**Molecular Mechanisms Underlying Cancer Prevention and Intervention with Bioactive Food Components**

Editor

**Anupam Bishayee**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editor* Anupam Bishayee Lake Erie College of Osteopathic Medicine Bradenton United States

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Cancers* (ISSN 2072-6694) (available at: www.mdpi.com/journal/cancers/special issues/Compound).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-8383-9 (Hbk) ISBN 978-3-0365-8382-2 (PDF)**

Cover image courtesy of Anupam Bishayee

© 2023 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.

## **Contents**

Reprinted from: *Cancers* **2022**, *14*, 3988, doi:10.3390/cancers14163988 ................ **199**

## **About the Editor**

#### **Anupam Bishayee**

Dr. Bishayee is a Professor of Pharmacology at the Lake Erie College of Osteopathic Medicine (Bradenton, Florida). He received his Ph.D. (cancer prevention) from Jadavpur University, Kolkata, India. Dr. Bishayee performed post-doctoral research at Rutgers University, Newark, New Jersey (formerly University of Medicine and Dentistry of New Jersey). Dr. Bishayee's primary research interest during last two decades encompasses natural products and dietary agents in health and disease. His laboratory is involved in investigating cancer preventive and therapeutic effects of medicinal plants, natural products, dietary and synthetic agents using various pre-clinical models of cancer and underlying mechanisms of action. Various projects of Dr. Bishayee are funded by the National Institutes of Health as well as private pharmaceutical and biotechnological companies. Dr Bishayee has been awarded a United States patent on development of a potential drug for breast cancer prevention and treatment. Dr. Bishayee has published more than 290 peer-reviewed original research papers and authoritative review articles, mostly in high-impact journals, 22 book chapters, and around 84 conference abstracts, and delivered more than 37 invited presentations at various national and international scientific meetings. Dr. Bishayee has been serving various reputed journals as Section Editor-in-Chief, Consulting Editor, Editorial Board Member, and reviewer. Dr. Bishayee edited an Elsevier book "Epigenetics of Cancer Prevention" and guest edited several special issues on natural products and cancer published by leading publishers, such as Elsevier, Springer Nature, MDPI, and Bentham. Dr. Bishayee is a "Highly Cited Researcher" in 2022 and his papers rank in the top 1% by citations for a field or fields (Clarivate, Web of Science). His Scopus h-index is 73 and scholars from all over the world have cited Dr. Bishayee's published work more than 17,000 times.

## *Editorial* **Molecular Mechanisms Underlying Cancer Prevention and Intervention with Bioactive Food Components**

**Anupam Bishayee**

College of Osteopathic Medicine, Lake Erie College of Osteopathic Medicine, Bradenton, FL 34211, USA<EMAIL_ADDRESS>or<EMAIL_ADDRESS>
Cancer is the second-leading cause of death in the world, and it represents a major health challenge. According to the International Agency for Research on Cancer/GLOBOCAN, more than 19 million new cancer cases and almost 10 million cancer deaths occurred in the year 2020 [1]. The overwhelming evidence, which is based on preclinical and clinical studies, clearly indicates that diet can modify cancer outcome. Natural dietary bioactive compounds, present in fruits, vegetables, spices, whole grains, and herbs, have shown enormous potential for cancer prevention and treatment due to their easy availability, relatively low cost, high margin of safety, widespread acceptability, and human consumption.

During the last few decades, an extraordinary number of bioactive food components have been investigated, employing cell culture assays, animal tumor models, and human subjects to understand their potential for cancer prevention and treatment. I am pleased to introduce this Special Issue, which captures recent advances in our knowledge on cancer preventive and therapeutic efficacy of putative food-derived substances with understanding of the underlying cellular and molecular mechanisms of action. This thematic issue contains five original research papers and eight review articles.

Phannasorn and coinvestigators [2] evaluated the chemopreventive effects of riceberry bran oil, containing phytosterols, γ-oryzanol, and γ-tocotrienol, in chemically induced liver and colon carcinogenesis in rats. Oral administration of riceberry bran oil suppressed preneoplastic hepatic lesions and colorectal aberrant crypt foci, induced hepatocellular and colorectal cell apoptosis, and reduced the expression of proinflammatory cytokines. Additionally, the oil promoted the alteration of gut microbiota in both tumor models. This outcome of these experimental results indicates the potential health benefits of the consumption of rice constituents in preventing hepatic and colorectal cancers.

β-Caryophyllene is the primary sesquiterpene present in black pepper, cloves, hops, rosemary, copaiba, and cannabis. It has been recognized as the first known "dietary cannabinoid," a common component of food with a "Generally Recognized as Safe" status. It is approved by the United States Food and Drug Administration as a taste enhancer, food additive, and flavoring agent. According to a study conducted by Mannino and colleagues [3], β-caryophyllene has been found to suppress cell proliferation and to induce apoptosis by impacting the crosstalk between Akt, β-catenin, and cyclin D/cyclin-dependent kinase 4/6 signaling in a concentration-dependent manner in multiple myeloma. These results indicate that β-caryophyllene may represent an interesting alternative or additional therapeutic option to conventional chemotherapy for the treatment of multiple myeloma.

Polygodial, a natural sesquiterpene, can be extracted from water pepper (*Persicaria hydropiper*), Dorrigo pepper (*Tasmannia stipitata*), and mountain pepper (*Tasmannia lanceolata*). The results of a study conducted by Venkatesan et al. [4] showed that polygodial effectively inhibited the viability, cell cycle progression, and migration of taxane-resistant prostate cancer cells, possibly by increasing the generation of reactive oxygen species, disrupting the mitochondrial membrane, and activating the intrinsic cell death pathway.

Parupathi et al. [5] investigated the potential of gnetin C, a phytocompound found in the melinjo plant and commonly used in Indonesian foods, to block prostate cancer progres-

**Citation:** Bishayee, A. Molecular Mechanisms Underlying Cancer Prevention and Intervention with Bioactive Food Components. *Cancers* **2023**, *15*, 3383. https://doi.org/ 10.3390/cancers15133383

Received: 19 June 2023 Accepted: 25 June 2023 Published: 28 June 2023

**Copyright:** © 2023 by the author. 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/).

sion. Their findings demonstrate that a gnetin C-supplemented diet effectively suppresses metastasis-associated protein 1-promoted tumor progression in high-risk premalignant prostate cancer transgenic mouse model. This study underscores the potential of gnetin C as a novel nutritional agent for prostate cancer prevention.

The study by Raina et al. [6] focused on elucidating the "stage-specific" efficacy of the bioactive food component inositol hexaphosphate (IP6, also known as phytic acid) against prostate cancer initiation, growth, and progression in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Results indicated that IP6 feeding during the initial stages of cancer development prevents the progression of prostatic intraepithelial neoplasia lesions to adenocarcinoma, and IP6 feeding during the late stage of the disease reduces tumor growth and prevents its progression to the advanced stage of the disease. It has also been indicated that the anti-prostate cancer effects of IP6 are associated with its potential to eradicate the prostate cancer stem cell pool in the TRAMP model. Accordingly, IP6 intervention could have a therapeutic benefit during all stages of prostate tumorigenesis.

In this Special Issue, three review articles capture recent developments regarding research, elucidating the role of dietary phytochemicals on gastrointestinal tract cancers. Kang et al. [7] summarized the potential therapeutic effects of bioactive food components on the prevention and treatment of gastric cancer, with special focus on molecular mechanisms of action, bioavailability, and safety aspects. De et al. [8] reviewed the literature on phenolic phytocompounds endowed with anti-colorectal cancer activities, which are based on animal and human studies, to understand the impact of these results on the prevention and treatment of this cancer, which represents a significant cause of death worldwide. Dacrema et al. [9] presented an overview of the reciprocal interactions between spice-derived bioactive compounds and the gut microbiota to understand the role of dietary spices in the prevention of colorectal cancer.

There are two reviews dedicated to the impact of food-derived phytochemicals on hormone-related neoplasms. Prajapati and colleagues [10] dissected the concept of targeting luminal A-derived breast cancer stem cells with dietary phytocompounds by summarizing the signaling pathways implicated in therapy resistance. In their review, Kumar et al. [11] discussed the role of green tea catechins in the prevention of prostate cancer, presented evidence on the associations of microbiomes with prostate cancer, and evaluated the concept of utilizing the microbiome to identify biomarkers for the efficacy of green tea-derived constituents.

*Nelumbo nucifera* Gaertn., also known as the lotus, sacred lotus, Indian lotus, or Chinese water lily, is a recognized dietary and medicinal plant. Bishayee and colleagues [12] critically evaluated the potential of *N. nucifera*-derived products and phytoconstituents in cancer prevention and intervention with in-depth understanding of cellular and molecular mechanisms of action. Sulforaphane represents a metabolite of the phytochemical glucoraphanin, which is present in cruciferous vegetables, such as broccoli, Brussels sprouts, cabbage, and watercress. A review by Kaiser et al. [13] evaluated the recent state of knowledge on the efficacy of sulforaphane in preventing or reversing a variety of neoplasms based on preclinical and clinical studies. The authors also discussed the current limitations and challenges associated with sulforaphane research, and they suggested future research directions.

Finally, the work of Bouyahya et al. [14] focuses on recent advances in using dietary phenolic phytocompounds to sensitize various cancer cells towards chemotherapeutic agents and their values, in combination therapy, along with conventional anticancer drugs. Several phenolics, including caffeic acid, curcumin, gallic acid, resveratrol, rosmarinic acid, and sinapic acid, exhibit encouraging anticancer activities through sub-cellular, cellular, and molecular mechanisms, and they can increase the effectiveness of the approved cancer chemotherapeutic agents.

In conclusion, it is my hope that this Special Issue, featuring high-quality articles written by recognized leaders in the field, as well as young investigators from all over the world, would accelerate the translational impact of mechanism-based cancer prevention and intervention using multi-targeted dietary phytocompounds, identify current knowledge gaps, challenges, and pitfalls, as well as galvanize future research.

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

#### **References**

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

## *Review* **Phenolic Phytochemicals for Prevention and Treatment of Colorectal Cancer: A Critical Evaluation of In Vivo Studies**

**Samhita De 1,† , Sourav Paul 2,† , Anirban Manna 1,‡, Chirantan Majumder 1,‡, Koustav Pal 3 , Nicolette Casarcia 4 , Arijit Mondal 5 , Sabyasachi Banerjee 6 , Vinod Kumar Nelson 7, Suvranil Ghosh 1 , Joyita Hazra 8, Ashish Bhattacharjee 2, Subhash Chandra Mandal 9, Mahadeb Pal 1,\* and Anupam Bishayee 4,\***

	- Balisha 743 234, India

**Simple Summary:** Colorectal cancer (CRC) is a significant cause of death worldwide. The inefficacy of the current treatment regimens is reflected in the frequent recurrence and emergence of a drugresistant form of CRC. Numerous published reports from independent investigators around the globe have shown the great potential of natural products as a source of anti-CRC drug-leads with novel functions. Here, we have reviewed the literature on phenolic phytochemicals carrying anti-CRC activity in various in vivo models and analyzed their molecular basis of action to understand the implications of these findings in the future treatment and prevention of CRC.

**Abstract:** Colorectal cancer (CRC) is the third most diagnosed and second leading cause of cancer-related death worldwide. Limitations with existing treatment regimens have demanded the search for better treatment options. Different phytochemicals with promising anti-CRC activities have been reported, with the molecular mechanism of actions still emerging. This review aims to summarize recent progress on the study of natural phenolic compounds in ameliorating CRC using in vivo models. This review followed the guidelines of the Preferred Reporting Items for Systematic Reporting and Meta-Analysis. Information on the relevant topic was gathered by searching the PubMed, Scopus, ScienceDirect, and Web of Science databases using keywords, such as "colorectal cancer" AND "phenolic compounds", "colorectal cancer" AND "polyphenol", "colorectal cancer" AND "phenolic acids", "colorectal cancer" AND "flavonoids", "colorectal cancer" AND "stilbene", and "colorectal cancer" AND "lignan" from the reputed peer-reviewed journals published over the last 20 years. Publications that incorporated in vivo experimental designs and produced statistically significant results were considered for this review. Many of these polyphenols demonstrate anti-CRC activities by inhibiting key cellular factors. This inhibition has been demonstrated by antiapoptotic effects, antiproliferative effects, or by upregulating factors responsible for cell cycle arrest or cell death in various in vivo CRC models. Numerous studies from independent laboratories have highlighted different plant phenolic compounds for their anti-CRC activities. While promising anti-CRC activity in many of these agents has created interest in this area, in-depth mechanistic and well-designed clinical studies are needed to support the therapeutic use of these compounds for the prevention and treatment of CRC.

**Citation:** De, S.; Paul, S.; Manna, A.; Majumder, C.; Pal, K.; Casarcia, N.; Mondal, A.; Banerjee, S.; Nelson, V.K.; Ghosh, S.; et al. Phenolic Phytochemicals for Prevention and Treatment of Colorectal Cancer: A Critical Evaluation of In Vivo Studies. *Cancers* **2023**, *15*, 993. https:// doi.org/10.3390/cancers15030993

Academic Editor: Antonio V. Sterpetti

Received: 30 December 2022 Revised: 30 January 2023 Accepted: 30 January 2023 Published: 3 February 2023

**Copyright:** © 2023 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/).

**Keywords:** colorectal cancer; phenolic compounds; prevention; treatment; molecular mechanisms; in vivo

#### **1. Introduction**

The diagnosis of colorectal cancer (CRC) is a death sentence to many. CRC is the third most diagnosed and second leading cause of cancer mortality worldwide [1]. In the United States alone, there were 149,500 new cases and 52,980 deaths in 2021, with an estimated 151,030 new cases for 2022 [1]. Globally, there were 1.9 million new cases and 935,000 deaths in 2020 [2]. These numbers have risen since 2018, as at that time statistics were noted to be 1.8 million new cases and 861,000 deaths [3]. Analyses predicted the global CRC burden to rise by 60% to 2.2 million new cases and 1.1 million deaths by 2030 [3–6]. Rising cases are attributed to a more sedentary lifestyle and altered dietary habits, such as consuming processed foods, tobacco usage, and heavy alcohol consumption. India's incidence of colon cancer in 2016 was estimated to be 63,000, with a sizeable interstate variation [7,8].

