The reciprocal effects of autophagy and the Warburg effect in pancreatic ductal adenocarcinoma: an in vitro study.

Abstract

Autophagy and the Warburg effect are two common pathways in pancreatic ductal adenocarcinoma (PDAC). To date, the reciprocal effects of these pathways have not yet been elucidated. Therefore, this study was designed to investigate the relationship between these factors in vitro and may provide therapeutic targets in the future. The Mia-Paca-2 and AsPc-1 cell lines were cultured under normal conditions. To achieve autophagy, starvation was induced by Hank's balanced salt solution (HBSS), whereas autophagy was inhibited by 3-methyladenine (3-MA). The Warburg effect is mimicked by lactic acid, and the Warburg effect is inhibited by oxamate, the main inhibitor of lactate dehydrogenase. Cell viability was checked through the MTT assay method. Autophagy was checked via evaluation of Beclin-1 via western blotting. The amount of lactic acid was also measured with a lactate dehydrogenase (LDH) assay kit. The cells were incubated with different concentrations of 3-MA, lactic acid and oxamate. The viability of AsPc-1 cells decreased, and the IC₅₀ values were 1195 µM, 23.06 mM and 8.617 mM for 3-MA, lactic acid and oxamate, respectively. Similarly, the IC₅₀ values of Mia-Paca-2 were 873.9 µM, 35.9 mM and 26.74 mM for 3-MA, lactic acid and oxamate, respectively. Our data revealed that starvation increased the expression of the autophagy-related protein Beclin-1 (P value < 0.05); however, 3-MA significantly reduced its expression (P value < 0.05). In addition, lactic acid alone did not affect the expression level of Beclin-1 (P value > 0.05), but oxamate treatment increased its expression (P value < 0.05). We also showed that starvation reduced lactic acid levels, but an autophagy inhibitor, 3MA, significantly increased lactic acid production (P value < 0.05). Our findings showed that lactic acid alone has no significant effect on autophagy and that oxamate induces autophagy, possibly because of caloric restriction. On the other hand, autophagy inhibits lactic acid production, whereas the inhibition of autophagy leads to increased lactic acid production.