吉非替尼对三阴性乳腺癌细胞MDA-MB-231迁移运动的影响

Effect of gefitinib on the migration of triple-negative breast cancer cell line MDA-MB-231 cells

目的观察吉非替尼对人表皮生长因子受体（EGFR）高表达的三阴性乳腺癌（TNBC）细胞系MDA．MB-231迁移能力的影响。方法采用Westernblot法检测不同浓度吉非替尼作用MDA-MB-231细胞后EGFR和Akt磷酸化水平；采用划痕实验和Boyden小室趋化实验观察吉非替尼对细胞迁移、趋化能力的影响；采用免疫荧光染色观察吉非替尼对细胞骨架重构及极性变化的影响。结果与对照组（OμmoVL吉非替尼）相比，不同浓度的吉非替尼可有效抑制EGFR及其下游通路关键蛋白的磷酸化水平，呈明显的量效关系。划痕实验24h后，0、0．1、1、10、20μmoL／L吉非替尼组细胞迁移距离分别为（36．3．4-4．0）μm、（30．3±3．8）μm、（26．8±3．3）μm、（17．0±2．6）μm和（11．04-2．5）μm；Boyden小室趋化实验3．5h后，0、0．1、1、10、20Dmol/L吉非替尼组的穿膜细胞数分别为（69．2±7．0）个、（51．8±7．5）个、（43．8±8．7）个、（30．6±4．8）个和（28．4±3．4）个。吉非替尼可明显延长划痕愈合时间，减少趋化穿膜细胞个数，差异有统计学意义（P〈0．05）。吉非替尼可明显减少细胞片状伪足的形成，抑制细胞骨架的重构及极性变化。结论吉非替尼可以通过抑制TNBC细胞系MDA-MB-231中EGFR／P13K／Akt通路的磷酸化水平，抑制细胞骨架蛋白（微丝）的重构及极性改变，从而降低细胞的迁移运动能力。

Objective To investigate the effect of gefitinib on the migration of triple-negative breast cancer cell line MDA-MB-231 ceils. Methods Gefitinib was used in concentrations of 0 μmol/L, 0. 1 μmol/L, 1 /μmol/L, 10 /μmol/L and 20 /μmol/L, respectively. Phosphorylation levels of EGFR and Akt were analyzed by Western blot. The capability of migration was measured by scratch test and Boyden chamber assay. Microfilaments （cell skeleton ） remolding and polarization were evaluated by immunofluorescence microscopy. Results Comparing with the control group （0 μmol/L gefitinib ）, gefitinib effectively inhibited the phosphorylation of EGFR and its downstream key proteins, and the effect displayed an obvious dose-effect relationship. At 24 hours after wound scratch, the cell migration distance of each group with 0, 0. I, 1, 10, 20μmol/L gefitinib was （36.3±4.0）μm, （30.3±3.8）μm, （26.8±3.3） μm, （ 17.0±2.6） μm, and （ 11.0±2.5 ） μm, respectively. At 3.5 hours after Boyden chamber assay, the cell count of each group with 0, 0.1, 1, 10, 20 μmol/L gefitinib was 69.2±7.0, 51.8±7.5, 43.8±8.7, 30.6±4.8, and 28.4±3.4, respectively. Compared with the control group （0 μmol/L gefitinib）, gefitinib could significantly prolong the wound-healing time and decrease the migrating cell count （ P 〈 O. 05 ）, and significantly inhibit the lamellipodium formation, cell skeleton remolding and changes of the cytoskeleton polarization. Conclusions Gefitinib can reduce the migration capacity of triple-negative breast cancer cells through inhibiting phosphorylation of EGFR/PI3K/Akt pathway, suppressing the cell skeleton （ microfilaments） remolding and changes of its polarization.