Since the implementation of a screening program in the United States in 1990, CRC incidence has consistently decreased in the population of those older than 50 years [9,10]. In contrast, CRC incidence has shown a significant and steady increase (2% per year) in the population of those less than 50 years of age, which is called young-onset CRC (yCRC) [9,11,12]. While yCRC comprises only 10% of total CRC incidence, 75% of yCRC incidence affects the population of those between 40 and 49 years of age [9,11–15]. A study undertaken between 1975 and 2010 predicted that yCRC would double by 2030 in the U.S. population of those younger than 35, indicating racial disparity [9,11–15].

Current treatment options available for colorectal cancer include laparoscopic surgery, resection, palliative, neoadjuvant chemotherapy, and radiotherapy [15–22]. Chemotherapy causes undesirable side effects. In addition to being frequently ineffective, current treatments are expensive.

Utilizing phytochemicals for cancer treatment and prevention has been a matter of serious discussion for decades [3,23]. Plants have been used to treat many diseases in traditional medicine and have been a forefront in alternative approach. Over 3000 plant species have anticancer activities, with thirty plant-derived compounds undergoing preclinical testing [5]. Anticancer activity in citrus fruits, allium vegetables, and medicinal plants has demonstrated preclinical success [5,8]. Secondary plant metabolites have been shown to decrease inflammation and increase apoptosis in addition to possessing antioxidant, anticarcinogenic, and antimetastatic properties [8,23,24]. The attraction to phytochemicals arises from relatively safer and cost-efficient natural products, and their consumption by humans is widespread [5]. While research is being conducted, often with promising results, only a limited number of natural compounds have been approved for clinical use, while the clinical application of many is hindered due to low bioavailability [5,23].

Numerous literature reviews and studies on natural compounds in CRC were dissected and sorted thoroughly for relevant and vital information. It was noted that very few articles reviewed CRC and the therapeutic prospects with polyphenols [25,26]. There is no review literature explaining all classes of phenolic compounds and their signaling pathways in contrast with CRC. We have also noted that few previous reviews have focused on using plant extracts and fractions rich in phenols and pure phenolic compounds [25,26]. Some have examined flavonoids and their effects on CRC [27–36], yet no such reviews consider other classes of phenolic compounds and their effects on CRC. In contrast, numerous reviews were dedicated to discussing the deadly disease of CRC, but did not examine natural products for its treatment. A few reviews that included CRC studied general nutrition and dietary effects, but the literature examined dietary products, such as calcium, fiber, processed meats, or medicinal plants, rather than plant phenolic compounds [37–41]. Furthermore, a review was noted to include the effects of phytochemicals on CRC, but only mentioned specific biochemical properties and pathways of cancer development [42]. In

view of the aforementioned limitations, our present review is up-to-date and offers the most recent information compared to previously published works. In this review, we first evaluated pertinent literature to present the characteristics of CRC and identify common risk factors and current treatment options. Then, we evaluated various in vivo studies on different phenolic phytochemicals to understand the potential of these natural agents for CRC prevention and treatment. We hope these phenolic phytocompounds spark interest in conducting new studies to eventually aid in decreasing the prevalence and lowering the risk of CRC.

#### **2. Risk Factors**

Familial, hereditary, and lifestyle factors are independent risk factors for developing CRC [43]. Genetic syndromes comprise 20–30% of CRC cases and can be divided into non-polyposis and polyposis types (Table 1). Lynch syndrome, an alternate term for the non-polyposis syndrome, is an autosomal dominant disease associated with a defect in DNA mismatch repair genes, such as *hMLH1*, *hMSH2*, *hMSH6*, or *hPMS2* [44,45]. This mutation results in microsatellite instability (MSI) regions, which is also associated with ~15% of sporadic CRC cases. As expected, individuals with MSI regions carry an increased risk for other cancers, such as endometrial carcinoma [44].

**Table 1.** Genes involved in different CRC syndromes and associated clinical symptoms.

Abbreviations: MUTYH, mutY DNA glycosylase; STK11, serine/threonine kinase; 11SMAD4, mothers against decapentaplegic homolog 4; PTEN, phosphate and tensin homolog; BMPR1A, bone morphogenic protein receptor type 1A; MLH, MutL homolog; MSH, MutS homolog; MMR, mismatch repair.

Familial adenomatous polyposis syndrome (FAP), which is characterized by multiple polyp formations in the gastrointestinal tract, is caused by a germline mutation in the adenomatous polyposis coli (APC) gene [67–69]. Inheriting a polyposis syndrome can increase an individual's risk of developing colon cancer up to 100% [70]. Furthermore, these patients carry the risk of developing other gastrointestinal cancers and desmoid tumors. MUTYH-associated polyposis (MAP), Peutz-Jeughers syndrome (*STK11*), Juvenile polyposis syndrome (*SMAD4* and *BMPR1A*), hyperplastic polyposis (HPP), familial CRC (FCC) syndrome X, and Cowden syndrome (*PTEN*) are other polyposis syndromes that predispose individuals to an increased risk of developing CRC [50,71,72].

Chronic inflammatory bowel diseases, which encompass both ulcerative colitis and Crohn's disease, predispose individuals to CRC [73]. Additionally, previous abdominopelvic radiation is a potent risk factor for CRC, especially for childhood cancer survivors [74]. Furthermore, individuals receiving prostate cancer-related radiation therapy are at a higher risk of developing rectal carcinoma, supporting previous radiation therapy as a risk factor for CRC [75]. Cystic fibrosis is also implicated in CRC, as there is a 5–10 times greater risk of acquiring CRC in these patients. As a result, they have a separate management for CRC screening, especially post-transplant [76].

Lifestyle patterns, such as smoking, consumption of alcohol, obesity, sedentary lifestyles, and chronic diseases, pose a potent overall risk of developing sporadic CRC [77–79]. A westernized diet, rich in processed foods and red meat and deficient in fruits, fiber, and leafy vegetables, can contribute to CRC development [16,80]. Conversely, consuming more vegetables, fruits, and fiber is protective against CRC. A meta-analysis has elucidated the risk of CRC with food's dietary inflammatory index (DII). A higher DII correlating with a proinflammatory state increases CRC risk [81]. Numerous studies have explored the opposite end of the spectrum, examining anti-inflammatory foods and drugs for CRC chemoprevention and treatment. This is supported by a case-control meta-analysis where a higher intake of calcium, magnesium, and potassium lowered the occurrence of CRC [82].

The risk of CRC is low in vegetarians compared to meat eaters with an HR ratio of 0.49 [95% confidence interval (CI): 0.36 to 0.66], and 0.73 [95% CI: 0.54 to 0.99] when not adjusted and adjusted (for sociodemographic and lifestyle factors, multimorbidity, and body mass index) respectively. When CRC was subcategorized, the HR of 0.69 [95% CI: 0.48 to 0.99] for the colon and 0.43 [95% CI: 0.22 to 0.82] for the proximal colon was observed in vegetarians, which is much less compared to meat eaters [83]. Adherence to the Mediterranean diet was found to be associated with a low risk of rectal cancer with RR of 0.82 [95% CI: 0.71 to 0.95] for rectal cancer, 0.94 [95% CI: 0.87 to 1.02] for proximal colon cancer, and 0.91 [95% CI: 0.79 to 1.04] for distal colon cancer [84]. The unhealthy diet pattern is associated with CRC-specific mortality with RR/HR of 1.52 [95% CI: 1.13 to 2.06] [85]. The high intake of dietary calcium and magnesium is negatively associated with CRC risk with HR of 0.76 [95% CI: 0.72 to 0.80] and 0.80 [95% CI: 0.73 to 0.87], respectively. The higher intake of dietary heme, however, was positively correlated to colon cancer incidence with HR of 1.01 (95% CI: 0.82 to 1.19) and rectal cancer incidence with HR of 1.04 [95% CI: 0.67 to 1.42] [82]. The increase in DII score, and CRC are found to be positively associated with an overall increased risk of CRC by 40% with RR of 1.40 [95% CI: 1.26 to 1.55] [81]. Smoking and CRC shows a positive association with ever smoker versus never smoker, the pooled RR was 1.18 [95% CI: 1.11 to 1.25], and the pooled risk estimate was 1.25 [95% CI: 1.14 to 1.37] [77]. Alcohol consumption is also associated with an increased risk for CRC mortality. In comparison, the pooled RR was 1.03 [95% CI: 0.93 to 1.15] for any, 0.97 for light drinkers who consume ≤12.5 g of ethanol/day, 1.04 [95% CI: 0.94 to 1.16] for moderate drinkers who consume 12.6–49.9 g ethanol/day), 1.04 [95% CI: 0.94 to 1.16] for heavy drinking men (who consume ≥50 g ethanol/day), which is higher than heavy drinking women [pooled RR = 0.79 (95% CI: 0.40 to 1.54)] [78].

#### **3. Pathogenesis**

Overall, the pathogenesis of colon cancer involves three main pathways: the chromosomal instability (CIN)/classic adenoma-carcinoma sequence [86,87], the CpG island methylator phenotype (CIMP), and the microsatellite instability (MSI) pathway [88]. While these are separate pathways, there is potential overlap within them. Moreover, they involve the stepwise accumulation of multiple mutations, eventually leading to CRC development [89].

The classic adenoma-carcinoma sequence accounts for 65–70% of sporadic diseases commonly observed as left-sided CRCs [90]. This mechanism involves a dysfunctional/ inactivated APC gene located on chromosome 5q21. APC, a "gatekeeper" of colonic neoplasia, has been implicated in familial adenomatous polyposis (FAP) syndrome. The onset of CRC is inevitable in a population with an inactivating mutation in both copies of the APC gene [91,92]. APC controls cell growth and differentiation through the Wnt/ β-catenin signaling pathway. The Wnt pathway is an essential cellular signaling system by which several developmental events for embryological and tissue homeostasis occur, involving cellular proliferation and differentiation. Deregulation of the Wnt pathway can lead to the development of cancer. When the Wnt/β-catenin pathway is suppressed, there is a lower rate of cellular proliferation and fewer intestinal stem cells [93]. Activating mutations of Wnt/β-catenin leads to the pathogenesis of CRC. Over 90% of CRC cases carry mutations within this pathway [94]. It has been found that APC deletion/loss of function leads to CRC development, while restoring APC function can regress adenomas by reducing Wnt activity [93].

Apart from APC, there are other Wnt activating mutations, such as mutations in the CTNNB1 gene encoding β-catenin. R-spondins are another module of Wnt signal activators, which are associated with up to 10% of CRC mutations. Antagonism of RSPO3 with paclitaxel effectively targeted Wnt signaling in CRC [95]. A higher expression of ß-catenin in CRC cells is associated with a worse prognosis and advanced stage of the disease. Because of this, CRC metastasis was determined by the combined β-catenin odds ratio in the nucleus [96].

In the absence of APC function, β-catenin translocate to the nucleus. In cooperation with the DNA binding factor TCF, it promotes the growth of colonic epithelium via uncontrolled overexpression of its targets c-Myc and cyclin D1 [93]. Next, a mutation in KRAS contributes to molecular pathogenesis by promoting adenoma formation [97]. Finally, a mutation in p53 facilitates the progression of CRC [98]. Although important roles of p53 and KRAS were implied in the adenoma-carcinoma pathway, mouse knockout of APC develops carcinoma irrespective of its KRAS and p53 status, and re-introduction of APC restores cellular differentiation and normal crypt formation [43,93].

The microsatellite instability pathway occurs due to the inactivation of DNA mismatch repair genes, which includes ATPases hMSH2, hMSH6, hMSH3, hMLH1, hPMS2, hPMS1, and hMLH3, as involved in Lynch syndrome [99]. The MSI pathway is involved in roughly 15% of CRCs, 3% of which are Lynch syndrome while the rest are sporadic, mainly caused by MLH1 hypermethylation. Finally, the CpG island methylator phenotype (CIMP) is involved in silencing genes by hypermethylation of CpG islands on their promoters [100,101]. CIMP has been associated with older patients, female patients, and right-sided lesions with high MSI and BRAF mutations. CIMP is also associated with PI3K mutations but lacks KRAS and p53 mutations. A clearer insight and greater understanding of CIMP is required to better study the treatment and prevention of CRC [102].

#### **4. Chemoprevention**

Chemoprevention aims to intervene, prevent, suppress, and reverse the initiation and progression of carcinogenesis. It further attempts to decrease the recurrence of cancer through the usage of drugs, vitamins, and nutritional supplements [66]. Various agents, including nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, and other agents, such as metformin, statins, minerals, and vitamins, have been previously studied for their chemopreventive benefits regarding CRC (Table 2). There is little doubt that a significant stride has been made into the unventured territories for the chemoprevention of CRC.

*Cancers* **2023**, *15*, 993

 **References**

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In CRC involving the APC/β-catenin pathway, cyclooxygenase-2 (COX-2) is often implicated in the early and later stages of the adenoma sequence, driving the formation into a carcinoma [120–123]. Furthermore, COX-2 overexpression produces vascular endothelial growth factor (VEGF), which promotes tumor angiogenesis [124,125]. Hence, by targeting COX-2, various studies have shown that NSAIDs, ranging from aspirin and sulindac to the more selective COX-2 inhibitors, such as celecoxib, have proven benefits in reducing disease risk [126,127]. In the 1990s, the U.S. Preventive Services Task Force recommended aspirin to prevent non-high-risk CRC [128–130].

Other drugs, such as metformin, showed promising effects in reducing the risk of CRC development. Recent meta-analyses showed that metformin could reduce CRC risk by 22% [131]. In an ongoing ASAMET trial for the tertiary prevention of stage I–III CRC, patients were administered low doses of aspirin combined with metformin for a potential synergistic chemo-preventive action [132]. Statins, a specific inhibitor of HMG-CoA reductase in the mevalonate synthesis pathway, have been recommended to lower serum lipid levels [133]. Statins were shown to reduce CRC alone and in combination with NSAIDS [134,135]. Further investigations on multiple agents, such as antioxidants, minerals, such as selenium, and vitamins, including A, C, E, and β-carotene, were previously believed to have benefits in decreasing the risk of CRC, yet they have yielded mixed results [130,136,137]. Studies on folate's use to lower CRC risks also yielded mixed results [130]. Fiber, alcohol, monounsaturated fatty acids, polyunsaturated fatty acids, omega-3, omega-6, niacin, thiamine, riboflavin, vitamin B6, vitamin B12, zinc, magnesium, selenium, vitamin A, vitamin C, vitamin D, vitamin E, folic acid, β-carotene, anthocyanin, flavonoids, garlic, ginger, onions, thyme, oregano, saffron, turmeric, rosemary, eugenol, caffeine, and tea have all demonstrated anti-inflammatory benefits, and therefore reduce the risk of CRC development [138,139]. A higher intake of dietary fiber, pertaining to whole grains, was associated with a lower CRC risk in men [140].

