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

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 5 mM 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.