Efficacy of resveratrol against breast cancer and hepatocellular carcinoma cell lines

Objectives: To evaluate the anti-cancer effect of resveratrol on Michigan cancer foundation-7 (MCF-7) and hepatoblastoma cell line (HepG2) cells. Methods: The study was carried out at the Department of Botany and Microbiology, Prince Sattam bin Abdulaziz University, Al-kharj, Saudi Arabia, from August 2022 to October 2022. Different concentrations of resveratrol were added to the MCF-7 and HepG2 cell lines. Cell death and proliferation were measured with MTT and Trypan blue exclusion assays. Apoptosis markers were assessed by using a quantitative PCR assay (qPCR). Results: The resveratrol was shown to suppress the proliferation of MCF-7 and HepG2 cells at dose- and time-dependent. The cytotoxic effect of resveratrol was observed even at 100 μM after 24 hours. In comparison to untreated cells, resveratrol treatment reduced the viability of MCF-7 cells to roughly 57.5% with a half maximal inhibitory concentration (IC50) of 51.18 μM and HepG2 cells to 56.2% with an IC50 of 57.4 μM. Furthermore, in the tested cell lines, resveratrol was able to induce apoptosis mediated by elevated apoptosis markers. Conclusion: Resveratrol appears to be an excellent candidate agent in anticancer therapy in various human cancers.


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C ancer continues to be the most common cause of mortality globally.In 2020, breast cancer was responsible for almost 685,000 deaths in women globally. 1Currently, the most widely used traditional treatment methods are surgical intervention, radiotherapy, chemotherapy, and immunotherapy, which represent conventional treatment strategies, or a combination of these options to cure cancer, shrink cancer, or stop the progression of cancer, but the availability of effective therapy for cancer is still elusive. 2,3Curative or primary surgery is usually carried out when cancer is found in only one part of the body.Possible complications during surgery may be caused by the surgery itself, drugs used, and the overall health status. 4 Cancer chemotherapy agents are an important alternative to surgery and radiation to successfully treat some types of solid tumors that work primarily by interfering with either macromolecular synthesis or the function of neoplastic cells by binding with DNA and preventing RNA synthesis, leading to the death of cancer cells. 3,5Over the last decades, approximately 50% of all anti-cancer drugs used worldwide are either natural product derivatives or supplemented with natural products. 6esveratrol is widely distributed in grapes, berries, peanuts, and red wine. 7,8Resveratrol appears to have the potential for the treatment of cardiovascular disease and protection against inflammatory disorders.][15] It also triggers apoptotic signalling via p53-dependent or p53-independent apoptosis. 16,17These studies highlight that resveratrol has pro-apoptotic potential in cancer cells needs further elucidation.However, the resveratrol-induced apoptosis via a possible intracellular signalling mechanism needs further elucidation.In the current study, the anti-cancer effect of resveratrol was tested on MCF-7 and HepG2 cells.The MCF-7 and HepG2 cells were grown at 1×103 cells/well )200 μl( in a 96-well microplate.After incubated for 24 hours at 37 °C with 5% CO2, 100 μl of fresh DMEM containing different concentrations of resveratrol )100 μl, 25, 50, 100, 200, and 300 μM(.Plate was then incubated for an additional 24 hours.

Methods
The MTT assay was used to estimate the cytotoxicity.Briefly, treated cells were exposed to 10 μl of MTT working solution in each well.Following 24 hours of incubation at 37 °C with shaking to generate formazan crystals.The cell culture medium of cells was gently aspirated, and 100 μl/well of 100% DMSO per well was added.The absorbance was measuered after further 10 minutes-incubation at 37 °C, with a microplate reader at 590 nm, ELX-808 )Biotek, USA(.Cell viability )%( for each drug concentration was calculated and was determined by comparing the absorbance of the treated cells to the untreated. 18The half maximal inhibitory concentration )IC 50 ( was defined as the concentration of the compound that causes 50% of cell proliferation inhibition and was determined by the regression probit model.
