Proline metabolism supports the hepatocellular carcinoma cell survival by mitigating ROS generation under nutrient starvation.

[BACKGROUND] Proline supplementation promotes proliferation and invasion in cancers under acute nutrient deprivation. However, its molecular basis in hepatocellular carcinoma is not fully understood yet. This study investigates how proline metabolism promotes hepatocellular carcinoma (HCC) cell proliferation and survival during nutrient starvation.

[METHODS] Proline metabolism-related protein expression in HCC versus paracarcinoma tissues was analyzed using The Cancer Genome Atlas database. HCC cell viability was quantified via Cell Counting Kit 8 assays, and colony formation capacity was evaluated. Intracellular ROS levels were measured by flow cytometry. Autophagic flux was assessed by GFP/mCherry fluorescence ratio and autophagy-related proteins by immunoblotting. Lipid droplet deposition was visualized using Hoechst 33342 and BODIPY 493/503 staining.

[RESULTS] Proline metabolism-related proteins were significantly upregulated in HCC tissues compared to paracarcinoma controls. Under acute nutrient stress, exogenous proline reduced cellular ROS levels, attenuated lipid droplet accumulation, and suppressed excessive autophagy in HCC cells. Proline rescued nutrient deprivation-induced tumor growth inhibition, which could be reversed by HO and rapamycin.

[CONCLUSION] Proline metabolism sustains HCC cell survival during nutrient restriction by reducing ROS accumulation, thereby inhibiting lipid droplet formation and autophagy.