptosis.

**Figure 3.** Effects of MMP generation in Ivalin-treated cells. (**A**,**B**) After Ivalin treatment for 24 h, flow cytometry and fluorescence microscope were used to detect cellular mitochondrial membrane potential. (**A**) Ivalin treatment decreased the red fluorescence intensity (aggregates) and increased green fluorescence intensity (monomers) in SMMC-7721 cells, indicating that Ivalin reduced the mitochondrial membrane potential, thereby leading to mitochondrial dysfunction. (**B**) Ivalin induced the loss of mitochondrial membrane potential as shown by flow cytometry. \*\*\* *p* < 0.01 vs. the control group.

**Figure 4.** Ivalin trigged apoptosis by means of the mitochondria activation. (**A**) The cytochrome c in mitochondria with the stimulation of Ivalin inflowed into the cytosol. (**B**) Cleaved caspase-3 was increased with the treatment of Ivalin. \*\*\* *p* < 0.001 vs. the control group.

### *2.3. The Generation of ROS Appeared in Ivalin-Treated Cells*

Intracellular ROS generation always appears during the process of mitochondrial obstruction. Therefore, we detected the fluorescence produced by dichlorofluorescein (DCF) by evaluating the possession of ROS by the flow cytometric assay to determine whether Ivalin can induce the accumulation of ROS. The group of treated and untreated cells were incubated with DCF-DA for 30 min and analyzed via flow cytometry. The results are shown in Figure 5 with a greater generation of ROS in the experimental groups in contrast to the untreated group.

**Figure 5.** Ivalin induced the generation of intracellular ROS. Graph shows the fluorescence intensities of DCF in the SMMC-7721 cells exposed to Ivalin in contrast to control. The fluorescence intensity of control group was set as 1. \* *p* < 0.05; \*\* *p* < 0.01 vs. the control group.

### *2.4. Ivalin Modulated the Protein and mRNA Levels of NF-*κ*B, I*κ*B, and p53*

Currently, a large member of anticancer compounds take effect in coping with hazardous substances by targeting transcription activators like nuclear factor-κB (NF-κB) and p53 regulating ROS [19–21]. In order to further perceive and recognize the role of Ivalin in apoptotic induction, we experimented with the expression of NF-κB, IκB, and p53 through western blot analysis. Ivalin-treatment enhanced the expressions of NF-κB and p53, but decreased that of IκB protein (Figure 6A).

We also performed real-time PCR to evaluate if there had been any alteration in the NF-κB, p53, and Bax mRNA levels in the presence of Ivalin treatment. The data revealed that Ivalin time-dependently induced the gene expressions of NF-κB, p53, and Bax (Figure 6B). NF-κB was activated before p53, and the activation of NF-κB mRNA reached the highest levels early, about 4 h. Therefore, the effective activation of NF-κB was associated with the apoptotic effect of Ivalin.

**Figure 6.** Trends of Ivalin on the p53, NF-κB and IκB expression. (**A**) The expressions of relative proteins were measured by western blotting. \* *p* < 0.05; \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control group. (**B**) Relative mRNA expression values were calculated by real-time PCR. The quantity of each mRNA was relative to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels.

### **3. Discussion**

In our previous work, we reported the ideal microtubule depolymerization activities of Ivalin in SMMC-7721 cells [13]. In this study, we found that Ivalin treatment may lead to obviously apoptotic features including apoptotic body formation and nuclear condensation in the same cells. Furthermore, in the presence of an indicated concentration of Ivalin, a significant increase in the proportion of

apoptotic cells was observed through double staining by annexin V-FITC and PI. In this connection, we presumed the apoptotic effect of Ivalin in SMMC-7721 cells.

Apoptosis can be induced by the extrinsic (death receptor pathway) and intrinsic pathways (mitochondria-mediated pathway). During the mitochondria-mediated pathway, the increase in mitochondria membrane permeability led to the subsequent release of mitochondria cytochrome c to cytosol, activated the caspase-9 and its downstream effector caspases including caspase-3, and finally induced apoptosis [22]. The Bcl-2 family proteins such as Bax and Bcl-2 mediate the intrinsic pathway by changing the mitochondria outer membrane permeabilization [23]. In particular, a common gateway for the mitochondria-mediated apoptotic pathway is the required level of Bax protein [24]. Ivalin treatment resulted in inducing the expression of Bax and attenuating that of Bcl-2 protein in SMMC-7721 cells. Additionally, treatment with Ivalin also caused the increase in the mitochondria membrane permeability, which was confirmed by the loss of MMP and an obvious release of cytochrome *c* from mitochondria into cytosol. Furthermore, the induction of cleaved caspase-3 expression in Ivalin-treated cells was also observed in the experiment. All of these results confirmed the effective mitochondria-mediated apoptotic effect of Ivalin among SMMC-7721 cells.

NF-κB, a transcription factor first identified in 1986, provides an effective goal for the progress of inflammation and apoptosis [25]. Nowadays, more and more evidence suggests the positive role of NF-κB in apoptosis, while the negative role of it in the apoptosis process has been discussed for many years [26]. In fact, the function of NF-κB in mediating apoptosis strongly relies on the type of cancer, the stimulation, and the related subunit [27–30]. When under non-stimulated status, NF-κB is in a complex formed with IκB in the cytoplasm without activation. Extracellular stimuli bring about rapid phosphorylation of IκB, which will be further degraded by ubiquitinase or protease [31]. When the degradation of IκB is finished, NF-κB activates rapidly and subsequently translocates into the nucleus to react with the downstream targets [32–34]. A key example was a report that confirmed the important role of NF-κB in the induction of wild type p53 expression to initiate pro-apoptotic signaling in response to ROS accumulation [21].

Here, Ivalin treatment led to a greater generation of ROS in SMMC-7721 cells. In this connection, furtherinvestigation was performed to confirm whether NF-κB wasinvolvedin themitochondria-mediated apoptotic effect of Ivalin. Western blot analysis revealed that Ivalin increased the expression of NF-κB and p53, but decreased the expression of IκB. Moreover, rt-PCR revealed that the mRNA levels of NF-κB, p53, and Bax increased after Ivalin treatment in a time-dependent manner. NF-κB was activated first, followed by the activation of p53 and Bax, and the expression of NF-κB mRNA reached the highest levels as early as about 4 h. This finding indicates that the NF-κB signaling pathway is involved in Ivalin-induced mitochondria-mediated apoptosis in SMMC-7721 cells.

To sum up, this study is the first to report that Ivalin induced mitochondria-mediated apoptosis associated with the NF-κB activation in SMMC-7721 cells. Ivalin treatment resulted in a significant generation of ROS in SMMC-7721 cells. In response to this, NF-κB was activated and served as a transcription factor to induce p53 activation, which subsequently induced Bax while decreasing Bcl-2 protein expression, which eventually led to mitochondria-mediated apoptosis in SMMC-7721 cells. However, the detail mechanisms responsible for the pro-apoptotic activity of Ivalin need to be explored further. Hence, Ivalin deserves further research to develop it into a promising chem-therapeutic agent or leading compound for anti-cancer agent searching.

