Cisplatin-dependent modulation of transport proteins in an ATP7B knockout cell line

Resistance of platinum-based anticancer agents like cisplatin (cp) was recently associated with transport proteins of copper homeostasis. In hepatocytes, excess copper is eliminated in bile via copper transporter ATP7B. High expression of ATP7B was proposed to confer resistance to cp. Knowledge of cp transporter genes is indispensable for improvement of cancer therapy and overcoming resistance. The aim of the present study was the identification of transporters other than ATP7B that are involved in hepatic cp resistance.

A recently established HepG2 ATP7B knockout (KO) cell line (Chandhok et al. PlosOne (2014)9:e98809) was used to generate a cisplatin resistant hepatoma cell line (CpR). CpR cells were characterized via cell viability (MTT), apoptosis (Annexin V), growth and intracellular cp level. RT-qPCR was used for analysis of gene modulation following cp exposure. Overexpression and transient knockdown (siRNA) of genes was employed to assess the impact of individual transporters.

Cultivation of KO cells in the presence of increasing concentrations of cp resulted in CpR cells. Viability and growth were increased as compared to parental cells when cp concentrations up to 4µM were used. Apoptosis and intracellular cp were significantly reduced. Analysis of CpR cells by RT-qPCR using a panel of genes involved in copper homeostasis and metal transport revealed a downregulation of organic cation transporter 3 (OCT3) (5.17 ± 2 fold). Using overexpression of OCT3, it was shown that resistance could be almost completely reversed. In addition, a cross-resistance was observed when cells were exposed to toxic copper.

The data suggest that OCT3 has a major role for establishment of cp and copper resistance in hepatocytes, at least in the absence of functional ATP7B.