A systematic review of the therapeutic effects of resveratrol in combination with 5-fluorouracil during colorectal cancer treatment: with a special focus on the oxidant, apoptotic, and anti-inflammatory activities

5-fluorouracil (5-FU), an effective chemotherapy drug, is commonly applied for colorectal cancer treatment. Nevertheless, its toxicity to normal tissues and the development of tumor resistance are the main obstacles to successful cancer chemotherapy and hence, its clinical application is limited. The use of resveratrol can increase 5-FU-induced cytotoxicity and mitigate the unwanted adverse effects. This study aimed to review the potential therapeutic effects of resveratrol in combination with 5-FU against colorectal cancer. According to the PRISMA guideline, a comprehensive systematic search was carried out for the identification of relevant literature in four electronic databases of PubMed, Web of Science, Embase, and Scopus up to May 2021 using a pre-defined set of keywords in their titles and abstracts. We screened 282 studies in accordance with our inclusion and exclusion criteria. Thirteen articles were finally included in this systematic review. The in vitro findings showed that proliferation inhibition of colorectal cancer cells in the groups treated by 5-FU was remarkably higher than the untreated groups and the co-administration of resveratrol remarkably increased cytotoxicity induced by 5-FU. The in vivo results demonstrated a decrease in tumor growth of mice treated by 5-FU than the untreated group and a dramatic decrease was observed following combined treatment of resveratrol and 5-FU. It was also found that 5-FU alone and combined with resveratrol could regulate the cell cycle profile of colorectal cancer cells. Moreover, this chemotherapeutic agent induced the biochemical and histopathological changes in the cancerous cells/tissues and these alterations were synergized by resveratrol co-administration (for most of the cases), except for the inflammatory mediators. The results obtained from this systematic review demonstrated that co-administration of resveratrol could sensitize the colorectal cancer cells to 5-FU treatment via various mechanisms, including regulation of cell cycle distribution, oxidant, apoptosis, anti-inflammatory effects.


Introduction
Colorectal cancer is the third and second most frequently diagnosed cancer among males and females worldwide, respectively [1]. Furthermore, it is the second most lethal malignancy worldwide [2]. Colorectal cancer is affected by both different genetic and environmental factors, each to a different degree in various patients [3]. The choice of first-line treatment for colorectal cancer patients currently comprises a multimodal approach based on cancer-related features and patient-related factors [4].
One of the therapeutic modalities for colorectal cancer is using different chemotherapy regimens; especially, it is considered as the mainstay of treatment in metastatic colorectal cancer. There are various chemotherapeutic drugs for managing colorectal cancer patients, including 5-fluorouracil (5-FU), capecitabine, oxaliplatin, cetuximab, irinotecan, bevacizumab, etc. [5,6]. 5-FU is a common and conventional treatment for colorectal cancer which is used for five decades. Nevertheless, its toxicity to normal tissues and appearance of colorectal cancer chemoresistance are main obstacles to successful cancer chemotherapy and hence, its clinical application is limited [7,8]. Therefore, identification of new therapeutic agents for their potential application combined with 5-FU during colorectal cancer treatment is warranted to improve patient survival and alleviate adverse effects.
In the current study, a systematic search was carried out on the potential therapeutic role of resveratrol during colorectal cancer chemotherapy by 5-FU. Additionally, we tried to answer the following issues: (1) the underlying mechanisms of toxicities induced by 5-FU chemotherapeutic agent on colorectal cancer cells, (2) the role of resveratrol on 5-FU-induced toxicities on colorectal cancer cells, and 3) the underlying mechanisms related to the chemo-sensitization role of resveratrol on colorectal cancer cells during 5-FU chemotherapy.

