Resveratrol as sensitizer in colorectal cancer plasticity

The phytoalexin resveratrol was extracted for the first time from the herbaceous plant Veratrum grandiflorum by the Japanese scientist Takaoka in 1939 [46]. It is a solid, alcohol-soluble ingredient alternatively known as 3,5,4′-trihydroxystilbene [47], with the sum formula being C₁₄H₁₂O₃ [48]. Structurally, both cis- and trans-isomer exist, with the trans-form (Fig. 1) being more abundant and convertible to the cis-form [49, 50]. Numerous different targets of resveratrol have been identified, including integrin-receptors, estrogen-receptors, and sirtuins (SIRT) [51‐53].

The best-known source of naturally occurring resveratrol is grapes of any color (Fig. 1), and the ingredient has been detected in bilberries, cranberries, and strawberries [54]. Consequently, this secondary phytochemical is also found in juices produced from these fruits and grape-based alcoholic beverages such as wine and champagne [54]. Furthermore, nuts such as peanuts and pistachios contain resveratrol, as does dark chocolate (Fig. 1) [55], which the presence of this plant component in cocoa beans could explain. In addition, some plants like Polygonum cuspidatum are particularly resveratrol-containing, leading to the occurrence of the Itadori tea brewed from it in Asia [56].

The legitimate inferential question is: Why has resveratrol been detected in many natural products? The answer is astonishing: The production of resveratrol is stimulated in plants whenever they are exposed to the stress of different genesis, for example, UV-radiation [22], ozone-exposition, or pathogenic confrontation [57]. Overall, this plant-native polyphenol balances the consequences of harmful environmental influences, protects the vegetables from fungal attack, parasite infestation, over-ripening, and rot [56], and thus represents a natural survival strategy. As humans are also permanently exposed to environmental influences of various kinds and often live a lifestyle that is detrimental to their health, research is constantly being conducted to determine whether and how to use resveratrol’s protective properties for the benefit of humankind.

According to previous publications, the absorption of resveratrol by the oral route in man is around 75-80% and presumed to be predominantly by transepithelial diffusion in vitro and in vivo [58, 59]. It has already been reported that resveratrol is distributed in high concentrations in tissues. Indeed, resveratrol accumulated nearly 40-fold in the human CRC cell line Caco-2 in vitro versus medium [60], concluding that intestinal cells are a primary target for this polyphenol. Because of the strong metabolism in the digestive tract and hepatobiliary system, with a short half-life of about 1.5 hours [60], bioavailability by oral ingestion is relatively limited, less than 1%, which does not change with increasing intakes. Biochemical studies have shown that resveratrol’s major degradation products in blood plasma and urine are glucuronides, di-hydro-resveratrol conjugates, as well as sulfates [61, 62] and about 50–60% of the ingested phytopharmaceutical is eliminated from the body in urine [58]. Encouragingly, the ingestion of 500mg resveratrol in tablet form, resulting in plasma concentrations of about 70ng/ml, was reported to be well-tolerated and safe [63]. However, some adverse effects, such as diarrhea, nausea, anemia, vomiting, and flatulence, were reported when resveratrol was administered in high doses [64, 65].

Optimizing this topic, a higher bioavailability of this compound has been achieved by creating more sophisticated preparations, including nanoparticles and nano-constructed lipid vehicles incorporating resveratrol, including verification of the efficacy of the association in recovering chemosensitivity. In addition, the oral bioavailability of resveratrol incorporated in casein nanoparticles is tenfold increased compared to the administration of the polyphenol in the form of an oral suspension [66, 67]. Furthermore, enhancement of resveratrol’s bioavailability in rats has already been targeted by treatment with 3,5,4’-tri-O-acetyl-resveratrol, an acetylated resveratrol precursor that can be hydrolyzed in cells to free trans-resveratrol [68, 69]. Research in this area is still in the preliminary stages, but it is anticipated and promising that these novel and innovative nano-transporters will yield significant benefits.

Moreover, in addition to organs such as the intestine and the hepatic system, the vital colon microflora is suspected of playing a central role in resveratrol metabolism [61]. Considering the significant and indispensable importance of the intestinal microbiota for many essential body functions, and here for the metabolism of resveratrol, it could be of fundamental and crucial relevance, as it has already been demonstrated for other natural products such as soy isoflavone and lignans [70, 71]. Altogether, the high accumulation and concentration of resveratrol in the enterocytes of the colon epithelium suggest that this area of the colon [72], because of the intestinal microflora, probably plays an essential role in the bioactivity of resveratrol metabolism.

