Autophagy Inhibitor3-methyl-adenine Induces Cell Cycle Arrest And Apoptosis Of Human Pancreatic Cancer Cell Line PANC-1Cells

Objectives: To investigate the expressions of autophagy-related genes LC3andBeclin-1in pancreatic carcinoma, and explore their correlations to tumorigenesis anddevelopment of pancreatic carcinoma; To study the effects of autophagy inhibitor3-methyl-adenine (3-MA) on proliferation and apoptosis of human pancreatic cancer cellline PANC-1cells and explore underlying mechanisms.Methods: Tissue microarrays and immunohistochemistry were used to detect theexpressions of autophagy-related proteins LC3and Beclin-1in88specimens of pancreaticcarcinoma and11specimens of normal pancreas. The correlations of LC3and Beclin-1expression to the clinical pathological parameters were also analyzed. MTT and Colonyformation assays were used to determine the cytotoxic effects of3-MA. Changes in cellmorphology were observed with Giemsa staining. Apoptosis and cell cycle were analyzedby flow cytometry assay (FCM). Molecular mechanism was determined by Westernblotting analysis.Results: Although the expressions of LC3and Beclin-1were detected both inpancreatic carcinoma and normal pancreas, the positive rates of LC3and Beclin-1inpancreatic carcinoma were significantly lower than that in normal pancreas. Theexpression of LC3was associated with clinical TNM stages and metastasis of lymph nodeswhile the expression of Beclin-1was related to clinical TNM stages. The survival analysisshowed that the survival time of patients with positive-expression of LC3or Beclin-1wassignificantly shorter. Treatment with3-MA could induce growth inhibition, G0/G1arrestand apoptosis in a time and dose-dependent manner in PANC-1cell line.3-MA also activated p38MAPK pathway and down-regulated cell cycle-related protein PCNA、CyclinD1, which could be partially abolished by SB203580(a specific p38MAPK inhibitor).Conclusion: The autophagy-related genes LC3and Beclin-1may play an importantrole in tumorigenesis and development of pancreatic carcinoma. Examination of theseproteins may predict prognosis of pancreatic cancers.Remarkable growth inhibition wasobserved in PANC-1cells after treatment with3-MA. G0/G1arrest and increasedapoptosis were both involved in the inhibitory effects of3-MA on PANC-1cells.Treatment with3-MA might down-regulate the expressions of PCNA and Cyclin D1partlyvia activation of p38MAPK pathway, which then led to G0/G1arrest.