Search strategy
According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline [30], we performed a systematic search to assess all relevant studies on "the effects of resveratrol in combination with 5-FU against colorectal cancer" in both medical subject heading (MeSH) or advance at four electronic databases including Web of Science, Embase, PubMed, and Scopus up to May 2021 using the keywords "Resveratrol" AND "Chemotherapy" OR "5-Fluorouracil" OR "5Fluorouracil" OR "5-FU" OR "5FU" OR "Fluorouracil" OR "Adrucil" OR "Carac" AND "Colorectal cancer" OR "Bowel cancer" OR "Colon cancer" OR "Rectal cancer" OR "Rectum cancer" OR "Colorectal malignancy" OR "Bowel malignancy" OR "Colon malignancy" OR "Rectal malignancy" OR "Rectum malignancy" OR "Colorectal neoplasm" OR "Bowel neoplasm" OR "Colon neoplasm" OR "Rectal neoplasm" OR "Rectum neoplasm" OR "Colorectal carcinoma" OR "Bowel carcinoma" OR "Colon carcinoma" OR "Rectal carcinoma" OR "Rectum carcinoma" OR "Colorectal tumor" OR "Bowel tumor" OR "Colon tumor" OR "Rectal tumor" OR "Rectum tumor" in keywords, titles or abstracts.

Study selection
The inclusion criteria considered for this systematic review were full-text articles with (1) English language, (2) our per-defined purpose on the role of resveratrol during colorectal cancer chemotherapy by 5-FU (based on the aforementioned keywords), (3) sufficient information, (4) no restriction on publication year, and (5) no restriction in publications with clinical, in-vivo, or invitro studies. For exclusion criteria, we excluded (1) not related papers, (2) review articles, (3) oral presentations, (4) posters, (5) case reports, (6) editorials, (7) letters to the editors, and (8) book chapters.

Data extraction
Each eligible study was reviewed by two researchers and the following data were then extracted: (a) author name and year of publication, (b) models (clinical, in-vivo or/ and in-vitro), (c), 5-FU dosage and route of administration type, (d) outcomes of colorectal cancer chemotherapy, (e) resveratrol dosage and route of administration type, and (f ) resveratrol co-administration outcomes. Figure 2 shows the process of study selection.

Literature search and screening
Two hundred and eighty-two articles were acquired by a comprehensive search on Web of Science (n = 112), Embase (n = 56), PubMed (n = 60), and Scopus (n = 54) electronic databases up to May 2021. After removing the duplicated papers (n = 155), the remaining ones (n = 127) were screened in their titles and abstracts, and 96 of them were omitted. Thirty-one articles were qualified for assessment of their full-texts. Thirteen articles were included in the current systematic review in accordance with the inclusion and exclusion criteria. The data of each eligible article were extracted and listed in Table 1.

The effects of resveratrol in combination with 5-fluorouracil against colorectal cancer Cell proliferation inhibition
The results obtained from some studies showed that proliferation inhibition of colorectal cancer cells in the groups treated by 5-FU was remarkably higher than the untreated groups [31][32][33][34][35][36][37][38]. Additionally, the inhibited cell proliferation following 5-FU treatment had timeand dose-dependent manners. In detail, it was observed that the colorectal cancer cell viability increases over time; while, the colorectal cancer cell viability decreases by increasing the chemotherapy dosage [31,33,35,37,38]. Furthermore, the type of colorectal cancer cell had a considerable effect on 5-FU-induced cell growth inhibition. For instance, it was shown that the SW-620 cells were more resistant to 5-FU than the HT-29 cells [33]. In addition, it was reported that the parental colorectal cancer cells (such as HCT116 and SW480) were more sensitive to 5-FU treatment than their corresponding isogenic FU-chemoresistant derived clones (i.e., HCT116R and SW480R) [35]. The resveratrol co-administration significantly increased cytotoxicity induced by 5-FU treatment on the colorectal cancer cells (synergistic effect) [31][32][33][34][35][36][37][38].

Tumor volume and tumor weight changes
The in vivo results demonstrated that tumor volume and tumor weight of mice were decreased in the 5-FU group than the untreated group. When both resveratrol and 5-FU were administered to the mice, dramatic decreases in the tumor volume and tumor weight were found compared to the 5-FU-treated group [39].