The viability of MCF-7 and HepG2 cells were carried out using Trypan blue exclusion assays, the cells were counted using an inverted light microscope.
The expression of genes coding was evaluated with resveratrol )100 μM(.Ribonucleic acid isolation of cells cultured was carried out by using a RNeasy Micro Kit )QIAGEN, Hilden, Germany(.The quantification of apoptosis markers in cancer cells was determined using qPCR by using one step RT² SYBR® Green/ROX™ qPCR Master Mix For each sample, a master mix was prepared by mixing the following components: Go Taq Qpcr master mix )2x( )10 μl(, one-step RT mix )0.4 μl(, CXL )0.33 μl(, F primer )0.4 μl(, R primer )0.4 μl(, RNase-free water )8.5 μl(, and RNA template )4 μl(.The mix was thoroughly mixed by gentle flicking at the bottom of the tube, centrifuged, and then transferred to the RT2 PCR Array Loading Reservoir.The one-step rRT-PCR was carried out in 7500 fast real-time PCR.The results confirmed the specificity of each primer set using melting curve analysis.The results were obtained using the 2 −ΔΔCq method, and delta Cq )ΔCq( values obtained for the different genes were normalized based on the value of glyceraldehyde 3-phosphate dehydrogenase )GAPDH( amplified from the same genes. 19The fold-change in expression was calculated Disclosure.Author has no conflict of interests, and the work was not supported or funded by any drug company.Saudi Med J 2023; Vol.44 )3( https://smj.org.sa as referenced to the expression of the untreated control cells.The primers used in PCR are presented in Table 1.
Statistical analysis.The Statistical Package for the Social Sciences, version 22.0 )IBM Corp., Armonk, NY, USA( with posthoc Dennet's test were used for data analysis.Each experiment was carried out in duplicate.Date were reported as the means ± standard deviation )SD(.A p-value of <0.05 was considered significant.
Results.We found that resveratrol significantly )p<0.05(reduced cell growth of MCF-7 cells to roughly 57.5% with an IC 50 of 51.18 μM and HepG2 cells to 56.2% with an IC 50 of 57.4 μM )Figure 1(.
We also found 100 μM of resveratrol for 24 hours, decreased in percentage of viable MCF-7 cell to approximately 57.5% )p<0.05( and HepG2 cells to 56.2% )p<0.05(than untreated cells )Figure 2(. Figure 3 indicated that there was a significantly increase in the caspase-3, caspase-8, and caspase-9 expression in MCF-7 and HepG2 resveratrol-treated cells than untreated control cells )p<0.05(.Resveratroltreated MCF-7 and HepG2 cells had a highly significant Bax mRNA level and low Bcl-2 and Bcl-xL anti-apoptotic genes than control )p<0.05; Figure 4(. Figure 5 demonstrates that resveratrol-treated MCF-7 and HepG2 cells showed higher p53 and p21 mRNA levels than control cells )p<0.01(.