Research into resveratrol’s effects and detailed mechanisms of action on different cancer cell types and lines has been of high scientific interest for decades. To illustrate this with some examples, a treatment with this natural polyphenol disrupts the cell cycle flow of cervical cancer cells [73], reduces the inflammation-related invasiveness of gastric cancer cells [41, 74], and initiates apoptosis in prostate cancer cells [75‐77]. Moreover, as a multitargeting agent in CRC cells, this active plant compound prevents invasion, proliferation, and metastasis [52]. It averts EMT-associated plasticity, necessary for metastasis, with a parallel promotion of apoptosis and being a chemosensitizer for treating cancers [78‐81]. Resveratrol modulates epigenetic changes in tumor cells, as it can stimulate the ATP2A3 gene, leading to down-regulation of histone deacetylase (HDAC) and thus HDAC2 expression in the nucleus, or through deoxyribonucleic acid (DNA) methylation, histone modification, non-coding messenger ribonucleic acid (mRNA), and telomerase levels, leading to suppression of cancer spread [81, 82]. In line with this, a clinical study showed that a daily oral intake of 0.5–1g resveratrol could have anti-carcinogenic effects in the human gastrointestinal tract and, at the same time, be well-tolerated by cancer patients [35].

Resveratrol has also been shown to inhibit cellular processes associated with the development of tumors through mutation. Indeed, it has been reported that resveratrol possesses several active anti-oxidant capacities [83], acts as an active scavenger of hydroxylenes/superoxides [84] and significantly blocks the formation of free radicals caused, for example, by 12-O-tetradecanoylphorbol-13-acetate (TPA) in cancer cells [85]. Furthermore, resveratrol also contributes to protection against lipid peroxidation in cell membranes and DNA damage caused by the release of reactive oxygen species (ROS) [86, 87]. In addition, the phytopharmaceutical has been found to have mutation-inhibiting and anti-carcinogenic effects, such as preventing the mutagenicity of N-methyl-N’-nitro-N-nitrosoguanidine in Salmonella typhimurium [88].

Cancer cells can become chemoresistant to various chemotherapeutic agents due to modifications in diverse biological processes in the subcellular signaling pathways [7, 8]. Resveratrol has great promise for targeting several molecular and cell signaling pathways. It has already been explored in various preclinical and clinical approaches as a chemosensitizer drug for combined therapy with standard drugs for diverse types of cancer [89, 90]. Figure 2 provides an overview of the beneficial properties of the combination treatment of standard chemotherapeutic substances with resveratrol and its various potential biological pathways involved in the chemosensitization of tumor cells. Table 1 lists recent research findings on the different subcellular signaling pathways and their mechanisms of resveratrol having anti-inflammatory, anti-proliferative, anti-metastatic, anti-oxidative, immunomodulatory, and pro- or anti-apoptotic effects in various in vitro or in vivo studies in cancer and healthy cells.

Acute inflammation is activated by immune-specific cells, especially in infections and allergies. Thus, it is part of the healthy immune system in the body, and it lasts only for a short time. However, if the inflammation lasts longer, it becomes chronic [185]. Key mechanisms such as chronic inflammation and the associated induction of angiogenesis, metabolizing enzymes, oxidation, cell cycling, cell plasticity, and anti-apoptotic proteins are among the most important prerequisites for the development of chronic diseases, including colitis and CRC, making agents to prevent and inhibit inflammation in the tissues, like colon and thus prevent colitis and CRC of particular interest. Well-documented inflammatory signaling pathways associated with the pathogenesis of colitis-related CRC include NF-κB, IL-6/STAT3, cyclooxygenase (COX)-2/prostaglandin E₂ (PGE₂), and IL-23/T helper 17 cells (Th17) [186]. More specifically, pro-inflammatory chemokines and cytokines, such as IL-1, -6, -8, TNF-α, and TNF-β, are produced very rapidly by injured body tissues and can trigger a variety of inflammatory responses and the expression of pro-inflammatory transcription factors, such as NF-κB, mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription (STAT) 3, HIF-1α, activator protein-1 (AP-1), and nuclear factor erythroid 2–related factor 2 (Nrf2), and their secondary inflammatory substances such as inflammatory mediators, such as matrix metalloproteinases (MMPs), 5-lipoxygenase (5-LOX), COX-2, and also the production of ROS [187‐191]. Inflammatory bowel disease (IBD) and Lynche’s syndrome have been shown to contribute to significantly increased development and pathogenesis of CRC, suggesting an intense interaction between inflammation and cancer development. In addition, experimental animal models of IBD have clearly demonstrated that resveratrol is a beneficial agent for the management of IBD [192].

