| Background Hepatocellular carcinoma (HCC) is one of the most common malignancies in china, about 6% of the worldwide incidence of type in all human cancers is on the rise. The overall survival in patients with liver cancer is a very short period, the overall five-year survival rate is less than 10%. Apoptosis resistance is one of the characteristics of HCC, and it has played a key role in the incidence of HCC development. Apoptosis resistance of hepatocellular carcinoma is also an important reason for treatment failure. The possible mechanism of apoptosis resistance is a very complicated thing.The endoplasmic reticulum (ER) stress induced insignificant apoptosis in tumor cells It was reported that ER stress, which indicated that tumor cells could adapt to ER stress and were resisted to the ER stress-induced apoptosis. Many evidences suggest the pivotal role for cyclooxygenase-2 (COX-2) in apoptotic resistance. Upregulation of COX-2 has been demonstrated in various tumor tissues. Many evidences suggest the pivotal role for cyclooxygenase-2 (COX-2) in apoptotic resistance. Upregulation of COX-2 has been demonstrated in various tumor tissues. The present study was designed to investigate the possible mechanism that hunman hepatoma cell line HepG2 resisted to the ER stress-induced apoptosis, in order to develop an effective therapy for HCC.Objective: To clarify the sensitivity and the pathway of apoptosis of endoplasimic reticulum stress induced hepatoma carcinoma cells, and investigate the role of Melatonin reticulum stress induced apoptosis.Methods: Cell viability was analyzed by the MTT assay. Cell apoptosis was evaluated by TUNEL method and flow cytometry. Glucose regulated protein78 (GRP78) and cycloxygenase-2 (COX-2) expressions were measured by western bloting.Results1. Inhibitory effect of TM or combination of TM and Melatonin on hepatoma carcinoma cells and hepatocyte cells proliferation1.1 Inhibitory effect of TM on hepatoma carcinoma cell line HepG2 and hepatocyte cell line HL-7702 cells proliferationHepatoma carcinoma cell line HepG2 and normal hepatocyte cell line HL-7702 cells were treated with different concentrations of TM for 48 h and cell survival was analyzed by conventional MTT assays. TM insignificantly reduced viability of HepG2 cells, HL-7702 cells displayed more sensitive to TM, the inhibitory rate was markly increased on HL-7702 cells compared with that the same concentration of TM.1.2 Inhibitory effect of combination of TM and Melatonin on hepatoma carcinoma cell line HepG2 and hepatocyte cell line HL-7702 cells Melatonin proliferationHepatoma carcinoma cell line HepG2 and normal hepatocyte cell line HL-7702 cells were treated with different concentrations of combination of TM and 10-7umol/L Melatonin for 48 h and cell survival was analyzed by conventional MTT assays. The combination of Melatonin and TM significantly increased the efficacy of treatment compared with the single agent TM only in HepG2 cells. However, in HL-7702 cells we didn't found significant increase efficacy.2. Proapoptotic effect of TM or TM combined with Melatonin on 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 and10-7umol/L Melatonin treated group. However, the cell density were decreased significantly and the number of apoptotic cells increased obviously in combination of 3umol/L TM and 10-7umol/L Melatonin treated group. 2.2 The apoptotic assay by FCMA Flow Cytometry (FCM) assay was performed to analyze apoptotic rate. The sub-G1 peak, which appeared before the G0/G1 phase that represents apoptotic cell population, was slightly increased in HepG2 cells treated with TM or 10-7umol/L Melatonin alone. The apoptotic peak was dramatically increased when the cells were exposed to combined TM and10-7umol/L Melatonin in HepG2 cells. Then, the apoptotic peak was markly increased with TM alone and no difference with the combination group in HL-7702 cells.2. ER stress induced the expression of COX-2 and GRP78HepG2 cells were treated with 3unol/L TM for 3, 6, 9, 12, 24 h, and the expression of COX-2 was determined by Western blotting. The expression of GRP78, an unfolded protein response chaperone, was enhanced in response to TM. COX-2 protein started to increase 3h after treatment with TM, while this effect was not detected in HL-7702 cells. Melatonin inhibited the expression of COX-2 induced by ER stress in HepG2 cells.ConclusionMelatonin can sensitize hepatoma carcinoma cells to ER stress-induced apoptosis.
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