| BackgroudPrimary liver cancer is a malignant tumor derived from epithelial tissue in the liver,and hepatocellular carcinoma(hepatocellular carcinoma HCC)is the highest degree of malignancy accounts for more than 90%.It is estimated that half of the global annual new cases and deaths of liver cancer occur in China.Therefore,research on HCC is significance.Studies have shown that cyclooxygenase-2(COX-2)plays a considerable effect on tumorigenesis,thus the targeted therapy of COX-2 in HCC patients may be a perspective treatment method.However,the conventional chemotherapy agents for HCC is substantially ineffective.Studies have demonstrated that autophagy is one reason of promoting tumor cell resistance to chemotherapeutic agents.Therefore,autophagy inhibition may increase tumor cells sensitive to chemotherapeutic agents.HBV is the most important pathogenic factors of HCC.Hepatitis B virus X gene(HBx)is a multifunctional regulatory proteins of hepatitis B virus and plays an important role in hepatocarcinogenesis.HBx protein can prevent p53-induced apoptosis by the way to activation COX-2/PGE2 path,but COX-2 inhibitors can prevent this effect and promote apoptosis.Moreover autophagy has significantly relate to hepatitis B virus replication.Accordingly,the inhibition of autophagy combined with COX-2 inhibitors can be used as a therapeutic target of HBV-related liver cancer.PurposeCOX-2 inhibitor combined with inhibition of autophagy increase apoptosis of HCC cells,especially for the treatment of HBV-related liver cancer.Through down regulation of HBx/Beclin1 autophagy pathway combined with HBx/COX-2 pathway inhibition the express of COX-2,and then upregulate the level of endoplasmic reticulum stress(ERS)via HBx/COX-2 pathway,cause the apoptosis of HCC.Method1.Observe the effection of COX-2 inhibitor celecoxib acting on the proliferation and apoptosis of HepG2 cells and HepG2 2.15 by MTT and Annexin-V + PI double staining method2.Observe the cisplatin-induced morphological changes of liver cancer autophagy inverted byMDC staining,transmission electron microscopy and inverted phase contrast microscope.Observe the effect of the proliferation and apoptosis with cisplatin alone,cisplatin combined with COX-2 inhibitor celecoxib,cisplatin combined with autophagy inhibitor 3-MA,and cisplatin combined with celecoxib united autophagy inhibitor 3-MA respectively.Compare the expression of autophagy-related gene Beclinl,LC3,apoptosis-related genes Bax,ERS-related gene GRP78 and CHOP and COX-2 by RT-PCR and Western blotting from mRNA levels and protein levels.3.Immunohistochemical staining method to detect the protein expression of HBx,COX-2 and Beclinl in 81 patients with HCC tissue samples,analyze the relationship between clinical pathological features.4.Construction of eukaryotic expression vector pEGFP-N1-HBx,transfected hepatoma cell line HepG2 and induce exogenous HBx protein overexpression.Oberseve the effection of the proliferation and apoptosis of HBX protein by MTT,Annexin-V + PI double staining.Compare the gene expression of HBx/Beclinl pathway,HBx/COX-2 pathway,ERS-related gene CHOP and apoptosis-related gene Bax by RT-PCR and Western blotting from mRNA levels and protein levels.Result1.Celecoxib dose and time dependent inbibition rate and apoptpsis rate of liver cancer cells2.The morphologica of liver cancer cells are shrinkaged,round,and the number of the adherent cells are decreasing observed by inverted phase contrast microscope after different concentrations of cisplatin.Autophagosome detected by MDC staining and TEM further confirmed that cisplatin-induced liver cancer autophagy.After the effect of cisplatin alone,cisplatin combined with COX-2 inhibitors celecoxib,cisplatin combined with autophagy inhibitor 3-MA,and cisplatin,celecoxib combined with autophagy inhibitor 3-MA,it can significantly increase the inhibition rate and apoptosis rate of liver cancer cell detected by MTT and Annexin-V + PI double staining method.However,autophagy inhibitor increase significantly in apoptosis of hepatoma cells.COX-2 inhibitor celecoxib can up-regulate the expression of the ERS-related gene GRP78 and CHOP protein,d ERS system induce the apoptosis of liver cancer cells by the induction of the apoptosis execution protein CHOP and further up-related the expression of apoptosis gene Bax.3.Immunohistochemical staining showed that HBx,Cox-2,Beclinl co-expressed in the tumor samples of HCC patients.In the cases of positive expression of HBX,Beclinl correlated to the pathology classify,and it not so sure in the negative.The expression of Beclinl correlated to size of the turmors(P<0.05).HBX,COX-2,Beclinl independent on the factors of age,gender,number of tumors,history of cirrhosis,AFP,vascular invasion,HBsAg,clinical stageConstruction of eukaryotic expression vector pEGFP-N1-HBx,transfected heatoma cell line HepG2,It is observed HBx can enhance cisplatin-induced autophagy.It also observed the HBx protein regulate liver cancer cell survival is dependent autophagy by MTT assays and Annexin-V+ PI double staining.It confirmed that inhibition of COX-2 and autophagy,final through the up-regulatr executive protein CHOP of ERS apoptosis to enhance the apoptosis of HCC,from mRNA levels and protein levelsConclusion1.As the target of liver cancer treatment,inhibition COX-2 has significant anti-proliferative and enhancing apoptosis roles in liver cancer cells.2.Inhibition of autophagy can significantly increase the sensitivity of apoptosis induced by cisplant.And HBx is the factor of promoting HCC survival in the induce HCC.3.It confirmed by the clinical pathology that HBx related to the expression of COX-2 and Beclinl4.The inhibitor of COX-2 combined with the inhibitor of autophagy can be the targeted therapy of HCC.Inhibition of autophagy down-regulate the level of autophagy through HBx/Beclinl pathway,inhibitor of COX-2 up-regulate the level of ERS through HBx/COX-2 pathway,which led to "cross-talk" of autophagy with ERS,ultimately induces liver cancer cells apoptosis... |
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