The Mechanism And Role On The Enhanced Anti-tumor Effect Of Pirarubicin And Dichloroacetate In Hepatocellular Carcinoma Cells

Hepatocellular carcinoma(HCC)is one of the most common malignancies with high recurrence rate.Due to local infiltration and intrahepatic metastasis,the prognosis of patients with advanced hepatocellular carcinoma is very poor.Besides surgery and radiotherapy,chemotherapy including some molecular targeted drugs play important roles in the treatment of HCC.However,the exsisting strategies are not satisfactory in clinic.Therefore,it is urgent to investiagte the methods to enhance the effectiveness of chemotherapy for HCC.Pirarubicin Hydrochloride(THP)is a new generation of anthracycline anticancer drugs.THP presents the advantages of wide antineoplastic spectrum,strong anti-tumor activity and small toxicity.THP has been widely used in TACE embolization in clinic for the treatment of primary liver cancer.Improving the killing effect of THP on HCC cells would be beneficial for HCC patients.Dichloroacetate(DCA)is a pyruvate dehydrogenase(PDH)activator,which could reduce the lactic acid in clinic.As a classic therapeutic metabolite,more and more experiments show that DCA can also kill a variety of tumor cells in vitro and in vivo.In addition,DCA can significantly enhance the anti-tumor effect of multiple chemotherapeutic drugs.However,wether DCA could enhance the cytocidal effect of THP has not been reported.Therefore,the purpose of this study is to investigate the synergetic effect of DCA and THP on HCC cells,and the related molecular mechanisms,which may provide some theoretical basis for clinical treatment for HCC.Contents and methods:Part ? DCA enhances the antitumor effect of THP in HCC cells1.The cell viability of HCC was detected with or without DCA and THP treatments.2.Studying the type of cell death after treatment with DCA and THP on HCC cells.Part ? The combinated treatment of DCA and THP activates ROS/JNK pathway1.To investigate the mechanism of apoptosis induced by combination of DCA and THP,the level of ROS in HCC cells treated with DCA and THP was determined using DCFH-DA probes.2.The activation of JNK was detected by Western blot to research the potencial signaling pathway related to the synergetic effect of DCA and THP.3.To further elucidate the relationship between ROS and JNK activation,the cell viability and cell death were measured under the combited treatments of DCA and THP in the presence or absence of antioxidant NAC and JNK kinase inhibitor SP600125.Part ? Autophagy attenuates the synergetic Effects effect of DCA and THP1.Besides inducing apoptosis,ROS / JNK pathway can also triger autophagy.To investigete the effect of the co-treatments of DCA and THP on autophagy,the protein level of LC3 and P62 were detected using western blot assay.2.The autophagy inhibitor CQ was added to test the role of autohagy on cell viability and cell death.According to the above expriments,the main methods used in this study are as follows:1.Cells culture and passage of HCC cell lines HepG2,Hep3 B and 7721.2.Identification of growth inhibition with CCK-8 assay.3.Identification of the cell death proportion with trypan blue exclusion assay.4.Detection cell apoptosis with flow cytometry and nucleus stained assay.5.Western blot was used to detect the expression of total proteins.6.Construction of plasmids with molecular cloning technology.7.Transient tranfection to change the gene expression level.8.Observation and calculation of the GFP punctate in GFP-LC3 transfected cells.Results:1.DCA significantly enhanced the anti-tumor effect of THP on HCC cells.2.DCA promoted THP-induced apoptosis of HCC cells.3.DCA combined with THP activated ROS-JNK pathway in HCC cells.4.ROS-JNK pathway contributes to the synergetic effect of DCA and THP.5.ROS-JNK pathway may also trigger autophagy that attuate the response of HCC cells to the co-treatments of THP and DCA.Conclusion:DCA significantly enhanced the anti-HCC effect of THP,the associated mechanism may refer to the activation of ROS/JNK pathway.DCA combined with THP can significantly promote the THP-induced ROS levels,which promotes the activation of JNK and subsequent cell death.In addidtion,the activation of ROS/JNK pathway may coinstantaneously trigger autophagy,which attenuats the synergetic anti-tumor effect of DCA plus THP.The study provides a new theoretical basis for the clinical treatment of HCC.