The Effect And Mechanisms Of Melatonin On Resistance To Apoptosis And Chemotherapy In Hepatocellular Carcinoma Cells

Background Hepatocellular carcinoma (HCC) is the most common malignant hepatobiliary disease and one of the leading causes of cancer mortality worldwide. Risk factors for HCC development include infection with hepatitis virus B or C, cirrhosis, nonalcoholic steatohepatitis, and the presence of genetic metabolic diseases. In the early stages of HCC, surgical resection, local ablation, or liver transplantation is able to cure a proportion of patients.In advance stages, treatments are limited.It is imperative to develop more effective and low-toxic chemotherapy agents for HCC. Melatonin (N-acetyl-5-methoxytryptamine) is a key regulator for the control of circadian rhythms in humans; In addition to this critical function, melatonin also has other biological functions, including an immunomodulatory effect and an antioxidant role. Recently, several studies have shown that melatonin has an oncostatic activity in cancer cells.The present study was designed to investigate the reversing mechanisms of melatonin on resistance to apoptosis and chemotherapy in hepatocellular carcinoma.Part I Melatonin overcomes apoptosis resistance in human hepatocellular carcinoma by targeting Survivin and XIAP via COX-2/PI3K/AKT pathway.Methods:(1) To evaluate the reversing effect of melatonin on apoptosis resistance in human hepatocellular carcinoma. Growth inhibition rate of each group was detected using MTT assay.The apoptosis rate was deteceted by flow cytometry and the morphological change of apoptosis was observed by TUNELstaining.(2) To clarify the reversing mechanism of melatonin on apoptosis resistance in human hepatocellular carcinoma.100cases of HCC tissue samples were collected.Expression of IAPs and COX-2were detected using immunohistochemistry. The association of COX-2expression with IAPs was analysed by Pearson chi-square test. The apoptosis rate was deteceted by flow cytometry and the morphological change of apoptosis was observed by TUNEL staining.The expression of COX-2,p-AKT,XIAP,Survivin and cIAP-1were assayed by Western-blot.Results:1. The reversing effect of melatonin on apoptosis resistance in human hepatocellular carcinoma (1) To evaluate the effect of melatonin on cell growth, two widely used human HCC cell lines, HepG2and SMMC-7721, were employed. The number of viable cells in culture was examined after melatonin treatment (10-9-10-3mol/L) at24and48hr using the MTT assay. Treatment with melatonin at the dose of concentration (10-7-10-3mol/L) significantly inhibited cell proliferation in a concentration-dependent manner and time-dependent manner whereas melatonin at10-9mol/L did not decrease cell viability in either HepG2or SMMC-7721cells.(2)To examine the effect of melatonin on apoptosis,induction in HepG2and SMMC-7721cells was assessed by TUNEL staining and FACS assay at48hr after treatment. Consistent with the results of the MTT assay, treatment of cells with melatonin, at the doses of10-7-10-3mol/L significantly induced apoptosis, leading to increased induction of the apoptotic TUNEL-positive cells. Similarly, the PI staining based FCM assay also showed that melatonin, at the doses of10-7-10-3mol/L markedly increased induction of the apoptotic sub-G1cell population in HepG2and SMMC-7721cells. 2. The reversing mechanism of melatonin on apoptosis resistance in human hepatocellular carcinoma (1) The formalin-fixed, paraffin embedded hepatocellular carcinoma specimens were first analyzed for the expression of IAPs and COX-2by immunohistochemical staining. Of the100hepatocellular carcinoma specimens, Members of IAPs,XIAP,Survivin,cIAP-1,cIAP-2,Livin were positively stained in the nuclei and/or cytoplasm of tumor cells in76%,79%,83%,63%,17%of the hepatocellular carcinoma respectively.COX-2were positively stained in the nuclei and/or cytoplasm of tumor cells in71%of the hepatocellular carcinoma.It was revealed that cIAP-1, Survivin, XIAP,and cIAP-2staining was positive in91.55%,84.51%,81.69%,64.79%of COX-2-positive cases, respectively,whereas it was negative in37.93%,34.48%,37.93%,41.38%of COX-2negative cases, respectively. XIAP, Survivin and cIAP-1was significantly associated with overexpression of COX-2.