Reversal of Adriamycin Resistance of Hepatocellular Carcinoma by Targeting with Recombined Adenovirus Caning Antisense mdr1 RNA

OBJECTIVE Chemotherapy is an important therapy for hepatocellular carcinoma (HCC). However, it is not effective in many cases due to recurrence and metastasis even if the initial treatment produces a response. Multidrug resistance (MDR) is considered to be one of the considerable causes. The aim of this study was to reverse MDR of HepG2/ADM cells by blocking mdr1 with an adenovirus vector carrying antisense mdr1 in a tumor transplantated in athymic mice. METHODS pCMV IE was removed from the pshuttle vector. A 0.3 kb AFP promoter was inserted into the pshuttle vector and pCMV changed into pAFP. The pAFP and asmdr1 PCR products were doubly digested with Kpnl and Apal, the digested products were ligated by T4 ligase, the asmdrl gene was inserted into pAFP and a newly plasmid pAFP-asmdr1 was constructed. Following digestion with PI-Scel/l-Ceu l, pAFP-asmdr1 was ligated with Adeno-X genome DNA and amplified in E.coli XL 1-Blue. The HEK293 cells were transfected and virus collected. The HepG2 MDR cells (HepG2/ADM) were induced by graded resistance to ADM and were inoculated into athymic mice. After adeno-asmdr1 was injected, the expression of mdr1 -mRNA and the volume of the transplantated tumor and its cells were observed. RESULTS Following injection with Adeno-asmdr1, the tumor volume in the ADM+Adeno-asmdr1 group did not increase. However the tumor volume in the PBS plus ADM group did significantly increase (P<0.05). In the tumor xenograft cells, mdr1 mRNA in the xenografts was assessed by RT-PCR and was found to be reduced at 1 week and 4 weeks in the ADM+asmdr1 group, but it was stable in the ADM group. It was only 20% in the ADM+asmdr1 group compared to the ADM group at the 4th week (P<0.05). Evidence of apoptosis was observed in the tumor xenograft cells treated with Adeno-asmdr1, but there was rare or no apoptosis in the group treated with ADM and PBS. CONCLUSION Adenovirus carrying antisense mdr1 RNA can partially reverse the MDR of HepG2/ADM cells and inhibit tumor growth by down-regulating mdr1 mRNA resulting in tumor cell apoptosis.

OBJECTIVE Chemotherapy is an important therapy for hepatocellular carcinoma （HCC）. However, it is not effective in many cases due to recurrence and metastasis even if the initial treatment produces a response. Multidrug resistance （MDR） is considered to be one of the considerable causes. The aim of this study was to reverse MDR of HepG2/ADM cells by blocking mdr1 with an adenovirus vector carrying antisense mdr1 in a tumor transplantated in athymic mice. METHODS PCMV IE was removed from the pshuttle vector. A 0.3 kb AFP promoter was inserted into the pshuttle vector and pCMV changed into pAFP. The pAFP and asmdr1 PCR products were doubly digested with Kpnl and Apal, the digested products were ligated by T4 ligase, the asmdr1 gene was inserted into pAFP and a newly plasmid pAFP-asmdr1 was constructed. Following digestion with PI-SceI/I-Ceu I, pAFP-asmdr1 was ligated with Adeno-X genome DNA and amplified in E.coli XL1-Blue. The HEK293 cells were transfected and virus collected. The HepG2 MDR cells （HepG2/ADM） were induced by graded resistance to ADM and were inoculated into athymic mice. After adeno-asmdr1 was injected, the expression of mdr1-mRNA and the volume of the transplantated tumor and its cells were observed. RESULTS Following injection with Adeno-asmdr1, the tumor volume in the ADM＋Adeno-asmdr1 group did not increase. However the tumor volume in the PBS plus ADM group did significantly increase （P〈0.05）. In the tumor xenograft cells, mdr1 mRNA in the xenografts was assessed by RT-PCR and was found to be reduced at 1 week and 4 weeks in the ADM＋asmdr1 group, but it was stable in the ADM group. It was only 20% in the ADM＋asmdr1 group compared to the ADM group at the 4th week （P〈0.05）. Evidence of apoptosis was observed in the tumor xenograft cells treated with Adeno-asmdr1, but there was rare or no apoptosis in the group treated with ADM and PBS. CONCLUSION Adenovirus carrying antisense mdr1 RNA can partially reverse the MDR of HepG2/ADM cells and inhibit tumor growth by down-regulating mdr1 mRNA resulting in tumor cell apoptosis.