Furthermore, one of the most critical pro-inflammatory transcription factors in inflammatory tissues is NF-κB, expressed by multiple cancers such as CRC [193]. This transcription factor induces genes involved in cell survival, cell adhesion, inflammation, differentiation, and growth. NF-κB is activated by various influences such as carcinogens, phorbol ester, pro-inflammatory agents, cigarette smoke, and cytokines such as IL-1β and TNF-α or TNF-β [194]. These factors promote the dissociation of NF-κB inhibitor alpha (IκBα) through phosphorylation, and the activated NF-κB migrates from the cytoplasm to the nucleus, leading to the binding and activation of transcription of target genes essential for the development of aggressive cancers. The expression of proliferation proteins (cyclin D1, Kiel-antigen 67 (Ki-67)), apoptosis suppressor proteins (Bcl-2 and B-cell lymphoma extra-large (Bcl-xL)), and proteins responsible for metastasis (MMPs, COX-2, CXCR4) as well as angiogenesis (vascular endothelial growth factor (VEGF)) are promoted by NF-κB [194]. Similarly, IL-6/JAK/STAT3 signaling pathway potently activates inflammatory response via tumor-infiltrating immune cells in the tumor immune microenvironment in CRC. Moreover, IL-6/JAK/STAT3 signaling up-regulates downstream target genes with anti-apoptotic and proliferative effects, promotes plasticity, invasion, and metastasis of cancer cells and angiogenesis, and induces cancer resistance [195, 196]. The COX-2/PGE₂ signaling has been closely associated with all stages of colorectal carcinogenesis. The role of COX-2 and its product PGE₂ in the pathogenesis of CRC is based on the function of fibroblasts from the mesenchymal (stromal) layer, which are the main target of cytokines e.g. TNF-α and IL-1β. Fibroblasts from non-neoplastic colorectal tissue are an important source of COX-2 expression that is well-validated as one of the most important risk factors of CRC [197]. Finally, IL-23 receptors play a crucial role in chronic inflammatory diseases due to their function in the processes of differentiation of Th17. IL-23 up-regulates PGE₂ levels and Th17 cell function that include expression increase of inflammatory cytokines, such as IL-17A, IL-17F, IL-21, and IL-22. For these reasons, the IL-23/Th17 signaling is strongly included in the pathogenesis of colitis-associated CRC [198].

Chemopreventive phytochemicals such as resveratrol inhibit several pro-inflammatory-related activations of transcription factors, cytokines, chemokines, proteins, and enzymes [199‐205]. Much evidence suggests that resveratrol is a multi-factorial bioactive phytochemical with numerous beneficial preventive effects on subcellular biological pathways, especially anti-inflammatory effects by inhibiting pro-inflammatory cytokines (IL-1β, TNF-α, and TNF-β), the pro-inflammatory transcription factor NF-κB and thus NF-κB-promoted end-proteins. A recent molecular docking study documented that resveratrol could be effective against CRC by targeting NF-κB signaling [206], and in this regard, our group described resveratrol’s NF-κB suppression by resveratrol associated with an anti-inflammatory mode of action in CRC [207]. Furthermore, a seven-day treatment with resveratrol (10mg/kg body weight) suppressed the dextran sulfate sodium-induced inflammatory colon injury via down-regulation of NF-κB, STAT3, ERK, and iNOS expressions in IRC mice [208]. Combinational application of resveratrol with 5-FU inhibited Akt/STAT3 signaling, which was associated with pro-apoptotic effects and increased anti-telomerase activity in human CRC cells [209]. Using DLD1 and HCT15 CRC cells, resveratrol inhibited cancer growth by targeting the Akt/STAT3 signaling pathway. These anti-cancer effects of resveratrol correlated with pro-apoptotic effects and blockage of the G1 phase cell cycle in cancer cells [123]. In addition, the resveratrol treatment sensitized HT-29 and SW620 CRC cell lines to 5-FU (via increased oxidative stress) through the down-regulation of Akt and STAT3 signal proteins [210]. Another study showed the inhibitory effect of resveratrol on the COX-2/PGE₂ signaling pathway (decreased both miRNA and protein levels) and consequent anti-cancer efficacy in HCT-116 human CRC cell lines [211]. A similar result was found in a Serra et al. study describing the inhibition of COX-2/PGE₂ signaling by resveratrol (also isoenzymes of NO synthase (iNOS) expression) in HT-29 colon epithelial cells [212]. The combination of resveratrol and ginkgetin synergistically attenuated the 5-FU-induced inflammation in HT-29 colon cancer xenograft nude mice through decreased expressions of COX-2 and inflammatory cytokines [213]. A micro-immunotherapy sequential medicine including resveratrol showed significant immunomodulatory effects on human macrophages via several cytokine-induced signaling pathways (including IL-23) and consequent tumor-suppressive efficacy using in vitro 2D and 3D spheroid models and animal xenograft colon carcinoma experimental approach [214]. Furthermore, the phytopharmaceutical possesses potential anti-cancer functions, such as CRC cell survival reduction, activation of apoptosis (caspase-3), inhibition of invasion, and preventing of EMT-plasticity in the CRC tumor microenvironment (TME) in vitro and in vivo (Table 2) [215‐234].