### **4. Materials and Methods**

### *4.1. Chemicals and Reagents*

Ivalin (>98%), provided by Dr. Xie (Shandong University, Weihai, China) [12], was dissolved and its concentration adjusted by dimethylsulfoxide (DMSO) when required. DAPI (4 ,6-diamidino-2 phenylindole) was acquired from Sigma-Aldrich Corp. (St. Louis, MO, USA). An Annexin V-fluorexcein isothiocyanate (FITC) Apoptosis Detection Kit was purchased from BD Biosciences (San Jose, CA, USA). Caspase-3, cytochrome c, p53, and NF-κB antibodies were obtained from Cell Signaling Technology (CST, Inc, Beverly, MA, USA). Bcl-2, Bax, and GAPDH antibodies were obtained from Abcam Inc. (Cambridge, MA, USA). Beyotime Institute of Biotechnology (Shanghai, China) supplied the cell mitochondria/cytosol isolation kit, JC-1 and DCFH-DA, to us.

### *4.2. Human Hepatocellular Carcinoma and Cell Culture*

Shanghai Institute for Biological Sciences (SIBS) of Chinese Academy of Sciences (Shanghai, China) supplied the SMMC-7721 cell line (human hepatocellular carcinoma cell line) and we cultured the cells according to the supplier's instructions.

### *4.3. DAPI Staining*

DAPI staining assay, as previously described [35], was used to observe the change in the nucleus after Ivalin treatment.

### *4.4. Mitochondrial Membrane Potential*

We performed JC-1 staining to measure the changes in mitochondrial membrane potential (ΔΨm) in SMMC-7721 cells after Ivalin treatment. Cells were treated with Ivalin (0 μM to 8 μM) for 24 h and dealt with JC-1 in the light of the instructions from the manufacturer. Stained cells were collected for flow cytometric analysis or fluorescence microscope observation. The results contained three independent experiments.

### *4.5. Apoptosis Detection*

The apoptosis rate of SMMC-7721 cells after Ivalin 24 h treatment was evaluated by Annexin V-FITC and Propidium Iodide (PI) double staining, according to our previously described study [13].

### *4.6. Measurement of Intracellular ROS Levels*

Cells were treated with Ivalin, as described above, for 24 h. The working principle of the kit was that with an increase in ROS, DCFH-DA can transform into DCFH, which reacts with ROS, presenting the fluorescence property. We took advantage of this to measure the change of intracellular ROS content via flow cytometry as previously described [35].

### *4.7. Western Blot Analysis*

Western blot analysis, performed as previously described [36], was used to detect the expression levels of indicated protein. In the assay for cytochrome c measurement, we used a mitochondria/cytosol isolation kit to separate the mitochondrial proteins from cytosol proteins.

### *4.8. Real-Time PCR Analysis*

Real-time PCR assay, as described in our previous work [36], was used to detect the mRNA levels of the NF-κB, p53, and Bax genes. We designed all needed primers using primer premier 5 and synthesis by Sangon Biotech Co Ltd. (Shanghai, China) for the NF-κB gene (sense primer: 5 -TAGAAACAGACCGAGGAG-3 and anti-sense primer: 5 -ACTGGCTAATAAAGTGAATG-3 ), p53 gene (sense primer: 5 -GTTTCCGTCTGGGCTTCT-3 and anti-sense primer: 5 -CCTCAGGCGGCTCATAG-3 ), and Bax gene (sense primer: 5 -TCAACTGGGGCCGGGTTGTC-3 and anti-sense primer: 5 - CCTGGTCTTGGATCCAGCC-3 ).

### *4.9. Statistical Analysis*

All data are presented as mean ± SD (standard deviation) if appropriate. A comparison between two groups was performed with the Student's *t*-test. \* *p* < 0.05; \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control group.

**Author Contributions:** Conceptualization, X.L.; Formal analysis, Z.H. and F.L.; Funding acquisition, X.L.; Investigation, Z.H., F.L., S.L., C.Z., J.M., C.G., F.J. and Q.Z.; Resources, W.X. and X.L.; Paper writing, S.L.; Paper review and editing, X.L.

**Funding:** This work was supported by Natural Science Foundation of Shandong Provincial under Grant (No. ZR2019MH001), the Fundamental Research Funds for the Central Universities under Grant (No. 2019ZRJC004), and the National Natural Science Foundation of China under Grant (No. 81273532).

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

### **References**

**Sample Availability:** Samples of the compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Apple Peel Flavonoid Fraction 4 Suppresses Breast Cancer Cell Growth by Cytostatic and Cytotoxic Mechanisms**

### **Chao-Yu Loung 1,**†**, Wasundara Fernando 1,**†**, H.P. Vasantha Rupasinghe 1,2 and David W. Hoskin 3,\***

Received: 7 June 2019; Accepted: 11 September 2019; Published: 13 September 2019

**Abstract:** Many dietary flavonoids possess anti-cancer activities. Here, the effect of apple peel flavonoid fraction 4 (AF4) on the growth of triple-negative (MDA-MB-231, MDA-MB-468), estrogen receptor-positive (MCF-7), and HER2-positive (SKBR3) breast cancer cells was determined and compared with the effect of AF4 on normal mammary epithelial cells and dermal fibroblasts. AF4 inhibited breast cancer cell growth in monolayer cultures, as well as the growth of MCF-7 spheroids, without substantially affecting the viability of non-malignant cells. A sub-cytotoxic concentration of AF4 suppressed the proliferation of MDA-MB-231 cells by inhibiting passage through the G0/G1 phase of the cell cycle. AF4-treated MDA-MB-231 cells also exhibited reduced in vitro migration and invasion, and decreased Akt (protein kinase B) signaling. Higher concentrations of AF4 were selectively cytotoxic for MDA-MB-231 cells. AF4 cytotoxicity was associated with the intracellular accumulation of reactive oxygen species. Importantly, intratumoral administration of AF4 suppressed the growth of MDA-MB-231 xenografts in non-obese diabetic severe combined immunodeficient (NOD-SCID) female mice. The selective cytotoxicity of AF4 for breast cancer cells, combined with the capacity of sub-cytotoxic AF4 to inhibit breast cancer cell proliferation, migration, and invasion suggests that flavonoid-rich AF4 (and its constituents) has potential as a natural therapeutic agent for breast cancer treatment.

**Keywords:** apoptosis; breast cancer; cell cycle; flavonoids; reactive oxygen species; tumor suppression

### **1. Introduction**

The regular consumption of fruits and vegetables that are rich in flavonoids and other bioactive molecules is associated with a reduced risk of developing various types of cancers [1–5]. It is unlikely that any single natural source-derived compound is responsible for this beneficial effect since multiple bioactive molecules in flavonoid-rich foods are likely to act in a synergistic fashion. Numerous studies on phytochemicals as potential anti-cancer agents have found that these natural compounds affect many different signaling pathways involved in cancer development and progression [6,7]. It is hoped that further research on the anti-cancer properties of phytochemicals will lead to the development of plant-based therapeutics for the prevention or treatment of cancer.

Apples, which are a common source of dietary flavonoids, have been widely investigated for their disease-fighting properties [8–11]. Apple peel flavonoid fraction 4 (AF4) is a flavonoid-rich ethanolic extract of the Northern Spy apple cultivar [12]. AF4 contains a number of polyphenolic compounds, including flavonols, anthocyanins, dihydrochalcones, phenolic acids, and flavan-3-ols. Quercetin glycosides (quercetin-3-*O*-galactoside, quercetin-3-*O*-rutinoside, quercetin-3-*O*-glucoside and quercetin-3-*O*-rhamnoside) comprise approximately 70% of the phenolic content of AF4. Previous studies have established the neuroprotective and anti-inflammatory properties of AF4 in different mouse models [12,13]. AF4 also inhibits the growth of hepatocellular carcinoma (HepG2) cells by causing cell cycle arrest and apoptosis, as well as acting as a topoisomerase toxicant [14].

