Ouabain Exhibited Strong Anticancer Effects in Melanoma Cells via Induction of Apoptosis, G2/M Phase Arrest, and Migration Inhibition

Abstract

Background Malignant melanoma was characterized by insensitive chemotherapy, drug resistance, and high metastatic ability, which resulted in the main reason for the mortality among skin-related cancers. The current agents were not sufficient to improve the treatment status of melanoma patients, and it was still needed to develop new chemotherapeutic drugs for melanoma. Our study aimed to study the anticancer effects and potential mechanisms of ouabain on melanoma cells. Methods The inhibitory effects of ouabain were determined by CCK8 and colony formation assays, and the morphological changes of melanoma cells were observed by inverted microscope. The apoptosis induction and cell cycle distribution were detected by annexin V/PI double staining and PI staining, respectively. The expression of the biomarker proteins in apoptosis and G2/M phase were determined by Western blotting analysis. The effects of ouabain on the migration of melanoma cells were measured by transwell migration assay and wound closure analysis. The potential mechanisms of ouabain in melanoma cells were analyzed by transcriptome sequencing. Results Our present study demonstrated that ouabain exhibited strong inhibitory effects on cell proliferation and triggered dramatical morphological changes of melanoma cells. Moreover, ouabain induced significant apoptosis in A375 rather than SK-Mel-28 cells via upregulation of bax expression and downregulation of bcl-2 expression. Consistently, ouabain treatment induced cell cycle arrest at G2/M phase in both A375 and SK-Mel-28 cells via upregulation of cyclin B1 and downregulation of cdc2 and cdc25c. Importantly, ouabain suppressed the migration of A375 and SK-Mel-28 cells. Furthermore, the transcriptome sequencing demonstrated that p53 and MAPK signaling pathway might play important roles in the inhibitory effects of ouabain. Conclusion Our study revealed that ouabain exhibited dramatical anticancer effects, which provided a novel application for cardiac glycoside drugs in the clinical treatment of melanoma.

The cardiac glycoside ZINC253504760 induces parthanatos-type cell death and G2/M arrest via downregulation of MEK1/2 phosphorylation in leukemia cells

“…Moreover, ZINC253504760 arrested CCRF-CEM leukemia cells in the G2/M phase of the cell cycle. This result is comparable to reports from many other CGs, such as proscillaridin A-treated glioblastoma cells (Denicolaï et al 2014 ), ouabain-treated melanoma cells (Wang et al 2021 ), and lanatoside C-treated breast, lung, or liver cancer cells (Reddy et al 2019 ). Based on this fact, the induction of parthanatos and G2/M phase arrest in cell cycle are the best evidence to support that ZINC253504760 leads to DNA damage.…”

Effects of Ouabain in Ehrlich Tumor Development in vitro and in vivo

“…This does not exclude the fact that the OUA has previously reported antitumor activity. However, the OUA antitumor activity has been demonstrated only in human tumor cell lines as A549 cells, MCF7 cells, DU 145 cells, U-2 OS cells, A375 cells, SK-Mel-28 cells and U-87MG cells (Chang et al, 2019;Chou et al, 2018;Wang et al, 2021;Xiao et al, 2017;Yang et al, 2018). Here we evaluated the effect of OUA on murine cells that are about 1000 times more resistant to the cytotoxic effects of OUA compared to human cells due to a difference in the isoform of the α1 subunit of the Na+/K+ ATPase pump that makes murine cells less sensitive to the OUA (Akimova et al, 2015).…”

“…In addition, OUA has been described as a substance capable of inhibiting neuroinflammation. Moreover, in vitro studies have shown the antineoplastic potential of OUA in different human tumor cell lines as lung A549 cancer cells, breast MCF7 cancer cells, prostate DU 145 cancer cells, osteosarcoma U-2 OS cells, melanoma A375 and SK-Mel-28 cells, and glioma U-87MG cells (Chang et al, 2019;Chou et al, 2018;Wang et al, 2021;Xiao et al, 2017;Yang et al, 2018).…”