艾拉莫德增强丝裂霉素C诱导的人食管癌Eca109细胞凋亡

doi:10.3877/cma.j.issn.2095-1221.2019.01.003

目的

探讨艾拉莫德增强丝裂霉素C（MMC）诱导的人食管癌Eca109细胞凋亡的作用及其机制。

方法

将人食管癌Eca109细胞分为五组：对照组、MMC组、25，50，100 μg/ml浓度艾拉莫德+MMC组；通过CCK-8和DCFH-DA染色法分别检测25，50，100 μg/ml浓度艾拉莫德联合MMC对人食管癌Eca109细胞活力和细胞活性氧（ROS）生成的影响，流式细胞术检测艾拉莫德联合MMC对人食管癌Eca109细胞凋亡的影响，并通过Western Blot检测艾拉莫德联合MMC对TNF-α和cleaved caspase3蛋白表达的影响。采用单因素方差分析和t检验进行统计学分析。

结果

CCK-8结果显示，与0 μg/ml T-614+MMC组A450值（0.85±0.03）比较，25、50、100 μg/ml T-614+MMC组A450值（0.73±0.02，0.52±0.02，0.33±0.02）均降低，差异具有统计学意义（F = 127.60, P < 0.01）；DCFH-DA染色检测结果显示，与0 μg/ml T-614+MMC组DCFH荧光值（130.00±10.00）比较，25、50、100 μg/ml T-614+MMC组DCFH值（219.67±9.50，280.33±10.50，338.33±16.07）均升高，差异具有统计学意义（F = 170.20，P < 0.01）；流式细胞术检测结果显示，与对照组的细胞凋亡率（5.33±0.35）﹪比较，T-614和MMC组的凋亡率（20.30±2.00）﹪，（25.60±3.00）﹪均升高。与MMC组细胞凋亡率（25.60±3.00）﹪比较，艾拉莫德与MMC联用组（T-614+MMC）食管癌Eca109细胞的细胞凋亡率（56.20±3.00）﹪升高，差异均具有统计学意义（F = 247.50，P < 0.01）；Western Blot结果显示，与MMC组细胞TNF-α和cleaved caspase3蛋白表达（0.87±0.06，0.25±0.03）比较，艾拉莫德与MMC联用组（T-614+MMC）食管癌Eca109细胞的TNF-α和cleaved caspase3蛋白表达（1.28±0.08，0.59±0.03）升高，差异均具有统计学意义（F = 96.90，P < 0.01）。

结论

艾拉莫德能够增强MMC对食管癌Eca109细胞活力的抑制作用，其机制可能通过促进ROS的生成，激活线粒体凋亡通路，最终导致食管癌Eca109细胞凋亡。

关键词: 食管癌, 艾拉莫德, 细胞因子, 细胞凋亡, 丝裂霉素

Objective

To investigate the effect of the combination of Iguratimod (T-?614) and mitomycin (MMC) on apoptosis of human esophageal cancer Eca109 cells, and to explore the possible mechanism.

Methods

Eca109 cells were exposed to MMC in the presence or absence of difference concentrations of T-614. Eca109 cell viability and reactive oxygen species (ROS) production were detected by CCK8 and DCFH-DA staining. Apoptosis of Eca109 cells was detected by flow cytometry. Expression of TNFα and cleaved caspase3 in Eca109 cells was detected by Western Blot. Statistical analysis was performed using one-way analysis of variance and t test.

