ABSTRACT
BACKGROUND: 17ßH-neriifolin, a cardiac glycoside compound had been successfully isolated from Cerbera odollam leaves based on the bioassay guided-isolation procedure. The aim of these studies were to determine the in vitro anti-cancer and binding effects of 17ßH-neriifolin on Na+, K+-ATPase. METHODS: The in vitro anti-cancer effects were evaluated using Sulphorhodamine B and Hoescht 33342 assays. The Na+, K+-ATPase assay was carried out using Malachite Green assay. In silico molecular docking studies and in vitro malachite green assay were used to predict the binding activities of 17ßH-neriifolin on Na+, K+-ATPase and ouabain was also included as for comparison studies. RESULTS: The compound was tested against breast (MCF-7, T47D), colorectal (HT-29), ovarian (A2780, SKOV-3) and skin (A375) cancer cell lines that gave IC50 values ranged from 0.022 ± 0.0015 to 0.030 ± 0.0018 µM. The mechanism of cell death of 17ßH-neriifolin was further evaluated using Hoescht 33342 assay and it was found that the compound killed the cancer cells via apoptosis. 17ßHneriifolin and ouabain both bound at α-subunit in Na+, K+-ATPase and their binding energy were - 8.16 ± 0.74 kcal/mol and -8.18 ± 0.48 kcal/mol respectively. CONCLUSION: The results had confirmed the anti-proliferative effects exerted by 17ßH-neriifolin in the breast, colorectal, ovarian and skin cancer cell lines. 17ßH-neriifolin had shown to cause apoptotic cell death in the respective cancer cell lines.17ßH-neriifolin and ouabain both bound at α-subunit in Na+, K+-ATPase and their binding energy were -8.16 ± 0.74 kcal/mol and -8.18 ± 0.48 kcal/mol respectively. This is the first report to reveal that 17ßH-neriifolin managed to bind to the pocket of α-subunit of Na+.K+-ATPase.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Cardenolides/pharmacology , Colorectal Neoplasms/metabolism , Ovarian Neoplasms/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Apocynaceae , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/drug therapy , Female , Humans , Molecular Docking Simulation , Ovarian Neoplasms/drug therapyABSTRACT
AIM: Abnormalities in apoptotic signalling pathways often occur in cancer cells and limit the successful chemotherapy outcomes in cancers. Therefore, there is an urgent need to discover new anticancer agents with novel mechanisms of action to overcome the resistance effect in chemotherapy. MATERIALS AND METHODS: In the present study, the anticancer effects and the mechanisms of action of 17ßH-neriifolin (cardiac glycoside) were evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and a proteomic approach in treated and non-treated SKOV-3 ovarian cancer cells. RESULTS: 17ßH-neriifolin was found to be active with IC50 values of 0.01 ± 0.001 in SKOV-3 ovarian cancer cell line, as evaluated by the sulforhodamine B (SRB) assay. RESULTS from TUNEL assay indicated that 17ßH-neriifolin caused apoptosis in SKOV-3 cells in a dose-dependent manner. Based on differential analysis of treated and non-treated SKOV-3 two-dimensional electrophoresis (2-DE) profiles, four proteins, namely vimentin (VIM), pyruvate kinase, muscle (PKM), heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) and transgelin (TAGLN1) were identified to be involved in apoptosis. Other proteins including piggybac transposable element derived 5 (PGBD5), DENN/MADD domain containing 2D (DENND2D) and formin-like 1(FMNL) have also been identified to be associated in SKOV-3 cell death induced by 17ßH-neriifolin. CONCLUSION: These findings may provide new insights on the potential of 17ßH-neriifolin's mechanism of action in killing ovarian cancer cells.