Breast cancer is the most common cancer among North American women and the second highest cause of cancer-related deaths [15]. The poor prognosis of metastatic breast cancer mandates the development of novel treatment strategies. The impact of AF4 treatment on breast cancer cells has not yet been determined. In the current study we used in vitro and in vivo approaches to elucidate the selective cytotoxic, anti-proliferative, anti-migratory and tumor suppressor effects of AF4 on breast cancer cells.

### **2. Results**

### *2.1. Apple Peel Flavonoid Fraction 4 (AF4) Selectively Inhibits the Growth of Breast Cancer Cells*

Exposure to AF4 reduced the number of viable triple-negative (MDA-MB-231, MDA-MB-468), estrogen receptor-positive (MCF-7), and HER2 receptor-positive (SKBR3) breast cancer cells in monolayer cultures in a dose- and time-dependent manner, as indicated by reduced cellular metabolic activity measured by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Figure 1A). Sensitivity to AF4 (100 μg/mL) at 72 h was as follows: MDA-MB-231 (39% ± 3% viable) and MDA-MB-468 (38% ± 3% viable) > SKBR3 (48% ± 2% viable) > MCF-7 (56% ± 2% viable).  In comparison to quercetin, AF4 was a less potent inhibitor of breast cancer cell growth but had greater selectivity (Figure S1). MCF-7 spheroids that were grown in the presence of AF4 (100 μg/mL) were smaller than vehicle-treated control spheroids (Figure 1C). In addition, numerous floating cells with morphology characteristic of dead or dying cells were present in AF4-treated cultures, and there was a significant decrease in the number of viable MCF-7 cells, as indicated by reduced phosphatase activity, within spheroids treated with AF4 (100 μg/mL) relative to vehicle-treated controls. Therefore, AF4 inhibited breast cancer cell growth in both 2-dimensional and 3-dimensional culture systems. Subsequent experiments focused on the effect of AF4 on MDA-MB-231 cells as this breast cancer cell line was most sensitive to AF4 and readily forms tumors in immune-deficient mice.

**Figure 1.** Apple peel flavonoid fraction 4 (AF4) is selectively cytotoxic to breast cancer cells. (**A**) MDA-MB-231, MDA-MB-468, MCF-7 and SKBR3 cells were cultured in the absence or presence of the indicated concentrations of AF4 for the indicated times. The % metabolic activity of AF4-treated cells was determined using an MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Data are expressed as mean ± standard error of the mean (SEM). (**B**) The % metabolic activity of AF4-treated (100 μg/mL) breast cancer cells was compared to MCF-10A cells (#) and HMECs (\*) cells. Data from MTT assays are expressed as mean ± SEM. (**C**) MCF-7 spheroids were cultured for 72 h in the absence or presence of the indicated concentrations of AF4. Relative cell number of viable cells was determined by acid phosphatase assay. Data are shown as mean ± SEM. (A–C) Statistical analysis of 3 independent experiments was performed using analysis of variance (ANOVA) and Tukey's multiple comparisons test; \* *p* < 0.05, \*\* and ## *p* < 0.01, \*\*\* and ### *p* < 0.001.

### *2.2. AF4 Suppresses the Proliferation of MDA-MB-231 Cells*

Next, we determined whether a non-cytotoxic concentration of AF4 could impact breast cancer cell growth. As shown in Figure 2, flow cytometric analysis of Oregon Green 488-stained MDA-MB-231 cells that were treated with a sub-cytotoxic dose of AF4 (40 μg/mL) revealed a significant reduction in the number of cell divisions (Figure 2A). In addition, cell cycle analysis showed that AF4-treated MDA-MB-231 cells accumulated in the G0/G1 phase of the cell cycle, with a corresponding reduction in the number of MDA-MB-231 cells in the S phase of the cell cycle (Figure 2B). The same effect was observed in AF4-treated MDA-MB-468 cells (Figure S2). Consistent with an AF4-induced partial block

at G0/G1, there was reduced expression of CDK4 and cyclin D3 in AF4-treated MDA-MB-231 cells (Figure 2C).

**Figure 2.** AF4 inhibits breast cancer cell proliferation. (**A**) MDA-MB-231 cells were stained with Oregon Green 488 dye and then cultured for 72 h in the absence or presence of the indicated concentrations of AF4. Fluorescence was measured by flow cytometry. Data are shown as representative histograms (filled peak, non-proliferating cells; black peak, vehicle; blue peak, AF4) and mean number of cell divisions ± SEM. (**B**) MDA-MB-231 cells were cultured for 72 h in the absence or presence of the indicated concentrations of AF4. Cells were stained with propidium iodide (PI) and cell cycle analysis was performed by flow cytometry. Data are shown as representative histograms and mean % number of cells ± SEM in each phase of the cell cycle. (**C**) MDA-MB-231 cells were cultured for 24 h in the absence or presence of the indicated concentrations of AF4. The relative expression of CDK4 and cyclin D3 was determined using Western blot analysis. Equal protein loading was confirmed by probing for β-actin. Data shown are representative blots and mean % relative expression ± SEM. (A–C) Statistical analysis of 3 independent experiments was performed using ANOVA and Tukey's multiple comparisons test; \* *p* < 0.05, \*\* *p* < 0.01.

### *2.3. AF4 Inhibits the Migration and Invasion of MDA-MB-231 Cells*

Gap closure and trans-well migration assays were used to determine the effect of sub-cytotoxic AF4 on the migration and invasion capacity of MDA-MB-231 cells. These experiments used AF4 at a final concentration of 20 μg/mL in order to ensure that there would be no AF4-associated cytotoxic activity. As shown in Figure 3, a sub-cytotoxic concentration of AF4 (20 μg/mL) inhibited the migration of MDA-MB-231 cells by 65% in gap closure assays and by 87% in trans-well migration assays (Figure 3A and B, respectively). In addition, the invasion of MDA-MB-231 cells through a fibronectin-coated porous membrane was reduced by 80% in the presence of sub-cytotoxic AF4 (Figure 3C). Expression of invasion-promoting matrix metalloproteinase 2 (MMP2) was also significantly reduced when MDA-MB-231 cells were cultured in the presence of sub-cytotoxic AF4 (Figure 3D).

### *2.4. AF4-Induced Apoptosis Is Associated with Oxidative Stress*

To determine the mechanism by which AF4 killed breast cancer cells, MDA-MB-231 cells were stained with Annexin V-488 and propidium iodide (PI) prior to culture for 24 h in the presence of a cytotoxic concentration of AF4 (100 μg/mL). As shown in Figure 4A, flow cytometric analysis revealed that a high concentration of AF4 caused MDA-MB-231 cells to die by apoptosis (Figure 4A). In contrast, neither MCF-10A epithelial cells nor dermal fibroblasts were sensitive to AF4, suggesting a selective cytotoxic effect on neoplastic cells. As shown in Figure 4B,C, treatment of MDA-MB-231 cells with an apoptosis-inducing concentration of AF4 resulted in the generation of reactive oxygen species (ROS), as indicated by Amplex Red assays and flow cytometric analysis of cells stained with the ROS-sensitive dye 5-(and-6)-chloromethyl-2 ,7 -dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA). MCF-10A cells that were cultured in the presence of AF4 (100 μg/mL) also exhibited increased levels of intracellular ROS (Figure S3). Oxidative stress was at least in part responsible for the cytotoxic action of AF4 since apoptosis of AF4-treated MDA-MB-231 cells was reduced in the presence of the antioxidant N-acetyl cysteine (NAC). In comparison to MDA-MB-231 cells, non-malignant MCF-10A epithelial cells were relatively resistant to oxidative stress (Figure S4).