An intense functional collaboration between tumor cells and immune cells is known to exist in the TME, which is essential for the further growth and progressive spread of tumors. Therefore, it is also important that the interaction between both systems is altered in favor of anti-tumor immunity [235]. T-lymphocytes (CD4+), which are among the most important immune cells of the entire defense system, have the ability to differentiate into other T-lymphocytes such as CD8+ and regulatory T-cells (Tregs) in TME in a variable and cytokine-dependent manner, making this interaction a critical factor in slowing tumor growth and providing a good prognosis for patients [236, 237]. Interestingly, resveratrol has been shown to modulate T-cells (CD4+) by excreting interferon (IFN)-γ and thereby up-regulating its biotarget Sirt-1 in CD4+ T-cells [238]. Moreover, these tumor-specific cytotoxic cells (CD8+ T-cells) can attack cancer cells through apoptosis-specific factors (IFN-γ, IL-4, and TNF-α) and mechanisms (perforation of the membrane) and initiate active suicide of tumor cells [239], promoted by resveratrol by participating in anti-tumor immunity, as reported by Choi and colleagues [240].

As previously reported, resveratrol suppresses HDACs, which correlates with the formation of various anti-inflammatory lymphocytes and Treg cells in the gut. In addition, a study in The Cancer Genome Atlas (TCGA) reported that an increase in the activity of the Treg-specific transcription factor FoxP3 or the anti-inflammatory IL-10 contributes to improved survival in patients with CRC. This suggests that changes in the gut microbiome may lead to an anti-inflammatory T-cell response that attenuates inflammation-related CRC [12, 216].

In addition, several investigations showed a significantly enhanced expression and stimulation of NO by iNOS in CRC cells, indicating an essential role of NO in tumorigenesis in the colon cells [241]. Interestingly, resveratrol decreases specific iNOS expression in CRC cells [242]. One of the most essential and primary mechanisms of action of resveratrol is its anti-inflammatory potential, the suppression of the p38-MAPK signaling pathway, which is involved in the production of inflammatory mediators such as cytokines, COX-2, p53, and iNOS [243] and thus is an important requirement for the prevention and inhibition of colitis and CRC (Table 2). For this reason, specific targeting and modulation of pro-inflammatory metabolic pathways, such as NF-κB have significant implications for preventing and eliminating serious diseases [193].

Surgical intervention and chemotherapeutic drug administration remain the main treatment options for CRC patients. However, the decision to use one or more of these approaches in treating CRC subjects depends on the tumor’s location, the cancer stage at diagnosis, and the case-by-case characteristics of the patient [244]. The challenge often arises when a patient (a) shows inherent resistance to the drug due to intrinsic drug resistance capabilities of the cancer cell or (b) develops resistance to a chemotherapeutic drug through exposure to the drug, called acquired resistance [245]. Chemotherapeutic drug resistance leads to lower sensitivity to the drug, compromised drug inefficacy, cancer cell plasticity, tumor relapse, poor prognosis, and higher mortality rates among CRC patients [246‐248].

One or more non-cellular (such as limited vascular accessibility and TME) and cellular (such as drug targets, levels and activity of detoxifying enzymes, levels, and activity drug uptake and extrusion transporters) factors play vital roles in CRC drug resistance [244]. Detailed discussions on the various mechanisms of chemotherapeutic drug resistance are beyond the scope of this current review article. Several articles have reported the multiple aspects and mechanisms of chemoresistance in CRC (illustrated in Fig. 3) and novel strategies to reverse resistance extensively and thoroughly [249‐251].