Cell cycle distribution
The findings revealed that the use of 5-FU chemotherapeutic agent regulated the cell cycle profile of colorectal cancer cells [32,37,40]. Chung et al. showed that upon 5-FU treatment, the HCT116 cells (%) in G 0 /G 1 phase decreased, while the rate of these cells in S phase increased compared to the control group [37]. The combined treatment of resveratrol and 5-FU had a reverse manner on the cell cycle distribution compared with the chemotherapy group alone [37]. However, Mohapatra et al. showed that treatment with 5-FU alone and combined with resveratrol halted the cell cycle progression at the S phase in the HCT-116 cells [32].
It is noteworthy that the effect of combined treatment of 5-FU and resveratrol (compared with 5-FU alone) on the cell cycle distribution depends on the type of colorectal cancer cell and can provide different results.

Histological changes
The histopathological results from the colon tissues of rats which received methylnitrosourea (as a tumor   TNF-α, Tumor necrosis factor alpha inducer) demonstrated moderate to severe inflammation, epithelial hyperplasia determined by crypt injury, and inflammatory cell infiltration. Nevertheless, the colon tissue sections from methylnitrosourea-induced colon cancer rats treated with 5-FU alone and 5-FU combined with resveratrol had more intact surface epithelium, less inflammatory, and normal colon cells than the methylnitrosourea-induced colon cancer group [7]. In another study, the histopathological findings obtained from mice with untreated colon tumors revealed blood vessels with a high vascular density; nevertheless, the vascular network of 5-FU-treated group illustrated significantly lower vascular density and branches and this decreased vascular density was severe in the group treated with resveratrol and 5-FU [39].

Discussion
In the current systematic review, we aimed to investigate the cytotoxic effects induced by 5-FU treatment on colorectal cancer cells/tissues. Additionally, the potential therapeutic effects of resveratrol in combined with 5-FU were assessed. A summary of the results has been presented in Table 1. Moreover, the effects of resveratrol co-treatment during colorectal cancer treatment by 5-FU chemotherapeutic agent are depicted in Fig. 3. Chemotherapy drugs is widely applied to treat different malignant tumors, such as head and neck, breast, gastric, and colorectal, etc. [42][43][44][45]. These drugs are able to modulate cell cycle that results in apoptosis [46][47][48]. The treatment of colorectal cancer with 5-FU (especially advanced cases) has two major problems: severe toxicity Fig. 3 The molecular mechanisms of resveratrol co-treatment during colorectal cancer treatment by 5-fluorouracil (5-FU) chemotherapeutic agent. Resveratrol exerts the synergistic anti-tumoral effects through increments in oxidant and apoptosis activities, and reduction in inflammatory effects. ↑↑synergistically increased by 5-FU plus resveratrol compared to 5-FU alone; ↑↑synergistically decreased by 5-FU plus resveratrol compared to 5-FU alone; ↓decreased by 5-FU plus resveratrol compared to 5-FU alone; Bcl-xL, B-cell lymphoma-extra large; BAX, Bcl-2-associated X protein; GPx, glutathione peroxidase; IL-6, interleukin 6; IKK, IκBα kinase; LPO, lipid peroxidation; ROS, reactive oxygen species; NF-κB, nuclear factor-kappa B; COX-2, cyclooxygenase-2; TNF-α, tumor necrosis factor alpha.; MMP-9, matrix metalloproteinase-9 to normal tissues and development of tumor resistance [33]. As its dose increases, the unwanted adverse effects of the chemotherapeutic agent also increase and resistance to the agent develops frequently [8]. In addition, concomitant use of other chemotherapy drugs with 5-FU in colon cancer patients with advanced stages develops drug resistance against chemotherapy and these patients suffer from severe adverse effects [49,50]. In view of the above, clinical application of 5-FU is limited during colorectal cancer treatment. These limitations highlight the requirement for the development of novel anti-tumoral agents in order to enhance the chemosensitivity of colorectal cancer cells and reduce unwanted adverse effects. In this regard, the combination treatment of 5-FU with less toxic substances derived from plants, such as resveratrol, has received more attention in recent years.
The anti-tumoral activity of resveratrol has been reported in some cancers, such as breast [51], skin [52], gastric [53], liver, and colorectal [54] cancers. More importantly, it has been shown that a combination of resveratrol and chemotherapeutic drugs not only can mitigate adverse drug reactions but can also reduce drug resistance (synergistic effect) [38,55,56]. Resveratrol exerts its anti-tumoral effects through several mechanisms, including oxidant, apoptotic, anti-inflammatory actions, etc. In the following, the mechanistic effects of 5-FU treatment on the colorectal cancer cells/tissues as well as the anti-tumoral effects of resveratrol co-treatment during colorectal cancer treatment by 5-FU chemotherapeutic agent are discussed.

