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Dihydromotuporamine C increases Na⁺/H⁺exchange activity via a Rho-kinase-dependent pathway and induces disruption of intercellular junctions in mammary epithelia Sinotte, Ryan Richard

Abstract

Invasion and angiogenesis are two major steps in the progression towards metastatic cancer. Dihydromotuporamine C (dh-MotC) inhibits both of these processes. To better characterize the mechanism of dh-MotC action, I examined the compound's effects on Na+/H+ exchange (NHE) activity and normal mammary epithelial cell viability and junctional architecture. In contrast to squalamine, an anti-angiogenic compound that inhibits Na+/H+ exchanger isoform 3 (NHE3), dh-MotC (5 or 10μM) increased the rate of recovery from an imposed acid load in nominally bicarbonate free conditions. This increased rate of recovery was inhibited by HOE694, a selective NHE inhibitor, and the Rho-kinase (ROCK) inhibitor Y-27632. Dh-MotC also induced an upward shift in steady state intracellular pH (pHi) of 0.14 +/- 0.02 pH units (n=6), which was not sensitive to ROCK inhibition. Therefore, dh-MotC activates Na+/H+ exchange via a ROCK-mediated cascade, and increases steady-state pHj in a ROCK-independent manner. Dh-MotC has been described as a Rho activator and is known to induce stress fibre and focal adhesion formation in serum-starved Swiss 3T3 cells. Inhibition of Na+/H+ exchange by HOE694 did not prevent dh-MotC-induced formation of stress fibres or focal adhesions. Similarly, the dh-MotC-mediated inhibition of tumour cell migration was not prevented by co-treatment with HOE694. These data suggest that increased Na+/H+ exchange activity is not required for dh-MotC induced stress fibres/focal adhesions and that dh-MotC inhibits tumour cell migration via a pathway that is independent of Na+/H+ exchange. Concentrations of dh-MotC that are effective at preventing tumour cell migration were toxic to normal mammary epithelial cells cultured on three dimensional basement membrane gels as evidenced by increased levels of active caspase-3 staining in dh-MotC treated mammary spheroids. Adherens and tight junction architecture was also disrupted under these conditions as evidenced by the loss of junctional E-cadherin and apically polarized ZO-1, components of adherens and tight junctions, respectively. In two-dimensional monolayer mammary epithelial cell cultures, dh-MotC did not affect cell morphology or localization of Ecadherin and ZO-1. Thus, there appears to be a differential response to dh-MotC between monolayer and spheroid cell culture.

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