The most widely used chemotherapeutic drugs for treating CRC include 5-FU, oxaliplatin, doxorubicin, cetuximab, irinotecan, and various combinations of the same. Drug resistance has been reported against one or more of these drugs and is summarized in Table 3 [252‐276]. In some instances, however, CRC resistance to drugs such as oxaliplatin enhanced the sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via the up-regulation of death receptor 4 and localization of lipid rafts [277]. Interestingly, a significant reduction in viable circulating tumor cells (CTCs) was observed in metastatic CRC patients treated with TRAIL liposomes [277].

Although chemotherapy is the mainstay of cancer treatment, the lack of efficacy of this treatment is a major concern. For many years, researchers believed that the failure of cancer treatments might be due to intrinsic genomic mechanisms, such as the development of mutations in the drug target that prevent its binding [144, 278]. Nevertheless, the primary cause of the ineffectiveness of chemotherapeutic agents is the acquisition of drug resistance by tumor cells throughout treatment [279]. Therefore, it is vital to understand the mechanisms of drug resistance to develop more efficacious treatments that can reduce the risk of relapse. An important factor that is implicated in drug resistance is the plasticity of tumor cells [143]. Cellular plasticity, concerning tumorigenesis, refers to the capacity of terminally differentiated cancer cells to undergo drastic changes in their cell phenotypes in response to oncogenic drivers or external stimuli [145]. The different types of plastic behaviors that help tumor cells acquire drug resistance are EMT, otherwise called epithelial-mesenchymal-plasticity, attaining properties of cancer stem cells (CSCs), and transdifferentiation into other cell types [143, 144]. A deeper insight into the molecular mechanisms underlying CRC drug resistance has revealed a convincing link between these tumor cell plasticity hallmarks and CRC progression [280, 281].

EMT is a developmental process during embryogenesis, tissue remodeling, and wound healing that allows epithelial cells to attain mesenchymal phenotype to enable its motility and invasiveness [282‐284]. However, molecular pathways comparable to development have been observed in cancer cells leading to EMT-plasticity [284]. This transition occurs through transcriptional repression of E-cadherin (cell adhesion molecule) by Snail, Slug, zeb 1/2, smad interacting protein 1 (SIP1) or Twist1, and elevated expression of vimentin and N-cadherin, through a complex network of signaling cascade which facilitates collective cell migration and invasion. Cellular growth factors, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), and transforming growth factor beta (TGF-β), are potent stimulators of EMT, which upon binding to their corresponding receptors, initiate signaling pathways including Notch, β-catenin/Wnt and PI3K/Erk signaling [284]. Further research has demonstrated the link between several EMT markers, apoptosis evasion, and enhanced cancer cell survival [285, 286]. This correlation has stimulated interest among researchers to investigate the connection of these EMT markers with resistance to anti-neoplastic treatment modalities. Apart from initiating tumor and inducing metastasis, EMT also confers resistance to cancer treatment interventions, including radiotherapy [285, 287, 288]. For instance, Liu and co-workers demonstrated that vincristine-resistant colon adenocarcinoma cells showed increased expression of Twist1 and thus exhibited elevated migratory and invasive ability. More importantly, this study showed that up-regulation of Twist1 markedly increased chemoresistance to vincristine by up-regulating the ATP-binding cassette transporters, ABCB1 and ABCC1 [288]. Nonetheless, the mechanism of how EMT-plasticity contributes to drug resistance is not yet fully understood. However, it is believed that the cells become more resistant to pro-apoptotic signals and excessive drug efflux by membrane transporters, leading to cell survival despite treatment [289].

An interesting study identified circulating tumor microemboli (CTMs) and three subpopulations of CTCs, namely, E-CTCs, M-CTCs, and E/M-CTCs based on the expression of epithelial cell adhesion molecule (EpCAM), the mesenchymal cell marker vimentin, or both EpCAM and vimentin respectively from blood samples of 126 CRC patients. However, the results showed that M-CTCs and CTMs were highly detected in patients with lymph node metastasis of CRC [290]. Another investigation explored the active involvement of reactive stroma in the modulation of EMT-plasticity in CRC and TNF-α produced by macrophages accelerated the process of TGF-β-induced EMT. This study also demonstrated an interplay between TNF-α and TGF-β signaling in the morphological conversion of organized colon epithelial cells to scattered mesenchymal cells. Besides, TNF-α stimulated Erk activation, which causes increased production of this cytokine by tumor cells. Hence, the role of stroma in the EMT-plasticity of CRC was further elucidated [291].

