Isovitexin Suppresses Cancer Stemness Property And Induces Apoptosis Of Osteosarcoma Cells By Disruption Of The DNMT1/miR-34a/Bcl-2 Axis

Xiao Liang,1 Chang Xu,2 Xiaocheng Cao,2 Wanchun Wang1 1Department of Orthopaedics, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China; 2Department of Pharmaceutical Science, Medical College, Hunan Normal University, Changsha 410013, ChinaCorrespondence: Wanchun WangDepartment of Orthopaedics, The Second Xiangya Hospital, Central South University, 139 Renmin Road, Changsha, Hunan 410011, People’s Republic of ChinaTel +86-13607482026Email wanchun.wang@csu.edu.cnXiaocheng CaoDepartment of Pharmaceutical Science, Medical College, Hunan Normal University, Changsha 410013, People’s Republic of ChinaTel +86-18773127496Fax +86-0731-88912434Email caocheng268@163.comBackground: Isovitexin (apigenin-6-C-glucoside, ISOV) is a natural flavonoid that exhibits tumor suppressive activity on various types of cancer. However, it is unknown whether the mechanism of its action in osteosarcoma (OS) is associated with epigenetic regulation and whether it involves DNA methyltransferase 1 (DNMT1), microRNAs and their targets.Materials and methods: The present study investigated the effects of ISOV on DNMT1 activation and miR-34a and Bcl-2 expression levels in order to explain the mechanism underlying ISOV-mediated repression of proliferation and stemness. In addition, the induction of apoptosis in the spheres derived from OS cells was investigated.Results: The results indicated that ISOV significantly repressed survival, induced apoptosis and decreased the level of CD133, CD44, ABCG2 and ALDH1 mRNA in the spheres derived from U2OS (U2OS-SC) and MG63 cells (MG63-SC). ISOV further reduced the sphere formation rate of U2OS-SC and MG63-SC. It is important to noted that, ISOV inhibited tumor growth and reduced tumor size of U2OS-SC xenografts in nude mice, which was accompanied by decreased CD133 protein levels, elevated apoptotic index, downregulation of proliferating cell nuclear antigen (PCNA) expression, reduced DNMT1 activity and expression, increased miR-34a and decreased Bcl-2 levels. We identified that Bcl-2 as a direct functional target of miR-34a. Furthermore, ISOV exhibited a synergistic effect with 5-aza-2′-deoxycytidine, the miR-34a mimic or ABT-263 in order to repress cell survival, induce apoptosis, downregulate CD133, CD44, ABCG2 and ALDH1 mRNA expression levels and reduce sphere formation rates of U2OS-SC and MG63-SC cells.Conclusion: The findings suggested that ISOV-mediated epigenetic regulation involved the DNMT1/miR-34a/Bcl-2 axis and caused the suppression of stemness and induction of apoptosis in the spheres derived from OS cells. The data indicated that ISOV exhibited a novel efficient potential for the treatment of OS.Keywords: osteosarcoma, cancer stem cell, isovitexin, DNMT1, miR-34a, Bcl-2