4ß-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells.

Reactive oxygen species (ROS) induction had been previously reported in 4ß-hydroxywithanolide (4ßHWE)-induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4ßHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8-oxo-2'deoxyguanosine [8-oxodG]) were detected in 4ßHWE-treated oral cancer (Ca9-22) and/or normal (HGF-1) cells. 4ßHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N-acetylcysteine (NAC). For immunofluorescence, 4ßHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4ßHWE-treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine-DNA glycolyase (Fpg)-based comet assay and 8-oxodG-based flow cytometry, the 8-oxodG expressions were higher in 4ßHWE-treated oral cancer cells than in oral normal cells. All the 4ßHWE-induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4ßHWE selectively induced DSB and oxidative DNA damage for the ROS-mediated selective killing of oral cancer cells.

Golden berry-derived 4ß-hydroxywithanolide E for selectively killing oral cancer cells by generating ROS, DNA damage, and apoptotic pathways.

BACKGROUND: Most chemotherapeutic drugs for killing cancer cells are highly cytotoxic in normal cells, which limits their clinical applications. Therefore, a continuing challenge is identifying a drug that is hypersensitive to cancer cells but has minimal deleterious effects on healthy cells. The aims of this study were to evaluate the potential of 4ß-hydroxywithanolide (4ßHWE) for selectively killing cancer cells and to elucidate its related mechanisms. METHODOLOGY AND PRINCIPAL FINDINGS: Changes in survival, oxidative stress, DNA damage, and apoptosis signaling were compared between 4ßHWE-treated oral cancer (Ca9-22) and normal fibroblast (HGF-1) cells. At 24 h and 48 h, the numbers of Ca9-22 cells were substantially decreased, but the numbers of HGF-1 cells were only slightly decreased. Additionally, the IC50 values for 4ßHWE in the Ca9-22 cells were 3.6 and 1.9 µg/ml at 24 and 48 h, respectively. Time-dependent abnormal increases in ROS and dose-responsive mitochondrial depolarization can be exploited by using 4ßHWE in chemotherapies for selectively killing cancer cells. Dose-dependent DNA damage measured by comet-nuclear extract assay and flow cytometry-based γ-H2AX/propidium iodide (PI) analysis showed relatively severer damage in the Ca9-22 cells. At both low and high concentrations, 4ßHWE preferably perturbed the cell cycle in Ca9-22 cells by increasing the subG1 population and arrest of G1 or G2/M. Selective induction of apoptosis in Ca9-22 cells was further confirmed by Annexin V/PI assay, by preferential expression of phosphorylated ataxia-telangiectasia- and Rad3-related protein (p-ATR), and by cleavage of caspase 9, caspase 3, and poly ADP-ribose polymerase (PARP). CONCLUSIONS/SIGNIFICANCE: Together, the findings of this study, particularly the improved understanding of the selective killing mechanisms of 4ßHWE, can be used to improve efficiency in killing oral cancer cells during chemoprevention and therapy.