Oxidant actions
Free radicals are normally generated in the cells and several defense mechanisms protect the cells against them [57,58]. In oxidative stress conditions, a lack of balance between the free radical amounts and these defense systems occurs, leading to increased free radical amounts [59,60]. Following the chemotherapy drug administration, the oxidative stress condition happens [61]. The generated ROS following 5-FU treatment can induce DNA damage either directly or indirectly, leading to cancerous cell death [33,36,62]. It was also showed that LPO levels were increased in both the plasma and erythrocyte samples of 5-FU-treated colorectal cancer patients, whereas GPx and glutathione (GSH) levels were decreased [63]. Additionally, 5-FU significantly inhibited the catalase activity and increased SOD activity [33]. These findings reflect that 5-FU treatment impairs hydroperoxide scavenging capacity in colon cancer cells. It is noteworthy that non-radical ROS such as H 2 O 2 through the activity of GPx enzyme and consuming GSH (as an important intracellular anti-oxidant agent) generates 2H 2 O [64]. Additionally, the catalase enzyme decomposes H 2 O 2 to H 2 O and O 2 [65]. O 2− also is one of the ROS molecules and it is turned to H 2 O 2 by the SOD enzyme [66]. Moreover, the marked increases in advanced oxidation protein products and MDA levels of 5-FU-treated colon cancer rats were observed [7]. In contrast, the colorectal cancer cell lines resistant to 5-FU showed increments in glutathione transferases (GST) level and Nrf2 expression following 5-FU treatment [67,68]. The activated Nrf2 also led to an increment in the protein expression and activity of heme oxygenase-1 (HO-1) [68]. Nrf2 is a transcription factor for several molecules involved in anti-oxidant activities, including NAD(P)H Quinone Dehydrogenase 1, HO-1, GST, and γ-glutamylcysteine synthetase (γ-GCS) [69]. Hence, an increased Nrf2 expression or Nrf2-regulated cytoprotective genes (such as GST) may play an important role in 5-FU resistance of cancerous cells.
Resveratrol through oxidant actions increased ROS level in colorectal cancer cells [33,36]. It has been also shown that this herbal agent through the interaction with the mitochondria of malignant cells is able to induce an imbalance in cellular anti-oxidant activities, which results to a remarkable increment in the levels of both intracellular ROS and lipid peroxides [33]. It is noteworthy that free radicals are normally created in the cells, particularly through electrons leakage from the mitochondrial electron transport chain and this process is enhanced during mitochondria damage [70]. Resveratrol can also inhibit oxidation-reduction (redox) system in malignant cells [33]. Moreover, it can downregulate catalase and GPx expressions and upregulate MDA, SOD, and LPO levels in colorectal cancer cells [7,33]. Resveratrol can also suppress Nrf2 activity, which may sensitize cancer cells resistance to chemotherapy agents in this way [71]. Besides, it was found that resveratrol combined with 5-FU chemotherapeutic drug against colorectal cancer synergistically increased the generated ROS, LPO, and SOD levels and decreased the catalase and GPx levels [33,36].

