姜黄素对肺癌A549细胞迁移和侵袭的影响

doi:10.3877/cma.j.issn.1674-6902.2020.03.005

目的

探讨姜黄素对肺癌A549细胞侵袭迁移力的影响及其机制。

方法

利用不同浓度姜黄素处理正常肺上皮细胞，台盼蓝法检测药物细胞毒性；不同浓度的姜黄素作用于A549细胞，MTT法测定A549细胞增殖水平；细胞划痕实验和Transwell实验分别测定药物处理后A549细胞的迁移和侵袭能力变化情况；Werstern blot测定A549细胞中于侵袭迁移有关的表皮生长因子受本(EGFR)、E-型钙黏蛋白(E-cadherin)、N-型钙黏蛋白(N-cadherin)蛋白的表达。

结果

姜黄素浓度在0～40 μg/ml时正常肺上皮病死率低，差异无明显统计学差异(P>0.05)，表明姜黄素对正常肺上皮细胞的不良反应小；MTT实验结果显示姜黄素以浓度依赖的方式抑制A549细胞的增殖，各组与对照组相比均具有统计学意义(P<0.01)；细胞划痕实验显示姜黄素以浓度依赖的方式抑制A549细胞的迁移能力，各组与对照组相比均具有统计学意义(P<0.01)；Transwell实验显示姜黄素以浓度依赖的方式抑制A549细胞的侵袭能力，各组与对照组相比均具有统计学意义(P<0.01)；Western blot显示姜黄素处理A549细胞后EGFR蛋白(P<0.01)和N-cadherin蛋白表达降低(P<0.01)，E-cadherin蛋白表达升高(P<0.01)。

结论

一定浓度内姜黄素细胞毒性低，姜黄素能够抑制A549的增殖、迁移和侵袭，可能是通过调节EGFR、N-cadherin、E-cadherin蛋白从而逆转上皮间质转化过程。

关键词: 姜黄素, 支气管肺癌, A549细胞, 迁移, 侵袭, EMT

Objective

To explore the effect and mechanism of curcumine on the migration and invasion of A549 cells.

Methods

Trypan blue dye exclusion assay was used to determine the effect of various concentrations of curcumine on the cell death rate. The proliferation of A549 cells was detected by MTT assay. The migration and invasion of A549 cells were determined by wound scratch assay and Transwell assay, respectively. The expression levels of EGFR, E-cadherin and N-cadherin in A549 cells affected by curcumine were detected by Western blotting method.

Results

When the curcumine concentration was 0-40 μg/ml, the mortality rate of the normal lung epithelial cells was low, and the difference was not statistically significant (P>0.05), indicating that curcumine had little toxic and side effects on the normal lung epithelial cells. The MTT results showed that curcumine inhibited the proliferation of A549 cells in a concentration-dependent manner, compared with the control group, and statistical significant difference was found between the two groups (P<0.01). The cell scratch experiment showed that the migration ability of A549 cells was reduced after treatment of curcumine for 24 hours, compared with the control group, and statistical significant difference was found between the two groups (P<0.01). Transwell assay showed that the invasiveness of A549 cells was decreased with the least amount of curcumine, compared with the control group, and statistical significant difference was found between the two groups (P<0.01). Western blotting showed that the expression of epidermal growth factor receptor (EGFR, P<0.01) and N-cadherin protein were decreased (P<0.01) and the expression of E-cadherin protein was increased (P<0.01) after treatment of 40 μg/ml of curcumine on A549 cells, compared with the control group, and statistical significant difference was found between the two groups.

Conclusion

Curcumine can inhibit the proliferation, migration and invasion of A549 cells probably through down-regulating the expression of EGFR and N-cadherin and regulating the expression of E-cadherin, and therefore reversing the process of epithelial stromal transformation.

Key words: Curcumine, Bronchogenic carcinoma, A549 cells, Migration, Invasion, EMT

图1 姜黄素对人正常肺上皮细胞病死率的影响；注：*P<0.01，与对照组比较

图2 姜黄素对A549细胞增殖的影响；注：*P<0.05，#P<0.01，与对照组比较

表1 姜黄素对A549细胞增殖的影响(±s，n＝5，48 h)

浓度(μg/ml)	A570	抑制率(%)
0	0.571±0.033	-
10	0.469±0.031^a	17.86
20	0.369±0.042^b	35.37
40	0.280±0.035^b	50.96
80	0.170±0.039^b	70.35

表1 姜黄素对A549细胞增殖的影响(±s，n＝5，48 h)

浓度(μg/ml)	A570	抑制率(%)
0	0.571±0.033	-
10	0.469±0.031^a	17.86
20	0.369±0.042^b	35.37
40	0.280±0.035^b	50.96
80	0.170±0.039^b	70.35

图3 A549细胞划痕实验光镜照片(HE×100)；注：*P<0.01，与对照组比较；#P<0.01，与较低剂量组比较

图4 A549细胞Transwell侵袭实验光镜照片(HE×100)；注：*P<0.01，与对照组比较；#P<0.01，与较10 μg剂量组比较

图5 Western blot显示各组中EGFR、E-cadherin以及N-cadher蛋白的表达变化(±s，n＝3)；注：*P<0.01 vs. 1; #P<0.01 vs 2; ∧P<0.01 vs. 3

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Effect of curcumine on migration and invasion of A549 cells

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Effect of curcumine on migration and invasion of A549 cells