Titanium dioxide nanoparticles induce apoptosis through ROS-Ca²⁺-p38/AKT/mTOR pathway in TM4 cells

Titanium dioxide nanoparticles (TiO₂ NPs) can cause apoptosis in TM4 cells; however, the underlying mechanism has not been entirely elucidated. The purpose of this study was to investigate the effects of TiO₂ NPs on ROS, Ca²⁺ level, p38/AKT/mTOR pathway, and apoptosis in TM4 cells and to evaluate the role of Ca²⁺ in p38/AKT/mTOR pathway and apoptosis. After exposure to different concentrations (0, 50, 100, 150, and 200 μg/mL) of TiO₂ NPs for 24 h, cell viability, ROS, Ca²⁺ level, Ca²⁺-ATPase activity, p38/AKT/mTOR pathway-related proteins, apoptosis rate, and apoptosis-related proteins (Bax, Bcl-2, Caspase 3, Caspase 9, and p53) were detected. The ROS scavenger NAC was used to determine the effect of ROS on Ca²⁺ level. The Ca²⁺ chelator BAPTA-AM was used to evaluate the role of Ca²⁺ in p38/AKT/mTOR pathway and apoptosis. TiO₂ NPs significantly inhibited cell viability, increased ROS level, and elevated Ca²⁺ level while suppressing Ca²⁺-ATPase activity. TiO₂ NPs regulated the p38/AKT/mTOR pathway via increasing p-p38 level and decreasing p-AKT and p-mTOR levels. TiO₂ NPs significantly enhanced the apoptosis. NAC attenuated Ca²⁺ overload and reduction in Ca²⁺-ATPase activity caused by TiO₂ NPs. BAPTA-AM alleviated TiO₂ NPs-induced abnormal expression of p38/AKT/mTOR pathway-related proteins. BAPTA-AM assuaged the apoptosis caused by TiO₂ NPs. Altogether, this study revealed that TiO₂ NPs elevated intracellular Ca²⁺ level through ROS accumulation. Subsequently, the heightened intracellular Ca²⁺ level was observed to exert regulation over the p38/AKT/mTOR pathway, ultimately culminating in apoptosis. These results provides a complementary understanding to the mechanism of TiO₂ NPs-induced apoptosis in TM4 cells.