Inflammatory actions
The inflammatory process is a biological phenomenon which occurs in response to tissue damage induced from several harmful stimuli such as chemotherapy, radiation, microbial pathogen infection, and/or wounding [116][117][118][119][120][121]. Among the characteristics of inflammation are: elevation in leukocyte migration to injured area, upregulation of pro-inflammatory cytokines, and leukocyte chemotaxis [122,123]. The chemotherapy-induced inflammation has a main role in tumor resistance and the incidence of various adverse effects. Additionally, chronic inflammation may develop second cancer during years after treatment [124]. Moreover, inflammatory mediators play an important role in angiogenesis and tumor growth. For instance, NF-κB is considered as one of the most significant links between inflammation, tumor resistance, and cell death [124]. It was found chemotherapy by 5-FU increases NF-κB phosphorylation in colorectal cancer cells [7,35,41,124]. In addition, 5-FU markedly induced phosphorylation of IκBα subunit, expression of MMP-9, and activation of IKK [35,41,125]. The MMP-9, as an enzyme involved in the inflammatory pathway, is regulated by NF-κB [125]. IkB inhibits nuclear translocation of NF-κB and also activation of IKK induces phosphorylation of NF-κB and IκB [85]. NF-κB can also stimulate the release of prostaglandins and resistance of cancerous cells to apoptosis through upregulation of COX-2 [124]. The findings obtained from 5-FU-treated mice also showed an increased expression of COX-2 than the control group [39]. COX-2 is a pro-inflammatory enzyme that is overexpressed at the inflammatory site of cancer [126]. It is a marker representing a worse prognosis and a stimulator for various cancers through multitasking roles (recently discussed in detail by Hashemi Goradel et al. [127]). It is noteworthy that suppression of NF-κB and COX-2 by novel therapeutic agents could provide a promising approach for cancer treatment, particularly when they are applied as an adjuvant with appropriate chemotherapy drugs. It has been also reported that the phosphorylated STAT3 level decreases in response to 5-FU treatment than the control levels of colorectal cancer cells [33,37]. One of the reasons for the resistance of cancerous cells is the upregulation of transcription factors such as STATs [16]. These enzymes are considered as transcription factors for cytokine signaling which are constitutively activated in many cancer types [128][129][130][131]. STAT3, as one of the subfamilies, involves in the modulation of angiogenesis and metastasis, suppression of apoptosis, and regulation of cell cycle progression via stimulation of VEGF, MMP-2, MMP-9, and IAP-1 [16,132]. Of note, targeting the STATs by chemotherapy agents can be considered as a strategy for overcoming tumor resistance. Moreover, the increased levels of IL-6 and TNF-α following 5-FU-treated colorectal cancer cells have been reported [39].
As mentioned earlier, inflammation is an inducer of carcinogenesis and can significantly affect the therapeutic outcome of chemotherapy [127,133]. In this regard, resveratrol, through its anti-inflammatory activities, can increase therapeutic efficiency and reduce the resistance of cancer cells to chemotherapy drugs. According to the findings obtained from combined treatment of resveratrol and 5-FU, it was found that resveratrol reduces phosphorylated NF-κB, IκBα, and IKK levels as well as MMP-9, COX-2, IL-6, and TNF-α levels in colorectal cancer cells [7,35,39,41]. Resveratrol also synergistically reduced phosphorylated STAT3 levels in 5-FU-treated colorectal cancer cells [33,37].

Perspective of future research
In addition to its anti-tumoral effects, resveratrol is able to mitigate chemotherapy-induced toxicities in normal cells/tissues. Resveratrol exerts the chemo-protective effects through anti-oxidant, anti-apoptotic, anti-inflammatory actions. Hence, it is proposed to conduct studies on the chemo-protective effects of resveratrol in normal cells/tissues during colorectal cancer chemotherapy by 5-FU.
It is noteworthy that the results represented in this systematic review are in accordance with in vitro and in vivo models. The use of resveratrol as a chemo-protector agent or/and chemo-sensitizer agent combined to 5-FU in colorectal cancer patients needs further studies because sometimes results are different between the in vitro and in vivo models and clinical studies.

Conclusion
The obtained findings revealed that combined treatment of resveratrol and 5-FU chemotherapeutic drug significantly increases the effectiveness of 5-FU treatment alone in colorectal cancer cells through increment of chemosensitizer and/or reduction of chemoresistance effects. Resveratrol, as an anti-tumoral agent, exerts the synergistic effects through various mechanisms such as regulation of cell cycle distribution, increments in oxidant and apoptosis activities, and reduction in inflammatory effects.