OncoTargets and Therapy (Nov 2020)
Simultaneous Inhibition of Ornithine Decarboxylase 1 and Pyruvate Kinase M2 Exerts Synergistic Effects Against Hepatocellular Carcinoma Cells
Abstract
Zhirui Zeng,1,* Jinzhi Lan,1,* Shan Lei,1,* Yushi Yang,2 Zhiwei He,3 Yan Xue,1 Tengxiang Chen1 1Guizhou Provincial Key Laboratory of Pathogenesis & Drug Research on Common Chronic Diseases, Department of Physiology, School of Basic Medical Sciences, Guizhou Medical University, Guiyang, Guizhou 550009, People’s Republic of China; 2Department of Pathology, Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 550009, People’s Republic of China; 3Department of Hepatobiliary Surgery, Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 550009, People’s Republic of China*These authors contributed equally to this workCorrespondence: Tengxiang ChenGuizhou Provincial Key Laboratory of Pathogenesis & Drug Research on Common Chronic Diseases, Department of Physiology, School of Basic Medical Sciences, Guizhou Medical University, Guiyang Guizhou 550009, People’s Republic of ChinaTel +86-18908507525Email [email protected]: Previously, we showed that lactate promoted the proliferation and mobility of hepatocellular carcinoma (HCC) cells by increasing the expression of ornithine decarboxylase 1 (ODC1). In this study, we determined the relationship between ODC1 and pyruvate kinase M2 (PKM2, a key lactate metabolism enzyme), and determined the combined effects of difluoromethylornithine (DFMO; an ODC1 inhibitor) and compound 3k (a PKM2 inhibitor) on HCC cells.Methods: First, the relationship between PKM2 and ODC1 was analyzed using Western blotting, Cell Counting Kit (CCK)-8 assays, transwell assays, bioinformatics, quantitative real-time fluorescent PCR (qRT-PCR), and immunohistochemical staining. Thereafter, the ODC1 inhibitor DFMO and the PKM2 inhibitor compound 3k were employed. Their combined effects on HCC cell proliferation and mobility were evaluated via CCK-8 assay, flow cytometry, a subcutaneous xenograft tumor model in mice, wound healing assays, and transwell assays. Additionally, the effects of DFMO and compound 3k on the epithelial–mesenchymal transition phenotype and the AKT/GSK-3β/β-catenin pathway were explored using Western blotting and immunofluorescence.Results: PKM2 knockdown significantly decreased the ODC1 expression, and the proliferation and invasion of HCC cells, while ODC1 overexpression reversed the inhibitory effects of PKM2 knockdown. Similarly, inhibition of ODC1 also decreased the expression of PKM2 via reducing the c-myc-induced transcription. PKM2 was co-expressed with ODC1 in HCC samples, while simultaneously upregulated PKM2 and ODC1 led to the poorest survival outcome. DFMO and compound 3k synergistically inhibited HCC cell proliferation, induced apoptosis, and suppressed cell mobility, as well as the EMT phenotype and the AKT/GSK-3β/β-catenin pathway. The AKT activator SC79 reversed the inhibitory effects.Conclusion: PKM2/ODC1 are involved in a positive feedback loop. The simultaneous inhibition of ODC1 and PKM2 using DFMO and compound 3k exerts synergistic effects against HCC cells via the AKT/GSK-3β/β-catenin pathway. Thus, DFMO combined with compound 3k may be a novel effective strategy for treating HCC.Keywords: hepatocellular carcinoma, ornithine decarboxylase 1, pyruvate kinase M2, DFMO, compound 3k, synergistic effect
