Cisplatin in Combination with Apigenin Inhibits Human Osteosarcoma Cells Proliferation by Promoting Osteogenic Differentiation: A Mechanistic Insight

Abstract

Background: Osteosarcoma, the predominant primary bone cancer in the pediatric and adolescent population globally, exhibits a relatively low annual incidence but contributes significantly to morbidity and mortality.Objectives: The current study aims to explore the impact of cisplatin, apigenin, and their combination on human osteosarcoma cell line (MG-63) osteosarcoma cells, given the challenges in clinical treatment such as chemotherapy’s adverse effects, resistance development, and relapses.Materials and Methods: Cytotoxicity assessment using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted for cisplatin, apigenin, and their combination. Apoptotic potential was evaluated through ethidium bromide (EtBr)/acridine orange (AO) dual staining for the combination treatment. Reactive oxygen species (ROS) production and cell cycle arrest were analyzed posttreatment with the cisplatin + apigenin combination using 2′-7′-dichlorodihydrofluorescein diacetate (DCF-DA) staining and fluorescence-activated cell sorting (FACS) analysis, respectively. Proapoptotic and antiapoptotic gene expression was assessed via quantitative polymerase chain reaction (qPCR) analysis. Additionally, messenger ribonucleic acid (mRNA) and protein expression of B-cell-specific Moloney murine leukemia virus integration site 1 (BMIL1) were analyzed in both untreated and cisplatin + apigenin treated cells. Enzymelinked immunosorbent assay (ELISA) analysis was performed for p16INK4a, E-cadherin, and Notch1.Results: Results indicated that the combination treatment significantly inhibited MG-63 cell proliferation compared to individual treatments. Enhanced apoptosis and ROS production were observed in the combination treatment, along with cell cycle arrest at the subG0-G1 phase. Furthermore, the combination downregulated BMIL1 and Bcl-2 expression while upregulating mRNA expression of Bax, Caspase-3, Caspase-9, Apaf-1, and p53. Gene expression results corroborated BMIL1 downregulation in the combination treatment. Additionally, protein levels of p16INK4a and E-cadherin were significantly decreased in the combination treatment compared to individual treatments and untreated control.Conclusions: In conclusion, cisplatin and apigenin combination exhibited superior efficacy against osteosarcoma.

Keywords: MG-63; osteosarcoma; apigenin; cisplatin; combination; apoptosis