Discussion.The limitations of conventional cancer therapies have pushed the field of chemotherapeutic drugs toward exploring other novel strategies to improve cancer therapy to inhibit cancer growth.Chemotherapeutic drugs are sometimes correlated with undesirable side effects.The plant-derived drugs were related to used as significant effective anti-cancer drugs with reduced side effects against cancer. 24][27] However, resveratrol-induced apoptosis is not fully understood. [22][23]

Gene names Primers sequence
Caspase-3 F: 5′-GCTGGATGCCGTCTAGAGTC-3′ R: 5′-ATGTGTGGATGATGCTGCCA-3′ Caspase-8 F: 5′-AGAAGAGGGTCATCCTGGGAGA-3′ R: 5′-TCAGGACTTCCTTCAAGGCTGC-3′ Caspase-9 F: 5′-ATTGCACAGCACGTTCACAC-3′ R: 5′-TATCCCATCCCAGGAAGGCA-3′ of 57.4 μM using the MTT assay.The MTT assay is a widely used method to determine the cytotoxic effects of anti-cancer compounds on cell lines. 28These observations are consistent with a previous study that reported that the MCF-7, HepG2, and A549 cancer cell viability was moderately inhibited by 100 μM of resveratrol for 24 hours using the MTT assay. 29A previous study also suggested that curcumin, resveratrol, or curcumin combined with resveratrol significantly inhibited the MCF-7 cell growth using the MTT assay after 24 hours of treatment. 30A 5 μg/mL resveratrol with paclitaxel )10 μg/ml( decreased the cells on HepG2 cells compared to other treatment groups. 23Similarly, another study reported that the MCF-7, HeLa, and HepG2 treated with resveratrol were reduced cell growth of MCF-7 cells to an IC 50 ranging from 35.1-83.8μM using the MTT assay. 31Another study reported that >1 μM resveratrol caused reduced cell proliferation in MCF-7 cells. 32Resveratrol was found to inhibit the HepG2 cells viablity at 24 hours with an IC 50 value of 100 μM.These observations are in agreement with previous studies indicating that 24 hours of treatment resulted in an IC 50 value of 60 μm resveratrol for HepG2 cells. 33On the other hand, resveratrol appears to have some toxic effects at high concentrations, thus, an effective strategy to decrease their chemotherapeutics concentrations may improve their anti-cancer effects. 34,35he apoptosis or the physiological process of cell death is characterized by distinct morphological characteristics and death. 36Apoptosis plays a crucial role in cancer therapy.7][38] The granule exocytosis pathway is an additional pathway that involves natural killer )NK( and T-cell-mediated killing of target cells. 37he extrinsic apoptotic pathway triggers apoptosis by the binding of ligands )namely, first apoptosis signal ligand, tumor necrosis factor, and tumour necrosis factor-related apoptosis-inducing ligand( to their death receptors. 39Apoptosis is also mediated by caspases.The exogenous apoptosis pathway can be activated by caspase-8 in response to the activation of cell surface death receptors. 40The intrinsic or mitochondrial pathway can be activated by caspase-9. 41Caspase-3 is involved in both exogenous and endogenous apoptosis, by interacting with caspase-8 and caspase-9, ultimately to induce cell apoptosis. 42n the present study, the mRNA caspase-3, caspase-8 caspase-9, p53, p21, and Bax were significantly increased in responses to 100 μM resveratrol in the MCF-7 and HepG2 cells, than control cells, whereas, there was a significantly decrease in the Bcl-2 and Bcl-xL antiapoptotic mRNA expression than control noninfected cells.Consistent with these results reported for HepG2 cells, a previous study reported that 5 μg/mL resveratrol activated caspase-3, caspase-8, and caspase-9.Similarly, 10-80 μM of resveratrol for 48 hours inhibited the MCF-7 and MDA-MB-231 breast cancer cells by binding to and activating estrogen receptor. 43In addition, in vitro study used in an animal model showed that resveratrol inhibited the tumor growth at a dose of 625 mg/kg. 44][47][48] Study limitations.Its flaws or shortcomings in terms of the methodology we used in our present study.Attempts to correlate protein abundance with mRNA expression level are not fully established, even though mRNA expression levels are widely employed as a proxy for estimating functional differences that occur at the protein level. 49Since in vitro studies are not sufficient for proving the anti-cancer effects, in vivo proof-ofconcept studies can contribute to these findings.
In conclusion, resveratrol showed to have a significant cytotoxic effect on MCF-7 to roughly 57.5% with an IC 50 of 51.18 μM and 56.2% of HepG2 cells with an IC 50 of 57.4 μM, which may be a significant anti-cancer effect on various types of cancer such as breast cancers.Resveratrol also showed to elevate caspase-3, caspase-8, caspase-9, Bax, p53, and p21 and reduce Bcl-2 and Bcl-xL mRNA levels.Our results suggested that resveratrol could be a potentially effective approach for treating breast and liver cancer.However, synergistic anti-tumor effects of resveratrol combined with another chemotherapeutic agent are needed to reduce the IC 50 .Further, in vitro studies are not sufficient for proving the anti-cancer effects, in vivo studies can contribute to these findings.