Moreover, Bates and colleagues revealed a significant role of the integrin αvβ6 (a receptor for fibronectin and tenascin) in CRC progression and metastasis. Their results showed that αvβ6 activated autocrine TGF-β, which causes EMT-plasticity. Clinical analysis of 488 CRC patient samples exhibited a reduction in survival of patients with increased expression of αvβ6, compared to the patients with low or no β6 expression and suggested that β6 expression could be a potential prognostic variable for CRC [292]. Another study reported that caspase-3 gene knockout caused reduced expression of EMT markers such as N-cadherin, Snail, Slug, and zeb1 and elevated E-cadherin expression and chemosensitivity compared to parental CRC cells. Furthermore, the critical role of caspase-3 in cancer cell invasion and metastasis was indicated [293]. Additionally, another examination demonstrated pre-mRNA processing factor (PRP4) as an essential factor in inducing EMT-plasticity and drug resistance in CRC cells by directly binding to p53 and causing its phosphorylation and up-regulating HIF-1α and miR-210, which activates p53 [294].

Focusing chronic oxaliplatin treatment of CRC cells, this therapy resulted in phenotypic changes associated with cellular plasticity, such as loss of polarity, spindle shape, and increase in mobility of these cells along with a decrease in E-cadherin expression and an increase in the expression of Snail and vimentin. This study showed that chronic oxaliplatin resistance in CRC cells leads them to switch to an invasive phenotype and initiates EMT [295]. Long-term exposure of CRC cells to 5-FU enabled these cells to overcome S-phase arrest, evade apoptosis and activate autophagy which is evident by the up-regulation of LC3B, vimentin, Twist1, Slug, and zeb2 mRNA levels and down-regulation of E-cadherin and Claudin-3 [296]. These results suggested that CRC cells respond to chemotherapy-induced cell stress by undergoing EMT-plasticity as an adaptive mechanism, leading to cell survival, plasticity, and evasion of apoptosis.

Another critical determinant of plasticity in CRC cells is CSC. CSCs are a subset of cells found in tumors that can self-renew, differentiate, and produce all cancer cell types. They are responsible for tumor initiation, maintenance, and recurrence [297]. CSCs are isolated and enriched from different tumors by identifying the CRC-specific expression of cell surface markers, including CD44, CD133, CD166, Lgr5, ALDH1, and EpCAM [298]. Further, they initiate aberrant expression of several cellular signaling pathways to maintain their stemness and self-replenishing properties. For instance, Wnt/β-catenin, Notch, TGF-β, and Hedgehog pathways are well implicated in colon cancer CSC development [299‐303].

Colon CSCs are the initiators of tumor cell proliferation, invasion, and metastasis to distinct locations. They are attributed to showing high resistance to chemotherapy and are a fundamental reason for tumor relapse or recurrence [304]. For example, many studies have reported that colon CSCs exhibit increased expression of anti-apoptotic proteins and apoptotic inhibitors, as well as ABC transporter proteins that expel drugs out of cancer cells [305, 306]. Moreover, an interesting study revealed that most circulating or migratory CRC cells are Lgr5- and this plastic behavior has an immense capacity for distant metastasis [307]. Additionally known is that the CD133+ CRC cell population resists anti-angiogenesis therapy, and this resistance occurs through an anti-apoptotic pathway including PP2A, p38MAPK, MAPKAPK2, and Hsp27 [308]. In sum, understanding the biology of CSCs is vital to develop novel therapies that can effectively target this hallmark of tumor plasticity.

Transdifferentiation (lineage plasticity), which refers to the ability of tumor cells and CSCs to switch their phenotypic characteristics into a different cell type, represents a different type of cell plasticity. Accumulating evidence has demonstrated that tumor cells vulnerable to therapeutic drugs transdifferentiate into other specialized cell lineages that are not drug targets [144, 309, 310]. Overall, transdifferentiation is a complex, poorly understood process but is believed to be an essential factor in developing drug resistance to cancer. Therefore, research is needed to understand the differentiation mechanism and its impact on drug resistance in cancer.

Data from different studies have shown that resveratrol sensitizes CRC cells toward chemotherapeutic drugs by modulating their plasticity via many signaling pathways and transcription factors. For example, a fascinating study revealed resveratrol’s suppression of CRC cell invasion and migration by inhibiting the TGF-β1/Smads signaling pathway and EMT. Resveratrol elevated the levels of E-cadherin but down-regulated EMT-inducing transcription factors, Snail and vimentin [196].