**Figure 3.** AF4 inhibits breast cancer cell motility and invasion. (**A**) Mitomycin C-treated MDA-MB-231 cells were cultured in wells containing cell culture inserts, which were removed at 0 h. After 24 h culture in the absence or presence of 20 μg/mL AF4 cultures were photographed. Representative images and mean % migration ± SEM of 3 independent experiments are shown. (**B**) Serum-starved MDA-MB-231 cells were treated with 20 μg/mL AF4 for 24 h. Mean % migration ± SEM through an 8 μm porous membrane and (**C**) mean % invasion ± SEM through a fibronectin-coated 8 μm porous membrane were determined as described in the Methods. (**D**) MDA-MB-231 cells were cultured for 24 h in the absence or presence of the indicated concentrations of AF4. Relative expression of MMP2 was determined using Western blot analysis. Equal protein loading was confirmed by probing for β-actin expression. Data shown are representative blots and mean % relative expression ± SEM. Statistical analysis of 3 independent experiments was performed using (A–C) Student's *t*-test or (D) ANOVA and Tukey's multiple comparisons test; \* *p* < 0.01, \*\* *p* < 0.001.

**Figure 4.** AF4-induced apoptosis of breast cancer cells is reactive oxygen species (ROS)-dependent. (**A**) MCF-10A cells, fibroblasts and MDA-MB-231 cells were cultured for 24 h in the absence or presence of 100 μg/mL AF4, and then stained with Annexin-V-488 and propidium iodide (PI) for flow cytometric analysis. Data are shown as mean % cell number ± SEM of 3 independent experiments. (**B**) MDA-MB-231 cells were cultured in the absence or presence of the indicated concentrations of AF4 for 2 h or 24 h and relative ROS in cultures was determined by Amplex Red assay. Data are shown as mean absorbance at 570 nm ± SEM of 3 independent experiments. (**C**) CM-H2DCFDA-stained MDA-MB-231 cells were cultured in the absence or presence of the indicated concentrations of AF4 for 2 h or 24 h and the relative amount of intracellular ROS was determined by fluorescence at 529 nm. Data are shown as mean fluorescence intensity ± SEM of 3 independent experiments. (**D**) MDA-MB-231 cells were cultured for 24 h in the absence or presence of 100 μg/mL AF4 without or with 5 mM N-acetyl cysteine (NAC), and then stained with Annexin-V-488 and PI for flow cytometric analysis. Data are shown as mean % cell number ± SEM of 3 independent experiments. (A–D) Statistical analysis was performed using ANOVA and Tukey's multiple comparisons test; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

### *2.5. AF4 Inhibits Akt Signaling*

To determine a possible mechanism to account for the growth inhibitory effect of AF4, we examined the impact of AF4 on Akt (protein kinase B) signaling in MDA-MB-231 cells. A sub-cytotoxic concentration of AF4 (40 μg/mL) was used in these experiments in order to obtain sufficient protein for western blot analysis. Figure 5 shows that AF4 inhibited the phosphorylation-induced activation of Akt at Thr308. AF4 also suppressed Ser380 phosphorylation of phosphatase and tensin homolog (PTEN), which is the upstream inhibitor of Akt. Both PTEN and Akt phosphorylation were restored when AF4-treated MDA-MB-231 cells were cultured in the presence of the antioxidant NAC. The importance of the Akt signaling pathway for the growth and survival of MDA-MB-231 cells was confirmed using 2 different Akt inhibitors (MK2206 and SC66). Figure S5 shows that the percentage of apoptotic MDA-MB-231 cells increased significantly when Akt activation was inhibited.

**Figure 5.** AF4 inhibits Akt and phosphatase and tensin homolog (PTEN) phosphorylation in breast cancer cells. MDA-MB-231 cells were cultured for 24 h in the absence or presence of 40 μg/mL AF4 without or with 5 mM NAC. Relative expression of phospho-Akt (Thr308), total Akt, phospho-PTEN (Ser380), and total PTEN was determined using Western blot analysis. Equal protein loading was confirmed by probing for β-actin expression. Data are shown as representative blots and mean % relative expression ± SEM of 3 independent experiments. Statistical analysis was performed using ANOVA and Tukey's multiple comparisons test; \* *p* < 0.05.

### *2.6. AF4 Suppresses Growth of MDA-MB-231 Xenografts*

The effect of AF4 on in vivo tumor growth was determined by intratumoral administration of AF4 (0.5 mg/kg) to MDA-MB-231 xenografts grown in female non-obese diabetic severe combined immunodeficient (NOD-SCID) mice. Dosage that was predicted to suppress MDA-MB-231 tumor growth was based on the total concentration of quercetin, quercetin glycosides, catechin, epicatechin, cyanidin-3-*O*-galactoside, phloridzin, chlorogenic acid and cafeic acid within AF4. Consistent with our in vitro findings regarding the anti-proliferative and cytotoxic activities of AF4, Figure 6A shows that treatment with AF4 significantly slowed the growth of MDA-MB-231 xenografts. Examination of tumor sections stained with hematoxylin and eosin revealed larger areas of necrosis in AF4-treated tumors (Figure 6B). Moreover, in comparison to saline-treated tumors, expression of the endothelial cell marker CD31 was reduced in the interior and periphery of AF4-treated tumors. No adverse effects were noted in AF4-treated mice, including no significant difference in average body weight between the treatment groups at day 15 (saline-treated group, 24.5 ± 0.6 g; AF4-treated group, 26.3 ± 0.5 g; *p* > 0.05 by Student's *t*-test).

**Figure 6.** AF4 suppresses MDA-MB-231 xenograft growth. MDA-MB-231 cells were xenografted into the left flank of non-obese diabetic severe combined immunodeficient (NOD-SCID) female mice and AF4 (0.5 mg/kg) or saline was injected directly into the resulting tumors every second day (day 1, 3, 5, 7 and 9, indicated by arrows) for 9 days. The control group consisted of 10 animals and the AF4 treatment group was 7 animals. (**A**) Mean tumor volume ± SEM was determined every second day. Statistical analysis was determined by Student's *t*-test; \* *p* < 0.05. Excised tumors from each treatment group at day 15 are shown. (**B**) At day 15 mice were euthanized and tumors were excised, fixed, and sectioned for staining with hematoxylin and eosin (H&E) and detection of CD31 expression by immunohistochemistry (IHC). Representative sections are shown; N denotes areas of necrosis and L denotes live cells.