#### **5. Treatment**

CRC incidence and mortality have been efficiently controlled by the routine screening and removal of polyps through colonoscopy [141]. Surgery, chemotherapy, and immunotherapy are mainstay treatments for CRC; the stage of CRC progression in each patient determines an appropriate combination. The treatment of CRC depends upon the diagnosis through tumor/node/metastasis (TNM) staging of the lesion. Adjuvant chemotherapy with fluorouracil (5-FU) decreases death rates in patients with high-risk stage II colon cancer by 3–5% and 10–15% in stage III disease alone [142]. MSI/MMR protein levels determined by IHC aid in deciding the adjuvant therapy [143–145]. Furthermore, after primary tumor resection, TNM or immunoscore can be considered to assess the tumor recurrence risk [146].

Single-agent therapy with 5-FU or therapy with multiple agents composed of 5-FU and oxaliplatin (FOLFOX), 5-FU and irinotecan (FOXFIRI) (IRI), or capecitabine and oxaliplatin (CAPOX), capecitabine (CAP), and irinotecan (CAPIRI) as first line chemotherapy is recommended based on the sensitivity and the stage of the disease. In many cases, singleagent chemotherapies yielded better results than combination therapy, given the associated systemic toxicity and unsatisfying responses [147–149]. A combination of 5-FU or CAP with oxaliplatin (OX) is recommended for stage III CRC for three to six months. Patients with intermediate-risk stage II CRC are recommended either 5-FU or CAP, which are added to OX, if the patients are high risk (stage II), for three months [145]. The International Duration Evaluation of Adjuvant Chemotherapy (IDEA) collaboration helped investigate whether three or six months of adjuvant chemotherapy was necessary, as cumulative toxicity develops from fluoropyrimidines/oxaliplatin in the form of peripheral sensory neuropathy. Results show that the overall disease-free survival was similar at 74.6% and 75.5% for three months and six months, respectively. After three months of treatment, the overall sensory peripheral neuropathy reduced from 34% to 11%. However, per ESMO guidelines, stage III CRC should still be treated with six months of FOLFOX or CAPOX if

the patient falls within the high-risk category. For patients who do not tolerate oxaliplatin, capecitabine, or LVGFU2 can be acceptable alternatives [145].

Various forms of supplemental targeted immunotherapies are considered to aid chemotherapy. Monoclonal antibodies are used to attack various potential genes, such as ERFR, VEGF, and PDL-1/PDL-1. Cetuximab, an anti-EGFR chimeric monoclonal antibody, and bevacizumab, an anti-VEGF chimeric monoclonal antibody, both of which prolong OS, were the first line targeted drugs approved by the United States Food and Drug Administration (FDA) in 2004 [150,151]. An immune checkpoint blocker α-PD1/PDL-1 antibody, in combination with chemo- and radiation therapy, was approved by the FDA for MSI-H and dMMR classes of CRCs for sustained progression-free survival [152]. Cetuximab yielded a positive outcome for CRC that did not respond to single-agent IRI or fluoropyrimidine therapy. Combining cetuximab with IRI, fluorocytidine, or OX delivered promising results [151,153]. EGFR (epidermal growth factor receptor) is overexpressed in various cancers to different extents, including 25–75% in CRC [154]. Cetuximab, once bound, results in the internalization and degradation of EGFR [111]. However, cetuximab was inactive in CRCs carrying the RAS (KRAS) mutation. Like EGFR, the VEGF level is also elevated in CRC, predicting a poor prognosis [155]. Along with an elevated VEGF level, increased vascular endothelial growth factor receptor (VEGFR) activity is found in adenomas, as well as in the metastatic stage of CRC [147,156]. While cetuximab is not suitable as a second line agent, bevacizumab is often an excellent choice.

#### **6. Literature Search Methodology**

We have followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines [157] for this work. Four scholarly databases, namely PubMed, Scopus, ScienceDirect, and Web of Science, were utilized to screen the literature for the last 20 years (2002 to 2022 November) by searching the title, abstract, and key words section with the key words "colorectal cancer" AND "phenolic compounds", "colorectal cancer" AND "polyphenol", "colorectal cancer" AND "phenolic acids", "colorectal cancer" AND "flavonoids", "colorectal cancer" AND "stilbene", and "colorectal cancer" AND "lignan." All search results were gathered, and duplicate files were removed. Next, literature was scanned based on title and abstract. Selected articles were then searched for retrieval. After reading the full articles, preclinical studies (in vivo animal models) with polyphenols were selected and incorporated. The methodology for literature search and study selection is depicted in Figure 1.

**Figure 1.** The PRISMA flow chart summarizing the literature search. Here "n" represents the number of articles.

#### **7. Phenolic Compounds with In Vivo Anti-CRC Activities**

Plants synthesize phenolic compounds as secondary metabolites and carry multiple aromatic rings with two or more hydroxyl groups. Phenolic compounds carry a wide (~8000 different) variety of chemical structures. Based on chemical structures, phenolic compounds are divided into different classes, such as flavonoids (e.g., anthocyanidins, flavanols, flavanones, flavones, flavonols, and isoflavoniods) and non-flavonoids, including phenolic acids (e.g., hydroxycinnamic acids and hydroxybenzoic acids), coumarins, stilbenes, lignans, and tannins [158–160]. Significant sources of phenolic compounds are fruits and vegetables. Various phenolic compounds are known for their interesting pharmacological properties, including antioxidant, anti-inflammatory, neuroprotective, and anticancer properties [161,162].

While western medicines have significant effects on CRC chemoprevention and treatment, extracts of numerous plants and plant products are still currently in use, as humanity has used plants for thousands of years as traditional or ethnic medicines for the prevention and treatment of various ailments, including cancer. The primary reasons for their popularity are fewer side effects, easy availability, and low cost compared to synthetic drugs [163–165]. Over the last several decades, steady progress has been achieved in identifying the bioactive secondary metabolites of plants, such as phenolic compounds, and understanding their mode of action to explain their health benefits [166–169]. In the following sections, we aim to summarize the anti-CRC effects of phenolic compounds based on animal studies. Table 3 describes the in vivo CRC activity of the compounds as revealed by our literature search as depicted in Figure 1. We have selected 16 relatively well-studied compounds to describe their anti-CRC activities in a greater detail in the following sections. The chemical structures of various classes of phenolic compounds with in vivo anti-CRC activities are presented in Figures 2–5.

**3.**Anti-CRCeffectsandmechanismsofactionofbasedinvivo

**Table**

**3.**

*Cont*.

**Table3.**

 *Cont*.

 *Cont*.

weight protein tyro-sine

mammalian

ornithine chromosome

TNF-α, tumor necrosis factor-α; Top1,

 10; QR, quinone reductase;

decarboxylase;

 PCNA,

 target of rapamycin;

phosphatase;

 5-ND, proliferating

 cell nuclear antigen; PI3K,

 SCID, severe combined

topoisomerase

 1; VDI, vascular density index; VEGF, vascular endothelial

 MAM, methyl 5-nucleotidase;

 NEDD9,

azoxymethane;

 MAPK, developmentally

phosphoinositide

immunodeficient;

 SIRT, Sirtuin 1; SOD, superoxide

 3-kinase; PP2A, protein

downregulated

 9; NF-κβ, nuclear factor-κβ; Nrf-2, nuclear factor erythroid -2 related factor; ODC,

phosphatase

 dismutase;

 growth factor; γ-GT,

 STAT3, signal transducer and activator of

γ-glutamyl

transpeptidase.

 2A; PTEN,

phosphatase

 and TENsin homolog deleted on

transcription

 3;

mitogen-activated

 protein kinase; MDF,

mucin-depleted

 foci; MMP, matrix

metalloproteinase;

 mTOR,

**Figure 2.** *Cont*.

**Figure 2.** *Cont*.

**Figure 2.** Chemical structures of flavonoids with in vivo anti-CRC activities.

**Figure 3.** Chemical structures of phenolic acids with in vivo anti-CRC activities.

**Figure 4.** Chemical structures of lignans with in vivo anti-CRC activities.

**Figure 5.** Chemical structures of stilbenes and miscellaneous compounds with in vivo anti-CRC activities.

#### *7.1. Flavonoids*

#### 7.1.1. Baicalin

Baicalin (molecular weight: 446.4 g/mol), conjointly called baicalein 7-O-glucuronide and 7-D-glucuronic acid-5, 6-dihydroxyflavone or known by its chemical name, 5, 6 dihydroxy-4-oxo-2phenyl-chromen-7-yl) oxy-3, 4, 5-trihydeoxytetrahydropyran-2-carboxylic acid, is a glycosyloxyflavone. It is a key component of a variety of traditional medicine preparations, consisting of Sho-Saiko-To, Yangkun pills, Kushen decoction, and Shuanghuanglian injections. Scutellariae radix, Scutellaria planipes, Scutellaria rehderiana, and Scutellaria scandens are only a few of the Scutellaria species that contain the compound baicalin, which is extensively distributed throughout the genus [358].

Baicalein suppressed AOM/DSS-induced colon tumors in mice and induced apoptotic cell death. Baicalein suppressed inflammation by PARP1-mediated NF-κB inhibition [180]. A dose of 50 mg/kg baicalin suppressed the growth of highly metastatic SW620 tumor xenograft in BALB/c nude mice [181]. Baicalin inhibited the TLR4/NF-κB signaling and significantly suppressed CT-26 tumor growth, migration, and invasion. Anti-tumor immunity was also enhanced by an increase in CD4+ and CD8+ T cells in CT-26 tumors [182]. Baicalein treatment induced apoptosis in a p53-mediated Akt-dependent manner and suppressed HT-29 tumor xenograft [183]. In another study, baicalein suppressed MMP-2 and MMP-9 and inhibited DLD1 tumor growth and metastatic effects by inhibiting phosphorylation of ERK [184].

Dou et al. [185] showed that baicalein and baicalin can significantly inhibit the growth of HCT116 tumor xenograft by inducing tumor cell apoptosis and senescence through inhibiting the telomerase reverse transcriptase. It has also been hypothesized that the control of colon cancer cell apoptosis and senescence is caused by the MAPK, ERK, and p38 signaling pathways. Wang et al. [186] verified that baicalin application increased the expression of DEPP and triggered its downstream target Ras/Raf/MEK/ERK and p16INK4A/Rb pathways by serving as an antioxidant, resulting in senescence in colon carcinoma cells in HCT116 tumor model in BALB/c athymic nude mice. It was revealed that baicalin inhibited the HT-29 xenograft tumor in nude mice by suppressing c-Myc as the driver of miRNAs responsible for oncogenic development (oncomiRs). These findings demonstrated an association of c-Myc in baicalin-mediated inhibition of colon cancer growth [187]. In orthotopically transplanted tumors of CRC cells in BALB/c nude mice, baicalin administration lowered the levels of marker proteins for cell cycle, EMT, and stemness [188].

Wang et al. [189] observed that the baicalein therapy dramatically decreased tumor numbers in the small intestine and colon of ApcMin/+ mice. Furthermore, reduced levels of inflammatory cytokines, such IL-1, IL-2, IL-6, G-CSF, and GM-CSF B, in this mouse tumor model suggested that baicalein's anti-CRC action was mediated by reducing gut inflammation. Baicalin treatment suppressed HCT116 tumor xenograft growth by downregulation of CircMYH9 and HDGF, and upregulation of miR-761 [190].

#### 7.1.2. Curcumin

Curcumin, with the chemical name (1E, 6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6 heptadiene-3,5-dione(diferuloylmethane), is a hydrophobic polyphenol derived from the roots of a well-known Indian spice, turmeric (Curcuma longa). Consumption of turmeric is believed to provide protection from numerous ailments, including CRC [359–362]. Anti-CRC activities of curcumin were demonstrated by several independent groups. Curcumin reduced putative precursor colonic lesions, e.g., aberrant crypt foci (ACF), through suppressing the levels of proinflammatory cytokines, such as TNF-α and IL-6, and proinflammatory mediators, such as COX-2, in obese and diabetic (db/db) mice [197]. Adiponectin plays an important anti-inflammatory role in the gut [363–365]. Curcumin increased the adiponectin level in both AOM-treated and untreated C57BL/KsJ-db/db (db/db) mice [197]. Leptin levels are directly proportional to body fat. High serum leptin levels can cause inflammationmediated CRC [366,367]. Curcumin was able to reduce the body fat content along with serum leptin levels, and thus reduce the severity of CRC. This study also observed AMPK activation and COX-2 inhibition in those animals [197].

Curcumin reduced DSS-induced ACF and β-catenin accumulation. Due to its antiinflammatory properties, curcumin suppressed pro-inflammatory cytokines and COX-2 and iNOS in DSS-induced colonic tissue [194]. Curcumin suppressed the growth of HCT116 tumor xenograft in ICR SCID mice. Curcumin treatment led to proteasome inhibition and induction of apoptosis which, in turn, suppressed the HCT116 tumor growth [195]. In another study, curcumin inhibited AOM/DSS-induced tumorigenesis in mice. Curcumin also downregulated Axin2 and exerted its anticancer activity by Axin2 mediated inhibition of the Wnt/β-catenin pathway [196].

Curcumin was found to inhibit HCT116-induced xenografts in male nude mice, along with suppressing NF-κB regulated genes, including Bcl-2, c-FLIP, IAP1, and survivin. It further cleaved procaspase-3 and procaspase-9. Curcumin pretreatment sensitized the tumor xenograft to γ-radiation and suppressed NF-κB activity by inhibiting the binding of NF-κB to its response element on its target genes, thus minimizing invasion, migration, and angiogenesis. Curcumin ameliorated the γ-radiation mediated increase of cellular proinflammatory mediator COX-2 and c-Myc in a HCT116 xenograft tumor model [198,199].

Furthermore, curcumin was found to modulate gut microbiome habitat in AOM-injected IL10-/-mice and was implicated in the function of anti-inflammation and the maintenance of gut homeostasis. The aberrant cytoplasmic and nuclear localization of β-catenin in AOM-treated wild-type and AOM/Il-10-/-mice was significantly reduced by curcumin treatment [200].