(2)To examine the effect of NS-398/LY294002on apoptosis, induction in HepG2and SMMC-7721cells by a TUNEL reaction and FCM assay based at48hr after treatment. Treatment of cells with NS-398/LY294002significantly induced apoptosis, leading to increased induction of the apoptotic TUNEL-positive cells. The PI staining-based FACS assay also showed that NS-398/LY294002markedly increased induction of the apoptotic sub-G1cell population in HepG2and SMMC-7721cells.(3)The expression of COX-2, p-AKT, XIAP, Survivin and cIAP-1was conducted by western blot analysis.The COX-2selective inhibitor (NS-398) effectively blocked the phosphorylation of AKT and decreased the expression of XIAP and Survivin, but had no effect on the expression of cIAP-1in both HepG2and SMMC-7721cells. In addition, the effects of the PI3K inhibitor, LY294002, on the XIAP and Survivin expression were examined. Inhibition of AKT phosphorylation by LY294002also reduced the levels of XIAP and Survivin.(3) We evaluated the effect of melatonin on COX-2protein expression by western blot. Surprisingly, treatment for melatonin dramatically inhibited the expression of COX-2protein in a concentration-dependent manner concomitant with the dephosphorylation AKT in HepG2and SMMC-7721cells. To determine whether XIAP,Survivin,cIAP-1and cIAP-2could be altered by melatonin treatment, the expression of XIAP, Survivin, cIAP-1was assessed by western blot analysis. Melatonin at10-7-10-3mol/L significantly decreased the levels of XIAP and Survivin, but had no effect on the expression of cIAP-1in both HepG2and SMMC-7721cells.Part Ⅱ Melatonin reverses tunicamycin-induced endoplasmic reticulum stress in human hepatocellular carcinoma cells and improves cytotoxic response to doxorubicin by increasing CHOP and decreasing SurvivinMethods:(1)To examine effect of tunicamycin on cell viability and apoptosis induced by doxorubicin in HepG2andSMMC-7721cells.Growth inhibition rate of each group was detected using MTT assay.The apoptosis rate was deteceted by flow cytometry and the morphological change of apoptosis was observed by TUNEL.(2)To examine effect of co-pretreatment with tunicamycin and melatonin on cell viability and apoptosis induced by doxorubicin in HepG2and SMMC-7721cells.Growth inhibition rate of each group was detected using MTT assay.The apoptosis rate was deteceted by flow cytometry and the morphological change of apoptosis was observed by TUNEL(3) To clarify the reversing mechanism of melatonin on ER stress induced resistance to doxorubicin-induced apoptosis in hepatocellular carcinoma cells. The expression of p-AKT,CHOP and Survivin were assayed by Western-blot analysis.The apoptosis rate was deteceted by flow cytometry and the morphological change of apoptosis was observed by TUNEL.Results:1. Induction of ER stress protects hepatocellular carcinoma cells against apoptosis induced by doxorubicin (1) HepG2and SMMC-7721cells were pretreated with tunicamycin (3μmol/L) for8hr then exposed to different concentrations of doxorubicin(0,0.31,0.63,1.25,2.5and5mg/L) for24hr. Cell viability of HepG2and SMMC-7721cells was determined by the MTT assay. Pretreatment with TM significantly decreased doxorubicin-induced cytotoxicity in HepG2and SMMC-7721cells between0.31to5mg/L of doxorubicin.