Melatonin Enhances The Antiproliferative Effect Of Doxorubicin On HepG2 Cell Line And The Possible Mechanism

Background Hepatocellular carcinoma (HCC) is the third most common cancer in China where the incidence is 30.3/100000. Over the past twenty years, the mortality was increased 41.7% which constituted 42% of all death number in the world. In recent years, the incidence of HCC is increasing in several developed countries, as a result of increased prevalence of HCV infection. The optimal treatment of HCC is hepatic resection. However, more than 80% of patients present with advanced or unresectable disease, and for those patients who do undergo resection, the recurrence rates can be as high as 50% at 2 years. Unfortunately, chemotherapy which is a significant way in the treatment of cancer has not play a key role for HCC patients. Doxorubicin (Dox) is perhaps the most widely used agent in the treatment of HCC. However, the response rate of Dox is only 4%~10.5% and the dose-limited toxicity constrained essentially the clinical application further. At present, many physicians began to research Chinese herbal medicine and human existence itself within the anti-tumor substances in-depth exploration and study, melatonin is one of them. Melatonin, mainly secreted as a pineal indole amine hormone, the chemical composition of N-acetyl-5-mehtoxytrytamine, is an important physiological inhibitor of tumor that possesses a broad biological effect. Its secretion obvious circadian rhythm is conducive to sleep, and can also inhibit tumor cell growth, strengthen the immune system function. As the body of melatonin, a natural anti-tumor factors have been more and more attention. The animal experiments demonstrates that Mel can significantly inhibit the growth of transplanted liver cancer in mice and can inhibit cancer metastasis. This study was designed to investigate the growth-inhibitory effect of Mel combined with Dox in human hepatoma cell lines HepG2 and the possible mechanism, in order to develop an effective combination therapy for HCC.Objective To investigate the effect and elucidate the mechanism of the Mel combined with Dox on effect inhibitor, apoptosis and expression of Bcl-2, Bax and Caspase-3 of hepatocarcinoma cell lines.Methods The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 Cellviability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Cell apoptosis was evaluated using TUNEL method and flow cytometry. Apoptosis-related protein Bax, Bcl-2 and Caspase-3 expressions were measured by immunohisto-chemical staining.1 Effect of Mel combined with Dox on the proliferation of human hepatoma cell line HepG2Investigated the effect of Mel, Dox and Mel combined with Dox on the proliferation of HepG2 respectively by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide (MTT) assay. The results suggested that Mel at concentrations of 10-8~10-5mmol/L, had the inhibitory effect on the proliferation of HepG2 cells which was dose- and time-dependence. The r values of dose-effect curves for single-agent Mel on HepG2 cell was 0.993. The IC50 values was 7.623*10-5. Dox with the concentration of 0.31~2.5mg/L also had the analogical effect on the HepG2 cells which was dose- and time-dependence. The r values of dose-effect curves for single-agent Mel on HepG2 cell was0.952. The IC50 values was 4.693*10-4. Furthermore, various concentrations of Mel (10-8,10-7,10-6,10-5mmol/L) combining with different concentrations of Dox (0.31~2.5mg/L) showed synergistically inhibitory effect on the proliferation of the HepG2 cell line.2 The apoptotic effect of Mel combined with Dox on HepG2 cells2.1 The apoptotic assay by TUNELMorphological evidence of apoptosis was assayed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). The typically apoptotic changes include chromatin condensation and deformed and fragmented nuclei. The results suggested that the proliferation was prosperous in control group. However, the cell density were decreased significantly and the number of apoptotic cells increased obviously in treated groups, especially in the combination group.2.2 The apoptotic detection by FCMA Flow Cytometry (FCM) assay was performed to analyze apoptotic rate. The sub-G1 peak, which appeared before the G0/G1phase that represents apoptotic cell population, was observed clearly in the HepG2 cell line treated with Mel or Dox alone or both. The apoptotic peak was dramatically increased when the cells were exposed the Mel combined with Dox which indicated that Mel had synergistic effect with Dox on inducing apoptosis of HepG2.3 Possible mechanism of Mel combined with Dox on the inducing apoptosis effect on HepG2The expression of Bcl-2, Bax and Caspase-3 were assayed by immunocytochemical analysis. The results showed that Bcl-2, Bax and Caspase-3 were expressed in different extent of HepG2. But the Bax and Caspase-3 were up-regulated, the Bcl-2 were down-regulated after Mel or/and Dox treated, especially in combined group.Conclusion Mel at some concentration could enhance the antiproliferation and inducing apoptosis effect of Dox on HepG2 cell line, which may be associated with the increased expression of Bax and Caspase-3 and the decreased expression of Bcl-2.