### **3. Discussion**

Dietary phytochemicals have received unique attention in the search for novel and safer cancer treatment options in light of widely documented findings that many of these natural sourced compounds kill cancer cells but are relatively non-toxic to healthy cells [14,16–19]. In this study, we used MTT assays to show that AF4 suppressed the growth of triple-negative (MDA-MB-231 and MDA-MB-468), estrogen receptor-positive (MCF-7) and HER2 receptor-positive (SKBR3) breast cancer cells but had little effect on the growth of non-malignant mammary epithelial cells (HMECs and MCF-10A), suggesting a pronounced selectivity for neoplastic cells. In contrast, quercetin, which is a minor component of AF4 [12], exhibited less selectivity for breast cancer cells.

MDA-MB-231 cells that were exposed to a low concentration of AF4 tended to accumulate at G0/G1, likely as a result of reduced expression of cyclin D3 and CDK4 that promote gene expression needed for G1 progression [20]. AF4-treated MDA-MB-468 cells also arrested at G0/G1. This effect of AF4 on 2 different breast cancer cell lines differed from the G2/M cell cycle arrest seen in cultures of AF4-treated HepG2 hepatocarcinoma cells [14], suggesting that the anti-proliferative activity of AF4 may be cell type-dependent. Treatment with a higher concentration of AF4 induced MDA-MB-231 cells to undergo apoptosis; however, the viability of non-malignant fibroblasts and MCF-10A mammary epithelial cells was not affected. AF4-induced apoptosis of breast cancer cells was consistent with an earlier report of

apoptotic death of liver cancer cells following treatment with AF4 [14]. We demonstrate here, for the first time, that AF4-induced cytotoxicity was at least partially due to the accumulation of intracellular ROS because the antioxidant NAC protected MDA-MB-231 cells from the cytotoxic effect of AF4. In contrast, quercetin-induced apoptosis of MDA-MB-231 cells is reported to be ROS-independent [21], which argues against a major role for quercetin in the cytotoxic effect of AF4. Interestingly, non-malignant MCF-10A cells also showed increased levels of intracellular ROS following AF4 treatment, even though these cells were refractory to the cytotoxic effect of AF4. Multiple mechanisms exist to either prevent oxidative stress or manage the negative consequences of this condition; however, excessive production and accumulation of ROS can overwhelm these defenses [22]. In line with evidence that neoplastic cells are more sensitive to ROS than are their non-malignant counterparts [23], we found that MDA-MB-231 breast cancer cells were more sensitive to ROS than non-malignant MCF-10A mammary epithelial cells. The selective effect of AF4 on breast cancer cells may involve the capacity of healthy cells to defend against endogenous ROS [24]. Although certain components of AF4 possess anti-oxidant capabilities [25], the ability of unfractionated AF4 to protect against oxidative stress has not yet been demonstrated. In any case, a number of natural source antioxidants, at high doses, are capable of causing ROS production in cancer cells [26–28].

Importantly, AF4 inhibited the growth of MCF-7 breast cancer spheroids that, in comparison to monolayer cultures of breast cancer cells, more closely resemble the 3-dimensional structure of solid tumors [29]. In addition, sub-cytotoxic AF4 interfered with the migration of MDA-MB-231 cells in gap closure and trans-well cell migration assays, as well as inhibiting invasion of MDA-MB-231 cells through a fibronectin-coated membrane and suppressing the expression of MMP2. These findings suggest that AF4 may be able to interfere with breast cancer metastasis since tumor cell locomotion and the ability to degrade extracellular matrix components via the synthesis of proteolytic enzymes such as MMP2 play essential roles in the metastatic process [30].

Decreased phosphorylation of Akt in the presence of AF4 may account for its anti-proliferative and cytotoxic effects on MDA-MB-231 cells since the growth, survival, and metabolism of breast cancer cells involves activation of Akt [31]. The important role played by the Akt signaling pathway in the growth and survival of MDA-MB-231 cells was confirmed by the observation that Akt inhibition resulted in apoptosis of MDA-MB-231 cells. Phosphorylation of PTEN, which is a tumor suppressor protein that downregulates Akt signaling [32], was downregulated in AF4-treated MDA-MB-2312 cells. Decreased activation of inhibitory PTEN is consistent with the finding that AF4 did not completely block Akt activation. AF4-induced ROS production is likely to be involved in the effects of AF4 on Akt and PTEN since ROS can directly inhibit Akt via the phosphorylation of thiol groups within the protein, whereas phosphatases such as PTEN are deactivated by ROS [33]. Restoration of Akt and PTEN phosphorylation to control levels when NAC was added to cultures of AF4-treated MDA-MB-231 cells confirmed the involvement of AF4-induced ROS in modulation of the Akt signaling pathway.

Intratumoral administration of AF4 suppressed the growth of MDA-MB-231 xenografts in immune-deficient mice, most likely due to the combination of anti-proliferative and cytotoxic effects of AF4 that were revealed by our in vitro studies. In this regard, AF4-treated tumors contained larger areas of necrosis relative to saline-treated tumors. In addition, decreased expression of the endothelial cell marker CD31 in the periphery and interior of AF-4 treated tumors suggested that AF4 may inhibit angiogenesis. Notably, AF4 did not cause any distress or adverse side effects such as weight loss in treated animals. Injection of AF4 directly into the tumor was employed to eliminate potential issues of AF4 bioavailability following oral dosing. Therefore, it will be important in future studies to assess the effectiveness of oral or intraperitoneal administration of AF4 to tumor-bearing mice. Delivery of an optimal concentration of AF4 to the tumor microenvironment via the oral or intraperitoneal route will likely require a nanoparticle delivery vehicle of the type that has been reported to greatly enhance the bioavailability of other bioactive phytochemicals such as curcumin [34,35]. In this regard, nanoparticles have also been employed to deliver plant extracts with anticancer properties to various types of cancer cells [36]. Identification of the components of AF4 that are responsible

for its anticancer activity could allow for the development of nanoparticles containing one or more pure compounds with greater bioactivity relative to the AF4 extract; however, it is important to note that such an approach risks the loss of any potential synergy between major and minor components of AF4. Nevertheless, demonstration of AF4-mediated in vivo tumor suppressor activity, as well as reduced proliferation/survival and motility/invasion of MDA-MB-231 triple-negative breast cancer cells following AF4 treatment, suggests that AF4 and its bioactive components warrant further investigation as potential selective natural-source agents for the treatment of triple-negative breast cancer.

