Abstract 5377: A role for Ctr1 in the synergistic interaction between mild hyperthermia and cisplatin treatment

Abstract

Abstract The ability of mild hyperthermia to synergistically enhance the chemotherapeutic effects of cisplatin has long been recognized, but the mechanism underlying this process remains unclear. It is known that hyperthermia sensitizes cells to the cytotoxic effects of cisplatin by increasing drug accumulation and subsequent platinum-DNA adduct formation. Here we investigate the role of the copper transporter Ctr1 in this process. Under normal metabolic conditions, Ctr1 maintains a homeostatic balance between copper levels inside and outside of the cell; however, Ctr1can also function as a cisplatin transporter. We hypothesize that the observed synergistic interaction between heat and cisplatin is partially due to the effect of heat on Ctr1. We used wild type (WT) and Ctr1 knockout (Ctr1-/-) mouse embryonic fibroblasts (MEFs) to test this hypothesis. These cells were treated with or without concurrent hyperthermia (42°C) and 100 µg/mL cisplatin for 1 hour and drug incubation for an additional hour. Cells were then harvested and total intracellular platinum levels were measured via ICP-AES (inductively coupled plasma atomic emission spectroscopy) to indicate cisplatin uptake. We observed higher platinum levels in the WT cells compared to the Ctr-/- MEFs with or without hyperthermia. Interestingly, there was a further increase in platinum levels in the WT MEFs after hyperthermia treatment that was not observed in the Ctr1-/- cells. The main mode of action for cisplatin cytotoxicity is the formation of platinum-DNA adducts, so we also examined adduct formation in cells treated with or without concurrent mild hyperthermia (42°C) and 50 µg/mL cisplatin for 1 hour and drug incubation for 3 additional hours. Using immunofluorescence staining, we observed a higher percentage of adduct-positive cells in the WT compared to the Ctr1-/- MEFs. As with our ICP-AES results, we observed an increase in adduct formation after hyperthermia in WT MEFs, however this was not observed in the Ctr1-/- MEFs. Published work has demonstrated that cisplatin exposure results in increased Ctr1 multimerization, suggesting a potential role for the multimerization process in cisplatin uptake. Using HEK293 cells expressing myc-tagged Ctr1, we demonstrated that hyperthermia increased the rate and the level of multimerization in the presence of cisplatin and that hyperthermia alone resulted in Ctr1 multimerization. Our data indicate that the increases in cellular cisplatin accumulation and platinum-DNA adduct formation observed after hyperthermia treatment may in part be due to increased Ctr1 activity. Cisplatin is currently used in the clinic to treat multiple types of cancer. Increasing cisplatin uptake in tumor cells by increasing cell surface Ctr1 levels may be useful in clinical settings for future cancer treatments. This work was supported by a grant from the NIH/NCI CA42745. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5377. doi:10.1158/1538-7445.AM2011-5377