Results

CCK8 results showed that A450 values of 25, 50, and 100 μg/ml T-614+MMC group (0.73±0.02, 0.52±0.02, and 0.33±0.02) were significantly reduced compared with A450 value (0.85±0.03) of 0 μg/?ml T-614+MMC group (F?=?127.60, P?< 0.01) . The results of DCFH-DA staining showed that the DCFH values of the 25, 50, and 100 μg/ml T-614+MMC group (219.67±9.50, 280.33±10.50, and 338.33±16.07) were significantly increased compared with the fluorescence value of DCFH in the 0?μg/ml T-614+MMC group (130.00±10.00) . The difference was statistically significant (F?=?170.20, P?<?0.01) . The results of flow cytometry showed that the apoptosis rate of T-614 group (20.30±2.00) ﹪ and MMC group (25.60±3.00) ﹪ was significantly increased compared with the control group (5.33±0.35) ﹪. Compared with MMC group (25.60±3.00) ﹪, the apoptosis rate (56.20±3.00) ﹪of the T-614+MMC group was significantly increased (F?= 247.50, P?< 0.01) . Western Blot results showed that the expression of TNFα and cleaved caspase3 in T-614+MMC group (1.28±0.08, 0.59±0.03) was significantly increased compared with the expression in MMC group (0.87±0.06, 0.25±0.03) (F?= 96.90, P?< 0.01) .

Conclusion

T-614 significantly enhanced the inhibitory effect of MMC on the viability of Eca109 cells. The mechanism may be related to the production of ROS and the activation of mitochondrial apoptosis pathway, which ultimately leads to apoptosis of esophageal cancer Eca109 cells.

Key words: Esophageal cancer, Iguratimod, Reactive oxygen species, Apoptosis, Mitomycin

表1 不同浓度T-614和T-614+MMC处理后CCK8检测Eca109细胞A450值（ ± s）

T-614分组浓度	样本数	T-614（A450）	T-614+MMC（A450）
0 μg/ml	3	1.05±0.05	0.85±0.03
25 μg/ml	3	0.98±0.08	0.73±0.02^a
50 μg/ml	3	0.82±0.08^a	0.52±0.02^a
100 μg/ml	3	0.55±0.05^a	0.33±0.02^a
F值	?	35.73	127.60
P值	?	< 0.0001	< 0.0001

表1 不同浓度T-614和T-614+MMC处理后CCK8检测Eca109细胞A450值（ ± s）

T-614分组浓度	样本数	T-614（A450）	T-614+MMC（A450）
0 μg/ml	3	1.05±0.05	0.85±0.03
25 μg/ml	3	0.98±0.08	0.73±0.02^a
50 μg/ml	3	0.82±0.08^a	0.52±0.02^a
100 μg/ml	3	0.55±0.05^a	0.33±0.02^a
F值	?	35.73	127.60
P值	?	< 0.0001	< 0.0001

表2 不同浓度T-614和T-614+MMC处理后DCFH法检测Eca109细胞DCFH荧光值（ ± s）

T-614分组浓度	样本数	T-614（DCFH）	T-614+MMC（DCFH）
0 μg/ml	3	35.00± 5.00	130.00±10.00
25 μg/ml	3	41.67± 1.53	219.67± 9.50^a
50 μg/ml	3	90.00±10.00^a	280.33±10.50^a
100 μg/ml	3	136.67±15.28^a	338.33±16.07^a
F值	?	74.71	170.20
P值	?	< 0.0001	< 0.0001

表2 不同浓度T-614和T-614+MMC处理后DCFH法检测Eca109细胞DCFH荧光值（ ± s）

T-614分组浓度	样本数	T-614（DCFH）	T-614+MMC（DCFH）
0 μg/ml	3	35.00± 5.00	130.00±10.00
25 μg/ml	3	41.67± 1.53	219.67± 9.50^a
50 μg/ml	3	90.00±10.00^a	280.33±10.50^a
100 μg/ml	3	136.67±15.28^a	338.33±16.07^a
F值	?	74.71	170.20
P值	?	< 0.0001	< 0.0001

图1 流式细胞术检测艾拉莫德对人食管癌细胞Eca109细胞凋亡的影响

图2 艾拉莫德对人食管癌细胞Eca109细胞凋亡的影响

图3 艾拉莫德对人食管癌细胞Eca109细胞蛋白表达的影响

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Iguratimod enhances mitomycin C-induced apoptosis in human esophageal cancer Eca109 cells

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Iguratimod enhances mitomycin C-induced apoptosis in human esophageal cancer Eca109 cells