Curcumin enhanced the anti-CRC activity of capecitabine in HCT116 tumor xenografts in male athymic nu/nu mice through the induction of apoptosis and inhibition of angiogenesis, invasion, and metastatic factors, such as VEGF, ICAM-1, and MMP-9, and CXCR4. Inhibition of COX-2 and cell cycle progression mediators, cyclin D1 and c-Myc, was also observed in the curcumin-treated animals. The anti-CRC effects of liposomal curcumin alone and combined with oxaliplatin were tested on CRC xenografts induced by Colo205

and LoVo cells in athymic nu/nu mice. The combination therapy showed efficient tumor growth inhibition by apoptosis (PARP-1 cleavage). Liposomal curcumin also inhibited angiogenesis in consistence with the inhibition of VEGF, CD31, and IL-8 expression [201]. Phytosomal curcumin was tested for its ameliorative effects on an AOM/DSS model of colitis-associated CRC alone and in combination with 5-FU in in vivo. Curcumin, alone and in combination, functioned through modulating Wnt/β-catenin signaling and E-cadherin activities. Curcumin administered by oral gavage and in combination with 5-FU significantly inhibited GSK3 α/β and cyclin D1 expression. Curcumin was shown to reduce oxidative stress induced ACF and colon injuries induced by AOM/DSS by upregulating endogenous antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), thiolase, and inducing autophagy by upregulating beclin1 [200].

#### 7.1.3. Catechins

Catechins are a group of polyphenols abundantly present in tea, cocoa, berries, grapes, and apples. Catechins have a myriad of health benefits, and their anticancer properties have been extensively studied [368,369]. Kim et al. [370] examined the effects of green tea polyphenol (GTP) dosage on DSS-induced acute colitis and DMH and DSS-induced colon cancer developed in male ICR mice. GTP contained 70% of total catechins, half of which were EGCG and 3% being caffeine. This study showed that a specific dosage of GTP was effective in ameliorating the carcinogenic effect of DSS/DMH. The basis of this activity was implicated in the antioxidant properties of GTP. If the dosage was higher or lower than the effective dose, GTP was ineffective. This is potentially due to a loss of, or insufficient, antioxidant properties. Depending on the treatment conditions, GTFP can exhibit antioxidant or pro-oxidant properties [371].

The anticancer effect of EGCG was also tested on azoxymethane (AOM)-induced male C57BL/KsJ-db/db (db/db) mice. EGCG caused a significant reduction in the levels of IGF-IR, phospho-IGF-IR, phospho-GSK-3β, β-catenin, COX-2, and cyclin D1. There was also an increase in serum IGFBP3 and a decrease in serum IGF-I, insulin, triglyceride, cholesterol, and leptin in the treated mice [206].

Zhong et al. [207] investigated the acetylated-EGCG activity against protumorigenic inflammatory mediators in AOM-mediated colitis-induced CRC in a male mouse model. Acetylated-EGCG inhibited the expression of pro-tumorigenic inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and COX-2. iNOS is one of the enzymes that remain in ACF and causes the continuous formation of nitric oxide (NO), leading to the promotion of tumorigenesis [372–374]. Furthermore, COX-2 converts arachidonate to prostaglandin E2. A sustained overexpression of prostaglandin E2 in the tissues may lead to epithelial cell cancers, including CRC [207,375,376].

EGCG showed the antistemness and chemosensitizing effects on xenograft tumors of HCT116 spheroid-derived cancer stem cells in male nude mice. EGCG inhibited CRC stemness and sensitized 5-FU-resistant HCT116 cells. EGCG suppressed stemness markers, such as Notch-1, and upregulated the expression of tumor suppressive miRNAs, including miR34a, miR200c, and miR-145 [208].

Another study demonstrated the effects of green tea catechins alone and in combination with curcumin on DMH-induced colon cancer in male Wistar rats [209]. A 32-week-long dietary treatment with curcumin, green tea catechins, and their combination showed a significant reduction in the number of colorectal aberrant cryptic foci in these animals. Notably, the combinatorial treatment had a greater effect than that with either of the compounds acting alone. A significant decrease in the proliferation index and an increase in the apoptotic index were reported in the groups treated with a combination of the compounds, compared to the mock-treated group or those receiving only DMH [209].

The anticancer effect of polyphenol E (PPE) was tested on AOM-treated F344 rats. PPE is a standardized GTP mixture containing 65% EGCG and other catechins. After AOM treatment, the animals were given a 20% high-fat diet, with or without 0.24% PPE for 34 weeks. PPE treatment resulted in a significant reduction in tumor size and the

number of tumors in these animals. PPE was shown to decrease nuclear β-catenin levels, induce apoptosis, and increase the levels of RXR-α, RXR-β and RXR-γ expression in adenocarcinomas. This was accompanied by the lowering of proinflammatory eicosanoids, prostaglandin E2, and leukotriene B4 in the plasma [276].

#### 7.1.4. Fisetin

Fisetin is a hydroxy flavone under the subgroup of flavonoid found in various fruits and vegetables, such as strawberry, apple, persimmon, grapes, onion, and cucumber. In an AOM/DSS-induced colitis associated CRC model in BALB/c mice, fisetin suppressed dysplastic lesions through inducing apoptosis in the colonic tissue along with downregulation of Bcl-2 and STAT3, and upregulation of cleaved-caspase-3 and BAX. Fisetin treatment restored the level of enzymatic (SOD, CAT, GPx, and GR) and non-enzymatic (vitamin E, and vitamin C) antioxidants in DMH-induced colonic tissue back to normal [213].

Fisetin treatment resulted in activation of AMPKα and inhibition of PI3K/Akt/mTOR signaling pathway along with decreased expression of PI3K, reduced Akt phosphorylation in PIK3CA mutants. In FC13K1ApcMin/+ mice, fisetin decreased the occurrence of colonic tumor incidences. In combination with 5-FU, fisetin reduced the overall colonic tumor incidences [214].

Fisetin inhibited growth of LoVo tumor xenograft in athymic nude mouse model. Mechanistic study revealed that fisetin acted by inducing apoptosis in tumor tissue through activation of caspase-8 and increased cyt. c expression. In the tumor tissue of treated animals, inhibition of IGF1R and Akt activation was observed [215].

Although CT-26 tumor growth was suppressed upon the intratumoral injection of fisetin, HCT116 tumors were not sensitive to the similar treatment where a combination of radiation with fisetin was more effective. Fisetin suppressed the oncoprotein securin in CT-26 tumor in a p53-independent fashion, but securin null HCT116 tumors are more sensitive to fisetin treatment [216].

Fisetin suppressed HCT116 induced tumor growth in NOD/Shi-scid-IL2R gamma (null) (NOG) mice in a dose-dependent manner compared to control group [218]. Another study showed that due to poor water solubility, the fisetin micelles, composed of poly(ethylene glycol)-poly(ε-caprolactone), i.e., MPEG-PCL, are more efficient antitumor agents over free fisetin as tested in CT-26 tumor model. MPEG-PCL showed enhanced inhibition of angiogenesis through inducing apoptotic cell death [217].

#### 7.1.5. Genistein

Genistein, a naturally occurring isoflavone, was first isolated from Genista tinctoria. Its anticancer properties have been extensively studied [377]. Sekar et al. [222] examined genistein's role in regulating the tumor microenvironment in DMH-induced colon cancer in Wistar rats. This study revealed that genistein could regulate enzymatic (SOD, CAT, GPx, and GR) and non-enzymatic (vitamin E, vitamin C, vitamin A, and GSH) antioxidants in DMH-induced colonic tissue environments. It was found that the loss of mucin secretion in DMH-induced animals was restored by genistein. There was also a reduction of mast cell population and collagen deposition in genistein-treated animals compared to mock-treated animals. Argyrophilic nuclear organizer region and proliferating cell nuclear antigen, two prognostic markers, were decreased by genistein in DMH-treated rats. Genistein activated NRF2 and its downstream target, heme oxygenase-1, and alleviated DMH-induced oxidative stress. Higher expression of colonic stem cell markers, such as CD133, CD44, and β-catenin, was found to be reduced by genistein in DMH-treated animals [222].

It was shown that oral administration of genistein to mice carrying orthotopically implanted human CRC did not inhibit tumor growth. However, it did show inhibition of distant metastasis formation at a dose non-toxic to the animals. Subsequent biochemical analyses showed genistein-mediated downregulation of matrix metalloproteinase-2 (MMP-2) and FMS-related tyrosine kinase 4, also known as vascular endothelial growth factor receptor 3, suggesting its inhibitory role against neoangiogenesis in mouse tumors [224].

Chen et al. [378] conducted a study in which clinical signatures of the anti-CRC activity of genistein were tested in clinical samples of plasma, tumor tissue samples, and standard tissue samples isolated from patients. The expression of miR-95, serum glucocorticoid kinase 1 (SGK1), Bcl-2, and Erk1 was highly elevated in samples of CRC compared to the normal samples. Furthermore, genistein could sensitize CRC SW620 cells to apoptosis with increased LDH content in a concentration-dependent manner, accompanied by downregulation of endogenous miR-95, SGK1, and Erk1 activities [378].

Zhang et al. [223] studied the effect of genistein on AOM-induced colon carcinogenesis in male Sprague Dawley rats. The animals were given a control diet, soya protein isolate (SPI), and a genistein diet orally, starting from gestation to 13 weeks of age. Pre-AOM treatment analysis was performed by taking samples at seven weeks of age, and the remaining rats were AOM-treated at this time for six weeks for analysis. Compared to the control group, AOM injections did not cause aberrant nuclear accumulation of β-catenin in SPI and genistein-treated groups. Moreover, SPI and genistein suppressed the expression of cyclin-D1 and c-Myc. In addition, the expression of Wnt signaling genes (Wnt5a, Sfrp1, Sfrp2, Sfrp5) was decreased to a level comparable to that of pre-AOM treatment by SPI and genistein. Furthermore, the rats fed SPI and genistein had lower numbers of total aberrant crypts, which correlated with the reduction in Wnt/β-catenin signaling. Genistein also lowered the number of ACF [223].

The first clinical study to assess the safety and tolerability of genistein in combination with chemotherapy for the treatment of metastatic CRC was conducted by Pivota et al. [379]. Patients diagnosed with metastatic CRC but not previously treated were administered FOLFOX or FOLFOX-bevacizumab. Genistein (60 mg/day) was given orally for seven days every two weeks. Treatment was started four days before chemotherapy and continued through days one through three of infusion chemotherapy. In this trial, thirteen patients received combinatorial treatment. Treatment with genistein alone resulted in mild side effects, such as headaches, nausea, and hot flashes, with one subject experiencing grade 3 hypertension. There was no increase in chemotherapy-related adverse events when genistein was added to FOLFLOX. The best overall response rate for the genistein supplementation of the chemotherapy regimen was 61.5%. The median progression-free survival of the same study was 11.5 months [379].

#### 7.1.6. Kaempferol

Kaempferol, a dietary flavanol found in many plants, including apple, tea, broccoli, and grapefruit, has been demonstrated to carry antitumor effects based on preclinical studies [380]. Nirmala et al. [239] demonstrated the beneficial effects of orally administered kaempferol with intravenous irinotecan in 1,2-dimethyl hydrazine (DMH)-induced colorectal carcinoma in male Wistar rats. In the kaempferol-fed animal groups, levels of DMH-induced erythrocyte lysate levels and decreased liver thiobarbituric acid reactive substances. Levels of several antioxidant enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase, were recovered, and the most successful effects were achieved at a dose of 200 mg/kg body weight of kaempferol (which is comparable to irinotecan).

The combined effect of fluoxetine, an antidepressant drug, and kaempferol in alleviation of DMH-induced colon carcinoma in male Sprague Dawley rats was also analyzed. Compared to fluoxetine and kaempferol alone, combined treatment of these two agents caused greater reduction in multiple plaque lesions and preneoplastic lesions in the colonic tissues. This combinatorial treatment also reduced tissue concentration of malondialdehyde and NO. Both serum and tissue β-catenin levels were significantly decreased by the combinatorial treatment. There was also a significant increase in serum and tissue caspase-3 levels. PCNA and COX-2 positive cells in the colon of animals receiving the combinatorial treatment were lower when compared to fluoxetine and kaempferol treatments alone [240].

Hassanein et al. [241] studied the effect of sulindac in combination with either EGCG or kaempferol in DMH-induced colon carcinogenesis in male Sprague Dawley rats. The combinations of EGCG and kaempferol with sulindac, a nonsteroidal anti-inflammatory drug, caused great enhancement of sulindac's antioxidant, anti-inflammatory, antiproliferative, and apoptotic activities. Sulindac combined with both compounds caused a decrease in thiobarbituric acid-reactive substance, tissue NO, and both serum and tissue β-catenin. Downregulation of PCNA and COX-2 and a decrease in the number of ACF caused by DMH administration were also noted [241].

#### 7.1.7. Luteolin

Luteolin (3 ,4 ,5,7-tetrahydroxyflavone) was discovered in different fruits, vegetables, and medicinal herbs. Plants rich in luteolin are used for treating various ailments, such as hypertension, inflammation, and cancer in Chinese traditional medicine [381,382]. The anti-CRC activity, as well as the anti-angiogenic, anti-invasive, and antimetastatic effects of luteolin were studied using AOM-induced colitis models of male BALB/c mice. Upregulation of γ-glutamyl transferase (GGT), found in a number of human neoplasms, facilitates neoplastic progression and metastasis [246,383]. GGT and 5 -nucleotidase (5 ND) were inhibited in AOM-treated mice by luteolin. Furthermore, luteolin reduced other tumor markers in AOM-treated animals, such as cathepsin-D and carcinoembryonic antigen (CEA), which are correlated with poor prognosis [246]. Luteolin inhibited invasion and metastasis by reducing the expression of MMP-2 and MMP-9 along with enhancing expression of tissue inhibitor metalloproteinases 2 (TIMP-2) [246]. Mast cells were associated with enhanced angiogenesis and tumor malignancy [384]. It was found that luteolin also decreased giant mast cell and total mast cell populations in AOM-treated mice, compared to AOM-induced control animals [246].

Luteolin reduced the number and size polyps of DSS-treated mice. Upon luteolin treatment, DSS-induced oxidative stress, level of carcinoembryonic antigen and COX-2 were decreased in colonic tissue [242]. In another study, luteolin was shown to suppress AOM-induced CRC by downregulating iNOS and COX-2 expression level [243]. Luteolin also suppressed AOM-induced CRC by activating Nrf2/Keap1 pathway [244].

