Cytotoxicity of withasteroids: withametelin induces cell cycle arrest at G2/M phase and mitochondria-mediated apoptosis in non-small cell lung cancer A549 cells

Abstract

Considerable interest has been gained by withasteroids because of their structural uniqueness and wide spectrum of biological activities. However, limited systematic studies for proving their cytotoxic potential have so far been reported. Hence, an attempt was made to test the cytotoxicity of six withasteroids viz., withametelin (WM), withaphysalin D, withaphysalin E, 12-deoxywithastramonolide, Withaperuvin B, and physalolactone against A549, HT-29, and MDA-MB-231 cancer cell lines. Significant cytotoxic effect of WM against A549 cells (IC value of 6.0 μM), MDA-MB-231 cells (IC value of 7.6 μM), and HT-29 cells (IC value of 8.2 μM) was observed. Withaperuvin B and physalolactone were found to be effective against MDA-MB-231 cells. The significantly active WM arrested the A549 cells at G2/M phase and downregulated the expression of G2/M regulatory proteins such as cdc2, cyclin B1, and cdc25C. Apoptosis induced by WM in A549 cells was associated with the generation of ROS and depletion of MMP. Furthermore, WM treatment resulted in Bax upregulation, Bcl-2 downregulation, translocation of cytochrome c to mitochondria, activation of caspase-9 and -3, and PARP cleavage corroborating the apoptosis induction through intrinsic apoptotic pathway. Thus, WM possessing broader cytotoxic effect is a promising lead molecule which has the potential to be developed as a new therapeutic agent for NSCLC.

Sinularin induces oxidative stress‐mediated G2/M arrest and apoptosis in oral cancer cells

“…Its apoptosis was based on annexin V/PI analysis and reached to about 20% for annexin V positive. In contrast, several drugs were reported to G2/M arrest and apoptosis without detectable subG1 accumulation, such as erianin in human osteosarcoma cells, evodiamine in lung cancer H446 and H1688 cells, withametelin in lung cancer A549 cells, and diarylpyrazole derivative (AM251) in A375 cells . Similarly, we found that sinularin induced G2/M arrest and apoptosis without detectable subG1 accumulation in oral cancer cell at 24 h treatment, where the apoptosis was confirmed by flow cytometry (annexin V/PI and pancaspase analyses) and western blotting (caspases 3, 8, 9, and PARP).…”

Sinularin Selectively Kills Breast Cancer Cells Showing G2/M Arrest, Apoptosis, and Oxidative DNA Damage

“…Although, we could not confirm an increase for subG1 population, sinularin induced apoptosis in breast cancer cells as validated by annexin V/7AAD, pancaspase analyses and Western blotting. Drugs such as evodiamine [ 28 ], withametelin [ 29 ], and (−)-anonaine [ 30 ], also cause apoptosis without subG1 accumulation. Interestingly, the proportion of drug-induced apoptotic cells may change over time for subG1 accumulation [ 30 ].…”

Development of a Novel Multi-Isoform ALDH Inhibitor Effective as an Antimelanoma Agent

“…Cell viability assays of UACC 903 cells transfected with siRNA, and melanoma cells (UACC 903, 1205 Lu, C8161.CI9, MelJuSo), normal human fibroblasts (FF2441), and melanocytes (NHEM) treated with ALDH inhibitors were performed as described previously (43)(44)(45). Briefly, 5,000 cells per well were plated in a 96-well plate and incubated overnight at 37 C in a 5% CO 2 atmosphere.…”

“…To quantify ROS levels, the nonfluorescent dye DCFDA (Sigma) was used (43). DCFDA turns to highly fluorescent 2 0 ,7 0 -dichlorofluorescein upon oxidation by ROS generated in cells (43). Briefly, cells were treated with 5 mmol/L of ALDH inhibitor or DMSO for 24 hours.…”

“…The Annexin-V-PE/7-AAD kit (Millipore) was used to distinguish live cells from apoptotic cells as described previously (43). Briefly, cells were incubated with 5 mmol/L of ALDH inhibitor or DMSO for 24 hours.…”