Role of glutamine in autophagic lysosome reformation in oral cancer cells

Abstract

Autophagic lysosomal reformation (ALR), is a mechanism to regenerate functional lysosomes from autolysosomes to maintain lysosome homeostasis. Glutamine acts as an intracellular carbon and nitrogen source for cellular homeostasis, and its uptake, as well as metabolism, is essential for cancer cell survival. In this study, we have found that glutamine starvation (GS) led to an accumulation of a significantly higher number of LC3 puncta suggesting an increase in the formation of autophagosomes. Further, we performed the co-localization analysis using LC3 and LAMP1 (Lysosome-associated membrane protein 1). We found significantly higher colocalization (%) of LC3-LAMP1 in glutamine-starved Cal33 cells compared to control conditions, indicating that glutamine starvation promotes autophagosomelysosome fusion in oral cancer cells. Next, we transiently transfected mcherryLAMP1 in Cal33 cells to examine the role of glutamine starvation in ALR. Interestingly, we found that glutamine starvation (10hrs) led to the formation of a higher number of proto-lysosome, as indicated by a more extended tubular structure than the control condition. Pharmacological inhibition of mTOR (rapamycin) and genetic inhibition of clathrin (sh-CLTH) and Rab7 (si-RAB7) significantly abolished the formation of proto-lysosome in glutamine starved Cal33 cells. However, treatment with dynasore (dynamin 2 inhibitor) leads to the elongation of tubular structures, indicating complete inhibition of proto-lysosome scissions. Conclusively, glutamine is essential for regulating lysosomal homeostasis through ALR in oral cancer