Proteasome inhibitor MG132 enhances the sensitivity of human OSCC cells to cisplatin via a ROS/DNA damage/p53 axis

Abstract

Cis-diamine-dichloroplatinum II (cisplatin, CDDP) is a key chemotherapeutic regimen in the treatment of oral squamous cell carcinoma (OSCC). However, the therapeutic efficacy of cisplatin in OSCC may be hampered by chemoresistance. Therefore, the development of novel combination therapy strategies to overcome the limitations of CDDP is of great importance. The proteasome inhibitor MG132 exhibits anti-cancer properties against various types of cancer. However, our knowledge of its anti-cancer effects in combination with CDDP in OSCC cells remains limited. In the current study, the synergetic effects of MG132 and CDDP were evaluated in the human CAL27 OSCC cell line. CAL27 cells were treated with CDDP alone or in combination with MG132. The results showed that MG132 significantly reduced cell viability in a dose-dependent manner. Additionally, cell viability was significantly reduced in CAL27 cells treated with 0.2 µM MG132 and 2 µM CDDP compared with cells treated with MG132 or CDDP alone. In addition, MG132 significantly enhanced the CDDP-induced generation of intracellular reactive oxygen species and DNA damage in OSCC cells. Furthermore, treatment with CDDP or MG132 alone notably inhibited colony formation and proliferation of OSCC cells. However, co-treatment of OSCC cells with MG132 and CDDP further hampered colony formation and proliferation compared with cells treated with either MG132 or CDDP alone. Finally, in cells co-treated with MG132 and CDDP, the expression of p53 was markedly elevated and the p53-mediated apoptotic pathway was further activated compared with cells treated with MG132 or CDDP alone, as shown by the enhanced cell apoptosis, Bax upregulation, and Bcl-2 downregulation. Overall, the results of the current study support the synergistic anti-cancer effects of a combination of MG132 and CDDP against OSCC, thus suggesting that the combination of MG132 and CDDP may be a promising therapeutic strategy for the management of OSCC.

Keywords: MG132; chemosensitivity; cisplatin; oral squamous cell carcinoma.

Figure 1

CDDP, MG132, or CDDP + MG132 exert inhibitory effects of oral squamous cell carcinoma cells. CAL27 cells were treated with different concentrations of (A) CDDP and (B) MG132 for 48 h after which CCK-8 assays were performed. (C) The synergistic effects of 2 µM CDDP + 0.2 µM MG132 on cell viability was assessed by CCK-8 assay. (D) Representative images showing cell morphology were captured under a light microscope. x40 magnification, scale bar 500 µm. ^**P<0.01. CDDP, cis-diamine-dichloroplatinum II; CCK-8, Cell Counting Kit 8.

Figure 2

MG132 and CDDP synergistically enhance ROS generation and DNA damage. (A) Cells were treated with 2 µM CDDP, 0.2 µM MG132, or both together for 48 h after which intracellular ROS accumulation was assessed by flow cytometry. (B) An integrated image of (A) is shown. (C) Quantitative analysis of the percentage of DCF positive cells (n=3). (D) DNA damage was assessed by TUNEL assay under a fluorescence microscope. TUNEL staining (green) was indicative of DNA injury. Nuclei were counterstained with Hoechst (blue). x200 magnification, scale bar 100 µm. (E) Quantitative analysis of TUNEL assays was performed by measuring double TUNEL- and Hoechst-positive cells. Data are presented as the mean ± SEM from three independent experiments. ^**P<0.01. CDDP, cis-diamine-dichloroplatinum II; ROS, reactive oxygen species.

Figure 3

MG132 enhances CDDP-induced inhibition of OSCC proliferation. CAL27 cells were treated with 2 µM CDDP, 0.2 µM MG132 or both combined for 48 h. (A) Cell proliferation was assessed using colony formation assays. Representative images of colonies in a six-well-plate are shown. (B) Quantitative analysis from three independent colony formation assays are presented. (C) EdU proliferation assays were performed to determine the proliferative ability of OSCCs. Red spots indicate EdU-positive cells; cell nuclei were counterstained with Hoechst blue. x200 magnification, scale bar 100 µm. (D) Quantitative analysis of the cell proliferation ratio was performed by measuring double EdU- and Hoechst-positive cells (n=3). (E) Flow cytometry analysis for cell cycle distribution of CAL27 cells. (F) Histograms show the percentage of cells in each stage. Data are presented as the mean ± SEM of 3 independent experiments. ^*P<0.05 and ^**P<0.01. OSCC, oral squamous cell carcinoma cells; CDDP, cis-diamine-dichloroplatinum II; EdU, ethynyl-2-deoxyuridine.

Figure 4

CDDP-induced apoptosis of oral squamous cell carcinoma cells is enhanced by MG132. (A) Representative image of flow cytometry analysis of CAL27 cells treated with CDDP, MG132, or CDDP + MG132. Cell apoptosis was assessed using an Annexin V/PI kit. Annexin V-/PI- staining indicates viable cells, Annexin V⁺/PI^- early apoptotic cells, Annexin V⁺/PI⁺ late apoptotic cells, and Annexin V^-/PI⁺ necrotic cells. (B) Quantitative analysis of total apoptotic cells (early and late apoptosis) is shown. Data are expressed as the mean ± SEM of three independent experiments. (C) Representative images of p53, Bcl-2, and Bax protein expression levels detected by western blot. (D) Densitometry analysis of the p53, Bcl-2, and Bax protein expression levels relative to GAPDH. ^*P<0.05 and ^**P<0.01. CDDP, cis-diamine-dichloroplatinum II; PI, propidium iodide.