Purpose: Hyperthermia enhances cytotoxic effects of chemotherapeutic agents such as cisplatin. However, the underlying molecular mechanisms remain unclear. We hypothesised that hyperthermia increases cisplatin accumulation and efficacy by modulating function of copper transport protein 1 (Ctr1), a major regulator of cellular cisplatin uptake. We examined the significance of Ctr1 in the synergistic interaction between hyperthermia and cisplatin. We assessed the importance of cisplatin- and hyperthermia-induced Ctr1 multimerisation in sensitising cells to cisplatin cytotoxicity.
Materials and methods: Ctr1 protein levels and cisplatin sensitivities were assessed in bladder cancer cell lines with immunoblotting and clonogenic survival assays. Using Myc-tagged-Ctr1 HEK293 cells, we assessed the effect of hyperthermia on Ctr1 multimerisation with immunoblotting. The effect of hyperthermia on cisplatin sensitivity and accumulation was assessed in wild-type (WT) and Ctr1 knockout (Ctr1-/-) mouse embryonic fibroblasts (MEFs) with clonogenic assays and inductively coupled plasma-mass spectrometry (ICP-MS).
Results: Increased Ctr1 protein expression was observed for the most cisplatin-sensitive bladder cancer cell lines and MEFs. Heat-induced increase in Ctr1 multimerisation with cisplatin was observed in Myc-tagged Ctr1 cells. Hyperthermia enhanced cisplatin-mediated cytotoxicity in WT more than Ctr1-/- cells (dose modifying factors 1.75 versus 1.4, respectively). WT cells accumulated more platinum versus Ctr1-/- cells; this was further increased by hyperthermia in WT cells.
Conclusions: Hyperthermia enhanced cisplatin uptake and cytotoxicity in WT cells. Heat increased Ctr1 activity by increasing multimerisation, enhancing drug cytotoxicity. Furthermore, Ctr1 protein profiles of bladder tumours, as well as other tumour types, may predict their response to cisplatin and overall efficacy of treatment.