Apoptin Inhibits Glycolysis and Regulates Autophagy by Targeting Pyruvate Kinase M2 (PKM2) in Lung Cancer A549 Cells

Abstract

Background: Pyruvate kinase M2 (PKM2) is a key enzyme in aerobic glycolysis and plays an important role in tumor energy metabolism and tumor growth. Ad-apoptin, a recombinant oncolytic adenovirus, can stably express apoptin in tumor cells and selectively causes cell death in tumor cells.

Objective: The relationship between the anti-tumor function of apoptin, including apoptosis and autophagy activation, and the energy metabolism of tumor cells has not been clarified.

Methods: In this study, we used the A549 lung cancer cell line to analyze the mechanism of PKM2 involvement in apoptin-mediated cell death in tumor cells. PKM2 expression in lung cancer cells was detected by Western blot and qRT-PCR. In the PKM2 knockdown and over-expression experiments, A549 lung cancer cells were treated with Ad-apoptin, and cell viability was determined by the CCK-8 assay and crystal violet staining. Glycolysis was investigated using glucose consumption and lactate production experiments. Moreover, the effects of Ad-apoptin on autophagy and apoptosis were analyzed by immunofluorescence using the Annexin v-mCherry staining and by western blot for c-PARP, p62, and LC3-II proteins. Immunoprecipitation analysis was used to investigate the interaction between apoptin and PKM2. In addition, following PKM2 knockdown and overexpression, the expression levels of p-AMPK, p-mTOR, p-ULK1, and p-4E-BP1 proteins in Ad-apoptin treated tumor cells were analyzed by western blot to investigate the mechanism of apoptin effect on the energy metabolism of tumor cells. The in vivo antitumor mechanism of apoptin was analyzed by xenograft tumor inhibition experiment in nude mice and immunohistochemistry of tumors' tissue.

Results: As a result, apoptin could target PKM2, inhibit glycolysis and cell proliferation in A549 cells, and promote autophagy and apoptosis in A549 cells by regulating the PKM2/AMPK/mTOR pathway.

Conclusion: This study confirmed the necessary role of Ad-apoptin in the energy metabolism of A549 cells.

Keywords: Apoptin; PKM2; autophagy; energy metabolism.; glycolysis; lung cancer.