Tumor cell cholesterol depletion and V-ATPase inhibition as an inhibitory mechanism to prevent cell migration and invasiveness in melanoma

Abstract

Background: V-ATPase interactions with cholesterol enriched membrane microdomains have been related to metastasis in a variety of cancers, but the underlying mechanism remains at its beginnings. It has recently been reported that the inhibition of this H⁺ pump affects cholesterol mobilization to the plasma membrane.

Methods: Inhibition of melanoma cell migration and invasiveness was assessed by wound healing and Transwell assays in murine cell lines (B16F10 and Melan-A). V-ATPase activity was measured in vitro by ATP hydrolysis and H⁺ transport in membrane vesicles, and intact cell H⁺ fluxes were measured by using a non-invasive Scanning Ion-selective Electrode Technique (SIET).

Results: Cholesterol depletion by 5mM MβCD was found to be inhibitory to the hydrolytic and H⁺ pumping activities of the V-ATPase of melanoma cell lines, as well as to the migration and invasiveness capacities of these cells. Nearly the same effects were obtained using concanamycin A, a specific inhibitor of V-ATPase, which also promoted a decrease of the H⁺ efflux in live cells at the same extent of MβCD.

Conclusions: We found that cholesterol depletion significantly affects the V-ATPase activity and the initial metastatic processes following a profile similar to those observed in the presence of the V-ATPase specific inhibitor, concanamycin.

General significance: The results shed new light on the functional role of the interactions between V-ATPases and cholesterol-enriched microdomains of cell membranes that contribute with malignant phenotypes in melanoma.

Keywords: Antimetastatic target; Cholesterol rafts; Proton pumps.