Nashi Widodo, Didik Priyandoko, Navjot Shah, Renu Wadhwa, Sunil C. Kaul
Background and Purpose: Ashwagandha is a popular Ayurvedic herb used in Indian traditional home medicine. It has been assigned a variety of health-promoting effects of which the mechanisms remain unknown. We previously reported the selective killing of cancer cells by leaf extract of Ashwagandha (i-Extract) and its purified component Withanone. In the present study, we investigated its mechanism by loss-of-function screening (abrogation of i-Extract induced cancer cell killing) of the cellular targets and gene pathways. Methodology/Principal Findings: Randomized ribozyme library was introduced into cancer cells prior to the treatment with i-Extract. Ribozymes were recovered from cells that survived the i-Extract treatment. Gene targets of the selected ribozymes (as predicted by database search) were analyzed by bioinformatics and pathway analyses. The targets were validated for their role in i-Extract induced selective killing of cancer cells by biochemical and molecular assays. Fifteen gene-targets were identified and were investigated for their role in specific cancer cell killing activity of i-Extract and its two major components (Withaferin A and Withanone) by undertaking the shRNA-mediated gene silencing approach. Bioinformatics on the selected gene-targets revealed the involvement of p53, apoptosis and insulin/IGF signaling pathways linked to the ROS signaling. We examined the involvement of ROS-signaling components (ROS levels, DNA damage, mitochondrial structure and membrane potential) and demonstrate that the selective killing of cancer cells is mediated by induction of oxidative stress. Conclusion: Ashwagandha leaf extract and Withanone cause selective killing of cancer cells by induction of ROS-signaling and hence are potential reagents that could be recruited for ROS-mediated cancer chemotherapy. © 2010 Widodo et al.
National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan; Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, Malang, Indonesia; Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