Even more interesting, a co-treatment with 5-FU and resveratrol in HCT-116 cells significantly lowered the levels of EMT regulatory factors such as Slug and vimentin and the stemness of the treated cells compared to untreated cells [209]. In accordance therewith, Buhrmann et al. reported that resveratrol treatment chemosensitized HCT-116 CRC cells to 5-FU and induced apoptosis while suppressing NF-κB activation, EMT-plasticity (decreased slug and vimentin, increased E-cadherin) and CSC formation (decreased CD133, CD44, and ALDH1) via the modulation of the TNF-β signaling pathway [80]. Resveratrol (5μM) treatment in 5-FU-resistant CRC cell lines HCT-116R and SW480R and their parental forms (HCT-116 and SW480) blocked cell proliferation and synergistically inhibited 5-FU (0.01-1nM) mediated effects on cell invasion [79]. Resveratrol increased cell-cell contact via increased desmosomes, gap- and tight junctions, and increased the expression of E-cadherin cell adhesion protein in both the parental and 5-FU-resistant forms of the HCT-116 CRC cell line [79]. Interestingly, a significant decrease in the vimentin and Slug (plasticity-associated factors) and the down-regulation of the activation and nuclear translocation of NF-κB (abolishing NF-κB driven gene expression of MMP9 and caspase-3) correlated to the ability of resveratrol to attenuate drug resistance in the 5-FU-resistant CRC cells [79]. Beyond that, the role of oxyresveratrol, a natural derivative of resveratrol, in inhibiting EMT-plasticity and metastasis of CRC cells has also been proven [311].

Similarly, many studies focus on targeting aberrant signaling pathways leading to the proliferation and enrichment of colorectal CSCs. It is well established that mutations in the Wnt/β-catenin signaling pathway, which take place in the stem cells of the intestinal crypt, are crucial for the continued proliferation of cancerous cells and stemness activity in colorectal stem cells [312]. For instance, an intriguing study demonstrated that resveratrol potentiated the anti-cancer effects of grape seed extract against colorectal CSCs in a rodent model by suppressing the Wnt/β-catenin pathway. Besides, the compound also induced mitochondrial apoptosis of colon CSCs by up-regulating the Bax/Bcl-2 ratio, p53, and cleaved PARP [313].

Transdifferentiation of CSCs to endothelial cells is an essential event in vascular bed formation for angiogenesis and metastasis of tumor cells [314]. Pouyafar and colleagues reported that resveratrol, in combination with sulindac (a Wnt-3a inhibitor), reduced the clonogenicity potential of colon CSCs and prevented the differentiation of CSCs to endothelial cells. Moreover, resveratrol treatment reduced angiogenesis factor YKL-40 and autophagy-related genes in CRC cells [315].

The pathogenesis of CRC is a complex process that can be distinguished on the basis of three different phenotypes: Chromosomal instability (CIN), Microsatellite instability (MSI), and CpG island methylator (CIMP). Indeed, intracellular biotargets of resveratrol, including a protein with high binding affinity, quinone reductase 2 (QR2), were found to be significantly overexpressed in CRC defined by CIN, particularly in cells harboring a positive KRAS (Kirsten rat sarcoma viral oncogene homolog) mutation, and by the MSI but not the CIMP phenotype. Analysis of data from Oncomine showed very good agreement between mRNA expression of QR2 and specific CRC causes [316]. In addition, several genes involved in the regulation of apoptosis, such as PMAIP1, BID, ZMAT3, CASP3, CASP7, and FAS, have been shown to be novel targets for gene regulatory treatment with resveratrol [317].

Conclusively, it is evident that resveratrol can target different tumor cell plasticity markers, provides a novel approach for treating CRC, and can potentially be a potent therapeutic agent.

Several articles have extensively documented the synergistic effects of resveratrol and its ability to sensitize drug-resistant cancers, including CRC, to therapeutic intervention [318]. The chemosensitizing effects of this phytopharmaceutical in CRC are summarized in Table 4 [319‐324].

5-FU is one of the most frequently chemotherapeutic drugs in the treatment of CRC, either as a monotherapy or in the drug combinations such as FOLFIRI (Folinic acid + Fluorouracil l + Irinotecan hydrochloride) and FOLFOX (Folinic acid + Fluorouracil + Oxaliplatin) [325]. Due to several mechanisms, drug resistance to 5-FU has been observed in many CRC patients [325]. Resveratrol seems to be able to re-sensitize the 5-FU-resistant CRC cells, rendering them susceptible to 5-FU intervention. Inhibition of the Akt signaling pathway, induction of S-phase cell-cycle arrest, inhibition of cellular proliferation and migration, and activation of programmed cell death occurred when the HCT-116 CRC cells were treated with a combination of 5-FU (10μM) and resveratrol (25μM) [209]. Furthermore, the combination of 5-FU and resveratrol inactivated STAT3 and blocked STAT3 binding to its hTERT promoter site, thereby blocking telomerase activity in HCT-116 cells [209]. Another study, using HT-29 and SW620 CRC cell lines, reported that resveratrol (100μM) exposure synergistically potentiated the 5-FU (10μM) treatment-mediated inhibition of cellular growth via the induction of mitochondrial oxidative stress and created an imbalance in the intracellular anti-oxidant enzymes [210]. The combination (resveratrol and 5-FU) treatment-induced increase in cellular oxidative stress was attributed to the significant inhibition of oncogenic Akt and STAT3 in the treated cells [210].