Luteolin inhibited HT29 xenograft's growth in nude mice by an activity consistent with modulation of miR-384/pleiotrophin axis [247]. miR384 expression was found to be downregulated in the majority (83%) of CRC biopsy samples, correlating with the invasiveness and migratory abilities of CRC [385]. Pleiotrophin plays a major role in angiogenesis through upregulation of VEGF in CRC [386]. Luteolin treatment of HT-29 cell-induced xenograft tumor developed in female nude BALB/c mice efficiently suppressed the migration of CRC cells from the spleen to the liver and metastasis through upregulation of miR-384/pleiotrophin axis. Luteolin upregulated the expression of miR-384, which, by targeting pleiotrophin expression, inhibited the expression of MMP-2, MMP-3, MMP-9, MMP-16, as well as invasion and metastasis of CRC [247]. Luteolin, in synergy with adenovirus CD55-TRAIL, inhibited HT-29 xenografts in female BALB/c nude mice through increasing the apoptotic activity [248].

In another study, luteolin showed antimetastatic activity against CT-26 lung metastasis by downregulating MMP-2 and MMP-9. MEK and Akt phosphorylation was suppressed by the inhibition of Raf and PI3K by luteolin [245].

#### 7.1.8. Myricetin

Myricetin (3,3 ,4 ,5,5 ,7-hexahydroxyflavone), a naturally occurring flavonoid pigment, is typically present in fruits, herbs, and nuts. The presence of three hydroxyl groups at 3- , 4- , and 5 -carbon positions makes myricetin unique from other flavanols [387]. Studies by Nirmala and Ramachandran [257] showed the efficacy of myricetin on 1,2 dimethylhydrazine-induced rat colon carcinogenesis. They demonstrated that myricetin administration reduced the incidence of tumor-bearing rats and tumors in total. Furthermore, myricetin supplementation dramatically decreased intestinal tumorigenesis developed in adenomatous polyposis coli multiple intestinal neoplasia (APCMin/+) mice. Additionally, myricetin treatment improved the antioxidant enzymes, including catalase, glutathione peroxidase, and GSH, in a dose-dependent manner [257].

Li et al. [258] assessed the effectiveness of myricetin against intestinal tumorigenesis in adenomatous polyposis coli multiple intestinal neoplasia (APCMin/+) mice. Promoting apoptosis in adenomatous polyps, myricetin-fed APCMin/+ mice grew fewer, smaller polyps and did not appear to experience negative side effects. By modifying the GSK-3 and Wnt/ catenin pathways, lowering the levels of the proinflammatory cytokines IL-6 and PGE2, and downregulating the phosphorylated p38 MAPK/Akt/mTOR signaling pathway, myricetin prevents the growth of intestinal tumors [258].

AOM/DSS-induced mice were used by Zhang et al. [259] to test myricetin's effectiveness against chronic colonic inflammation and inflammation-driven carcinogenesis. Myricetin significantly decreased the levels of inflammatory factors, such as TNF-, IL-1, IL-6, NF-B, p-NF-B, COX-2, PCNA, and cyclin D1, to inhibit the development of colorectal tumors and shrink colorectal polyps [259].

M10, a new derivative of myricetin, was tested by Wang et al. [205] to show that M10 inhibits robust endoplasmic reticulum (ER) stress-induced autophagy in inflamed colonic mucosal cells of AOM/DSS-induced mice model. The decreased levels of proinflammatory mediators, such CSF/M-CSF, IL-6, and TNF-α, in colonic mucosa and the prevention of the NF-κB/IL-6/STAT3 pathway, were shown to be associated with the antitumor activity [260].

#### 7.1.9. Naringenin

Naringenin, a flavonoid found mostly in citrus fruits and vegetables with no taste or color, carries antioxidant, anti-inflammatory, antiviral, antimicrobial, and antitumor properties [388]. In addition, naringenin was found to reduce the number of high multiplicity aberrant crypt foci (HMACF) by 51% and the proliferative index by 32% in an AOM-induced rat model. Here, naringenin was implied to prevent CRC through decreasing proliferation and increasing apoptosis of luminal surface colonocytes [261].

Naringenin inhibited a dextran sulfate sodium (DSS)-induced murine colitis model. The inhibitory action was correlated with the inhibition of iNOS, ICAM-1, MCP-1, COX-2, TNF-α, and IL-6 transcript levels. The decrease in TNF-α and IL-6 levels was consistent with the suppression of TLR4 mRNA and protein in the colon mucosa. LPS-induced nuclear translocation of p65/RelA was also inhibited by naringenin in RAW264.7 cells, suggesting its action through TLR4 inhibition [262].

6-C-(E-phenylethenyl)-naringenin (6CEPN) inhibited anchorage independent growth of CRC cells, as well as in a CRC-induced xenograft in a dose-dependent manner through the inhibition of COX-1, an underlying cause of malignant character of CRC cells [263].

Naringin was shown to reduce tumor size and growth of AMO or DSS-induced CRC model in C57BL/6 mice by suppressing ER stress-induced autophagy in colorectal mucosal cells [265]. Another study showed naringin-mediated inhibition of tumor cell proliferation and AOM-induced CRC through inducing apoptosis in an AOM-injected Sprague-Dawley rat model [266,389].

#### 7.1.10. Quercetin

Quercetin (3,4,5,7-pentahydroxyflavone), a polyphenolic flavonoid, was isolated from vegetables, fruits, grain, seeds, and tea [282,390]. Quercetin was shown to carry various pharmacological properties, including anticancer properties. It was further found to be effective against AOM/DSS-mediated colitis induced CRC and showed a decrease in mucin-depleted foci and aberrant crypt foci development [391]. In addition, quercetin treatment was shown to efficiently reduce AOM/DSS-induced inflammation, a major cause of colon carcinogenesis [282,392,393]. In another study, quercetin was found to restore leukocyte levels lost by AOM/DSS treatment. It was also noted that quercetin efficiently downregulated various oxidative stress-related markers, such as lipid peroxide (LPO), NO, SOD, glucose-6-phosphate (G6PD), and glutathione (GSH), explaining its role in neutralizing inflammation. The metabolic profiling of sera demonstrated the effect of quercetin through the downregulation of biomarkers that are upregulated in AOM/ DSS-treated mice [282].

In a metastatic cancer model induced in BALB/c mice by CT-26 cells, quercetin was shown to be effective through inducing the intrinsic pathway of apoptosis, along with upregulating the p-38 MAPK pathway. Notably, quercetin function was correlated with modulation of the EMT markers, such as downregulation of N-cadherin, snail, MMP-2, and MMP-9, while E-cadherin, TIMP-1, and TIMP-2 were upregulated [283].

Quercetin augmented radio-sensitization of CRC cells observed in HT-29 tumor xenografts through induction of apoptosis. Combining quercetin with a low dosage of 5Gy radiation effectively suppressed CRC cell proliferation with little toxicity towards normal colonic epithelial cells, CCD-18Co. The combinational therapy was found to target cancer stem cells, as suggested by the reduction of cancer stemness factors, such as DCLK-1, CD24, Lgr5, CD29, and CD44, and the colonosphere formation. The proportion of CD133+ cells also decreased in DLD-1 and HT-29 cells under combinatorial treatments [284].

Li et al. [284] further observed that the combinational therapy of ionizing radiation and quercetin targets the notch-signaling pathway through the downregulation of γ-secretase. The combinational therapy of ionizing radiation and quercetin effectively reduced the expression of γ-secretase complex components nicastrin, PEN2, APH1, presenilin-1, and presenilin-2, which suppressed notch cleavage and thus notch signaling. The combination therapy also inhibited the expression of Jagged-1 and cleaved Notch-1 protein levels [284].

Quercetin induced antiproliferative activity and proapoptotic effects are mediated by the upregulation of cannabinoid receptor-1 (CB1-R) in AOM-treated mice. The downregulation of STAT3 and pSTAT3 was also observed [279].

When radiation therapy was used with quercetin treatment, it suppressed the tumor size of the DLD1 tumor xenograft in athymic nude mice, indicating that quercetin enhanced the radiosensitivity of DLD1 tumors [280].

#### 7.1.11. Rutin

Rutin, a glycosidic derivative of quercetin, is also known as quercetin-3-O-rutinoside or vitamin-P. It is known to carry antimicrobial, antifungal, anti-inflammatory, anticancer, and antiallergic properties, with poor solubility in water [394]. Rutin naturally occurs in various plants, including buckwheat, Mez, Labisia pumila, *Sophora japonica* L., Schum, *Canna indica* L., and *Ruta graveolens* L. [395,396]. In a dose-dependent manner, rutin suppressed SW480 cell-induced tumor growth in a tumor xenograft model without affecting the organ or body weight. In the same model, rutin was shown to enhance mean survival time by 50 days and suppressed angiogenesis through decreasing the serum VEGF level [285].

#### 7.1.12. Tangeretin

Fruits and vegetables contain a wide variety of flavonoids. Citrus fruit flavonoids exhibit various biological effects, such as anticancer and antitumor properties. For example, tangeretin, a polymethoxylated (5,6,7,8,4 -pentamethoxyflavone) flavone, is predominant in the peel of citrus fruits and is thought to operate as a natural resistance factor against pathogenic fungus. In addition, tangeretin has been demonstrated to have several biological properties, including the capacity to suppress cancer cell growth [397].

Bao et al. [291] sought to create a nano-system that included tangeretin (TAGE) and atorvastatin (ATST) and was embellished with RGD (cyclized arginine-glycine-aspartic acid sequences) to treat colon cancer. To assess the anticancer effects of these two drugs on colon cancer cells and in female BALB/c mice harboring cancer models, these researchers produced ATST and TAGE combination nanosystems; RGD-ATST/TAGE CNPs. Results indicated that the RGD-decorated nano-system was more hazardous to HT-29 cells than the undecorated nano-system and that the weight ratio of ATST to TAGE, at which the highest synergism was seen, was 1:1. The integrated nano-systems had a high in vivo biodistribution in the tumor site and effectively reduced in vivo tumor development without significantly harming the treated mice's primary organs and tissues [291].

#### 7.1.13. Wogonin

The medicinal plant *Scutellaria baicalensis* and the traditional Chinese medicine of Huang-Qin (Scutellaria radix) include a significant active monoflavonoid called wogonin (5,7-dihydroxy-8-methoxyflavone). Wogonin has many therapeutic possibilities, including anti-inflammatory and anticancer effects. It has also been observed to inhibit the development of several types of cancer cells with excellent specificity between normal cells and cancer cells [398,399].

To study wogonin's role in colitis-associated colorectal cancer (CAC), Yao et al. [298] developed the AOM/DSS-induced C57BL/6 mice paradigm. They discovered that wogonin markedly reduced the prevalence of tumors and prevented the growth of colorectal adenomas by lowering the expression and secretion of IL-6 and IL-1β, as well as decreasing the cell proliferation and expression of NF-κB in adenomas and adjacent tissues. Further, it increased Nrf2 nuclear translocation in those same tissues [298].

Feng et al. [299] evaluated wogonin's anti-colon cancer effect in an AOM-DSS-induced CRC animal model. They discovered that wogonin decreased tumor abundance and kept colon length within normal range without adversely affecting other organs. In addition, wogonin administration inhibited the SW480 cell-induced xenograft growth in BALB/c mice. Another study, by You et al. [300], further examined the effects of wogonin in mice with colon cancer. Treatment with wogonin abrogated the survival and metastasis properties of colon cancer cells in vivo. A detailed analysis revealed that wogonin-mediated upregulation of p-YAP1 level was responsible for the observed anti-colon cancer effect. This suggested the involvement of the Hippo signaling pathway in the process.

#### *7.2. Phenolic Acids*

#### 7.2.1. Caffeic Acid

Caffeic acid (3,4-dihydroxycinnamic acid) is a nonflavonoid catechol with potent antioxidant properties. It is found in almost all plants as an intermediate in the lignin biosynthesis pathway. The prime source of caffeic acid is coffee. Caffeic acid possesses various pharmacological properties, such as antioxidant, anti-inflammatory, anticancer, and neuroprotective effects [400]. Caffeic acid, by direct interaction, inhibited MEK1 and TOPK activity in an ATP non-competitive manner. Kang et al. [303] conducted experiments using caffeic acid on a mouse tumor model. It demonstrated action by inhibition of ERK and p90RSK activation. Caffeic acid suppressed the TPA-induced activation of AP1, NF-κB, and ERK signaling, and thus neoplastic transformation induced by TPA, EGF, and H-Ras. Through inhibition of ERK functions, caffeic acid inhibited lung metastasis of CT-26 cells. This study also indicated the usefulness of caffeic acid in reducing ERK activity in patient tumor samples.

Caffeic acid effectively inhibited cancer stem cells (CSC) and reduced radiationinduced sphere formation of CD133+ and CD44+ CSC in two patient-derived tumor xenograft (PDTX) models of human CRC in immune-suppressed mice. In vivo, the radiation-induced elevation of PI3K/Akt signaling pathway was also suppressed by caffeic acid. In caffeic acid-treated xenograft samples, the abundance of CD133+ and CD44+ subpopulations of CSC cells were decreased. In addition, CD44+ and CD133+ cells of CRC lost their ability for self-renewal, migration, and CSC-like properties due to caffeic acid in a PDTX xenograft model. Inhibition of PI3K/Akt signaling was described as a significant mode of action caffeic acid in inhibiting CSC proliferation [304].

Both caffeic acid phenethyl ester (CAPE) and caffeic acid phenylpropyl ester (CAPPE) could inhibit HCT116 cell-induced tumor xenograft in immune-compromised mice through inhibition of PI3K/Akt and inactivation of mTORC1 by AMPK activation. Treatment with CAPE and CAPPE reduced the MMP-9 level at a non-hepatotoxic concentration. In addition, CAPE and CAPPE suppressed expression of cyclin D1, Cdk4, cyclin E, c-Myc, and N-cadherin, and upregulated p21 in vivo. Expression of tumor biomarkers, such as PCNA and FASN, was also suppressed by CAPE and CAPPE in tumor tissue [305].

CAPE and caffeic acid p-nitro-phenethyl ester (CAPE-pNO2) upregulated the levels of p53, p27, p21, cytochrome c (cyt. C), and cleaved caspase-3, but downregulated procaspase3, Cdk2, and c-Myc in HT-29 tumor xenograft in mice. There was a dose-dependent inhibition of tumor growth and VEGF expression by these compounds, with no visible toxicity to normal cells [306].

Consumption of decyl caffeic acid inhibited tumor growth in mice with a HCT116-induced tumor xenograft. The mechanism of action involved the induction of cell cycle arrest at the S phase as well as autophagy [307].

