Cytotoxic Effect of Resveratrol on Colorectal Cancer Cell Line

This study aimed to examine the cytotoxic effect of resveratrol as an anticancer in human colorectal cancer (HRT) cell line by assessment of its half-maximal inhibitory concentration (IC50) and its ability to inhibit the growth of these cancerous cells. Resveratrol inhibited the proliferation of HRT cell lines when used at different increased concentrations in this study (25, 50, 100, 200, and 300 μmol). These increased concentrations of resveratrol caused a corresponding significant inhibition in the growth percentage of the tested cancerous cell line (13%, 31.33%, 53.66%, 63.66 %, and 76.33%, respectively) when compared with DMSO0.1% as negative control, in a concentration-dependent manner. Resveratrol at 300 μmol concentration showed the highest significant increase in the growth inhibitory percentage (76.33%). Moreover, resveratrol IC50 against HRT cell line was determined as 75.63 μmol. The study suggests a promising anticancer activity of resveratrol, which can interfere with many dysregulated signaling pathways in transformed cells which are proposed to be driving forces for its anticancer effect.


Introduction
Colorectal cancer (CRC) is the third most diagnosed cancer worldwide and ranks second among cancer-related deaths in many countries (1). The proportions of newly diagnosed CRC cases are 9.2% in men and 10% in women, which accounts for 9.7% of overall cancer incidence worldwide (2). The risk of CRC increases with age, hereditary genetic predisposition, chronic bowel inflammation, and diabetes, in addition, smoking, alcohol consumption, low vegetable intake, obesity, and sedentary lifestyle are associated with an increased incidence of CRC (3). In contrast, chronic intake of certain non-steroidal anti-inflammatory drugs, high calcium consumption, and post-menopausal hormone usage are associated with reduced risk of the disease (4). About 70% of CRC occurs without familial or inherited predisposition (sporadic disease), while less than 10% of patients inherit the tendency to develop the disease (5). The most common inherited conditions that predispose to CRC are familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome). FAP accounts for less than 1% of all CRC cases and is associated with autosomal dominant inheritance of a mutant adenomatous polyposis coli (Apc) gene. FAP patients develop hundreds of colonic adenomatous polyps that start malignant transformation when the patient is as young as 20 and almost all patients develop cancer by the age of 40 years unless the colon is removed. HNPCC is an autosomal dominant disease that accounts for 2-3% of all CRC cases. It is associated with alterations in human mutL homolog 1(MLH1) and MutS homolog 2(MSH2) genes that are important in DNA repair systems, thus they may also be associated with other malignancies such as cancer of the pancreas, kidney, stomach, ovary, and endometrium (6). Unlike FAP, HNPCC patients develop a few adenomas with 70-80% malignant transformation rate, and are usually diagnosed in their mid-forties (7). In addition, germline mutations of the liver kinase B1/Serine/threonine kinase 11(LKB1/STK11) gene are associated with Peutz-Jeghers syndrome and mutations of small mother cell against decapentaplegic (SMAD4) or activing like kinase 3(ALK3) result in juvenile polyposis (8). Resveratrol (3,5,4'-trihydroxy-trans-stilbene). is a polyphenolic natural product which belongs to the group of phytoalexins that are synthesized when plants are subject to environmental stress, such as exposure to pathogens. It can be found in widely consumed foods and beverages such as red wine, grapes, peanuts, pistachios, berries, and Itadori tea. In fact, resveratrol is produced by more than 70 species of plants in response to stress. Resveratrol is particularly thought to mediate some of the potential health benefits of red wine (9). Many studies have examined and described different actions of resveratrol, such as antioxidant and anti-inflammatory properties. For instance, the serum antioxidant activity is shown to be significantly elevated by up to 18% in normal individuals after ingestion of 300 ml of red wine (10). In addition, moderate intake of red wine has been revealed to influence by decrease level of lowdensity lipoprotein (LDL) in humans (11). Resveratrol has been shown to exert effects similar to dietary restriction, increasing the lifespan of animal experimental models (12). Moreover, resveratrol can act as an inhibitor of the proinflammatory cyclooxigenase-2 COX-2 enzyme, in addition to its inhibitory effect on human platelet aggregation and eicosanoid synthesis (13). The effects of resveratrol appear to be achieved through numerous cell signaling pathways, including induction of cell cycle arrest, stimulation of apoptosis and differentiation, suppression of inflammation and angiogenesis, and reduction of adhesion, invasion, and metastasis (14). Various in vitro studies have described antioxidant, antiinflammatory, and anticancer features of resveratrol, exerted through modification of signaling pathways such as downregulating WNT2 and reducing the nuclear accumulation of β-catenin and transcription factors involved in cellular processes such as apoptosis, stimulation of cell cycle arrest and lipid oxidation (15). Different studies have explained the effects of resveratrol in several human colorectal cell lines and the related mechanisms. For example, in the human colonic adenocarcinoma cell line Caco2 and the HCT-116 colon carcinoma cell line, resveratrol was revealed as a cell cycle inhibitor, indicated by low cyclin D1 and cdk4, and as an apoptosis inducer, shown by high caspase activity (16). Regarding the resveratrol significant biological role as cell cycle inhibitor, the current study was designated for detection its in vitro cytoxicity on cancer cell line as preliminary for future in vivo studies.

