The Effects Of SAHA Combined With Cisplatin On The Proliferation,apoptosis And Migration Of Human Osteosarcoma Cell Line 143B And Its Mechanism Research

BackgroundOsteosarcoma is the most common primary bone tumor with a high degree of malignancy in children,adolescents,and young adults.Cisplatin,as a first-line chemotherapy drug,has been widely applied for therapy of osteosarcoma.However,its application is limited by drug-resistance and serious side-effects,such as nephrotoxicity and ototoxicity.Suberoylanilide hydroxamic acid(SAHA)is a newly developed HDAC inhibitor,which is the first FDA-approved HDAC inhibitor for the treatment of cutaneous manifestations of cutaneous T-cell lymphoma(CTCL).However,SAHA as a monotherapy was shown to be limited,especially in solid tumors.ObjectivesTo observe the combination effects of SAHA combined with CDDP on the growth,apoptosis and migration of osteosarcoma cell line 143 B in vitro,and to investigate its underlying mechanisms.Methods1.The role of SAHA,CDDP and SAHA+CDDP on the proliferation,apotosis and migration of human osteosarcoma 143 B cells.Osteosarcoma 143 B cells were treated with different concentrations of SAHA or CDDP,either alone or combined for 48 h.The morphological characteristics of the treated cells were observed with an inverted microscope.The cell viability of the combination of SAHA and CDDP on 143 B cells was measured by MTT assay.Drug combination effects were evaluated by the Chou-Talalay method.The cell cycle arrest and apoptosis were detected by flow cytometer.The colony-forming ability was observed by colony formation assay.The cell migration ability was determined by wound healing assay.2.Bioinformatics screening of genes and signaling pathways regulated by SAHA combined with CDDP and functional network analysis.The common target genes and signaling pathways were enriched using Wayne on the basis of CTD database and drugs-targets network was accomplished by Cytoscape 3.4.0.3.The effect of SAHA combined with CDDP on apoptosis related proteins and pathways.Osteosarcoma 143 B cells were treated with SAHA or cisplatin,either alone or combined for 48 h.Western blot was used to detect the protein expression levels of Bax,Bcl-2,cleaved-Caspase-3,cleaved-Caspase-8 and cleaved-PARP.Results1.The necrosis of 143 B cells was obvious in the combination group and the inhibition of cell proliferation in the combination group was significantly higher than that in single drug groups.The effect of the combination of SAHA and CDDP on cell proliferation was evaluated by combination indices and shown to be synergistic.FCM datas indicated that the cell cycle was arrested in the S phase in the combination group and the early apoptosis rate of SAHA+CDDP group was significantly higher than those of single drug groups.The colony-forming ability and cell migration ability in SAHA+CDDP group was significantly lower than those in single drug groups.2.The results of bioinformatics analysis showed that there are 78 differential pathways enriched in the development and progression of osteosarcoma based on CTD.Meanwhile,twelve canonical genes were further identified to be co-targets of combination therapy in osteosarcoma.3.After being treated with SAHA+CDDP for 48 h,the expression level of pro-apoptotic protein(Bax)in the combination group was higher than that in the control group and individual groups(P<0.05,and P<0.05),but the expression level of anti-apoptotic protein(Bcl-2)in combination group were lower than that in the control group and individual groups(P<0.05,and P<0.05).In addition,the expression levels of cleaved-Caspase-3,cleaved-Caspase-8,cleaved-PARP in the combination group was significantly higher than those in the control group and individual groups(P<0.05,and P<0.05).Conclusions1.The combination of SAHA and CDDP inhibited the proliferation and induced apoptosis of 143 B cells synergistically.2.The combination of SAHA and CDDP inhibited the colony-forming ability and cell migration ability of 143 B cells significantly.3.Pathways regulation mediated by SAHA and CDDP were achieved in a synergistical manner.4.SAHA+CDDP could alter the expression of Caspase family and Bcl-2 family of apoptosis-related proteins in the process of apoptosis of osteosarcoma 143 B cells apoptosis induced by SAHA+CDDP.