Study on the Induction of Artesunate on Iron Death in T24 Cells of Bladder Cancer Based on GSH/GPX4

OBJECTIVE To investigate the inhibitory effect of artesunate(ART) on bladder cancer cells T24 and the mechanism of inducing ferroptosis.

METHODS Different concentrations of ART were used to treat T24 cells of bladder cancer, CCK-8 method was used to detect the cell proliferation inhibition rate, while the IC₅₀ was calculate, and selected the appropriate concentration range for subsequent experiments. Cell cycle changes were detected by flow cytometry; cell migration activity was detected by cell scratch method; Transwell invasion method was used to detect the invasion ability of cells. CCK-8 method was used to detect the changes of cell activity after ART was used alone or combined with ferroptosis inhibitordesferriamine mesylate(DFOM), apoptosis inhibitor(Z-VAD-FMK), necrosis inhibitor(Necrosin-1) and autophagy inhibitorchloroquine(CQ). The ultrastructural changes of ART treated cells were observed by transmission electron microscopy. The ROS level of cells after ART treated alone and in combination with DFOM was detected by fluorescence probe. The levels of Fe²⁺, GSH and MDA in cells after ART treated alone and in combination with DFOM were detected by colorimetric kit; Western blotting was used to detect the levels of FTH1, GPX4 and Nrf2 protein in the cells after ART treated alone and in combination with DFOM.

RESULTS Compared with blank control group, ART significantly inhibited the proliferation of T24 cells(P<0.01). It induced G₁ and G₂ phase arrest of T24 cells(P<0.05 or P<0.01), and inhibited cell migration ability(P<0.01) and invasion ability(P<0.01). Compared with ART treat alone, the decrease in cell viability caused by ART could be reversed to varying degrees when combined with DFOM, Z-VAD-FMK, CQ, and Nec-1(P<0.05 or P<0.01). After ART treatment, mitochondrial volume decreased, membrane thickened, ridge fracture or reduction. Compared with blank control group, ROS, Fe²⁺ and MDA levels in ART group were significantly increased(P<0.05 or P<0.01), GSH level was decreased(P<0.01), protein expression levels of FTH1 and GPX4 were decreased(P<0.05 or P<0.01), and Nrf2 level was increased(P<0.01). Compared with ART group, ROS, Fe²⁺ and MDA levels in ART+DFOM group were decreased(P<0.05 or P<0.01), while GSH level was increased(P<0.01). The protein expression levels of FTH1 and GPX4 were increased(P<0.05 or P<0.01), while the level of Nrf2 was decreased(P<0.05).

CONCLUSION ART can inhibit proliferation, migration and invasion of bladder cancer T24 cells, arrest cell cycle, induce ferroptosis by regulating GSH/GPX4, and may activate ferroptosis resistance.