Abstract
It has been demonstrated that numerous types of metastatic cancer overexpress vacuolar-type H+ (V)-ATPases. It may be possible to inhibit the growth and metastasis of human cancer cells by inhibiting V-ATPases. It was previously reported that diphyllin, a novel V-ATPase inhibitor, can inhibit the migration and invasion of SGC7901 human gastric cancer cells; however, the effects of cleistanthin A (CA), a diphyllin glycoside, on melanoma cells has not been demonstrated. The present study aimed to investigate the effect of CA as a V-ATPase inhibitor and its effects on the invasion and metastasis of A375 cells. The results of an MTT assay in the present study indicated that the growth inhibition of A375 cells by CA was induced in a dose- and time-dependent manner; however, A375 cell viability was not significantly affected by low concentrations (0.03, 0.1 and 0.3 µM) after 24 h. Similar results were obtained by viable cell counting with trypan blue. Therefore, these concentrations of CA were selected for the treatment of A375 cells in further experiments. It was demonstrated that CA inhibited the expression of V-ATPases in a dose-dependent manner and decreased the internal pH level of A375 cells. Alterations to the lysosomal pH were associated with the CA concentration. Furthermore, CA treatment induced a significant decrease in cell migration and invasion, as demonstrated with wound-healing and Transwell assays. Gelatin zymography and western blot analysis demonstrated that the expression levels of matrix metallopeptidase (MMP)-2 and -9 decreased following CA treatment. Therefore, CA can be characterized as a novel V-ATPase inhibitor for the treatment of melanoma that may inhibit invasion and metastasis by downregulating the expression of MMP-2 and -9.