Zeng Zhirui, Lan Jinzhi, Lei Shan, Yang Yushi, He Zhiwei, Xue Yan, Chen Tengxiang
Guizhou 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.
Department of Pathology, Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 550009, People's Republic of China.
Onco Targets Ther. 2020 Nov 17;13:11697-11709. doi: 10.2147/OTT.S240535. eCollection 2020.
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.
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.
knockdown significantly decreased the ODC1 expression, and the proliferation and invasion of HCC cells, while overexpression reversed the inhibitory effects of knockdown. Similarly, inhibition of also decreased the expression of PKM2 via reducing the c-myc-induced transcription. was co-expressed with in HCC samples, while simultaneously upregulated and 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.
/ 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.
此前，我们发现乳酸通过增加鸟氨酸脱羧酶1（ODC1）的表达促进肝细胞癌（HCC）细胞的增殖和迁移。在本研究中，我们确定了ODC1与丙酮酸激酶M2（PKM2，一种关键的乳酸代谢酶）之间的关系，并确定了二氟甲基鸟氨酸（DFMO；一种ODC1抑制剂）和化合物3k（一种PKM2抑制剂）对HCC细胞的联合作用。
首先，使用蛋白质免疫印迹法、细胞计数试剂盒（CCK）-8检测、Transwell检测、生物信息学、定量实时荧光PCR（qRT-PCR）和免疫组织化学染色分析PKM2与ODC1之间的关系。此后，使用ODC1抑制剂DFMO和PKM2抑制剂化合物3k。通过CCK-8检测、流式细胞术、小鼠皮下异种移植肿瘤模型、伤口愈合检测和Transwell检测评估它们对HCC细胞增殖和迁移的联合作用。此外，使用蛋白质免疫印迹法和免疫荧光法探讨DFMO和化合物3k对上皮-间质转化表型和AKT/GSK-3β/β-连环蛋白通路的影响。
敲低显著降低ODC1表达以及HCC细胞的增殖和侵袭，而过表达逆转了敲低的抑制作用。同样，抑制PKM2也通过减少c-myc诱导的转录降低了PKM2的表达。PKM2与ODC1在HCC样本中共同表达，同时上调PKM2和ODC1导致最差的生存结果。DFMO和化合物3k协同抑制HCC细胞增殖、诱导凋亡并抑制细胞迁移，以及上皮-间质转化表型和AKT/GSK-3β/β-连环蛋白通路。AKT激活剂SC79逆转了抑制作用。
PKM2/ODC1参与正反馈回路。使用DFMO和化合物3k同时抑制ODC1和PKM2通过AKT/GSK-3β/β-连环蛋白通路对HCC细胞发挥协同作用。因此，DFMO与化合物3k联合可能是治疗HCC的一种新型有效策略。
