Targeted Knockdown of Macrophage Migration Inhibitory Factor Enhances UVB Irradiation-Induced Apoptosis Via Increasing ROS Generation in Oral Squamous Cell Carcinoma

Abstract

Objectives: We investigated the effects of macrophage migration inhibitory factor (MIF) knockdown or overexpression combined with ultraviolet radiation B (UVB) irradiation on cell proliferation and apoptosis of oral squamous cell carcinoma (OSCC). Methods: MIF expression in OSCC and adjacent tissues was detected by immunohistochemistry. MIF expression in human immortalized oral epithelial cells (HIOEC) and OSCC cells was detected by western blotting. MIF was knocked down or overexpressed in OSCC cell lines (SCC-25 and CAL-27). OSCC cells were set up into control (CON), MIF overexpression/knockdown (oeMIF/shMIF), CON + UVB, and oeMIF + UVB/shMIF + UVB groups based on their exposure to UVB irradiation. Cell line proliferation was studied using a cell counting kit-8 (CCK-8) and colony formation assays. Flow cytometry was applied for determination of apoptosis, cell cycle, reactive oxygen species (ROS) abundance, and mitochondrial membrane potential. Apoptosis-related proteins were assayed by western blotting. Results: The expression of MIF was significantly higher in OSCC tissues and cell lines than in adjacent tissues and HIOEC. MIF knockdown accompanied by UVB irradiation significantly hampered cell viability and proliferation compared to MIF knockdown or UVB irradiation alone. Western blotting and flow cytometry showed that MIF knockdown combined with UVB irradiation not only induced apoptosis via the mitochondrial pathway but also mediated the cell cycle. Flow cytometry showed that ROS and mitochondrial membrane potential depolarization were increased in the combination treatment groups compared with the mono-treatment groups. Additionally, the ROS scavenger N-acetylcysteine significantly attenuated MIF knockdown combined with UVB irradiation-induced apoptosis and reversed MIF knockdown combined with UVB irradiation-induced MAPK activation. Conclusion: MIF knockdown combined with UVB irradiation significantly inhibited the proliferation of OSCC cells. MIF was involved in UVB-induced ROS generation and enhanced UVB irradiation-induced mitochondria-dependent apoptosis of OSCC cells by activating the MAPK pathway. This suggests that MIF-targeted therapy combined with UVB irradiation may be a novel approach for treating OSCC.

Keywords: apoptosis; macrophage migration inhibitory factor; oral squamous cell carcinoma; reactive oxygen species; ultraviolet radiation B irradiation.

Figure 1.

Expression of MIF in OSCC tissues and OSCC cell lines. (A) The expression of MIF in HIOEC, SCC-9, SCC-25 and CAL-27 was detected by western blotting. (B) Detection of MIF expression in OSCC and adjacent tissues by immunohistochemistry (×100 and ×400).

Figure 2.

MIF knockdown combined with UVB irradiation inhibited OSCC cell viability and proliferation. (A) SCC-25 and CAL-27 cells in the CON and shMIF groups were treated with different doses of UVB irradiation (0, 1, 2.5, 5, 7.5, 10, 12.5, and 15 mJ/cm²), and CCK-8 assay was performed for cell viability measuring. (B) The effect of UVB irradiation (5 mJ/cm²) on the proliferation of SCC-25 and CAL-27 cells in the CON and shMIF groups.

Figure 3.

MIF knockdown or overexpression combined with UVB irradiation induced apoptosis in OSCC cells and altered the cell cycle distribution. In SCC-25 and CAL-27 cells, the CON and shMIF groups were treated with or without UVB irradiation before being collected after 24 h. (A and B) Propidium iodide (PI) staining was performed for cell cycle distribution assesssing via flow cytometry. (C and D) Apoptosis was assayed using Annexin V PE/7-AAD staining.

Figure 4.

MIF knockdown combined with UVB irradiation triggers apoptosis via the mitochondrial pathway. (A) In CAL-27 cells, mitochondrial membrane potential was assayed with JC-1 staining in the CON and shMIF groups after treatment with or without UVB irradiation. (B and C) The effect of MIF knockdown and/or UVB irradiation on protein Bax, Bcl-2, caspase-3, Cleaved-caspase-3, caspase-7, Cleaved-caspase-7, and PARP in OSCC cells. (D and E) Effects of MIF overexpression and/or UVB irradiation on the expression of Bax, Bcl-2, caspase-3, Cleaved-caspase-3, caspase-7, Cleaved-caspase-7, and PARP in OSCC cells.

Figure 5.

MIF knockdown combined with UVB irradiation triggers apoptosis via the MAPK pathway. (A and B) The effect of MIF knockdown and/or UVB irradiation on the expression of P38, P-P38, JNK 1/2, and P-JNK 1/2 in OSCC cells. (C and D) The effect of MIF overexpression and/or UVB irradiation on the expression of P38, P-P38, JNK1/2, and P-JNK1/2 in OSCC cells.

Figure 6.

ROS generation appears as an upstream regulator in apoptosis induced by UVB irradiation. (A and B) ROS generation in OSCC cells treated with or without UVB irradiation was detected using flow cytometry. CAL-27 cells were incubated with or without 2 mM NAC for 1 h before being treated with or without UVB irradiation. The cells were collected after 24 h. (C) Intracellular ROS production was detected by flow cytometry. (D) Apoptosis detection was accomplished using an Annexin-V PE/7-AAD kit. (E) Protein PARP, P38, P-P38, Bax, and Bcl-2 were assayed by Western blotting.