()-Englerin A-evoked Cytotoxicity Is Mediated by Na  Influx and Counteracted by Na  /K  -ATPase * Received for publication, August 29, 2016, and in revised form, November 9, 2016 Published, JBC Papers in Press, November 14, 2016, DOI 10.1074/jbc.M116.755678 Melanie J. Ludlow ‡ , Hannah J. Gaunt ‡ , Hussein N. Rubaiy ‡ , Katie E. Musialowski ‡ , Nicola M. Blythe ‡ , Naveen S. Vasudev ‡ , Katsuhiko Muraki § , and David J. Beech ‡1 From the ‡ Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, Leeds LS2 9JT, United Kingdom and the § School of Pharmacy, Aichi-Gakuin University, 1-100 Kusumoto, Chikusa, Nagoya 464-8650, Japan Edited by Roger J. Colbran ()-Englerin A (()-EA) has a rapid and potent cytotoxic effect on several types of cancer cell that is mediated by plasma membrane ion channels containing transient receptor potential canonical 4 (TRPC4) protein. Because these channels are Ca 2 - permeable, it was initially thought that the cytotoxicity arose as a consequence of Ca 2 overload. Here we show that this is not the case and that the effect of ()-EA is mediated by a heteromer of TRPC4 and TRPC1 proteins. Both TRPC4 and TRPC1 were required for ()-EA cytotoxicity; however, although TRPC4 was necessary for the ()-EA-evoked Ca 2 elevation, TRPC1 was not. TRPC1 either had no role or was a negative regulator of Ca 2 entry. By contrast, both TRPC4 and TRPC1 were neces- sary for monovalent cation entry evoked by ()-EA, and ()- EA-evoked cell death was dependent upon entry of the mono- valent cation Na  . We therefore hypothesized that Na  / K  -ATPase might act protectively by counteracting the Na  load resulting from sustained Na  entry. Indeed, inhibition of Na  /K  -ATPase by ouabain potently and strongly increased ()-EA-evoked cytotoxicity. The data suggest that ()-EA achieves cancer cell cytotoxicity by inducing sustained Na  entry through heteromeric TRPC1/TRPC4 channels and that the cytotoxic effect of ()-EA can be potentiated by Na  /K  - ATPase inhibition. (-)-Englerin A ((-)-EA) 2 is a sesquiterpene from the bark of the African plant Phyllanthus engleri. Its isolation was first reported in 2009 alongside an NCI60 human tumor cell line screen demonstrating its rapid cytotoxic potential with potency in particular against renal cell carcinoma cell lines (1). Cur- rently approved treatments for renal cell carcinoma are of lim- ited efficacy and are often toxic. Therefore, there has been keen interest in the discovery of (-)-EA and methods for its synthe- sis (2–5). A larger screen revealed that (-)-EA kills cells from a wide range of lineages but again supported the absence of adverse effects on normal, non-cancerous cells (6). Efforts to identify the cellular target of (-)-EA have suggested that it acti- vates PKC and the Ca 2+ -permeable non-selective channels formed by assembly of transient receptor potential canonical (TRPC) proteins TRPC4 and TRPC5 (6 – 8). PKC was involved in (-)-EA-induced cell death in the 786-0 renal carcinoma cell line (8) but was barely detectable in other (-)-EA-sensitive cells (A498 renal carcinoma and A637 Ewing’s sarcoma-derived cell lines) (6, 7), and its proposed mechanism of action, promoting dependence on glucose while simultaneously starving cells of glucose (8), involves relatively slow gene regulatory events. Activation of an ion channel would be more consistent with the rapid (1 h) onset of cell death induced by (-)-EA (7), and analysis of 500 well characterized cancer cell lines revealed that TRPC4 mRNA abundance is the feature best correlated with sensitivity to (-)-EA (6). In support, knockdown of TRPC4 in A498 and A673 cells offered protection against (-)- EA-induced cell death (6). The related TRPC5 protein is, in contrast, rarely overexpressed in tumor cell lines (6). Notably, the latter study identified a few exceptions, cells with enhanced TRPC4 but low sensitivity to (-)-EA, indicating that up-regu- lation of TRPC4 is not the sole factor imparting (-)-EA sensi- tivity. Although ion channels are increasingly suggested to have important roles in cancer development and progression, inhib- itors, rather than activators, represent the more obvious approach for drug discovery. Therefore, it is important to better understand how activation of TRPC4-containing channels by (-)-EA results in rapid cancer cell death. Results To investigate mechanisms of (-)-EA-induced cytotoxicity, we studied two (-)-EA-sensitive cancer cell lines: A498 renal cell carcinoma cells and Hs578T triple negative breast carci- noma cells (Fig. 1, A and B). In both cell types, knockdown of TRPC4 suppressed (-)-EA-evoked cytotoxicity (Fig. 1, C and D). Current-voltage relationships of (-)-EA-evoked responses in A498 and Hs578T cells lacked the seatlike inflection of TRPC4 homomers, suggesting that the TRPC4 proteins are in heteromers with TRPC1 (Fig. 2, A and B) (7). To test the prin- ciple of whether (-)-EA can activate TRPC1/TRPC4 hetero- mers, a stable human embryonic kidney (HEK) 293 cell line inducibly expressing a TRPC4-TRPC1 concatemer was estab- * This work was supported by the Leeds Teaching Hospitals Trust Charitable Foundation and the University of Leeds. The authors declare that they have no conflicts of interest with the contents of this article. Author’s Choice—Final version free via Creative Commons CC-BY license. 1 To whom correspondence should be addressed: LIGHT Laboratories, Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, Leeds LS2 9JT, UK. Tel.: 44-113-343-4323; E-mail: D. J.Beech@leeds.ac.uk. 2 The abbreviations used are: (-)-EA, (-)-englerin A; TRPC, transient receptor potential canonical; NMDG, N-methyl-D-glucamine; EC 50 , half-maximal effective concentration; SBS, standard bath solution; ANOVA, analysis of variance. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 2, pp. 723–731, January 13, 2017 Author’s Choice © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. crossmark JANUARY 13, 2017 • VOLUME 292 • NUMBER 2 JOURNAL OF BIOLOGICAL CHEMISTRY 723 at UNIVERSITY OF LEEDS on February 22, 2017 http://www.jbc.org/ Downloaded from