(−)-Englerin A-evoked Cytotoxicity Is Mediated by Na+ Influx and Counteracted by Na+/K+-ATPase

Melanie J Ludlow,Hannah J Gaunt,Hussein N Rubaiy,Katie E Musialowski,Nicola M Blythe,Naveen S Vasudev,Katsuhiko Muraki,David J Beech

doi:10.1074/jbc.m116.755678

Abstract

(−)-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 Ca2+-permeable, it was initially thought that the cytotoxicity arose as a consequence of Ca2+ 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 Ca2+ elevation, TRPC1 was not. TRPC1 either had no role or was a negative regulator of Ca2+ entry. By contrast, both TRPC4 and TRPC1 were necessary for monovalent cation entry evoked by (−)-EA, and (−)-EA-evoked cell death was dependent upon entry of the monovalent 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.

Highlights

(Ϫ)-Englerin A ((Ϫ)-EA)2 is a sesquiterpene from the bark of the African plant Phyllanthus engleri
Activation of an ion channel would be more consistent with the rapid (Ͻ1 h) onset of cell death induced by (Ϫ)-EA [7], and analysis of Ͼ500 well characterized cancer cell lines revealed that transient receptor potential canonical 4 (TRPC4) mRNA abundance is the feature best correlated with sensitivity to (Ϫ)-EA [6]
To investigate mechanisms of (Ϫ)-EA-induced cytotoxicity, we studied two (Ϫ)-EA-sensitive cancer cell lines: A498 renal cell carcinoma cells and Hs578T triple negative breast carcinoma cells (Fig. 1, A and B)

Summary

Introduction

(Ϫ)-Englerin A ((Ϫ)-EA)2 is a sesquiterpene from the bark of the African plant Phyllanthus engleri. Knockdown of TRPC4 suppressed (Ϫ)-EA-evoked cytotoxicity (Fig. 1, C and D). Current-voltage relationships of (Ϫ)-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].

Results

Conclusion