(2) HepG2cells were pretreated with3μmol/L tunicamycin for8hr and then exposed to doxorubicin (2.5mg/L) for24hr. Sub-G1analysis were determined by FACS and cell morphology and percentage of apoptotic cells was examined by TUNEL staining.Treatment of HepG2and SMMC-7721cells with doxorubicin resulted in a dramatic increase in the sub-G1cell population, which was significantly reduced in the presence of tunicamycin. Similar results were observed through TUNEL staining.2. Endoplasmic reticulum stress-induced resistance to doxorubicin is reversed by melatonin treatment in human hepatocellular carcinoma cells(1)HepG2and SMMC-7721cells were treated with3μmol/L tunicamycin for8hr,either in the absence or\the presence of (10-7to10-3mol/L) melatonin and then exposed to doxorubicin (0.63,1.25,2.5,5,10mg/L) for24hr. Cell viability was determined by the MTT assay.Melatonin at(10-5to10-mol/L) significantly increased doxorubicin-induced cytotoxicity when co-pretreated with tunicamycin in HepG2and SMMC-7721cells whereas melatonin at10-7mol/L did not induce changes in doxorubicin-induced cytotoxicity.(2) HepG2and SMMC-7721cells were treated with3μmol/L tunicamycin for8hr,either in the absence or\the presence of (10-3mol/L) melatonin and then exposed to doxorubicin (2.5mg/L) for24hr. Apoptosis was assessed by FCM analysis and TUNEL staining.Apoptosis induced by doxorubicin was increased by co-pretreatment of tunicamycin and melatonin. In this group the sub-G1percentage and the number of TUNEL-positive HCC cells indicative of apoptosis were increased compared to cells pretreated with tunicamycin alone. 3. The reversing mechanism of melatonin on ER stress induced resistance to doxorubicin-induced apoptosis in hepatocellular carcinoma cells (1) HepG2and SMCC-7721cells were treated with3μmol/L tunicamycin (TM) for0(control),4and8hr. Equal protein amounts of cell lysates were subjected to western blot assay using specific anti-p-AKT and anti-GRP78antibody. Administration of tunicamycin to HepG2and SMMC-7721cells induced an early increase in GRP78expression in both cells, indicative of ER stress. Simultaneously, the expression of p-AKT in both HepG2and SMMC-7721cells rapidly increased after treatment with tunicamycin concomitant with increasing the levels of Survivin,but but had no effect on the expression of CHOP in both HepG2and SMMC-7721cells.(2) HepG2cells were pretreated with3μmol/L tunicamycin for8hr, either in the absence or the presence of LY294002(30μmol/L) and then exposed to doxorubicin (2.5mg/L) for24hr. Apoptosis was analyzed as the sub-G1fraction by fluorescence-activated cell sorting (FACS) and Cell morphology and percentage of apoptotic cells was examined by TUNEL staining, apoptosis induced by doxorubicin was increased by co-pretreatment of tunicamycin and LY294002. In this group the sub-G1percentage and the number of TUNEL-positive HCC cells indicative of apoptosis were increased compared to cells pretreated with tunicamycin alone.(3) HepG2and SMMC-7721cells were treated with3μmol/L tunicamycin in either the absence (control) or the presence LY294002(30μmol/L) for8hr. Equal amounts of cell lysates were subjected to western blot analysis using specific anti-Survivin and anti-CHOP antibody. HepG2and SMMC-7721cells treated with the PI3K inhibitor, LY294002, in the presence of TM decreased the expression of Survivin and increased the expression of CHOP protein.(4) HepG2and SMMC-7721cells were treated with3μmol/L tunicamycin in either the absence (control) or the presence of melatonin (10-3μmol/L) for8hr. Equal amounts of cell lysates were subjected to western blot analysis using specific anti-phospho (p)-Akt antibody. HepG2and SMMC-7721cells treated with melatonin, in the presence of TM decreased the expression of (p)-Akt. Equal amounts of cell lysates were also subjected to western blot analysis using specific anti-Survivin and anti-CHOP antibody. HepG2and SMMC-7721cells treated with the melatonin, in the presence of TM increased the expression of CHOP protein and decreased the expression of Survivin.Conclusions:1. Melatonin overcomes apoptosis resistance in human hepatocellular carcinoma by targeting Survivin and XIAP via COX-2/PI3K/AKT pathway.2. Melatonin reverses endoplasmic reticulum stress-induced resistance to doxorubicin by increasing CHOP and decreasing Survivin via PI3K/AKT pathway in human hepatocellular carcinoma cells.