Mechanism Of Proline To Maintain The Proliferation Of Hepatocellular Carcinoma During Acute Nutrient Stress

Objective: Malignant proliferation of tumor cells is accompanied by high nutritional requirements and leads to nutrient depletion in local tumor tissues.Thus,metabolic reprogramming under nutritional stress is essential for malignant tumor proliferation.Proline metabolism contributes to cancer proliferation and invasion,but its underlying mechanisms remain unclear.Here,we investigated the mechanism whereby proline metabolism maintains hepatocellular carcinoma(HCC)cell proliferation.Hepatocellular carcinoma is the most common primary liver cancer.Due to the difficulty in early diagnosis of the disease,the rapid progression of the disease,and the limited treatment options for advanced patients,the prognosis of hepatocellular carcinoma patients is poor,which poses a serious threat to the health of patients.Surgical treatment is the first choice for hepatocellular carcinoma.However,due to its insidious onset,rapid progression and high metastasis rate,most patients are diagnosed with unresectable advanced liver cancer.Although multi-target tyrosine kinase inhibitors,biologics and immunologic agents have been approved for inoperable primary liver cancer patients,recent studies have found that drug resistance leads to treatment failure in many patients.Drug therapy can also put liver cancer cells in a state of nutrient stress.Therefore,it is particularly important to explore the mechanism of tumor cell survival under nutrient stress and propose possible adjuvant therapy.Methods: The Cancer Genome Atlas(TCGA)database was used to analyze the bioinformatics of proline metabolism-related proteins in HCC patients.The expression difference of proline metabolism-related proteins in HCC tissues and para cancer tissues and their prognostic effects were compared.Firstly,Western blot assay was used to verify the differences in the expression of proline metabolism-related proteins in cancer tissue and para cancer tissue.Secondly,appropriate metabolic stress conditions were selected as acute nutrient stress program by cell viability experiment,and the effects of exogenous proline on the proliferation of hepatocellular carcinoma cells under acute nutritional stress were studied by cell viability experiment and platecloning experiment.Western blot assay was used to detect the expressions of energy metabolism-related proteins(AMPK)and autophagy related proteins(ULK,P62 and LC3 I /II).After that,the changes of intracellular lipid droplets,autophagy flow and reactive oxygen species were detected by flow cytometry and immunofluorescence assay under acute nutrient stress and after proline supplementation,and the correlation between the changes of intracellular reactive oxygen species and lipid droplets and autophagy flow was verified by western blot assay.Results:1.Through bioinformatics analysis of The Cancer Genome Atlas(TCGA)database,it was found that the expression of genes related to the metabolism of glutamine(GLS)and proline(ALDH18A1 and PYCR1)was up-regulated in the cancer tissues of HCC patients,and was associated with poor prognosis.2.The appropriate acute nutrient stress condition was determined as deprivation of glucose and inhibition of glutamine metabolism by cell viability test.Glucose deprivation requires the retention of 1m M glucose to maintain osmotic pressure and basic cell demand.Inhibition of glutamine metabolism is achieved by CB-839 or BPTES,the GLS allosteric inhibitor.3.The proliferation of HCC cells was significantly inhibited under acute nutrient stress,and the proliferation of HCC cells could be partially restored after proline supplementation.4.Autophagy level and the number of lipid droplets of hepatocellular carcinoma cells increased under acute nutrient stress,and could be restored to normal level after proline supplementation.5.Increased reactive oxygen species(ROS)in HCC cells under acute nutrient stress can be restored to normal or even lower levels after proline supplementation.6.Elimination of intracellular ROS under acute nutrient stress showed similar effects as proline supplementation,that is,the elimination of intracellular ROS can downregulate the level of autophagy and the number of lipid droplets to a normal level.Conclusion:1.Through bioinformatics analysis,proline metabolism may play an important role in hepatocellular carcinoma proliferation.2.Cell experiments confirmed that the proliferation of HCC cells was significantly inhibited under acute nutrient stress,and the level of intracellular reactive oxygen species,lipid droplets and autophagy were increased.After proline supplementation,cell vitality was partially restored,the level of intracellular reactive oxygen species was reduced to the normal level,lipid droplets were reduced,and autophagy was decreased.3.Proline metabolism may play an important role in the proliferation of nutrientdeficient HCC cells and may be a potential target for adjuvant cancer therapy.