Piperine enhances hyperthermia-induced apoptosis in AGS gastric cancer cells by increasing reactive oxygen species and attenuating heat-shock protein induction.

Thermotherapy can potentiate anticancer activity, yet heat-induced stress adaptation-largely involving the HSF1-HSP axis-may limit its efficacy. Here, we investigated whether piperine acts as a thermosensitizer in AGS gastric cancer cells and explored associations with oxidative stress, apoptosis, and the heat-shock response. AGS cells were treated with piperine and subjected to mild hyperthermia (43 °C), followed by evaluation of viability, migration, colony formation, apoptosis, and cell-cycle distribution. Piperine enhanced hyperthermia-induced growth suppression, reduced scratch closure, and increased apoptotic cell death, accompanied by an increase in the sub-G1 populations. The combination elevated intracellular ROS, promoted caspase activation, and was associated with attenuation of pro-survival signaling. Piperine attenuated hyperthermia-induced HSP expression, most prominently HSP27 and HSP90, without reducing HSF1 or its activating phosphorylation. To extend these observations, we validated the cytotoxic and apoptotic phenotype in a second gastric cancer cell line, NCI-N87: piperine plus hyperthermia increased cleaved caspase-3, and NAC pretreatment partially attenuated this increase, supporting a contribution of oxidative stress to the apoptotic response, whereas the heat-shock protein response in NCI-N87 cells was not uniformly reproduced, indicating a cell-line-dependent pattern. Collectively, piperine enhanced hyperthermia-associated cytotoxicity and apoptosis in both gastric cancer cell models, whereas attenuation of HSP expression was more evident in AGS cells and was not uniformly reproduced in NCI-N87 cells, supporting further study as an adjunct strategy for hyperthermia-based anticancer approaches.