Maintenance of Cell Cultures
Homo sapiens, human colon tissue (HRT cells) were provided by Iraqi Centre for Cancer and Medical Genetics Research (ICCMGR), Baghdad, Iraq, and maintained in Roswell Park Memorial Institute medium (RPMI-1640) (S,p.A., Italia) supplemented with 10% fetal calf serum (Capricorn, Germany). These are important because they act as growth factors, which facilitate cell survival, and proliferation, 100 units/ml of penicillin, and 100 µg/mL of streptomycin were added to the medium. Cells were passaged using Trypsin-EDTA (Ethylenediaminetetraacetic acid) (Capricorn, Germany) reseeded at 80% confluence twice a week, and incubated in CO2 incubator (Bio Tek, USA) 5% CO2 at 37 °C (17).

Cytotoxicity Assays
To determine the cytotoxic effect of resveratrol, the Methyl thiazolyl tetrazolium (MTT) cell viability assay was done using 96-well plates. Cell lines were seeded at 1 × 10 4 cells/well. After 24 h, or a confluent monolayer was achieved, cells were treated with resveratrol at different concentrations (25,50,100,200, and 300 µmol). Cell viability was measured after 72 h of treatment by removing the medium, adding 28 µL of 2 mg/mL solution of MTT stain and incubating the cells for 3 h at 37 °C. After removing the MTT solution, the crystals remained in the wells were solubilized by the addition of 130 µL of DMSO 2.5% (Dimethyl Sulphoxide) followed by 37 °C in CO2 incubator (Cypress Diagnostics, Belgium) at 5% of CO2 for 15 min with shaking (18). The absorbency was determined on a micro plate reader (Bio Tek, USA) at 492 nm, the assay was performed in triplicate. The inhibition rate of cell growth (the percentage of cytotoxicity) was calculated as the following equation: Cytotoxicity = A-B/A *100% Where A is the optical density of control and B the optical density of samples.

Materials and Chemicals
Trypsin-EDTA (0.5%) was purchased from Capricorn (Germany) causes cells to detach from the growth surface. Methyl thiazolyl tetrazolium (MTT) solution: was purchased from Sigma-Aldrich Co. Fetal calf serum was purchased from Capricorn (Germany). These are important because the growth factors facilitate cell survival and proliferation while antibodies could bind to the cells in culture. RPMI 1640 was purchased from S. p. A. (Italy) as a supplement media. The cell lines used in this study were supplied by tissue culture unit, Iraqi Centre for Cancer and Medical Genetics Research (ICCMGR) Baghdad, Iraq maintained in RPMI-1640. Resveratrol with chemical formula C14H12O3 g/mol. resveratrol purity 99% was purchased from SIGMA and ALDRICH. Resveratrol was dissolved in DMSO 0.1% as stock and stored at -20 °C. Stock solution of resveratrol (1000 μmol) was prepared in DMSO 0.1% and then five different concentrations were prepared (25,50,100,200, and 300 µmol).