### **4. Materials and Methods**

### *4.1. Reagents*

AF4 was extracted from the peels of the apple cultivar Northern Spy, as previously described [12]. AF4 in ethanol was filter-sterilized and stored at −80 ◦C. Prior to use in this study, ethanol was evaporated under nitrogen gas and the AF4 residue was dissolved in sterile pyrogen-free water and aliquots of the resulting AF4 stock (10 mg/mL) were stored at −20 ◦C. Amplex Red reagent, Annexin-V-488, Dulbecco's modified Eagle's medium (DMEM), DMEM/F12, horse serum, fetal calf serum, CM-H2DCFDA, and PI were from Life Technologies Inc. (Burlington, ON, Canada). Hydroxyethyl piperazineethanesulfonic acid (HEPES)- and bicarbonate-buffered mammary epithelial cell medium, mammary epithelial cell growth supplement, penicillin/streptomycin solution and poly-L-lysine were purchased from ScienCell Research Laboratories Inc. (Carlsbad, CA, USA). DMEM, phenol red-free DMEM, recombinant human insulin, hydrocortisone, phosphatase substrate, Triton X-100, mitomycin C, NAC, MTT, quercetin, MK2206, and SC66 were from Sigma-Aldrich (Oakville, ON, Canada). Paraformaldehyde was from Bioshop Canada Inc. (Burlington, ON, Canada). Fibroblast cell growth medium and supplements were from Lonza Inc. (Walkersville, MD, USA). DNase-free RNase A was from Qiagen Inc. (Mississauga, ON, Canada). Recombinant epidermal growth factor and basic fibroblast growth factor were from PeproTech (Rocky Hill, NJ). Diff-Quik staining kit was from Siemens Healthcare Diagnostics (Los Angeles, CA, USA). Rodent M block, anti-rabbit horse 6 radish peroxidase (HRP)-polymer and HRP/DAB detection system were from Biocare Medical (Markham, ON, Canada). HRP-conjugated anti-β-actin monoclonal antibody (Ab), anti-cyclin D3 monoclonal Ab, anti-CDK4 rabbit Ab, anti-phospho-PTEN (Ser380) monoclonal Ab, anti-PTEN monoclonal Ab, anti-Akt monoclonal Ab, and anti-phospho-Akt (Thr308) monoclonal Ab were from Cell Signaling Technology (Beverly, MA, USA). Anti-MMP2 Ab and anti-CD31 Ab were from Abcam Inc. (Toronto, ON, Canada). HRP-conjugated-goat anti-mouse IgG Ab and HRP-conjugated-donkey anti-rabbit IgG Ab were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

### *4.2. Cell Culture*

The MDA-MB-231 breast cancer cell line was provided by Dr. S. Drover (Memorial University of Newfoundland, St. John's, NL, Canada). MDA-MB-468, MCF-7, and SKBR3 breast cancer cell lines were from Dr. P. Lee, Dr. K. Goralski and Dr. G. Dellaire, respectively (Dalhousie University, Halifax, NS, Canada). Breast cancer cell lines were authenticated by short tandem repeat analysis conducted by ATCC (Manassas, VA, USA). All breast cancer cell lines were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum, 5 mM HEPES buffer (pH 7.4), 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, and were maintained at 37 ◦C in a humidified incubator supplied with 10% CO2. HMECs from ScienCell Research Laboratories Inc. were grown in serum-free, HEPES- and bicarbonate-buffered mammary epithelial cell medium supplemented with 1% mammary epithelial cell growth supplement and 1% penicillin/streptomycin solution, and maintained for a maximum of seven passages at 37 ◦C in a humidified incubator supplied with 5% CO2. The MCF-10A normal mammary epithelial cell line was from Dr. P. Marcato (Dalhousie University, Halifax, NS, Canada). MCF-10A cells were cultured in DMEM/F12 supplemented with 10% heat-inactivated horse serum, 10 μg/mL human insulin, 20 μg/mL EGF, 0.5 μg/mL hydrocortisone, 100 U/mL penicillin, and

100 μg/mL streptomycin. MDF-10A cultures were maintained at 37 ◦C in a humidified incubator supplied with 10% CO2. Human dermal fibroblasts from Lonza Inc. (Walkersville, MD, USA) were maintained as per the supplier's instructions.

### *4.3. MTT (3-(4,5-Dimethythiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) Assay for Cell Viability*

Cells were plated in quadruplicate into 96-well flat-bottom plates at a density of 5 × 103 cells/well and cultured in the absence or presence of the indicated concentrations of AF4 for desired time. At the end of culture, MTT was added to each well to a final concentration of 0.5 μg/mL. Plates were then incubated for 2 h at 37 ◦C, after which supernatant was removed and formazan crystals in each well were solubilized in 100 μL of dimethyl sulfoxide (DMSO). Absorbance was measured at 570 nm using an Asys Expert Microplate Reader (Biochrom Ltd., Cambridge, UK). Results are expressed as % metabolic activity relative to the medium control.

### *4.4. Acid Phosphatase Assay of Spheroid Growth*

MCF-7 cells in F12 medium containing 20 ng/mL basic fibroblast growth factor, 20 ng/mL epidermal growth factor, 100 U/mL penicillin, 100 μg/mL streptomycin and B27 serum-free supplement were cultured for 48 h in ultra-low adherent cell culture plates and then treated with the indicated concentrations of AF4 or vehicle for 72 h. Spheroids were photographed, washed with phosphate-buffered saline, resuspended in 100 μL acid phosphatase assay solution (0.1 M sodium acetate at pH 5.5, 0.1% Triton-X-100, 4 mg/mL phosphatase substrate) and incubated for 2 h at 37 ◦C in the dark. The reaction was stopped by adding 25 μL 1 N NaOH to each well. Absorbance was measured at 405 nm using an Asys Expert Microplate Reader and % acid phosphatase activity was determined.

### *4.5. Flow Cytometric Cell Proliferation Assay*

MDA-MB-231 cell cultures were synchronized by serum starvation for 20 h and then seeded into 6-well plates and allowed to form monolayers. Cells were stained with 1.25 μM Oregon Green 488 dye in serum-free DMEM and a sample of stained cells was retained for use as a non-proliferative control. The remaining cells were cultured for 72 h in the absence or presence of the indicated concentrations of AF4. Cellular fluorescence was then measured using a FACSCalibur instrument (BD Bioscience, Mississauga, ON, Canada). The fluorescence of control and AF4-treated cells was compared to that of the non-proliferative control and the number of cell divisions (n) was calculated as follows: MCFbaseline = (2n)(MCFsample); where n denotes the number of cell divisions and MCF denotes mean channel fluorescence.

### *4.6. Cell-Cycle Analysis*

MDA-MB-231 and MBA-MB-468 cell cultures were synchronized by serum starvation for 20 h and then cultured for 72 h in the absence or presence of the indicated concentrations of AF4. Cells were then collected, washed with ice-cold phosphate-buffered saline, and ice-cold 70% ethanol was added drop-wise to the cells under constant agitation. Cells were then stored at −20◦C for 24 h, washed, and resuspended in phosphate-buffered saline containing 0.02 mg/mL PI, 0.1% Triton X-100, and 0.2 mg/mL DNase-free RNase A. After incubation for 30 min at room temperature in the dark, cellular fluorescence was determined using a FACSCalibur instrument. Data were analyzed using ModFitLT V2.0 software (Becton Dickson, CA, US).

### *4.7. Gap-Closure Assay*

MDA-MB-231 cells were seeded at a density of 1 × 104 cells/mL into wells containing culture inserts. After 18 h of culture, cells were treated with 10 μg/mL of mitomycin C in serum-free DMEM for 2 h at 37 ◦C to prevent cell division. After 12 h, cells were washed with complete DMEM and cultured in the absence or presence of 20 μg/mL AF4. At 0 h and 24 h, culture inserts were removed and the gaps were photographed.

### *4.8. Trans-Well Migration*/*Invasion Assay*

MDA-MB-231 cells were treated with 20 μg/mL AF4 for 24 h and serum-starved for 6 h, after which 5 × 104 cells in serum-free DMEM were placed into wells of the upper chamber of the trans-well cell migration apparatus. The bottom chamber wells contained DMEM plus 10% fetal calf serum as a chemoattractant. Migration of cells through an 8 μm porous membrane (uncoated or coated with fibronectin) was detected by Diff-Quik staining. Migrated cells were imaged under a light microscope.

