Objective: To study the effects of octadecadienoic acid (ODA) on the proliferation and apoptosis of glioma cells and its mechanisms.
Methods: Cultured human glioma cells (cell density 2×106 cells/L) were divided into solvent control group (DMSO, 30 μl/L), 5-FU group (10 mg/L) and octadecadienic acid groups (0.3, 0.6 and 1.2 mg/L groups). The toxicity of ODA on glioma cells was detected by trypan blue and thiazolium blue (MTT). The expression levels of P53, PI3K, P21, PKB/Akt and Caspase-9 in glioma cells were determined by enzyme-linked immunosorbent assay (ELISA).
Results: ① Cell count under optical microscope showed that the inhibition rate of cell proliferation in ODA low, medium and high dose groups and 5-FU group was significantly higher than that in the solvent control group (P＜0.01), but there was no statistical significance compared with the 5-FU group (P＞0.05). ② MTT assay showed that the inhibition rate of cell proliferation was increased significantly in ODA low, medium and high dose groups and 5-FU groups (P＜0.01), compared with the solvent control group. Compared with 5-FU group, the inhibition rate of cell proliferation was increased significantly only in ODA high dose group (P＜0.01). ③ The number of G0/G1 phase cells in ODA low, medium and high dose groups and 5-FU group were increased significantly (P＜0.05, P＜0.01), the number of G2/M phase cells were decreased significantly (P＜0.01), and the apoptosis rate was increased significantly (P＜0.01),compared with the solvent control group. Compared with the 5-FU group, the number of cells in G2/M phase was decreased significantly (P＜0.01) and the apoptosis rate was increased significantly (P＜0.01) in ODA high dose group. ④ ELISA test results showed that the protein expression levels of P53, PI3K and PKB/Akt in ODA low , medium and high dose groups and 5-FU group were significantly lower than those in solvent control group (all P＜0.01), but the protein expression levels in ODA high dose group were significantly lower than those in 5-FU group (P＜0.01). The protein expression levels of P21 and caspase-9 in ODA low , medium and high dose groups and 5-FU group were significantly higher than those in solvent control group (P＜0.05, P＜0.01), but the protein expression levels in ODA high dose group were significantly higher than those in 5-Fu group (P＜0.01).
Conclusion: ODA can significantly inhibit the proliferation and promote apoptosis of glioma cells. The mechanisms are related to up-regulating the levels of P21 and caspase-9 to promote apoptosis, down-regulating the levels of P53, PI3K and PKB/Akt to inhibit the cell division cycle, and reducing the activity of PI3K-Akt signal transduction pathway.
目的: 探索十八碳二烯酸(ODA)抑制神经胶质瘤细胞增殖与促凋亡作用及其机制。方法: 取培养的人神经胶质瘤细胞(细胞密度2×106 cells/L)分为溶剂对照组(给予DMSO,含量为30 μl/L)、5-FU组(含量10 mg/L)和十八碳二烯酸组(设0.3、0.6、1.2 mg/L三个剂量组 )。用台盼蓝、噻唑蓝(MTT)检测ODA对神经胶质瘤细胞的毒性作用,用酶联免疫吸附法(ELISA) 检测神经胶质瘤细胞P53、PI3K、P21、PKB/Akt、caspase-9蛋白表达水平。结果: ①光学显微镜细胞计数显示: ODA低、中、高剂量组和5-FU组细胞增殖抑制率比溶剂对照组显著升高(P＜ 0.01),与5-FU组相比差异无统计学意义(P＞0.05)。②MTT检测结果显示:与溶剂对照组相比,ODA低、中、高剂量组和5-FU组细胞增殖抑制率显著升高(P＜0.01);与5-FU组相比,仅 ODA高剂量组细胞增殖抑制率显著增加(P＜0.01)。③流式细胞仪检测结果显示:与溶剂对照组相比,ODA低、中、高剂量组和5-FU组G0/G1期细胞数显著增加(P＜0.05,P＜0.01), G2/M期细胞数显著减少(P＜0.01),细胞凋亡率显著增加(P＜0.01);与5-FU组相比,仅 ODA高剂量组G2/M期细胞数显著减少(P＜0.01),凋亡率显著增加(P＜0.01)。④ELISA检测结果显示:ODA低、中、高剂量组和5-FU组P53、P13K、PKB/Akt蛋白表达水平比溶剂对照组均显著降低(均P＜0.01),仅ODA高剂量组蛋白表达水平均显著低于5-FU组(P＜0.01);ODA低、中、高剂量组和5-FU组P21、caspase-9蛋白表达水平明显高于溶剂对照组(P＜0.05,P＜0.01),仅ODA高剂量组蛋白表达水平显著高于5-FU组(P＜0.01)。结论: ODA对神经胶质瘤细胞增殖具有明显的抑制和促凋亡作用,其机制与上调细胞P21、caspase-9水平促凋亡,下调P53、PI3K、PKB/Akt水平抑制细胞分裂周期,降低PI3K-Akt信号转导通路活性有关。.
Keywords: cell apoptosis; glioma cells; inhibitory effect; octadecadienoic acid.