#### 7.2.2. Gallic Acid

Gallic acid (3,4,5-trihydoxy benzoic acid) is a naturally occurring polyhydroxy phenolic acid found as an active compound in various fruits, nuts, food compounds, vegetables, and numerous plants, such as green chicory, grapes, blackberries, raspberries, blueberries, and strawberries. Gallic acid is well known for its antimicrobial, antioxidant, anti-inflammatory, and anticancer potential [401,402]. In a dose-dependent manner, gallic acid was shown to inhibit DSS-induced colitis in mice through the inhibition of STAT3 phosphorylation [320]. This inhibitory mechanism includes reduced proinflammatory mediators Th1, TNF-α, and IL-6, and chemokines, such as KC and MCP-1 [320].

In another study, the inhibitory effects of gallic acid were tested in HCT116 and HT29 cells and tumor xenografts in BALB/c mice. The function of pro-oncogenic factors, such as Src, STAT3, EGFR, and Akt, along with key players in the apoptosis pathway were analyzed. The results demonstrated inhibition of STAT3 and Akt by inhibiting Src and EGFR functions. Furthermore, net enhancement of the cleaved caspase-3 and caspase-9 suggested the involvement of apoptosis as the mechanism behind cell death [321].

Gallic acid was shown to ameliorate ulcerative colitis-associated CRC induced in rats by TNBS treatment by modulating ferroptosis, an iron-dependent process of cellular necrosis [322]. Gene expression profiling interactive analysis (GEPIA) and bioinformatics analysis identified significant involvement of ferroptosis-related genes in CRC prognosis. This analysis indicated that eight ferroptosis-related genes are involved in cell survival. This docking study suggested that gallic acid could induce ferroptosis by modulating some of these genes [322].

#### *7.3. Stilbenes*

#### Resveratrol

Resveratrol (3,5,4 -trihydroxystilbene), a stilbenoid that can be found in peanuts, skin of red grapes, and blueberries, has been studied for its potential anticancer properties [403,404]. Saud et al. [350] used a mouse model with a knocked-out APC locus, and Kras activated specifically in the distant colon to study the effect of resveratrol on sporadic CRC. The mice received a diet supplemented with resveratrol (150 or 300 ppm) before the appearance of tumors. This resulted in a 60% inhibition of tumor production and loss of Kras expression in 40% of mice that developed tumors. Oral administration of resveratrol for tumor bearing mice resulted in complete tumor remission in 33% of mice and a decrease in tumor size in 97% of the remaining mice. Upregulation of miR-96, a negative regulator of Kras expression, in non-tumoral and tumoral colonic tissues suggested that resveratrol exerted its anti-CRC effects by downregulating Kras expression [350]. Alfaras et al. [351] examined the effects of oral administration of trans-resveratrol on DMH-induced precancerous colonic lesions in male Sprague-Dawley rats. This resulted in the reduction of aberrant cryptic foci by 52% and mucin depleted foci by 45% in the colon. In colonic contents, dihydroresveratrol was the most abundant compound detected, followed by trans-resveratrol and its derivatives [351]. Synergistic effects of resveratrol and curcumin on CRC were studied by Majumdar et al. [352].

One study analyzed the effects of resveratrol and its PLGA-chitosan based nanoformulation in animal models (both xenograft and orthotopic) of colon cancer. Both the compound and its nanoformulation caused an appreciable decrease in tumor growth and hemoglobin percentages of tumor mass, signifying reduced angiogenesis with nanoformulation exhibiting more bioavailability and functional efficacy than [353]. Resveratrol combined with ginkgetin, a phytochemical obtained from Ginkgo biloba, exhibited a synergistic effect in suppressing VEGF-induced endothelial cell proliferation, migration, invasion, and tube formation in HT29

cell-induced xenografts in mice. When administered together, these two compounds demonstrated a synergistic antitumor effect with 5-FU, causing a reduction in micro vessel density of the tumors. Furthermore, the combinatorial treatment relieved the 5-FU-induced inflammatory response by lowering the expression of COX-2 and inflammatory cytokines [354]. Resveratrol also suppressed TGF-β1/Smad signaling, downregulated Snail and vimentin, and upregulated E-cadherin expression, which in turn inhibited EMT [349].

#### **8. Phenolics in Clinical Trials for CRC Treatment**

Many of the compounds discussed here, such as curcumin, resveratrol, EGCG, genistein, and fisetin, entered into different phases of clinical trials. Curcumin, the most studied phytochemical in both preclinical and clinical studies, has been tested for its effectiveness as an anti-inflammatory agent as well as its potential in prevention, management and therapy of different cancer types, including CRC [405]. The anticancer potential of resveratrol has been documented through studying its efficacy, safety, and pharmacokinetics in more than 244 clinical trials, with additional clinical trials currently being carried out by independent groups [406,407]. Although the clinical utility of resveratrol is well documented, the rapid metabolism and poor bioavailability have limited its therapeutic use [406,408]. Clinical trials on green tea extract containing EGCG as the major active component were conducted, demonstrating the good tolerance of the agent with no significant advantage of its inclusion between the placebo and the treated groups [409]. The efficacy of flavonoid fisetin supplementation on the inflammatory status and MMP levels was tested in small groups of CRC patients, while several markers were measured to assess its therapeutic efficacy, treatment with this polyphenol primarily resulted in the significant changes in IL-8 concentrations compared to the placebo group [410]. The safety and tolerability of genistein in combination with a chemotherapy agent in metastatic CRC were studied in a clinical trial with a small group of patients receiving FOLFOX or FOLFOX-bevacizumab. The results demonstrated the safety and tolerability of the treatment with notable efficacy [379]. While the results of these studies are encouraging, additional studies are needed to assess the long-term use of these phytochemicals in the clinic.

#### **9. Conclusions and Future Perspectives**

CRC is the third most diagnosed and second leading cause of cancer-related death worldwide. According to recent statistics, CRC claims close to a million lives, which is about half of the population it affects globally every year. Although the CRC death rate has declined due to routine screening and early detection, CRC incidence is predicted to be doubled by the end of this decade due to various reasons, demanding an urgent need to overcome the limitations of current treatment strategies, including the development of alternative therapy regimens. This review aims to present a detailed account of the recent advances in studies on various phenolic phytochemicals with anti-CRC activities demonstrated in animal experiments with the underlying molecular basis of their actions (summarized in Table 3).

As discussed here, the phytochemicals were reported to act through inhibiting hallmarks of various CRC attributes, such as the potential of cell growth and proliferation, selfrenewal, invasion, migration, and angiogenesis through inducing apoptosis, ferroptosis, and autophagy-mediated cell death pathways (Figure 6). These activities involved the modulation of various pathways, such as the levels of proinflammatory cytokines and chemokines (IL-1, IL-6, ICAM-I, TNF, COX-2, iNOS, KC, and MCP1), oxidative stress markers and pathways (SOD, catalase, thiolase, glutathione peroxidase, GSH and Keap1/NRF2/GSK-3β/HO-1), cell cycle regulators (cyclin D1, cyclin E, and CDK 4/6), apoptotic/autophagy regulators (p21, p53, caspase-3, caspase-9, Bax, Bcl-2, Bak, and Beclin1), proliferative signaling pathways regulators (PI3K/Akt/mTOR/AMPK, Wnt/β-catenin, MAPK-p38, ERK, MEK, and c-Myc), regulators of invasion, migration, metastasis, and angiogenesis (Notch1, STAT-3, VEGF, CD31, MMP-2, MMP-3, MMP-9, MMP-16, EGFR, Twist1, Vimentin, FMS-related tyrosine kinase 4, endothelial growth receptor-3, Snail, N-cadherin, E-cadherin, TIMP-1, and TIMP-2), stemness

(CD133, CD44, ALDH1, CD29, DCLK-1, and LGR5) and expression of tumor suppressive miRNAs (miR34a, miR200c, and miR145). The downregulation of COX-2 levels can be achieved upon treatment with EGCG [206], curcumin [194,197], kaempferol [239], luteolin [242,243], myricetin [259], naringenin [262], piceatannol [342], pterostilbene [344], syringic acid [326], boeravinone B [191], hesperidin [227], isoliquiritigenin [235], orientin [268], quercetin [281], and xanthohumol [301]. Caffeic acid suppressed TPA-induced activation of AP1 and NF-κB signaling [303]. Many phytophenols can induce an antioxidant response, such as EGCG, gallic acid, boeravinone B, eriodyctyol, luteolin, and morin. Caffeic acid phenethyl ester and caffeic acid phenylpropyl ester-induced mTOR inhibition through the activation of AMPK [305]. Isoangustone A upregulated AMPK phosphorylation in vivo [234]. Pterostilbene inhibited EGFR in an AOM-induced colonic adenomas in mice [344].

**Figure 6.** Genetic and molecular basis of colorectal cancer along with the current treatment strategies where potentials of phenolic compounds were indicated. CIN, MSI and CIMP are the prime factors in CRC development. Besides the currently available chemotherapeutic treatment strategies, different polyphenols are reported to induce CRC cell death by apoptosis and/or autophagy and/or necrosis.

There is increasing evidence in favor of the idea that diet can influence the intestinal microbiome and thus the risk of CRC. Diets rich in fruits and vegetables can be associated with gut microbiome rich in Prevotella compared with Bacteroides associated with good colonic health while the consumption of diet with low plant-based food rich in processed food led to the opposite effects [411,412]. Diets rich in plant-based nutraceuticals could regulate host immune and inflammatory behavior and thus gut homeostasis through modulating the composition and functionality of the gut microbiome [413]. Therefore, CRC incidence and progression can be reduced by modulating gut microbiome by careful choice of diet and phytochemicals which could be a promising and efficient way to reduce the burden of CRC [413]. Gut microbiota can digest dietary phytochemicals by their unique ability to produce short chain fatty acids, such as butyric acid, with anti-inflammatory and antineoplastic activity [414]. Phenolic phytochemicals have served us as an important source of novel drugs/leads. While the studies discussed here provided encouraging results, several issues are needed to be considered to get a step closer to the end users, such as:

**Author Contributions:** Conceptualization, M.P.; methodology, S.D. and S.P.; investigation, S.D., S.P., A.B. (Ashish Bhattacharjee) and S.C.M.; data curation, S.D. and S.P.; writing—original draft preparation, S.D., S.P., K.P., A.M. (Anirban Manna), J.H., V.K.N., C.M., N.C. and A.B. (Anupam Bishayee); writing—review and editing, M.P., K.P., N.C. and A.B. (Anupam Bishayee); visualization, S.G., A.M. (Arijit Mondal) and S.B.; supervision, M.P. and A.B. (Anupam Bishayee); project administration, M.P. and A.B. (Anupam Bishayee), funding acquisition, M.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Science and Engineering Research Board, Department of Biotechnology (DBT), and Department of Science and Technology (DST) to M.P. S.D. is a University Grant Commission (UGC) Senior Research Fellow (SRF), A.M. (Anirban Manna) is a DBT SRF, and C.M. is a DST Inspire Fellow. S.G. was an SRF of the Council of Scientific and Industrial Research.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the writing of the manuscript.

#### **Abbreviations**

#### **References**

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## *Article* **Gnetin C Intercepts MTA1-Associated Neoplastic Progression in Prostate Cancer**

**Prashanth Parupathi 1,†, Gisella Campanelli 1,† , Rabab Al Deabel 2,‡ , Anand Puaar 1, Lakshmi Sirisha Devarakonda 1, Avinash Kumar 1 and Anait S. Levenson 3,\***

**Simple Summary:** The incidence of prostate cancer is increasing because of the aging population. Evidence suggests that diets rich in bioactive polyphenols can reduce the incidence of prostate cancer. The aim of this work was to investigate the potential of gnetin C, a compound found in the melinjo plant and commonly used in Indonesian foods, to block prostate cancer progression. To this end, we evaluated the anticancer efficacy of gnetin C-supplemented diets in a unique and adequate high-risk premalignant prostate cancer transgenic mouse model. Our results indicate that a gnetin C-supplemented diet reduces the progression of prostate cancer by reducing the proliferation of cells, inflammation, and the formation of blood vessels. The finding that a gnetin C-supplemented diet effectively blocks tumor progression in a preclinical mouse model may be exploited to initiate chemoprevention trials for novel nutritional interception for untreated patients under active surveillance.

**Abstract:** Nutritional chemoprevention is particularly suitable for prostate cancer. Gnetin C, a resveratrol dimer found abundantly in the melinjo plant (*Gnetum gnemon*), may possess more potent biological properties compared to other stilbenes. We examined the effects of gnetin C in a highrisk premalignant transgenic mouse model overexpressing tumor-promoting metastasis-associated protein 1 (MTA1) on the background of *Pten* heterozygosity (*R26MTA1*; *Pten+/f*; *Pb-Cre+*). Mice were fed diets supplemented with the following compounds: pterostilbene (70 mg/kg diet); gnetin C, high dose (70 mg/kg diet); and gnetin C, low dose (35 mg/kg diet). Prostate tissues were isolated after 17 weeks and examined for histopathology and molecular markers. Serum was analyzed for cytokine expression. Gnetin C-supplemented diets substantially delayed the progression of preneoplastic lesions compared to other groups. Prostate tissues from gnetin C-fed mice showed favorable histopathology, with decreased severity and number of prostatic intraepithelial neoplasia (PIN) foci, reduced proliferation, and angiogenesis. A decreased level of MTA1, concurrent with the trend of increasing phosphatase and tensin homolog expression and reduced interleukin 2 (IL-2) levels in sera, were also detected in gnetin C-fed mice. Importantly, gnetin C did not exert any visible toxicity in mice. Our findings demonstrate that a gnetin C-supplemented diet effectively blocks MTA1-promoted tumor progression activity in high-risk premalignant prostate cancer, which indicates its potential as a novel form of nutritional interception for prostate cancer chemoprevention.

**Keywords:** gnetin C; diet supplementation; transgenic mice; targeted interception; MTA1; prostate cancer

**Citation:** Parupathi, P.; Campanelli, G.; Deabel, R.A.; Puaar, A.; Devarakonda, L.S.; Kumar, A.; Levenson, A.S. Gnetin C Intercepts MTA1-Associated Neoplastic Progression in Prostate Cancer. *Cancers* **2022**, *14*, 6038. https:// doi.org/10.3390/cancers14246038

Academic Editor: Anupam Bishayee

Received: 13 October 2022 Accepted: 3 December 2022 Published: 8 December 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/).

