Cucurbitacin D Induces Apoptotic Cell Death via NOX4 and Overcomes Radioresistance in Colorectal Cancer

Colorectal cancer (CRC) incidence is a significant cancer globally, and radiotherapy resistance is a serious problem. Cucurbitacin D (CBD), extracted from many plants such as the tubers of <i>Trichosanthes kirilowii</i> and the fruits of <i>Ecballium elaterium</i> (squirting cucumber), has various therapeutic effects, such as anti-cancer, -inflammation, -diabetes, and -viral infection effects. Since reports have indicated that CBD exhibits effective anti-cancer activity across various cancer types, our hypothesis is that CBD will overcome radioresistance in CRC radiotherapy. In the present study, we identified that CBD, a triterpenoid compound isolated from <i>Trichosanthes kirilowii</i> and <i>Ecballium elaterium</i>, has an anti-cancer and anti-inflammatory effect in vivo and in vitro. In LPS-induced murine models, CBD suppresses LPS-mediated cytokines, including TNFα, IL-6, IL-1β, and COX-2. In CRC xenograft mouse models, CBD treatment results in significantly smaller tumor volumes than the control. In HCT116 and HT29 cells, CBD treatment suppresses cell viability and increases LDH cytotoxicity and caspase-3 activity and cleavage. However, combined treatment of CBD and Z-VAD-FMK inhibits caspase-dependent apoptosis and cell death. Since CBD induces intracellular calcium (Ca²⁺) and reactive oxygen species (ROS) generation, it mediates ER stress-induced apoptotic cell death through the PERK-ATF4-CHOP axis. Moreover, ER stress inducer thapsigargin (TG) mediates synergistic apoptotic cell death in CBD-treated HCT116 and HT29 cells. However, PERK or CHOP knockdown suppresses ER stress-mediated apoptosis in CBD-treated HCT116 and HT29 cells. CBD treatment induces oxidative stress through the NADPH Oxidase 4 (NOX4) and also increases ROS generation. However, NOX4 knockdown and ROS inhibitor NAC or DPI block ER stress-induced apoptotic cell death by inhibiting the suppression of cell viability and the elevation of caspase-3 activity, LDH cytotoxicity, and intracellular ROS activity in CBD-mediated HCT116 and HT29 cells. We established radioresistant CRC models (HCT116R and HT29R); subsequently, radiation (2 Gy) in combination with CBD treatment overcame radioresistance via the modulation of the epithelial-mesenchymal transition (EMT) phenomenon, including the increase in N-cadherin and vimentin and the reduction in E-cadherin. Thus, these results show that CBD may be a new powerful therapeutic approach for CRC radiotherapy.