Role of Copper Transporter ATP7B in Cisplatin Resistance in Human Hepatoma Cell Lines

Platinum-containing anticancer agents are widely used in the treatment of cancer. One of the major obstacles in effective treatment is the development of intrinsic or acquired drug resistance to chemotherapeutic agents. Overexpression of copper transporters ATP7A and ATP7B was associated with resistance of many tumors to cisplatin (DDP) treatment. ATP7B is primarily expressed in the liver and its role in hepatocellular carcinoma (HCC) remains unclear. The objective of the present study was to determine the effect of ATP7B expression in DDP resistance in different hepatoma cell lines. ATP7B was overexpressed in four different hepatoma cell lines HepG2, HuH-7, HepRG, and PLC/PRF/5. ATP7B mRNA expression was significantly increased in all ATP7B transfected cell lines. All four ATP7B overexpressing hepatoma cell lines did not show increased resistance to DDP. Drug sensitivity and induction of apoptosis was determined in HepG2 and ATP7B knockout (KO) cells. No significant differences were observed among the two cell lines. Next, DDP resistant cells (HepG2-R and KO-R) were generated by stepwise increase of DDP concentrations in the media. Both cell lines showed increased DDP resistance with normal growth rates compared to parental cell lines. Intracellular DDP accumulation and induction of apoptosis was significantly reduced in HepG2-R and KO-R. Gene expression analysis of copper homeostasis genes and other transporters revealed two candidate genes that were significantly affected in cisplatin resistant cells. Further experiments will explore DDP resistance mechanism by overexpression and downregulation of the candidate genes via plasmid and siRNA transfer. Our results show that native ATP7B and overexpressed ATP7B do not confer DDP resistance in HCC. Exploring mechanistic basis of transporter genes other than ATP7B may help explain resistance to chemotherapeutic drugs in HCC.