Statistical Analysis
The obtained data were statically analyzed using an unpaired t-test with GraphPad Prism 6. The values were presented as the mean±SEM of triplicate measurements (19).

Results and Discussion
The result showed that the IC50 of resveratrol on HRT colon cancer cell line is 75.63 µmol at 72 h of incubation. Growth inhibition of HRT cell line listed in Figure 1 and Table 1, showed positively proportional concentration-dependent effect of resveratrol (25,50,100,200, and 300 µmol) that caused significant (P≤0.05) growth inhibition percentage of 13%, 31.33%, 53.66%, 63.66 %, and 76.33%, respectively, for resveratrol when compared with DMSO (0.1%) as the vehicle control, where the growth of HRT cell line was 100%.  (20). In addition, these treatments (chemotherapy and radiation) are frequently toxic to normal host cells (21). Many plant-derived phytochemicals have been shown to be beneficial to human health like resveratrol and catechin compounds which were tested previously against each of four human tumor cell lines (MCF-7 breast carcinoma, HT-29 colon carcinoma, A-427 lung carcinoma and UACC-375 melanoma (22). In the present study, we sought to determine the effect of resveratrol on cancer cell growth. Antiproliferative effect of resveratrol was analyzed using a colon cancer cell line. Current results of the inhibitory effect of different resveratrol concentrations against in vitro growth of (HRT cell line) showed dose dependent inhibitory cancer growth for all concentrations used in this study against HRTcell line. The maximum effect of inhibiting cancer cell growth was seen with the concentration of (300) μmol of resveratrol. We speculate that this inhibitory effect of resveratrol might be due to many reasons. The first one is that it may be the mechanism of antioxidative or potent free radical scavenging effects of resveratrol and possibly other reported mechanisms like receptor mediated inhibitory which interfere and inhibit cancer cell growth (23). Other mechanisms could be hypermethylation or induction of suppressor gene, apoptosis, DNA repair or increasing cellular gap junction.
Interestingly results of previous published study indicated that resveratrol causes apoptosis and mitochondrial dysfunction by membrane depolarization in malignant human pancreatic cancer cell (24). Resveratrol has protective effects against lung cancer (25) and many more anticancer reports are present in the literature that strongly suggest a specific cytotoxic effect on cancerous rather than non-cancerous cells. These results suggest that resveratrol can induce cell cycle arrest and apoptosis independently of p53 status (26). Nevertheless, other researchers have reported that p53 upregulation could play an important role on the synergistic effect between resveratrol and etoposide, a topoisomerase II inhibitor used as an antineoplastic drug (27). Other researches showed that resveratrol also sensitizes HT-29 and SW620 colorectal cancer cell lines to cytotoxic oxidative stress induced by 5flourouuracil 5-FU, by inhibiting their endogenous antioxidant capacity (28). Moderate resveratrol concentration (15 μmol) in combination with very low 5-FU (0.5 μmol) concentration causes significant inhibition of cell proliferation, migration, and cell cycle arrest at S phase, leading to apoptosis in HCT-116 cells. The same study provides evidence suggesting that its mechanism of action may be related with the activation of the MAPK pathway through upregulation of p-JNK and p38, with no p-ERK changes (29). Similar results were found in a study with etoposide resistant HT-29 cells, where resveratrol was able to chemosensitize HT-29 cells promoting cell cycle inhibition, reactive oxygen species (ROS) generation, AMPK activation, and apoptosis induction (30). In conclusion, CRC is a prevalent cancer and one of the main causes of cancer mortality entire the world. Several factors from genetics to diet are involved in the incidence of this malignancy. Its pathophysiology is heterogeneous which multiple molecules and various signaling pathways including inflammation, oxidative stress, and apoptosis are implicated in its incidence and progression. Several studies have supported the potential effects of resveratrol in CRC treatment. This polyphenol compound represents different properties including antioxidant, antiinflammatory, apoptosis inducer, and antiangiogenesis efficacy. Due to these significant effects, resveratrol is suggested as a novel therapeutic agent for cancers. Moreover, some studies reported that consumption of resveratrol in combination with other anti-cancer drugs can increase their effects and decrease their side effects. Thus, this multi-tasking compound can be suggested as a new candidate in CRC treatment however, more human studies are needed.