#### **1. Introduction**

Prostate cancer remains the second highest type of cancer-related mortality in men [1]. According to the American Cancer Society, prostate cancer accounts for about 14% of newly diagnosed cancers, and about 5.7% of all cancer deaths in the United States. Although prostate cancer incidence remained stable over the past few years, there was an annual 4–6% increase in advanced prostate cancer, which accounted for the rise in cases of metastatic disease [2]. Recent updates from the National Comprehensive Cancer Network guidelines for prostate cancer highlight a new risk categorization system for patients with prostate cancer, which demands different approaches for the management of the disease [3]. For the three categories defined as "very low", "low", and "intermediate" risk groups, there is no treatment strategy in current practice to prevent prostate cancer progression. It has been accepted that diverse clinical lesions in these groups are represented by "large gland" morphology, including high-grade prostatic intraepithelial neoplasia (PIN) and PIN-like carcinoma, which are main precursors to invasive carcinoma [4]. We believe that nutritional interception may represent the most adequate intervention to protect not only the general population, but also the moderate- or high-risk subpopulation of patients under active surveillance.

Epidemiological studies have continually supported the argument that naturally occurring dietary polyphenols with anti-inflammatory, antioxidant, and anticancer properties may be considered for prostate cancer chemoprevention [5–8]. It is well-established that natural polyphenols of different classes have pleiotropic effects through various signaling pathways, including epigenetic mechanisms, in inhibiting the progression of prostate cancer [9–11]. Specifically, the potential application of stilbene polyphenols acting through multiple mechanisms in prostate cancer chemoprevention and treatment was demonstrated and summarized by several groups [12–17].

It is imperative to establish a relevant and adequate preclinical model system that represents heterogeneous lesions observed in the clinic, in which to determine the efficacy of natural compounds [18]. In our previous studies, we have shown that stilbenes, such as resveratrol and pterostilbene, can act through metastasis-associated protein 1 (MTA1) mediated mechanisms to prevent the progression of premalignant prostate cancer to adenocarcinoma [19–27]. MTA1 is a chromatin modifier and transcriptional regulator, and plays a cancer-promoting role in all stages of prostate cancer [19,27–29]. Aberrant alterations in the molecular levels of MTA1 trigger several downstream targets with significant roles in inflammation, cell survival, and invasion, ultimately causing metastasis [22,26,30,31]. Among the downstream pathways affected by the changes in MTA1 levels is the phosphatase and tensin homolog/v-akt murine thymoma viral oncogene (protein kinase B) (PTEN/Akt) pathway, the deregulation of which plays an essential role in prostate cancer [22,32,33]. We have previously shown an inverse association of MTA1 with PTEN, and a direct correlation of MTA1 with p-Akt [22,34]. In fact, we have demonstrated that MTA1 inhibition by resveratrol and pterostilbene promotes the acetylation and activity of PTEN, which in turn inhibits the activity of Akt [22,34]. Given that MTA1 can be targeted by stilbene polyphenols, these compounds are of great interest. We have recently reported that a pterostilbene-supplemented diet exerted beneficial effects by diminishing inflammatory pathways and accelerated PIN progression in *R26MTA1*; *Pten+/f* mice [27]. Since we have also previously demonstrated the more potent MTA1-targeted inhibitory activity of gnetin C, a dimer resveratrol (Figure 1A), compared to resveratrol and pterostilbene, both in vitro and in prostate cancer xenografts [31,35], we sought to determine the efficacy of gnetin C in an adequate preclinical model of prostate cancer.

**Figure 1.** (**A**) Chemical structure of gnetin C, a resveratrol dimer, isolated from melinjo plant (*Gnetum gnemon*). (**B**) Schema showing the experimental design used for prostate cancer chemoprevention by diet supplementation in a precancerous *R26MTA1*; *Pten+/f*; *Cre+* murine model. In total, 24 mice were fed with the control diet or diets supplemented with Pter70 (70 mg/kg diet), Gnetin C70 (70 mg/kg diet), or Gnetin C35 (35 mg/kg diet). At sacrifice, urogenital system and prostate tissues were isolated for histological and molecular analysis. Blood was also collected. (**C**) Effects of different diets on average body weight gain and food intake in mice. Values are mean ± SD, *n* = 6 per group.

We hypothesized that gnetin C may possess more potent biological effects compared to pterostilbene in a transgenic model of prostate cancer. Therefore, the current study was undertaken to examine the promising MTA1/PTEN/Akt-mediated chemopreventive and interceptive properties of gnetin C, a dimeric stilbenoid, using a clinically relevant model of murine prostate cancer representing high-risk premalignant neoplasia.

#### **2. Results**

#### *2.1. Effects of Gnetin C-Supplemented Diet on Prostate Cancer Progression*

For the current study we accumulated twenty-four *R26MTA1*; *Pten+/f* mice and randomized them into four groups (*n* = 6 per group) on the respective diets ad libitum for 17 weeks (Figure 1B), after which prostate tissues and blood samples were collected for analysis. Diet supplementation with either pterostilbene or gnetin C did not have any significant effect on either the body weight gain or food intake in these mice (Figure 1C). The effect of such treatment on the gross anatomy of the urogenital system (UGS) over this period is shown in Figure 2A, *upper panel*. Gnetin C-supplemented diets decreased the appearance of the prostate compared to the pterostilbene diet (Pter70-Diet)- and control diet (Ctrl-Diet)-fed mice. Consistent with our previous study [27], *R26MTA1*; *Pten+/f* mice in all the groups developed high-grade PIN characterized by disorganized glandular structures with pseudostratified epithelium and hyperproliferation, but with an intact basal layer of smooth muscle actin (SMA)-positive cells (Figure 2A). However, there were significant differences in pathological features, and some morphological differences, among

the groups. As shown in Figure 2A, presenting hematoxylin and eosin (H&E) images, mice fed diets supplemented with compounds demonstrated favorable histopathology, with restored normal ductal structures and fewer glands involved in PIN compared to mice on the Ctrl-Diet (Figure 2B, *top*). Moreover, mice on the gnetin C high-concentration diet (Gnetin C70-Diet) exhibited a significantly reduced number of glands involved in PIN compared to the Pter70-Diet, suggesting a more potent efficacy of gnetin C in restoring the histopathology of the prostate when used at the same concentration in the diet. The gnetin C low-concentration diet (Gnetin C35-Diet) worked at the same level as the Pter70-Diet, demonstrating, once more, the greater biological potency of gnetin C compared to pterostilbene. The changes in histopathology were accompanied by a corresponding reduction in proliferation and angiogenesis in treatment groups. Analysis of prostate tissues for the cellular protein marker of proliferation (Ki67) and cluster of differentiation (CD31) markers revealed a significant reduction in corresponding positively stained cells in all treatment groups compared to control mice (Figure 2A, *lower panels*)**.** Importantly, once again, the differences between reduced proliferation and angiogenesis in mice of the Gnetin C70-Diet and Pter70-Diet groups were statistically significant (Figure 2B, *lower panels*). Notably, gnetin C at half concentration (Gnetin C35-Diet) showed either the same or more potency compared to Pter70-Diet, indicating that gnetin C is more efficacious than pterostilbene. However, there were no significant differences in the anticancer histopathological activities between the lower and higher concentrations of gnetin C diets. Taken together, our data indicate a more potent anticancer activity of gnetin C compared to pterostilbene when provided as dietary supplementation in mice with premalignant neoplasia.

#### *2.2. Gnetin C Effectively Inhibits the MTA1-Associated PTEN/Akt Axis in a Transgenic Mouse Model of Early-Stage Prostate Cancer*

Our earlier studies using gnetin C in prostate cancer cell lines and xenografts had shown potent inhibition of the MTA1 and MTA1-associated downstream signaling targets, including proto-oncogene 2 (ETS2), Cyclin D1, and Notch 2 [31,35]. To further strengthen our previous in vivo findings on the effects of stilbenes on the MTA1/PTEN/p-Akt axis [22,34], we evaluated the expression of MTA1 and PTEN/pAkt in prostate tissues from *R26MTA1*; *Pten+/f* mice fed gnetin C diets. We found that MTA1 protein levels were significantly reduced in prostate tissues by diets supplemented with both pterostilbene and gnetin C (Figure 3A *top***,** B). Specifically, both pterostilbene and gnetin C diets inhibited MTA1 expression in prostate tissues compared to control prostates with high significance (*p* < 0.0001) (Figure 3B). Moreover, despite the heterogeneity of MTA1-stained tissues in the Pter70-Diet group, the differences between Pter70-Diet and Gnetin C70-Diet groups were statistically significant. Furthermore, cytoplasmic PTEN and p-Akt staining of tissues showed a concomitant PTEN increase and p-Akt reduction in mice fed with supplemented diets compared to mice fed the control diet. Subtle differences in PTEN and p-Akt levels between the treatment groups are also evident in selected immunochemistry (IHC) images (Figure 3A, *middle and bottom panels*).

Next, we evaluated the response to treatments by measuring levels of MTA1, PTEN, and p-Akt/Akt in prostate tissue lysates. The expression MTA1 protein levels in prostate tissues from mice fed diets supplemented with stilbenes was significantly reduced compared to control mice (Figure 4A,B). We were also able to detect differences among treatment groups, indicating the trend towards gnetin C's greater potency. In contrast to the inhibitory effects on MTA1, diets supplemented with pterostilbene and gnetin C restored PTEN levels in prostate tissues (Figure 4A,B). In general, prostate tissues from mice in the same treatment group demonstrated an expected trend compared to control mice, however, with noticeable heterogeneity, particularly with respect to p-Akt/Akt in mice in the Gnetin35-Diet group (Figure 4A, *right*) (Supplementary Figure S1). At the messenger RNA (mRNA) level, MTA1 and PTEN were respectively inhibited and upregulated by stilbene-supplemented diets compared to control mice (Figure 4C). The differences among treatments groups were not significant.

**Figure 2.** (**A**) *Top*, Representative images of UGS of mice from different groups: The anterior prostates (marked) in mice fed regular diet were larger compared to the treatment groups, but there were no differences in size between treatment groups. Representative images of H&E (scale bar, 100 μm), SMA (scale bar, 50 μm), and Ki67- and CD31-stained sections (scale bar, 20 μm) of the prostate tissues from mice in different groups. (**B**) *Top*, Quantitation of prostate glands involved in PIN formation: The glands were quantified in five randomly selected areas per sample (*n* = 3 per group), and the average count is expressed as a percent. *Middle*, Quantitative analysis of Ki67 immunostaining, expressed as a percent, showing the drastic effect of gnetin C supplementation on cell proliferation. *Bottom*, Quantitative analysis of CD31 immunostaining, expressed as area, showing the strong effect of gnetin C supplementation on angiogenesis. Values are mean ± SEM analyzed from five separate areas per sample. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\*\* *p* < 0.0001 (one-way ANOVA).

We have chosen the human prostate cancer cell line 22Rv1 that expresses wild-type PTEN for our in vitro experiments. Treatment of these cells for 24 h with the same equipotent concentrations resulted in a statistically significant reduction in MTA1 by gnetin C (*p* < 0.05) and an insignificant increase in PTEN by both compounds compared to control untreated cells, revealing, once again, the more potent activity of gnetin C compared to pterostilbene (Figure 5A,B). Inhibition of p-Akt/Akt under the treatments followed the same trend as MTA1.

Collectively, these data demonstrate a more potent MTA1/PTEN/Akt response to gnetin C than to pterostilbene treatment, both in a murine prostate model of early-stage prostate cancer and in a prostate cancer cell line. This emphasizes that MTA1-targeted interception by diet supplemented with gnetin C may have greater potential benefits compared to pterostilbene supplementation, which we have recently reported for prostate cancer chemoprevention [27].

**Figure 3.** (**A**) Representative images of MTA1 (*top*), PTEN (*middle*), and p-Akt (*bottom*)-stained sections of the prostate tissues from mice in different groups (*n* = 3 per group). Images are 20× (scale bar, 50 μm). (**B**) Quantitative analysis of MTA1 immunostaining. Values are mean ± SEM of cells counted in five separate areas per sample, and the average count is expressed as a percent. \* *p* < 0.05; \*\*\*\* *p* < 0.0001 (one-way ANOVA).

**Figure 4.** (**A**) Representative immunoblot images of MTA1, PTEN, and p-Akt/Akt levels in the prostate tissues from mice in different treatment groups (*n* = 3 per group). β-actin, GAPDH, or Hsp70 were used as loading controls (*left*). Immunoblot images of MTA1, PTEN, and pAkt/Akt levels in tissues from three mice (#1–3) of the Gnetin C35-Diet group (*right*). (**B**) Quantitation of the relative expression of these markers in prostate tissues from mice in different treatment groups (*left*). Values are mean ± SEM of data from three or more independent experiments. \* *p* < 0.05; \*\* *p* < 0.01; (one-way ANOVA). (**C**) Quantitation of relative MTA1 and PTEN mRNA levels in prostate tissues from mice in different treatment groups. β-actin amplification was used as a normalization control. Changes in mRNA expression were calculated by the 2−ΔΔCt method. Values are mean ± SEM of data from three independent experiments. \* *p* < 0.05; \*\* *p* < 0.01; (one-way ANOVA). ns, not significant.

**Figure 5.** (**A**) Representative immunoblot images of MTA1, PTEN, and p-Akt/Akt levels in 22Rv1 prostate cancer cells treated with pterostilbene and gnetin C at 25 μM concentration. β-actin was used as a loading control. (**B**) Quantitation of the relative expression of these markers in prostate cancer cells. Values are mean ± SEM of data from three independent experiments. \* *p* < 0.05; (one-way ANOVA). ns, not significant.

#### *2.3. Effects of Gnetin C-Supplemented Diet on Pro-Inflammatory Interleukin 2 (IL-2) and Interleukin 6 (IL-6) Cytokine Levels in Murine Serum*

To evaluate the systemic efficacy of gnetin C dietary intervention, we determined pro-inflammatory IL-2 and IL-6 levels in mouse sera by enzyme-linked immunosorbent assay (ELISA). Our results show significantly reduced levels of circulating IL-2 in sera of mice fed supplemented diets compared to control mice (68.34% reduction for Gnetin C70-Diet and 93.86% reduction for Gnetin C35-Diet) (Figure 6A). Interestingly, gnetin C supplementation at the lower 35 mg/kg diet concentration secured a greater attenuation of IL-2 levels compared to both Pter70 and Gnetin C70 diets. Moreover, the Gnetin C35 diet also inhibited IL-6 levels, while Gnetin C70-Diet mice showed a paradoxical increase in the levels of pro-inflammatory IL-6 (Supplementary Figure S2). In agreement with our previous data, which revealed that high-concentration pterostilbene supplementation (100 mg/kg diet) reduced serum IL-6 significantly (*p* < 0.01) [27], we now observe a downregulation of IL-6 in mice treated with Pter70-Diet, albeit without significance. Nevertheless, these data suggest that systemic inflammation was decreased in mice fed the low-concentration gnetin Csupplemented diet (Gnetin C35-Diet), which should ensure the beneficial anti-inflammatory effects of gnetin C, and validate the utilization of sera as a liquid biopsy strategy for the evaluation of gnetin C responsive, prognostic, and predictive noninvasive biomarkers.

