Combined Effects of Hesperidin and Quercetin on Apoptosis in HepG۲ Cell Line

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death with an increasing incidence worldwide. Many patients are not diagnosed until advanced stages and require systemic chemotherapy. Despite the significant role of chemotherapy for cancer patients, its therapeutic efficacy is limited serious reasons. Extensive studies have provided a promising insight into developing alternatives strategy fighting against cancer with minimal side effects. Managing cancer treatment by using nutraceuticals is one of these strategies. Natural dietary agents including fruits and vegetables have drawn attention of the scientific community due to their demonstrated ability to suppress cancers. Flavonoids are considered to be the major category of such dietary polyphenol. One of the most important flavonoids with promising anticancer potential is hesperidin, which is widely found in oranges and lemons. It has been reported that this compound can induce cell apoptosis. Several approaches including the combination of hesperidin with other phytochemicals could be utilized further to improve its bioavailability and efficacy as an anticancer agent. Quercetin, an important dietary flavonoid, has received increasing attention as a pro apoptotic flavonoid with specific, and almost exclusive, effects on tumor cells rather than normal, non-transformed cells. Methods: The human hepatocarcinoma HepG۲ cell line was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with ۱۰?tal bovine serum (FBS) and ۱% concentration ratio of penicillin and streptomycin. HepG۲ cells were grown in a humidified incubator containing ۵% CO۲ and ۹۵% air at ۳۷°C. The viability of cells was evaluated using the colorimetric MTT assay after ۲۴ and ۴۸ hours of incubation with different concentrations of Hesperidin and Quercetin (۰-۱۰۰۰ µM). Then Total RNA from treated and untreated HepG۲ cells was isolated. Using cDNAs as the template, quantitative real-time PCR was carried out using specific oligonucleotide primers for human BAX and BCL-۲ genes and mRNA expression levels were normalized the corresponding β-actin housekeeping gene expression levels. Relative mRNA levels were quantified by calculating the comparative ۲-ΔΔCt method. Flow cytometry analysis was also carried out to investigate proportion of cancer cells undergoing apoptosis. Results: The MTT showed that the IC۵۰ value after treatment with Hesperidin and Quercetin was ۲۵۰ µM at ۴۸ hours. Our experiment showed that mRNA expression levels of BCL-۲ were decreased in treated cells compared to untreated cells. In contrast, mRNA expression levels of BAX were increased in treated groups as compared with the untreated group. Flow cytometry data showed that Apoptosis was induced treated groups as compared to the control group. Furthermore in the combined treatment group of both flavonoids, more increases and decreases were observed than in each of alone. Conclusion: As expected, Hesperidin and Quercetin induced apoptosis in HCC cells by down-regulating Bcl-۲ expression and up-regulating BAX expression and increasing Bax-to-Bcl-۲ ratio, while this effect is greater when they are taken together. Our data indicate that combination of Hesperidin and Quercetin could improve the efficacy of chemotherapy for liver tumors and may be a potential drug candidate in anti-cancer therapy.

کلیدواژه ها:

Hesperidin ، Quercetin ، HepG۲ ، Apoptosis