Chidamide Inhibits Glioma Cells by Increasing Oxidative Stress via the miRNA-338-5p Regulation of Hedgehog Signaling

Abstract

Objective: Chidamide has a broad spectrum of antitumor activity but its function on glioma remains unknown. The increase of reactive oxygen species (ROS) and reactive nitrogen species (RNS) may control glioma risk by promoting its apoptosis and necrosis. Hedgehog pathway is crucial to glioma cell proliferation and controls ROS production. We aimed to explore the effects of chidamide on the levels of miR-338-5p (glioma cell inhibitor), which may regulate Hedgehog signaling, resulting in the changes of RNS. Materials and Methods. Migration and invasion activities of glioma cells were measured by using the Transwell chamber assay. The expression levels of Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), Desert Hedgehog (Dhh), miR-338-5p, and related molecules were detected by using real-time PCR (RT-PCR) and or Western Blot in U87 and HS683 glioma cells. The effects of chidamide on these molecules were measured by using the miR-338-5p inhibitor or mimics in U87 and HS683 glioma cell lines. ROS and RNS were measured by DCF DA and DAF-FM DA fluorescence. Biomarkers of oxidative stress were measured by using a corresponding kit. Apoptosis and necrosis rates were measured by using flow cytometry.

Results: Chidamide inhibited the growth rate, migration, and invasion of human malignant glioma cells and increased the level of miR-338-5p. miR-338-5p inhibitor or mimics increased or inhibited the growth rate of U87 and HS683 glioma cells. Chidamide inhibited the levels of Shh, Ihh, migration protein E-cadherin, and invading protein MMP-2. The increase in the level of Shh and Ihh led to the reduction in the ROS and RNS levels. miR-338-5p inhibitor or mimics also showed a promoting or inhibitory function for the levels of Shh and Ihh. Furthermore, miR-338-5p mimics and inhibitor inhibited or promoted the migration and invasion of the glioma cells (P < 0.05). Evaluated levels of miR-338-5p increased oxidative stress level and apoptosis and necrosis rate by regulating the levels of biomarkers of oxidative stress (P < 0.05). Evaluated levels of miR-338-5p increased oxidative stress level and apoptosis and necrosis rate by regulating the levels of biomarkers of oxidative stress (.

Conclusion: Chidamide inhibits glioma cells by increasing oxidative stress via the miRNA-338-5p regulation of Hedgehog signaling. Chidamide may be a potential drug in the prevention of glioma development.

Figure 1

³H-TdR incorporation analysis of the growth rate of U87 and HS683 glioma cells. (a) The effects of chidamide on the growth rate of U87 glioma cells. (b) The effects of chidamide on the growth rate of HS683 glioma cells. (c) The growth rate of U87 glioma cells among different groups. (d) The growth rate of HS683 glioma cells among different groups. CG: control group; CHG: chidamide group; MG: miR-338-5p mimic group; IG: miR-338-5p inhibitor group; CHMG: chidamide-treated miR-338-5p mimic group; CHIG: chidamide-treated miR-338-5p inhibitor group. N = 5 for each group and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. the CG group.

Figure 2

The effects of chidamide on the activity of HDAC in U87 and HS683 glioma cells. (a) Relative activity of HDAC in U87 glioma cells. (b) Relative activity of HDAC in HS683 glioma cells.

Figure 3

Flow cytometry analysis of the cell cycle, necrosis, and apoptosis. (a) The proportions of the U87 cells blocked at the G₀/G₁ phase after being treated with 0 μM chidamide. (b) The proportions of the U87 cells blocked at the G₀/G₁ phase after being treated with 4 μM chidamide. (c) The proportions of the U87 cells blocked at the G₀/G₁ phase after being treated with 8 μM chidamide. (d) The proportions of the HS683 cells blocked at the G₀/G₁ phase after being treated with 0 μM chidamide. (e) The proportions of the HS683 cells blocked at the G₀/G₁ phase after being treated with 4 μM chidamide. (f) The proportions of the HS683 cells blocked at the G₀/G₁ phase after being treated with 8 μM chidamide. (g) Phase fraction in HS683 cells after being treated with different concentrations of chidamide. (h) G₁ phase fraction in U87 cells after being treated with different concentrations of chidamide. (i) G₁ phase fraction in HS683 cells after being treated with different concentrations of chidamide. (j) The proportions of the U87 cells with apoptosis and necrosis after being treated with 0 μM chidamide. (k) The proportions of the U87 cells with apoptosis and necrosis after being treated with 4 μM chidamide. (l) The proportions of the U87 cells with apoptosis and necrosis after being treated with 8 μM chidamide. (m) The proportions of the HS683 cells with apoptosis and necrosis after being treated with 0 μM chidamide. (n) The proportions of the HS683 cells with apoptosis and necrosis after being treated with 4 μM chidamide. (o) The proportions of the HS683 cells with apoptosis and necrosis after being treated with 8 μM chidamide. (p) The total apoptosis and necrosis rates in U87 cells after being treated with different concentrations of chidamide. (q) The total apoptosis and necrosis rates in HS683 cells after being treated with different concentrations of chidamide. All data are expressed as mean ± SD (N = 6) and ^∗P < 0.05 and ^∗∗P < 0.01 vs. the 0 μM chidamide group.