The epidermal growth factor receptor (EGFR), frequently overexpressed in malignant cells, is a crucial contributor to cancer proliferation, angiogenesis, inhibition of apoptosis, and metastasis [320]. Hence, the EGFR pathway can be targeted to curb tumor growth. Cetuximab, a monoclonal antibody, targets EGFR, hinders endogenous ligand binding to EGFR, and thus suppresses the phosphorylation and activation of EGFR. Subsequently, disruption in the EGFR-related downstream pathways, such as the Ras-Raf-MAPK and PI3K-Akt pathways, have also been reported [320]. However, mutations caused by NRAS, KRAS, BRAF, PI3KCA, and Akt activation in cancers may confer the cancer cells’ therapeutic resistance to cetuximab and render it a less effective anti-cancer agent [320, 326]. Cancer cells develop resistance to cetuximab via the activation of Akt [320]. Wang et al. showed that resveratrol (5μg/ml) treatment in HCT-116 and CT-26 (murine colon adenocarcinoma) cells abolished the resistance to cetuximab and sensitized the cells to cetuximab (10μg/ml) exposure [320]. Resveratrol treatment in cetuximab-exposed cells up-regulated the expression and phosphorylation of connexin 43 with a resultant increase in cell-cell contact via gap junction function. It inhibited the activation of Akt and NF-κB which is related to the increase in cetuximab treatment associated with the suppression of the growth of the cancer cells [320].

Resveratrol (50–100μM) potentiated the oxaliplatin (1μM) mediated inhibition of Caco-2 CRC cell growth [323]. This growth inhibitory effect of the combination of resveratrol and oxaliplatin also increased the apoptotic death of Caco-2 cells [323]. Furthermore, the conditioned media from resveratrol and oxaliplatin-treated Caco-2 cells, when used to grow human-monocyte-derived macrophages, activated the tumoricidal potential of the macrophages while preventing their immunosuppressive characteristics [323].

The efficacy of a chemotherapeutic intervention and/or resistance to the drug quite often depends on levels of expression of drug extrusion membrane transporters (such as P-gp, MDR1, and BCRP) and the levels and activity of intracellular drug-metabolizing enzymes (such as CYP3A4 and GST) [321]. In a doxorubicin-resistant CRC cell line, Caco-2, increasing concentrations of resveratrol (1–500μM) inhibited the drug extrusion capabilities of P-gp and MDR1 proteins in a concentration-dependent manner [321]. The resveratrol (20μM) exposed Caco-2 cells were susceptible to doxorubicin treatment compared to non-resveratrol exposed doxorubicin treated cells [321]. Resveratrol also inhibited the activity of drug-metabolizing enzymes CYP3A4 and GST in these cells and increased the caspases-3, caspases-8, and caspases-9, indicating activation of apoptotic cell death in the treated cells [321]. The mRNA levels of P-gp, MDR1, BCRP, CYP3A4, and GST were significantly reduced upon resveratrol treatment in the drug-resistant Caco-2 cells [321]. In HCT-116 and HT-29 CRC cells, a combination of doxorubicin and resveratrol significantly reduced the IC₅₀ value of the doxorubicin in the cells [322]. The expression of the pro-apoptotic Bax gene and apoptosis significantly increased when the doxorubicin-exposed HCT-116 cells were treated with resveratrol while inducing S-phase arrest in these cells [322]. Resveratrol seems to sensitize the HCT-116 cells to the anti-cancer effects of doxorubicin by blocking the P-gp drug efflux mechanism in these cells [322].

It is, however, noteworthy that certain studies reported contradicting data; that resveratrol (30 or 50μM) reverses the inhibitory effect of oxaliplatin (2 or 5μM) on the mRNA expression and protein levels of anti-apoptotic survivin in HCT-116 CRC cells and thereby abolishes the cytotoxic effects of oxaliplatin [324].