Abstract: Objective To investigate the effect of rapamycin for enhancing the inhibitory effect of RSL3 on proliferation, invasion and migration of testicular cancer I-10 cells in vitro. Methods I-10 cells treated with 0-16 μmol/L RSL3 (Type II) either alone or in combination with 16 μmol/L rapamycin (RAPA) were examined for changes in proliferation using MTT assay and colony-forming assay, and the changes in cell migration and invasion abilities were detected with wounding-healing assay and Transwell assay. The changes in the levels of lipid reactive oxygen species in the treated cells were detected using flow cytometry. GSH and MDA contents in the cells were detected using commercial detection kits, and GPX4 protein expression level was determined with Western blotting. Results The cytotoxic effect of RSL3 increased dose-dependently in I-10 cells, and the combined treatment with rapamycin further enhanced its cytotoxicity. Treatment of I-10 cells with RSL3 alone significantly decreased cell colony numbers (P<0.05), wounding-healing rates (P<0.01), and invasion and migration cell numbers (P<0.05), increased lipid reactive oxygen species level and MDA content (P<0.05), and lowered GSH content and expression level of GPX4 protein in the cells (P<0.01). The inhibitory effects of RSL3 were significantly enhanced by co-treatment of the cells with rapamycin (P<0.05 or 0.01). Conclusion Rapamycin enhances the inhibitory effect of RSL3 on proliferation, invasion and migration of I-10 cells by enhancing RSL3-mediated cell ferroptosis.

Key words: testicular cancer; RAS-selective lethal 3; rapamycin; proliferation; migration; invasion; ferroptosis