**Figure 6.** Effects of diets supplemented with gnetin C on circulating IL-2 cytokine levels **measured by ELISA in murine sera** (*n* = 3–4 per group) Data represent the mean ± SEM of three independent experiments performed in duplicate. \*\* *p* < 0.01; \*\*\* *p* < 0.001; (one-way ANOVA). ns, not significant.

#### **3. Discussion**

Naturally occurring resveratrol oligomers, including gnetin C, have been proposed as potential cancer chemopreventive compounds [36]. Antitumor activities of gnetin C, a resveratrol dimer, have been reported in acute myeloid leukemia [37], colon cancer [17], and neuroblastoma [38]. Accumulated data indicate that gnetin C exhibits potent antitumor activity in prostate cancer. Using a panel of human prostate cancer cell lines (PC3, LNCaP, and DU145) and mouse prostate cancer cells derived from the adenocarcinoma of PTEN null mice (PTEN-CaP8), Narayanan et al. [17] first reported the significant proliferation inhibitory effects of gnetin C in cancer cells without affecting normal prostate epithelial RWPE-1 cells. Moreover, gnetin C was significantly more potent in inhibiting cell proliferation and apoptosis in prostate cancer cells compared to resveratrol [17]. Our own published in vitro studies with DU145 and PC3M cells showed more potent inhibition of cell proliferation with lower IC50 values for gnetin C compared to pterostilbene and resveratrol [35]. In fact, we have repeatedly shown gnetin C-concentration-dependent cell survival inhibition in a panel of prostate cancer cells (Supplemental Figure S3). Moreover, we have demonstrated that gnetin C was more potent in causing apoptosis and inhibiting metastatic potential of prostate cancer cells than resveratrol and pterostilbene [35]. In addition, we also demonstrated that gnetin C is a lead compound among stilbenes for effectively blocking tumor progression in immunodeficient mice implanted with PC3M [35]. Importantly, in a recent paper using genetically modified DU145 and PC3M prostate cancer cells, gnetin C was shown to have more potent MTA1-mediated cytotoxicity, apoptosis, inhibition of clonogenic cell survival, and motility compared to resveratrol and pterostilbene [31]. To evaluate gnetin C's clinical potential, in the current study, we assessed the efficacy of gnetin C-supplemented diets as MTA1-targeted interception using a unique transgenic mouse model (*R26MTA1*; *Pten+/f*; *Pb-Cre+*) representing high-risk early-stage prostate cancer.

The clinical significance of MTA1 in prostate cancer progression and metastasis has been reported [28–30,39]. We have also demonstrated that MTA1 is a molecular target for stilbene polyphenols, such as resveratrol, pterostilbene, and gnetin C, in prostate cancer, in vitro and in vivo [22,24–26,34,35,40,41]. Our group has tested the MTA1-targeted chemopreventive and therapeutic potential of stilbenes and grape extracts in murine prostate cancer models [22,24,27,42]. Particularly, our recent report showed that a pterostilbenesupplemented diet fed to mice with early-stage prostate cancer can block the progression of prostate cancer through the inhibition of MTA1-mediated signaling [27]. Since gnetin C showed improved pharmacokinetic parameters in mouse and human studies compared to both resveratrol and pterostilbene [35,43–46], we sought to compare, for the first time, the MTA1-targeted inhibitory efficacy of gnetin C and pterostilbene supplemented diets in a transgenic mouse model of early-stage prostate cancer.

For this study, we fed prostate-specific *R26MTA1*; *Pten+/f* mice reference diets (Ctrl-Diet and Pter-Diet) along with gnetin C-supplemented diets, and asked two major questions: (1) whether gnetin C at the same diet concentration has a more potent MTA1-mediated effect than pterostilbene; and (2) whether gnetin C at half diet concentration has beneficial efficacy in this model.

While both pterostilbene- and gnetin C-supplemented diets showed expected beneficial effects compared to Ctrl-Diet, the differences between Pter-Diet and Gnetin C-Diet (s) were considerable: mice treated with Gnetin C-Diet(s) exhibited favorable histopathology compared to mice fed Pter-Diet. Immunohistochemical results showed a statistically significant decrease in epithelial cancer cell proliferation and angiogenesis in mice fed Gnetin C-Diet(s) compared to the Pter-Diet. Furthermore, gnetin C at both concentrations had potent anticancer activity through targeting MTA1, and this effect was statistically significant compared to the Pter70-Diet group. In addition, consistent with apparent trends in inhibiting MTA1 expression after gnetin C treatment, levels of PTEN were increased and p-Akt were decreased in Gnetin C-Diet groups. Further analysis of prostate tissues and prostate cancer cell lines confirmed the potent MTA1 inhibitory potential of gnetin C compared to pterostilbene. The more potent biological effects of gnetin C could be explained by its improved pharmacokinetics [44].

It has been reported that stilbenoids can regulate cytokine expression in different cellular systems [47–49]. With regards to cytokine-mediated anti-inflammatory effects in cancer, resveratrol was found to inhibit metastasis and angiogenesis by reducing inflammatory cytokines, such as tumor necrosis factor α (TNFα), IL-6, and interleukin 1 beta (IL-1β), in vitro and in vivo in oral cancer [50], and decreasing levels of the pro-inflammatory cytokines in rat colon carcinogenesis [51]. In prostate cancer, we have shown the inhibitory effects of orally administered resveratrol, trimethoxy-resveratrol, and piceatannol on circulating IL-6 levels in LNCaP xenografts [41]. In addition, the inhibitory effects of dietary pterostilbene on IL-6 levels were also detected in sera from *R26MTA1*; *Pten+/f* transgenic mice [27]. The inhibitory effects of grape extract diet supplementation and pterostilbene treatment on the levels of pro-inflammatory and pro-angiogenic IL-1β were also reported in prostate-specific *Pten*-deficient mouse models [26,42].

Here, murine serum was used to evaluate the systemic cytokine-mediated immune response to the gnetin C-supplemented diets. Our results show that, consistent with our previous observations [27], pterostilbene reduced the levels of IL-6 compared to the control. However, interesting results were obtained with gnetin C: low diet concentration of gnetin C (35 mg/kg diet) significantly suppressed levels of pro-inflammatory IL-2, and to a lesser extent, levels of IL-6. Curiously, the effectiveness of high-concentration gnetin C (70 mg/kg diet) was profoundly diminished for both cytokines, even resulting in a rather opposite effect by drastically increasing levels of IL-6. Our unexpected results on the systemic anti-inflammatory effects of gnetin C are consistent with some reports that natural products or individual polyphenols at lower doses exert more potent anti-inflammatory/anticancer effects than at higher doses in vivo [17,52–54]. Relevant to our study, a low dose of melinjo seed extract (MSE) that contains gnetin C had more profound effects on tumor growth and angiogenesis in mice with colon tumors than it did at double the dose [17]. Further studies are needed to clarify the dose-and-effect relationship of natural polyphenols as anticancer and anti-inflammatory agents.

Melinjo fruit is consumed as a food in Southeast Asia as well as being traditional medicine [55]. Importantly, nontoxic effects of MSE and gnetin C in nonmalignant cells in culture [17,36,37,56] and in vivo toxicity studies in mice [17] and rats [57] has been demonstrated. Crucially, MSE and gnetin C appear to be safe in humans, as it has been demonstrated in clinical trials [44,45,58,59].

Based on an average food consumption of 4g diet per day for each mouse (Figure 1C, middle panel), doses in the current study were 0.28 mg per day for the Gnetin C70 diet and Pter70 diet, and was 0.14 mg per day for the Gnetin C35 diet. There are no other in vivo studies with these compounds in transgenic mouse models, except our own studies [22,26,27]. The dose that we used in our study is in line with the safe doses used in human studies.

In summary, to the best of our knowledge, the current study is the first to demonstrate the in vivo anticancer activity of gnetin C-supplemented diets in a clinically relevant transgenic mouse model of prostate cancer. Due to the known safety of gnetin C, and its improved bioavailability compared to pterostilbene and resveratrol, lower doses of gnetin C may become one of the most promising targeted strategies for prostate cancer interception. Our study also suggests that the evaluation of pro-inflammatory cytokines in the blood may provide noninvasive prognostic and predictive biomarkers. The curious mixture of gnetin C's ability to inhibit cancer cell proliferation and simultaneously have a complex immunomodulatory response at high concentrations needs further investigation.

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

#### *4.1. Animals*

We have previously generated prostate-specific MTA1-overexpressing mice (*R26MTA1*; *Pb-Cre+*) [27]. The generation of prostate-specific MTA1 overexpression on the background of *Pten* heterozygous mice was achieved by breeding our MTA1 transgenic mice (*R26MTA1*) with a C57BL/6J female mouse homozygous for the "floxed" *Pten* allele, which was purchased from Jackson Laboratories (Bar Harbor, ME, USA). Transgenic *R26MTA1*; *Pten+/f*; *Pb-Cre+* male mice (hereafter *R26MTA1*; *Pten+/f*) for this study were confirmed by PCR-based tail genomic DNA genotyping using primers, as previously described [27]. The animals were housed in cages with corn cob bedding in a temperature-controlled room with a 12 h light–dark cycle. Mice had free access to drinking water and designated irradiated AIN-76A diets (Envigo Teklad, Boyertown, PA, USA). All animal protocols were approved in advance by the Institutional Animal Care and Use Committee (IACUC) at Long Island University in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. Mice were monitored daily for their general health and signs of toxicity.

#### *4.2. Diets and Study Design*

Gnetin C was a generous gift from Hosoda SHC Co., Ltd. (Fukui, Japan). The purity of gnetin C and pterostilbene (Pter) (Sigma Aldrich, St. Louis, MO, USA) was determined to be ≥99%. Gnetin C and Pter powders were shipped to Envigo Teklad Diets (Boyertown, PA, USA) for the formulation of three different supplemented diets on the basis of an AIN-76A control diet (Ctrl-Diet). Based on our previous experience with a Pter diet at a concentration of 100 mg/kg diet [27], we formulated the following diets with gnetin C and pterostilbene, as a reference diet: gnetin C, high concentration, 70 mg/kg diet (Gnetin C70-Diet); gnetin C, low concentration, 35 mg/kg diet (Gnetin C35-Diet); and pterostilbene at 70 mg/kg diet (Pter70-Diet). For our calculations, we used the following formula: DD = (SD × BW)/FI (Research Diets Inc., New Brunswick, NJ, USA), where DD is diet dose (mg compound/kg Diet); SD is single dose (mg compound/kg bw/day); BW is body weight (g bw/animal), and FI is daily food intake (g Diet/day). For prospective clinical relevance of the doses used in this study, we determined equivalent doses for humans using the following human equivalent dose (HED) formula: HED (mg/kg) = Animal dose (mg/kg) × km ratio, where the Km ratio for mice is 0.081 [60]. Considering that the average human male BW is 70 kg, the doses used in this study for mice approximately translate into 53.2 mg/day (Gnetin C70-Diet), 26.6 mg/day (Gnetin C35-Diet), and 53.2 mg/day (Pter70-Diet) in humans. All these doses are well tolerated and safe in humans, as shown in respective clinical trials [45,58,59,61]. The diets were stored at 4 ◦C and protected from light. Fresh diets were weighed each week and added to cages. The nutritional composition of diets was published previously [27].

*Treatment groups:* Twenty-four 3-week-old *R26MTA1*; *Pten+/f* mice were randomized into four major groups, *n* = 6 per group: Ctrl-Diet; Pter70-Diet; Gnetin C70-Diet; and Gnetin C35-Diet. All mice were fed ad libitum. The animals were weighed weekly and monitored regularly for their food intake and general health. After 17 weeks on their respective diet, mice were euthanized by CO2 inhalation and cervical dislocation. Due to small proportions of mouse prostate, animals from the same treatment group were used for different purposes. From six mice in each treatment group, we used three mice for the UGS (seminal vesicle, prostate, and the urinary bladder) isolation to be processed for histology and IHC. The remaining three mice were used for the isolation of prostate tissues for molecular analysis (protein and RNA isolation). Prostate tissues were dissected, snap frozen, and kept at −80 ◦C until use for further molecular analyses. Blood was collected by cardiac puncture upon sacrifice at week 17; serum samples were prepared and stored at −80 ◦C.

#### *4.3. Histopathology and Immunohistochemistry*

Urogenital system tissues were fixed in 10% neutral-buffered formalin, processed, and embedded in paraffin, cut into 4 μm sections, and mounted onto slides (Reveal Biosciences, San Diego, CA, USA). H&E-stained sections were assessed for mouse PIN and/or adenocarcinoma. Slides were subjected to IHC analysis as described previously [22,27], using antibodies from Abcam (Boston, MA, USA) for Ki67 (1:50) and SMA (1:700), and from Cell Signaling Technology (Beverly, MA, USA) for CD31 (1:500), MTA1 (1:50), PTEN (1:150), and pAktSer473 (1:50). Images were taken using an EVOS XL Core microscope (Thermo Fisher Scientific, Somerset, NJ, USA). Ki67, MTA1, and CD31 positively stained cells were quantified in five randomly selected areas using Image J software.

#### *4.4. Tissue Processing and Western Blot Analysis*

Frozen prostate tissues were homogenized in RIPA buffer (Thermo Fisher Scientific, Somerset, NJ, USA), and Western blots were performed as described previously [22,26,27,42]. Briefly, samples were separated using 10–15% polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% milk/TBS/0.1% Tween, and then probed with primary antibodies for MTA1 (1:2500); PTEN (1:1000); and Akt and p-AktS473 (1:1000) from Cell Signaling Technology (Beverly, MA, USA). B-actin (1:5000), Hsp70 (1:1000), and GAPDH (1:1000) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and were used as loading controls. Signals were detected using enhanced chemiluminescence (Thermo Fisher Scientific, Somerset, NJ, USA). Band intensity was measured using Image J.

#### *4.5. Tissue Processing and Real-Time RT-PCR*