Knockdown of PKM2 induces apoptosis and autophagy in human A549 alveolar adenocarcinoma cells
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- Published online on: June 16, 2015 https://doi.org/10.3892/mmr.2015.3943
- Pages: 4358-4363
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Abstract
The M2 isoform of pyruvate kinase (PKM2), which has been identified as the predominant cause of the Warburg effect in cancer cells, is essential in tumor metabolism and growth. However, the role of PKM2 in autophagy remains to be elucidated. The present study investigated the effect of PKM2 knockdown on autophagy and apoptotic cell death in human A549 alveolar adenocarcinoma cells. Two short hairpin (sh)RNAs targeting human PKM2 mRNA were designed and lentiviral vectors were constructed. The A549 cells were infected with lentiviruses, containing shRNAs against PKM2, and the expression of PKM2 was examined by reverse transcription-quantitative polymerase chain reaction (RT‑qPCR) and immonoblotting analysis. A lactose dehydrogenase (LDH)‑coupled enzyme assay was used to detect the pyruvate kinase activity. RT‑qPCR was used to detect the mRNA expression level of glycolysis‑associated enzymes. The quantification of cells with punctate LC3 and expression of LC3II were examined to demonstrate autophagy. An MTT assay was used to detect cell viability and flow cytometry was used to determine cell apoptosis. The activity of caspase 3/7 and the expression of Bcl‑2 were also detected in A549 cells with PKM2 knockdown. The present study demonstrated that the two shRNAs efficiently downregulated the mRNA and protein expression levels of PKM2 in A549 cells. The knockdown of PKM2 decreased pyruvate kinase activity and glycolysis. Autophagy was induced in A549 cells with PKM2 knockdown. Inhibition of autophagy accelerated apoptotic death in PKM2‑knockdown cells and this was dependent on increased caspase 3/7 activity and decreased expression of Bcl‑2. In conclusion, the downregulation of PKM2 induced apoptosis and autophagy in A549 cells and this autophagy protected the cells from apoptotic cell death.
