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

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 drug-resistant 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.


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][4][5][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][12][13][14][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][12][13][14][15].
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][28][29][30][31][32][33][34][35][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][38][39][40][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.

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.
After one year of treatment, in the DFMO + aspirin arm vs. placebo, there was a significant reduction in rectal aberrant crypt foci (precursor of rectal carcinoma). (74% vs. 45%, p = 0.020). No statistically significant reduction of colorectal adenomas was observed. [110] Erlotinib + Sulindac RCT Erlotinib is an EGFR inhibitor; sulindac is a COX-2 inhibitor.
In 82 patients of familial adenomatous polyposis, Sulindac + Erlotinib was associated with a 69.4% decrease in those with an intact colorectum compared with placebo (95% CI, 28  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][121][122][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][129][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].

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][144][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][148][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.

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.

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, phenoli compounds are divided into different classes, such as flavonoids (e.g., anthocyanidins

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][159][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][164][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][167][168][169]. In the following sections, we aim to summarize the anti-CRC effects of phenolic compounds based on animal studies. Table 3     Reduced tumor incidences; inhibited nuclear translocation of β-catenin; downregulated TNF-α and interferon-γ; downregulated COX-2 and p53 [194] HCT116 tumor xenograft in female ICR SCID mice Intragastric administration, 500 mg/kg Suppressed tumor growth; inhibited proteasome; suppressed proliferation; induced apoptosis [195] AOM-DSS induced CRC in male C57BL/6 mice Oral gavage, 500 mg/kg Reduced CRC tumor number; downregulated IL-1β, IL-6, COX-2 and β-catenin; suppressed Axin2 by inhibiting Wnt/β-catenin pathway [196] AOM-induced colonic preneoplastic lesion in C57BL/KsJ-db/db obese mice Dietary supplement, 0.2% and 2.0% Inhibited colonic premalignant lesion [197] HCT116 tumor xenograft in athymic nu/nu nude mice Oral administration, 1 g/kg Enhanced the efficacy of radiation therapy; suppressed NF-κB activity and expression [198] Colo205 and LoVo tumor xenografts in athymic nu/nu mice Tail vein injection, 40 mg/kg Inhibited tumor growth; suppressed angiogenesis [199] AOM-induced colon carcinogenesis in Il10 −/− mice Oral administration, 1% Reduced colon tumors [200] AOM/DSS-induced colitis in C5757BL/6 mice Oral administration, 25 mg/kg Suppressed colitis-associated colon cancer and reduced tumor number [201]
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,  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 Apc Min/+ 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].
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].

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][373][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].
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].

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 FC 1 3K 1 Apc Min/+ 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].

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].

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].

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].
Li et al. [258] assessed the effectiveness of myricetin against intestinal tumorigenesis in adenomatous polyposis coli multiple intestinal neoplasia (APC Min/+ ) mice. Promoting apoptosis in adenomatous polyps, myricetin-fed APC Min/+ 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].
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].

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].

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].

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].

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].

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.  [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 procaspase-3, 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].

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].

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].

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.

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]. 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 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:

1.
Apparently, the functions of many phytochemicals are limited by their poor solubility, absorption, and bioavailability. Encapsulation by nano-formulation as well as chemical derivatization of the compound could resolve this issue.

2.
Some cases reproducing the activity observed in preclinical animal models into the clinic/human could be challenging due to several factors. Success in this endeavor requires careful optimization in administered doses to assess functional synergy, if any, with anti-CRC regimens used in the clinic. Once positive results are obtained in the preclinical settings, testing the validity of the finding, such as safety and efficacy, in clinical trials with appropriate controls will be important to move further.

3.
It is reasonable to think that a phenolic compound showing very weak and toxic activity can yield desirable effect when combined with another phytochemical. Therefore, a careful combination of selected polyphenols can yield unique anti-CRC activity. It is important to clearly determine the maximum tolerable dose of a phytochemical to better understand its therapeutic efficacy alone or in combination with another phytochemical or drug.

4.
Once a phenolic compound with unique anti-CRC activity is identified, it would be important to develop strategies to synthesize the compound in the laboratory, given the very low abundance of a secondary metabolite in the plants. A detailed understanding of the pharmacophore responsible for the observed function should be helpful for chemical synthesis or semi-synthesis, and cellular target identification of the compound. Given the structural complexity of the plant secondary metabolites, it is often a major challenge for natural product chemists and medicinal chemists to solve. Ideally, the simultaneous engagement of experts from interdisciplinary areas, such as ethnopharmacology, molecular biology, biochemistry, natural product chemistry, medicinal chemistry, bioinformatics, and pharmacology, will be necessary to achieve progress in real-time in harvesting the full potential of natural products as the source of novel drug leads.

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