### *4.9. Flow Cytometric Measurement of Apoptosis*

MDA-MB-231 cells were seeded at a density of 1 × 105 cells/well into 6-well plates and cultured in the absence or presence of the indicated concentrations of AF4 without or with 5 mM NAC for the desired time. In separate experiments, MDA-MB-231 cells were cultured for 48 h in the absence or presence of the Akt inhibitors MK2206 and SC66. At the end of culture cells were harvested, washed and stained with Annexin-V-488 and PI (1 μg/mL) for 15 min at room temperature. Flow cytometric analysis was performed using a FACSCalibur instrument.

### *4.10. Reactive Oxygen Species (ROS) Measurements*

Measurement of ROS by Amplex Red assay was performed in 96-well flat-bottom plates containing quadruplicate cultures of MDA-MB-231 cells without or with AF4 at 50 μg/mL. In the dark, 100 μL of master mix containing 25 μM Amplex Red reagent and 0.005 U/mL HRP in phenol red-free cDMEM was added to each culture. After incubation at 37 ◦C for 2 or 24 h, absorbance was measured at 570 nm with an Asys Expert Microplate Reader. Measurement of ROS by fluorescence was performed by staining quadruplicate cultures of MDA-MB-231 cells or MCF-10A cells with 5 μM CM-H2DCFDA in serum- and phenol-red free DMEM. Cells were then cultured for 2 or 24 h in the absence or presence of the indicated concentrations of AF4 in phenol-red free DMEM containing 1% fetal calf serum. Fluorescence at 529 nm was measured with a Spectramax M2 Microplate Reader (Molecular Devices, San Jose, CA, USA).

### *4.11. Western Blot Analysis*

MDA-MB-231 cells were cultured in the absence or presence of the indicated concentrations of AF4 for 24 h, and then placed in ice-cold lysis buffer (50 mM Tris at pH 7.5, 150 mM NaCl, 50 mM disodium hydrogen phosphate, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 100 μM Na3VO4, 10 mM NaF, 5 mM ethylenediaminetetraacetic acid and 5 mM ethylene glycol tetraacetic acid) containing freshly added protease inhibitors (1 mM phenylmethylsulfonylfluoride, 10 μg/mL aprotinin, 5 μg/mL leupeptin, 10 μM phenylarsine oxide, 1 mM dithiothreitol and 5 μg/mL pepstatin). After 15 min, cell lysates were cleared by centrifugation and the protein concentration was determined by Bradford assay. Equal amounts of protein (20 μg) were loaded into 12% or 15% sodium dodecyl sulfate polyacrylamide gels. Separated proteins were transferred onto nitrocellulose membranes, which were then blocked by 1 h incubation in 5% non-fat milk or 5% bovine serum albumin in Tween-Tris buffered saline (TBS) solution (0.25 M Tris at pH 7.5, 150 mM NaCl, 0.2% Tween-20). Blots were probed overnight at 4 ◦C with an optimal concentration of primary Ab, and then washed thoroughly with Tween-TBS and probed with HRP-conjugated donkey anti-rabbit IgG Ab or anti-mouse IgG Ab, as appropriate, for 1 h at room temperature. Even protein loading was confirmed by probing the blots with HRP-conjugated rabbit anti-β actin Ab. Proteins of interest were visualized by chemiluminescence.

### *4.12. Xenograft Breast Cancer Model*

Six to eight week-old female NOD-SCID mice were purchased from Charles River Canada (Lasalle, QC, Canada) and housed under sterile conditions and fed a sterilized rodent diet and water supplied ad libitum. Pathogen-free MDA-MB-231 cells (5 × 107) were implanted by subcutaneous injection into the right hind flank. Starting two weeks after xenografting, tumor sizes and body weights were recorded every other day until the last day (day 15) of the experiment. Tumor volume was calculated according to the equation, (L × P2)/2 where L is tumor length and P is perpendicular to tumor length. Intratumoral injection of AF4 commenced once the tumors reached a volume of 100 mm3 (recorded as day 1). A total of 5 intratumoral injections of AF4 at 0.5 mg/kg in 20 μL saline (7 mice) or saline alone (10 mice) were administered every other day for 9 days. Mice were monitored for an additional 6 days and their tumor sizes and body weights were recorded. Mice were euthanized at day 15, after which the tumors were excised and photographed. Tumors were fixed in buffered formalin, embedded in paraffin and cut into 5 μm thick sections. Tumor sections were mounted onto glass slides and stained with hematoxylin and eosin for detection of necrotic and live cells. Immunohistochemistry was performed to detect CD31 expression. Ethics approval for animal use was obtained from the Dalhousie University Committee on Laboratory Animals, and was in accordance with Canadian Council on Animal Care guidelines.

**Author Contributions:** Conceptualization, D.W.H. and H.P.V.R.; methodology, C.-Y.L. and W.F.; formal analysis, C.-Y.L. and W.F.; writing-original draft preparation, C.-Y.L. and W.F.; writing-review and editing, D.W.H.; supervision, D.W.H.; project administration, D.W.H. and H.P.V.R.; funding acquisition, D.W.H. and H.P.V.R.

**Funding:** This research was funded by the Canadian Cancer Society/Canadian Breast Cancer Foundation Endowed Chair in Breast Cancer Research (D.W.H.) and the Natural Sciences and Engineering Research Council of Canada (D.W.H. and H.P.V.R.).

**Acknowledgments:** W.F. was supported by a trainee award from the Beatrice Hunter Cancer Research Institute with funds provided by the Harvey Graham Cancer Research Fund as part of The Terry Fox Strategic Health Research Training Program in Cancer Research at the Canadian Institutes for Health Research.

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

### **References**

**Sample Availability:** Samples of the compound AF4 are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**2. Adhesion**

Urine of uninfected person is sterile due to urinary flow and antimicrobial activity of uric acid. Regular flow of urine does not allow microorganism to colonize inside the urinary tract. However, attachment of *E. coli* to uroepithelial cells allows them to overwhelm the effect of urine flow. For many pathogenic microorganisms, it is considered as the first step in the colonization process [13, 14], and both the host and *E. coli* function in this process. The ability of UPEC to colonize depends upon the expression of different fimbrial adhesins. For a successful adherence to the host cell surface, UPEC expresses many adherence factors which are crucial for attachment and thus regarded as virulence factors. Many bacterial adhesins are organized in a thin filamentous structure called fimbriae or pili although there are evidences of presence of adhesins in the cell surface of bacteria. Adhesins of fimbrial nature are important during attachment process [15]. Fimbriae, also known as pili, are long hair-like structures contained in the cell surface of bacteria that recognize specific compounds usually carbohydrates of the target host cells [11]. Pili are the short form of fimbriae and might be used interchangeably with fimbriae. Fimbriae consist of oligomeric pilin proteins. These proteins are arranged in such a manner that they form a helical cylindrical structure and are both thinner and shorter than flagellum. These proteinaceous structures are expressed in uropathogenic strains of *E. coli* and are considered as virulence factors [11]. Most of the receptors for these fimbriae are carbohydrates. They include type 1 fimbriae, P fimbriae, and thin aggregative fimbriae [16]. Many bacterial pathogens can produce an array of these adhesins, and often inhibition of a single adhesin may cost enough to a bacterium to lose its virulence. Functions of pili or fimbriae are not limited only to adhesion and can help in many other crucial pathways for the microbe to survive and evade the immune system of the host. Evolution of different types of adhesins plays a role in tissue tropism.

