Proteasome Inhibitor MG132 Enhances Cisplatin-Induced Apoptosis in Osteosarcoma Cells and Inhibits Tumor Growth

Abstract

Although cisplatin has been shown to be an integral part of chemotherapy regimen in osteosarcoma (OS) treatment, toxicity issues and chemoresistance have hindered therapeutic development for OS. Exploring novel combination therapy methods is needed to circumvent the limitations of cisplatin alone. The proteasome inhibitor MG132 has shown antitumor effects in many solid tumors. However, little is known about its effects in combination with cisplatin in OS cells. In this study, we examined the effects of MG132 in combination with cisplatin in human OS cells (MG-63 and HOS). MG132 and cisplatin were applied to OS cells, respectively or jointly. The results demonstrated that MG132 markedly inhibited cell viability in a dose- and time-dependent manner, whereas viability of osteoblast cells was not affected, suggesting a selective toxicity of MG132 to cancerous cells. Mechanistically, MG132 arrested cells in the G₂/M phase in association with increased p21^waf1 and induced cell apoptosis, which was accompanied by cleaved PARP. In addition to its apoptotic effect alone, MG132 significantly enhanced cisplatin-induced apoptosis in OS cells. Furthermore, cell viability of the combined application of 10 μM MG132 and 5 μg/ml cisplatin was markedly inhibited compared to that of the individual application. These events were accompanied by the downregulation of NF-κB, mitochondrial antiapoptotic protein Bcl-xL, and PI3K/Akt, which play a key role in cell survival. Finally, combination treatment of MG132 and cisplatin showed more antiproliferative effect than the single treatment in OS xenograft models. In summary, we concluded that MG132 interacted synergistically with cisplatin, which raised the possibility that combining the two drugs may represent a novel strategy in OS.

Figure 1

MG132 selectively inhibited cell viability in a dose- and time-dependent manner in osteosarcoma (OS) cells but not in osteoblast cells. OS cell lines MG-63 (A) and HOS (B) and osteoblast hFOB 1.19 cells (C) were treated with different concentrations (0.5, 1, 5, 10, and 20 μM) of MG132 for different times (12, 24, 36, 48, 60, and 72 h), and cell counting kit-8 (CCK-8) assays were carried out. Cell survival rate was calculated as a percentage: (A _MG132/A _DMSO).

Figure 2

MG132 induced cell cycle arrest at the G₂/M phase and apoptosis in OS cells. MG-63 and HOS cells were treated with dimethyl sulfoxide (DMSO) or 10 μM MG132 for 24 h. (A, B) Cell cycle distribution was analyzed by flow cytometry. *p < 0.05 versus control (DMSO) denotes a significant difference. (C, D) Apoptotic rates of the treated cells were detected by flow cytometry. *p < 0.05 versus control (DMSO) denotes a significant difference. (E) Expression of PARP and p21^waf1 proteins in treated OS cells was detected by Western blot.

Figure 3

MG132 increased cisplatin-induced apoptosis and proliferation inhibition in OS cells. MG-63 and HOS cells were treated with DMSO/5 μg/ml cisplatin/10 μM MG132/combined (5 μg/ml cisplatin + 10 μM MG132) for 24 h. (A–D) Morphological appearances of the cells were observed under an inverted phase-contrast microscope. (E, F) The CKK-8 assay was performed to determine the effect of MG132/cisplatin/combination on cell proliferation. *p < 0.05 versus control (DMSO) denotes a significant difference, #p < 0.05 versus MG132 or cisplatin denotes a significant difference.

Figure 4

MG132 inhibited nuclear factor κB (NF-κB), Bcl-xL, and PI3K/Akt activities in OS cells. (A) MG-63 and HOS cells were treated with DMSO or 10 μM MG132 for 24 h, and the expression of p-Akt, Akt, and Bcl-xL was detected by Western blot and was normalized to GAPDH. *p < 0.05 versus control (DMSO) denotes a significant difference. (B) NF-κB activity in nuclear extracts of the treated cells was determined by ELISA. A 50-fold of unlabeled wild-type (WT) consensus NF-kB oligonucleotide (50 × WT) was used as a competitor for NF-kB binding to monitor the specificity of the assay. The mutated consensus oligonucleotide (MUT) should have no effect on NF-kB binding and served as negative control. *p < 0.05 versus control (DMSO) denotes a significant difference.

Figure 5

Combined treatment with MG132 and cisplatin inhibited tumor growth in HOS xenograft mouse model. HOS cells (1 × 10⁷) were suspended in 200 μl of PBS and subcutaneously injected into the right flanks of the nude mice. When the tumors were visible, the mice were randomly divided into four groups (n = 5): control (0.2 ml of PBS), MG132 (2 mg/kg), cisplatin (2 mg/kg), and combination group (MG132: 2 mg/kg + 2 mg/kg). The mice were intraperitoneally injected with the above reagents every 3 days for 15 days. (A) Tumor volume was measured every 2 days using a caliper. *p < 0.05 versus control (PBS) denotes a significant difference, #p < 0.05 versus MG132 or cisplatin denotes a significant difference. (B) At the end, mice were sacrificed, and tumors were removed and weighed. *p < 0.05 versus control (PBS) denotes a significant difference, #p < 0.05 versus MG132 or cisplatin denotes a significant difference. (C) Mice body weights were measured once a week and were used as an indicator of the systemic toxicity of the treatment.