| Background Hepatocellular carcinoma(HCC)is the sixth most common cancer worldwide and the second leading cause of cancer-related mortality overall.It is reported that there are approximately 750,000 new cases of liver cancer per year.Molecularly targeted therapy has developed rapidly in recent years.The multikinase inhibitor sorafenib was the first systemic agent to prolong the median overall survival for patients with advanced HCC.However,sorafenib is beneficial in only approximately 30% of patients,and acquired resistance often develops within 6 months.Considering the current situation,it is valuable to investigate how to improve the effect of sorafenib.Melatonin,a derivative of tryptophan,was produced by the pineal gland,and other sources such as retina,gut,skin,platelets,lymphocytes,and bone marrow.The main physiological functions of melatonin are related with regulating circadian rhythm,immunomodulation,hematopoiesis,and antioxidation.In addition,a number of studies have reported that melatonin inhibits the growth of a variety of cancers including liver,lung,breast,prostate,and colon,and decreases the growth rates of tumors in vivo both in transplantable animal models and in animal models induced by the administration of carcinogens.Melatonin has been reported to be synergistic with other antitumor drugs.In our preliminary experiment,anti-tumor effects were enhanced by a combination treatment of melatonin and doxorubicin.Thus,it is imperative to study whether the effect of sorafenib could be enhanced by melatonin.Macroautophagy(also referred to as autophagy)is a bulk degradation system that recycles unnecessary or dysfunctional cellular components,such as proteins or organelles,for the maintenance of cellular homeostasis.However,programmed cell death instead of survival may be induced by excessively stimulation.Therefore,the role of autophagy may be a double-edged sword.Many studies have reported that sorafenib modulates autophagy in different in vitro and in vivo experiment models.It is reported that sorafenib-related cell resistance generation is correlated to autophagy,which may explain the unsatisfactory therapeutic efficacy of sorafenib.Inhibition of autophagy by the pharmacologic inhibitor-chloroquine(CQ)-could enhance antitumor effects or reverse resistance to targeted drugs.Therefore,changes in the levels of autophagy induced by a combination of sorafenib and melatonin areworth exploring.In the present study,our results revealed that sorafenib combined with melatonin have synergistic anti-tumor effects on HCC cell lines.We also found that autophagic activity induced by sorafenib was decreased after the addition of melatonin.Futhermore,inhibition of autophagy by CQ could further improve the anti-tumor effects of sorafenib alone or sorafenib combined with melatonin.Objective To evaluate whether the anti-tumor effects of sorafenib could be enhanced by melatonin on HepG2 and Bel-7402 cells.Moreover,the specific mechanisms of sorafenib combined with melatonin on tumor suppression was also investigatedMethods(1)Sorafenib at five concentrationsns for 24,48,72 hours was administrated to HCC cells,respectively.And the inhibition rate was detected by CCK-8 assay.(2)HCC cells were co-cultured either agent alone or combination 48 hours,then the inhitibiton rate was detected by CCK-8 assay.The coefficient of drug interaction(CDI) was used to assess the anti-proliferative effects.And the apoptosis rate of either agent alone or combination was measured by FCM assay.(3)Sorafenib(10μmol/L)and/or Melatonin(10-5mol/L)were administrated to HCC cells.The expression levels of Bcl-2、 Bax、LC3、P62 proteins were detected by Western-Blot assay.(4)HCC cells were treated with Melatonin(10-5mol/L)or/and Sorafenib(10μmol/L)for 48 h in the presence or absence of Chloroquine(5μg/ml).There are six groups:control group,melatonin group,sorafenib group,sorafenib combined with melatonin group,sorafenib with CQ,sorafenib combined with melatonin in the presence of CQ.The expression levels of LC3 and P62 were detected by Western-Blot assay.(5)The influence of sorafenib alone or combined with melatonin were explored by CCK-8 assay in the presence or absence of CQ.Apoptosis rate were detected by FCM.Results(1)Results showed that a dose-and time-dependent anti-proliferative effect was observed on both HepG2 and Bel-7402 cells.The IC50 of sorafenib were 13.21μM and11.83μM for HepG2 and Bel-7402 cells at 48 hours,respectively.(2)Combined treatment with the two agents increased tumor cell growth inhibition,as compared with treatment with either sorafenib or melatonin alone on HepG2 and Bel-7402 cells.We chosen sorafenib(10μM)and melatonin(10-5 mol/L)as the treatment concentrations,and CDI value was(0.827±0.09)for HepG2 cells at 48 hours.And CDI value was(0.91±0.05)when the concentration used was the same as for HepG2 cells.(3)Melatonin combined with sorafenib enhanced HepG2 and Bel-7402 cells apoptosis(52.5%±12.56%)compared with either melatonin(6.79%±2.34%)or sorafenib(36.8%±1.51%)alone by Annexin V/FITC-PI staining assay.Similar results were observed in Bel-7402 cells.The protein levels of Bcl-2 were reduced,while Bax were increased after exposing HepG2 cells to combination treatment compared with those in the control cells and those treated with melatonin or sorafenib alone.The ratio of Bcl-2/Bax in sorafenib combined with melatonin group was significantly decreased than that of control or either agent alone group.These data indicated that synergistic effects were induced by a combination of melatonin and sorafenib on HepG2 and Bel-7402 cells.(4)Sorafenib upregulated the activity of autophagy in HepG2 cells compared with the untreated cells.However,combination of melatonin decreased sorafenib induced autophagic activity compared with sorafenib alone.In addition,sorafenib decreased the expression levels of P62 proteins compared with either control or sorafenib combination with melatonin.While,the expression levels of P62 protein in the melatonin group and the control group showed no significant difference,which was consistent with the change in LC3-II/LC3-1 ratio.(5)Inhibition of autophagy by CQ increased the cytotoxicity of sorafenib alone or combined with melatonin compared with those in the absence of CQ.And Flow cytometry analysis showed that apoptotic cells were increased follwed by the addition of CQ.Conclusions(1)Sorafenib combined with melatonin have synergistic anti-tumor effects on HCC cell lines.(2)The synergistic effects may be attributed to melatonin which could reduce the activity of sorafenib induced autophagy.(3)Inhibition of autophagy by CQ could further improve the antitumor effects of sorafenib alone or combined with melatonin. |
PDF Full Text Request