Figure 4

Relative mRNA levels of miR-338-5p. (a) The effects of chidamide on the level of miR-338-5p in U87 cells. (b) The effects of chidamide on the level of miR-338-5p in HS683 cells. (c) Relative mRNA levels of miR-338-5p in U87 glioma cells. (d) Relative mRNA levels of miR-338-5p in HS683 glioma cells. CG: control group; CHG: chidamide group; MG: miR-338-5p mimic group; IG: miR-338-5p inhibitor group; CHMG: chidamide-treated miR-338-5p mimic group; CHIG: chidamide-treated miR-338-5p inhibitor group. N = 5 for each group and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. the CG group.

Figure 5

The effects of miR-338-5p on the function of chidamide (10 μM) for controlling cell invasion per field. CG: control group; CHG: chidamide group; MG: miR-338-5p mimic group; IG: miR-338-5p inhibitor group; CHMG: chidamide-treated miR-338-5p mimic group; CHIG: chidamide-treated miR-338-5p inhibitor group. (a) The invasion of U78 glioma cells. (b) The invasion of HS683 cells. Values are mean ± SD (N = 5) and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. the CG group.

Figure 6

The effects of miR-338-5p on the function of chidamide (10 μM) for controlling cell immigration. CG: control group; CHG: chidamide group; MG: miR-338-5p mimic group; IG: miR-338-5p inhibitor group; CHMG: chidamide-treated miR-338-5p mimic group; CHIG: chidamide-treated miR-338-5p inhibitor group. (a) The migration of U87 glioma cells. (b) The migration of HS683 glioma cells. Values are mean ± SD (N = 5) and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. the CG group.

Figure 7

Real-time qPCR analysis of the effects of chidamide (10 μM) on relative mRNA Hedgehog signaling and migration proteins. (a) U87 glioma cells. (b) HS683 glioma cells. CG: control group; CHG: chidamide group; MG: miR-338-5p mimic group; IG: miR-338-5p inhibitor group; CHMG: chidamide-treated miR-338-5p mimic group; CHIG: chidamide-treated miR-338-5p inhibitor group; Shh: Sonic Hedgehog; Ihh: Indian Hedgehog; Dhh: Desert Hedgehog; MMP-2: matrix metalloproteinase-2. N = 5 for each group and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. the CG group.

Figure 8

Western Blot analysis of the effects of chidamide (10 μM) on the relative protein level of the Hedgehog signaling and migration protein. (a) U87 glioma cells. (b) HS683 glioma cells. CG: control group; CHG: chidamide group; MG: miR-338-5p mimic group; IG: miR-338-5p inhibitor group; CHMG: chidamide-treated miR-338-5p mimic group; CHIG: chidamide-treated miR-338-5p inhibitor group; Shh: Sonic Hedgehog; Ihh: Indian Hedgehog; Dhh: Desert Hedgehog; MMP-2: matrix metalloproteinase-2. N = 5 for each group and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. the CG group.

Figure 9

The oxidative levels among different groups. Glioma cells were labeled with (a) DCF DA and/or DAF-FM DA, and intracellular fluorescence was measured using a plate reader to estimate ROS and RNS. (a) ROS levels in U87 glioma cells. (b) RNS levels in U87 glioma cells. (c) ROS levels in HS683 glioma cells. (d) RNS levels in HS683 cells. CG: control group; CHG: chidamide group; MG: miR-338-5p mimic group; IG: miR-338-5p inhibitor group; CHMG: chidamide-treated miR-338-5p mimic group; CHIG: chidamide-treated miR-338-5p inhibitor group. N = 5 for each group and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. the CG group.

Figure 10

Flow cytometry detection of apoptosis rate. (a) Apoptosis rate in U87 glioma cells. (b) Apoptosis rate in HS683 glioma cells. N = 5 for each group and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. the CG group.