Topotecan induces apoptosis via ASCT2 mediated oxidative stress in gastric cancer

Background: Topotecan (TPT) is a Topo I inhibitor and shows obvious anti-cancer effects on gastric cancer. Cancer cells reprogram their metabolic pathways to increase nutrients uptake, which has already been a hallmark of cancer. But the effect of TPT on metabolism in gastric cancer remains unknown. Purpose: To investigate the effect of TPT on metabolism in gastric cancer. Methods: ATP production was measured by ATP Assay kit. Glucose and glutamine uptake were measured by Glucose (HK) Assay Kit and Glutamine/Glutamate Determination Kit respectively. To detect glutathione (GSH) concentration and reactive oxygen species (ROS) generation, GSH and GSSG Assay Kit and ROS Assay Kit were adopted. Apoptosis rates, mitochondrial membrane potential (MMP) were determined by flow cytometry and protein levels were analyzed by immumohistochemical staining and western blotting. Results: TPT increased ATP production. TPT promoted glucose uptake possibly via up-regulation of hexokinase 2 (HK2) or glucose transporter 1 (GLUT1) expression, while decreased glutamine uptake by down-regulation of ASCT2 expression. ASCT2 inhibitor GPNA and ASCT2 knockdown significantly suppressed the growth of gastric cancer cells. Inhibition of ASCT2 reduced glutamine uptake which led to decreased production of GSH and increased ROS level. ASCT2 knockdown induced apoptosis via the mitochondrial pathway and weakened anticancer effect of TPT. Conclusion: TPT inhibits glutamine uptake via down-regulation of ASCT2 which causes oxidative stress and induces apoptosis through the mitochondrial pathway. Moreover, TPT inhibits proliferation partially via ASCT2. These observations reveal a previously undescribed mechanism of ASCT2 regulated gastric cancer proliferation and demonstrate ASCT2 is a potential anti-cancer target of TPT.