Abstract 843: Influx of thiosemicarbazone NSC73306 is mediated by the cisplatin and copper transporter SLC31A1 (CTR1)

Abstract

Multidrug resistance (MDR) mediated via the drug efflux pump P-glycoprotein (P-gp/ABCB1) overexpression is a major obstacle to the success of chemotherapy to combat many cancers. Clinically, P-gp expression correlates with poor clinical response to chemotherapy in patients, and its presence on the blood-brain barrier (BBB) can reduce the efficacy of otherwise promising agents targeted to the brain. Attempts to reverse drug resistance by inhibiting P-gp have not been successful, and we have been investigating alternative strategies for targeting multidrug-resistant (MDR) cancer cells. One such strategy is the use of NSC73306, a thiosemicarbazone compound that is selectively toxic towards P-gp-expressing cells. Here, we investigate the cellular uptake of NSC73306, and examine its interaction with P-gp and copper transporter 1 (SLC31A1, CTR1). In porcine kidney epithelial LLC-PK1 cells, cellular accumulation of NSC73306 is saturable, and overexpression of human P-gp in LLC-PK1 cells sensitizes them to NSC73306. Accumulation of [3H]NSC73306 in LLC-PK1 cells is inhibited by cyclosporin A (IC50 = 500 μM), verapamil (IC50= 700 μM) and cisplatin (IC50 = 77 μM). Among these compounds, we tested the effect of cisplatin on NSC73306. Selective toxicity of NSC73306 to P-gp-expressing LLC-PK1 cells was reversed by cisplatin in a dose-dependent manner (up to 40 μM). In contrast, the amount of cisplatin required to kill 50% of LLC-PK1 cells was not affected by increasing the amount of NSC73306 (up to 2μM). Since cisplatin could inhibit NSC73306 uptake, we hypothesize that these two compounds share a common uptake pathway, but cisplatin has additional routes of entry into cells. Cells transiently expressing CTR1 showed significantly higher [3H]NSC73306 accumulation. In contrast, CTR1 knockdown cells had lower [3H]NSC73306 uptake. Although cisplatin is a transport substrate of CTR1, we found that CTR1 protein was overexpressed in cisplatin-resistant KB-CP20 and BEL7404-CP20 cell lines. Compared to their parental cisplatin-sensitive cells, even though these resistance cells show decreased cisplatin accumulation, suggesting that CTR1 is not rate-limited for cisplatin uptake. These cell lines also showed higher [3H]NSC73306 uptake compared to their parental cells. In contrast, MDR cell lines overexpressing P-gp had unchanged CTR1 protein levels. Incubation with NSC73306 for 24 hours down-regulated CTR1 in a dose-dependent manner. These results suggest that NSC73306 is a transport substrate of CTR1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 843. doi:1538-7445.AM2012-843