Zerumbone increases oxidative stress in a thiol-dependent ROS-independent manner to increase DNA damage and sensitize colorectal cancer cells to radiation

Locally advanced rectal cancers are treated with neoadjuvant chemoradiation therapy followed by surgery. In a minority (~20%) of patients, no tumor is present at the time of surgery; these patients with a complete pathologic response (pathCR) to neoadjuvant therapy have better treatment outcomes. Unfortunately, the inherent radioresistance of colorectal cancer (CRC) cells dictates that the majority of patients do not achieve a pathCR. Efforts to improve these odds have fueled the search for novel, relatively less-toxic radiosensitizers with distinct molecular mechanism(s) and broad-spectrum anticancer activities. Here, we use zerumbone, a sesquiterpene from the edible ginger (Zingiber zerumbet Smith), to enhance radiosensitivity of CRC cells. Short exposure to zerumbone (7 h) profoundly sensitized CRC cells, independent of their p53 or k-RAS status. Zerumbone enhanced radiation-induced cell cycle arrest (G2/M), increased radiation-induced apoptosis, but induced little apoptosis by itself. Zerumbone significantly enhanced radiation-induced DNA damage, as evident by delayed resolution of post-irradiation nuclear γH2AX foci, whereas zerumbone treatment alone did not induce γH2AX foci formation. Zerumbone pretreatment inhibited radiation-induced nuclear expression of DNA repair proteins ataxia-telangiectasia mutated (ATM) and DNA-PKcs. Interestingly, zerumbone-mediated radiosensitization did not involve reactive oxygen species (ROS), but was mediated through depletion of cellular glutathione (GSH). Ability of only thiol-based antioxidants to abrogate zerumbone-mediated radiosensitization further corroborated this hypothesis. The α,β-unsaturated carbonyl group in zerumbone was found to be essential for its bioactivity as zerumbone analog α-Humulene that lacks this functional group, could neither radiosensitize CRC cells, nor deplete cellular GSH. Our studies elucidate novel mechanism(s) of zerumbone's ability to enhance CRC radiosensitivity.