Enhanced Inhibitory Effect Of Doxorubicin On Human Drug-Resistant Hepatocellular Carcinoma Xenograft In Nude Mice By Oxymatrine And Underlying Mechanism

Objective:The subaxillary skins of nude mice were subcutaneously inoculated with human doxorubicin-resistant hepatocellular carcinoma strain HepG2/ADM to establish subcutaneously tumor xenograft models in nude mice.Oxymatrine（OM）and doxorubicin（DOX）interventions were offered to explore whether oxymatrine can enhance the inhibitory effect of DOX on human drug-resistant hepatocellular carcinoma xenograft in nude mice and to explore its underlying mechanism.Methods:The subaxillary skins of nude mice were subcutaneously inoculated with human doxorubicin-resistant hepatocellular carcinoma strain HepG2/ADM.After the establishment of this model,twenty female nude mice were randomly allocated to four groups（five in each group）:normal saline（0.9%NS）group(0.2 ml·d^-1),doxorubicin group(6mg·kg^-1·d^-1),oxymatrine group(100 mg·kg^-1·d^-1),oxymatrine+doxorubicin group(100mg·kg^-1·d^-1+6 mg·kg^-1·d^-1).Those drugs were used by caudal vein injection in these nude mice(0.2 ml·d^-1).After the use of the drugs for 14 days,the tumors were taken out.（1）General conditions of the nude mice and growth situations of the tumors were recorded,and the inhibitory rates were calculated.（2）Hematoxylin-eosin staining was used to detect pathologic conditions of the tumors.（3）RT-PCR assay was applied to detect ABCB1 mRNA levels.（4）Immunohistochemical method and ELISA technique were used to examine the protein expressions of P-gp and Beclin 1.（5）SELDI-TOF mass spectrometry was used to test CpG island methylation status in ABCB1 promoter region.These research activities were approved by the ethics committee of our university.Results:（1）The subaxillary skins of nude mice were subcutaneously inoculated with humandoxorubicin-resistanthepatocellularcarcinomastrainHepG2/ADMand subcutaneous tumor xenograft models were established successfully in nude mice after 10days.OM could improve the quality of lives and minimize the impacts of DOX on weights in nude mice.The OM+DOX group had the strongest inhibitory effect on tumor xenografts in the four groups（P<0.05）.The OM group had the inhibitory effect that was second only to the OM+DOX group（P<0.05）on tumor xenografts.Nevertheless,comparison of tumor xenografts’volumes between DOX and NS group showed that there was no statistically significant difference（P>0.05）.So the DOX group had weaker inhibitory effect on tumor xenografts.The inhibitory rate in OM+DOX group was 74.5%and that in the OM was 47.5%.They were both better than that in the DOX group（0.2%）.（2）Hematoxylin-eosin staining showed that OM could promote necrosis in HepG2/ADM cells and this effect was enhanced after the use of DOX and OM.（3）From the results of ELISA and immunohistochemical method,the P-gp protein expressions in the OM+DOX and OM group were highly lower than those in the NS and DOX group（P<0.05）.And from the results of ELISA and immunohistochemical method,the Beclin 1 protein expressions in the OM+DOX and OM group were significantly higher than those in the NS and DOX group（P<0.05）.（4）From the results of RT-PCR,the ABCB1 mRNA expressions in the OM+DOX and OM group were significantly lower than those in the NS and DOX group（P<0.05）.（5）In most of the sites,the CpG island methylation status in ABCB1 promoter region showed that the methylation rates in the OM+DOX group and in the OM group were higher than those in the NS group（P<0.05）,but the methylation rates in the DOX group were lower than those in the NS group（P<0.05）.Conclusion:1.OM may improve the quality of lives of drug-resistant HepG2/ADM xenograft-bearing nude mice and minimize DOX side effects.2.OM can enhance the inhibitory effects of DOX on drug-resistant HepG2/ADM xenograft in nude mice.Its underlyingmechanismmaybeasfollow:（1）OMmayenhancethe CpG island methylation rates in ABCB1 promoter regions to reduce the ABCB1 mRNA and P-gp protein expressions.（2）OM may enhance the protein level of Beclin 1 to promote the autophagic death of HepG2/ADM cells.