Abstract
Withaferin A (WFA) is one of the most active steroidal lactones with reactive oxygen species (ROS) modulating effects against several types of cancer. ROS regulation involves selective killing. However, the anticancer and selective killing effects of WFA against oral cancer cells remain unclear. We evaluated whether the killing ability of WFA is selective, and we explored its mechanism against oral cancer cells. An MTS tetrazolium cell proliferation assay confirmed that WFA selectively killed two oral cancer cells (Ca9-22 and CAL 27) rather than normal oral cells (HGF-1). WFA also induced apoptosis of Ca9-22 cells, which was measured by flow cytometry for subG1 percentage, annexin V expression, and pan-caspase activity, as well as western blotting for caspases 1, 8, and 9 activations. Flow cytometry analysis shows that WFA-treated Ca9-22 oral cancer cells induced G2/M cell cycle arrest, ROS production, mitochondrial membrane depolarization, and phosphorylated histone H2A.X (γH2AX)-based DNA damage. Moreover, pretreating Ca9-22 cells with N-acetylcysteine (NAC) rescued WFA-induced selective killing, apoptosis, G2/M arrest, oxidative stress, and DNA damage. We conclude that WFA induced oxidative stress-mediated selective killing of oral cancer cells.
Highlights
Most anticancer drugs effectively kill cancer cells; they non-selectively kill normal cells, which limits their therapeutic value
MTS assays showed that the relative cell viability (%) of the Ca922 and CAL 27 oral cancer cells were significantly lower than control after 24 h Withaferin A (WFA) treatments of 0.5, 1, 2, and 3 μM in a dose-dependent manner (Figure 1A)
HGF-1 normal oral cells treated with WFA showed no reduction in viability
Summary
Most anticancer drugs effectively kill cancer cells; they non-selectively kill normal cells, which limits their therapeutic value. When the balance between proliferation and apoptosis is interrupted during tumor development, cell proliferation is deregulated (Scully et al, 2000; Evan and Vousden, 2001). Therapeutics that target oral squamous cell carcinoma (OSCC) cell proliferation and apoptosis regulators can enable these cancer cells to evade the regulatory system (Evan and Vousden, 2001). Several drugs that modulate ROS have been reported (Nicco et al, 2005; Trachootham et al, 2006; Wu and Hua, 2007; Widodo et al, 2010) to regulate apoptosis for selective killing. ROS might mediate the selective activation of apoptosis for selective killing in cancer chemotherapy (Pollack et al, 2001; Daniel et al, 2003; Real et al, 2004)
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