Biochimica et biophysica acta. General subjects · 2017
Tumor cell cholesterol depletion and V-ATPase inhibition as an inhibitory mechanism to prevent cell migration and invasiveness in melanoma.
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Costa Gildeíde Aparecida, de Souza Sávio Bastos, da Silva Teixeira Layz Ribeiro, Okorokov Lev A, Arnholdt Andrea Cristina Vetö, Okorokova-Façanha Anna L, Façanha Arnoldo Rocha
Аннотация
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.
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.