Synergistic antitumor effect of suberoylanilide hydroxamic acid and cisplatin in osteosarcoma cells

Abstract

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, including nephrotoxicity and ototoxicity. Suberoylanilide hydroxamic acid (SAHA) is a newly developed histone deacetylase (HDAC) inhibitor, which is the first Food and Drug Administration-approved HDAC inhibitor for the treatment of cutaneous manifestations of T-cell lymphoma. However, SAHA as a monotherapy was revealed to be limited, particularly in solid tumors. In the present study, 143B osteosarcoma cells were treated with multiple concentrations of SAHA or cisplatin, either alone or combined. The morphological characteristics of the treated cells were observed using an inverted microscope. The cytotoxicity effects of the combination of SAHA and cisplatin on 143B cells were analyzed by MTT assay, colony formation assay, wound healing cell migration assay, cell apoptosis assay and cell cycle analysis. Western blot analysis was performed to detect the protein expression levels of B cell lymphoma-2 (Bcl-2)-associated X protein (Bax), Bcl-2, cleaved-caspase-3, cleaved-caspase-8 and cleaved-poly (ADP-ribose) polymerase (PARP). The experimental data indicated that the inhibition of cell proliferation in the combination group was significantly increased compared with that in single drug groups. Expression levels of pro-apoptotic protein were upregulated, whereas anti-apoptotic Bcl-2 was downregulated significantly in 143B cells following SAHA/cisplatin treatment. Taken together, the results revealed that the combination of SAHA and cisplatin inhibited the proliferation of 143B cells and induced their apoptosis synergistically, and this effectiveness may be mediated by caspase activation.

Keywords: cisplatin; drug combination; osteosarcoma; suberoylanilide hydroxamic acid; synergistic effect.

Figure 1.

Effect of SAHA or cisplatin monotherapy on cell viability of 143B osteosarcoma cells. (A) Exposure to SAHA resulted in significant proliferation inhibition and cytotoxicity in 143B cells in a concentration-dependent manner. (B) Exposure to cisplatin resulted in significant proliferation inhibition and cytotoxicity in 143B cells in a concentration-dependent manner. (C) Exposure to SAHA resulted in significant proliferation inhibition and cytotoxicity in 143B cells in a time-dependent manner. (D) Exposure to cisplatin resulted in significant proliferation inhibition and cytotoxicity in 143B cells in a time-dependent manner. SAHA, suberoylanilide hydroxamic acid.

Figure 2.

Combination effect of SAHA and cisplatin on the cell viability of osteosarcoma cells. Combination of SAHA and cisplatin inhibited the cell viability of 143B cells more significantly compared with the control and the individual groups (*P<0.05). SAHA, suberoylanilide hydroxamic acid.

Figure 3.

Morphological changes of osteosarcoma cells following exposure to SAHA, cisplatin or SAHA+cisplatin. (A) Necrosis of 143B cells was marked in the SAHA/cisplatin group (magnification, ×100). (B) The number of colonies decreased significantly in the SAHA/cisplatin group in 143B cells compared with the control and the individual groups (magnification, ×100; *P<0.05). (C) The combination of SAHA and cisplatin impaired migration in 143B cells significantly compared with the control and the individual groups. *P<0.05 vs. control; **P<0.05 vs. monotherapy. SAHA, suberoylanilide hydroxamic acid.

Figure 4.

Flow cytometry analysis of osteosarcoma cells following exposure to SAHA, cisplatin or SAHA+cisplatin. (A) The percentage of apoptotic cells in the combination group was significantly increased compared with the control and the monotherapy groups. (B) The cell cycle distribution was altered in the combination group compared with the control and the monotherapy groups. *P<0.05 vs. control; **P<0.05 vs. single treatment. FITC, fluorescein isothiocyanate; SAHA, suberoylanilide hydroxamic acid; PI, propidium iodide.

Figure 5.

Western blot analysis of osteosarcoma cells treated with SAHA, cisplatin or SAHA+cisplatin. (A) Western blot analysis demonstrated that in 143B cells that were treated with SAHA/cisplatin, the protein expression level of Bax was increased whereas the protein expression level of Bcl-2 was decreased compared with the control and the monotherapy groups. (B) Following treatment with SAHA/cisplatin, the expression levels of cleaved-caspase-3, cleaved-caspase-8 and cleaved-PARP were increased. *P<0.05 vs. control; **P<0.05 vs. single treatment. Bcl, B cell lymphoma; SAHA, suberoylanilide hydroxamic acid.