In gram-negative bacteria like UPEC, adhesins are unveiled by chaperone-usher-assisted pathway. This pathway involves two proteins, one is a periplasmic chaperone, and the other is a protein called usher. Usher act as the base of the structure, and the function of chaperone is folding and recruitment of the subunits [17, 18]. In absence of the chaperone, pilin proteins are degraded and misfolded and thus cannot be assembled in the form of a mature pilus. On the other hand, usher helps to mature the fimbriae and its transportation through the outer assuring integrity of the outer membrane. The constituents of usher proteins are an N-terminal domain (NTD), 24-stranded beta-barrel channel, a plug domain, and two C-terminal domains (CTD). In uropathogenic *E. coli* strains, chaperone-usher family fimbriae are more abundant.

#### **2.1. Type 1 fimbriae**

vitamin K2

factors of uropathogenic *E. coli* will be discussed.

tems which are important virulence factors of UPEC.

**2. Adhesion**

. Strains of *E coli*, however, obtaining ability to colonize inside the urinary tract

and to make themselves safe from the host immune system, become uropathogenic *E. coli*. UPEC causes >80% of UTI [2]. Urinary tract infections are very common, and approximately 10% of people [3] and half of all women (at least one time) become infected throughout their life. According to a study, more than 100,000 patients in the United States are hospitalized annually due to urinary tract infections [4], and in the year 2011, 400,000 patients were hospitalized, and the estimated cost was about 2.8 billion USD [5]. Infections can occur in both upper and lower urinary tracts. Lower urinary tract infection is known as cystitis, and in the case of upper urinary tract infection, it is called pyelonephritis. Without distinction of site, in order to cause infection, the causative agent must at first dodge the host's immune system and colonize in the urinary tract [6]. Several different virulent factors are needed for the bacterial population to cause infections [7]; for instance, pathogenic strains of *E. coli* express adherence factors which form pili or fimbriae of different types for their attachment in the sites where they usually do not live [7]; these are structural virulence factors and predominantly include P fimbriae and type 1 fimbriae [1]. Fimbrial adhesins such as PapG and CsgA are virulence factors that facilitate the attachment of *E. coli* [8]. In animal models, type 1 fimbriae aggrandize the chance survival of *E. coli* [9]. Beside these, UPEC can impair host immune system by a variety of ways [10], such as toxins and iron acquisition systems, and these are called secreted virulence factors. The production of these virulence factors by UPEC may cause an inflammatory response which makes a possible pathway for UTI symptoms [1]. However, both the host and the uropathogenic *E. coli* strain have different roles in the establishment and colonization process in the urinary tract [11]. Here in this chapter, different types of important virulence

8 Microbiology of Urinary Tract Infections - Microbial Agents and Predisposing Factors

In Gram-negative and some Gram-positive bacteria, virulence genes are allocated in particular segments (about 10–200 kilo bases in size) of their genome which have different G + C content than the other parts of the genome that are termed as pathogenicity islands. They are present in the virulent strains but present rarely in the nonpathogenic strains of the same species. These sequences can be transferred horizontally from species to species [12]. Pathogenicity islands encode virulence factors such as adherence factors, toxins, and iron acquisition sys-

Urine of uninfected person is sterile due to urinary flow and antimicrobial activity of uric acid. Regular flow of urine does not allow microorganism to colonize inside the urinary tract. However, attachment of *E. coli* to uroepithelial cells allows them to overwhelm the effect of urine flow. For many pathogenic microorganisms, it is considered as the first step in the colonization process [13, 14], and both the host and *E. coli* function in this process. The ability of UPEC to colonize depends upon the expression of different fimbrial adhesins. For a successful adherence to the host cell surface, UPEC expresses many adherence factors which are crucial for attachment and thus regarded as virulence factors. Many bacterial adhesins are organized in a thin filamentous structure called fimbriae or pili although there are evidences of presence of adhesins in the cell surface of bacteria. Adhesins of fimbrial nature are important during attachment process [15]. Fimbriae, also known as pili, are long hair-like structures contained in the cell In 99% of *E. coli* strains, genes to encode type 1 fimbriae are present [19], and during urinary tract infections, they damage urinary tract cells by mediating an increased inflammation [20]. In order to enter into the host cells of the urinary tract, type 1 fimbriae play a great role. Type 1 fimbriae are remarkably versatile virulence factors of UPEC that can stabilize the attachment of the bacteria to different type of cells throughout the urinary tract. Though in Bowman's capsules and glomerulus their binding sites could not be identified, a strong affinity of type 1 fimbriae was found in proximal tubules and vessel walls. In the bladder, they bind strongly to muscular layers and moderately to vessel walls. Receptors for type 1 fimbriae were also found in the distal tubules and collecting ducts. They can also induce their binding to the surface of macrophages [9]. These fimbriae recognize uroplakin from bladder epithelial cells and mannoside-containing host proteins. Unlike many other important types of adhesins, these are encoded by the bacterial backbone DNA [21] and are mainly composed of FimA proteins along with FimF, FimG, and FimH [17]. FimA proteins are most in number but are not pivotal for virulence. Among other subunits of type 1 fimbriae, allelic variations of FimH determine the sugar specificity and deletion of *fimH* results in less amount of colonization in mouse models of ascending UTI, and colonization could be restored by expression of plasmid with *fimH* gene [20]. FimH alone or in association with LPS can stimulate toll-like receptor 4 (TLR4) to initiate particular signaling cascade that may activate the humoral immune response. Many studies revealed that expression of type 1 fimbriae results in virulence and loss of expression results in loss of expression but their presence cannot be correlated with UTI as normal fecal strains also have equally expressed type 1 fimbriae [22]. However, type 1 fimbriae-mediated attachment is a crucial stage for cystitis. Adhesins of these fimbriae are mannose sensitive.


**4. Climate change impacts on land suitability in KRB**

Considering the CPWF objectives, few studies were done on river basin climate change (CC) issues. The study done by Ghaffari et al. [17] on land suitability under current and CC scenarios in the KRB was the main study, and the results are summarized as follows;

Assessing the suitability of an area for crop production requires considerable effort in terms of information collection that presents both opportunities and limitations to decision-makers. Land suitability is assessed as part of a 'rational' cropping system, and optimizing a piece of land for a specified use should be based on its attributes. Furthermore, land may be considered either in its present condition or after specified improvements. Although criteria may vary, they are essentially based on climate, soil, topography, and water availability which are the most important categories of natural environmental information required for assessing land suitability.

The CC study in KRB describes a climate-soil-site model to assess CC impacts on land suitability for dryland winter wheat, focusing on the potential effects of temperature increase and rainfall variables on the land suitability in KRB. Assessments were made for the current climate condition and future climate scenarios by 2025 with GIS maps generated through a Simple Limitation Approach (SLA). Ghaffari et al. [17] used topography maps (10 m resolution), 25 years of climate data (1973–1998), physiological and phonological crop parameters, CC scenarios, soil management domain (SMD), and a simple limitation approach (SLA).

In **Table 5**, results of the projection of impacts of CC on land suitability classes for dryland winter wheat in upstream areas of KRB are provided.

It was concluded that by increasing temperature alone, it would be expected that highly and moderately suitable areas increases in the KRB. Increasing temperature and increasing precipitation increase highly and moderately suitable areas. Decreasing precipitation alone or increasing temperature will lower highly and moderately suitable areas. The main reason for this is water stress risk, not the direct effect of temperature [17].