淋巴细胞趋化因子对乳腺癌MCF-7 细胞的表柔比星药物敏感性及侵袭转移能力的影响

doi:10.3877/cma.j.issn.1674-0807.2017.06.007

目的

探讨淋巴细胞趋化因子(lymphotactin, XCL1)对人乳腺癌MCF-7 细胞的表柔比星药物敏感性及侵袭转移能力的影响。

方法

根据是否进行表柔比星及XCL1 干预, 将MCF-7 细胞分为4 组:对照组(MCF-7 NC),表柔比星单药组(MCF-7 E),XCL1 单药组(MCF-7 XCL1),联合干预组(MCF-7 XCL1+E)。用细胞计数试剂盒8(CCK-8)及平板克隆实验观察XCL1 干预后MCF-7 细胞对表柔比星药物敏感性的影响。用Transwell 法观察XCL1 对MCF-7 细胞侵袭转移能力的影响,并用免疫荧光法检测对照组和XCL1 单药组埃兹蛋白(ezrin)及磷酸化埃兹蛋白(p-ezrin)的表达。用Western blot 实验检测4 组E 钙黏蛋白(E-cadherin)、ezrin 及p-ezrin 的表达水平。细胞生长抑制率的比较采用析因设计的方差分析,2 组均数比较采用t 检验,多组均数比较采用单因素方差分析,两两比较用LSD 法。

结果

经XCL1处理2 周后,表柔比星对MCF-7 细胞的抑制作用降低(组间比较F=23.780,P＜0.001;不同浓度比较,F=160.602,P＜0.001;浓度因素和分组之间无交互作用,F=1.565,P=0.208)。 XCL1 刺激MCF-7 细胞后,各组(MCF-7 NC、MCF-7 XCL1、MCF-7 E 和MCF-7 XCL1+E)细胞克隆形成率分别为(15.63±0.61)%、(19.47±1.94)%、(7.77±0.71)%和(12.37±1.55)%,差异有统计学意义(F=41.925, P＜0.001)。迁移实验中MCF-7 XCL1 组的细胞穿膜数高于MCF-7 NC 组(180.00±20.42 比88.00±7.00,t=-7.382, P=0.002),侵袭实验中MCF-7 XCL1 组的细胞穿膜数也高于MCF-7 NC 组(176.33±8.02 比90.33±12.90, t=-9.808, P=0.001)。免疫荧光实验发现XCL1 刺激后的MCF-7 细胞中p-ezrin 蛋白表达明显升高(MCF-7 XCL1:1.08±0.12, MCF-7 NC:0.65±0.11,t=-4.528, P=0.011)。 Western blot 检测发现,4 组比较E-cadherin 和p-ezrin 表达的差异均有统计学意义(F=6.317、48.517,P 均＜0.050),ezrin表达差异无统计学意义(F=0.868,P=0.497)。与MCF-7 NC 组比较(1.10±0.09), MCF-7 E 组、MCF-7 XCL1 组和MCF-7 XCL1+E 组E-cadherin 表达均降低(0.65±0.22、0.67±0.14、0.71±0.11,P 均＜0.050);与MCF-7 NC 组比较(0.37±0.07),MCF-7 XCL1 组和MCF-7 XCL1+E 组p-ezrin 表达升高(0.93±0.10、1.28±0.15、P 均＜0.050)。

结论

XCL1 可能通过增强ezrin 蛋白磷酸化来增强乳腺癌MCF-7 细胞的侵袭转移能力,同时降低其对表柔比星的药物敏感性。

关键词: 乳腺肿瘤, 淋巴细胞趋化因子, 表柔比星, 抗耐药, 肿瘤, 埃兹蛋白

Objective

To explore the effect of lymphotactin (XCL1) on epirubicin sensitivity of human breast MCF-7 cells and invasion and metastasis ability of cells.

Methods

The MCF-7 cells were divided into four groups: the control group (MCF-7 NC) and three experimental groups (MCF-7 E group treated with epirubicin, MCF-7 XCL1 group treated with XCL1 and MCF-7 XCL1+E group treated with XCL1 plus epirubicin). CCK-8 assay and plate clone formation assay were used to measure the epirubicin sensitivity of MCF-7 cells after XCL1 treatment. Transwell assay was used to evaluate the migration and invasion ability of MCF-7 cells. The expressions of ezrin and phosphorylated ezrin (p-ezrin) in the control group and MCF-7 XCL1 group were determined by immunofluorescence assay. The expressions of E-cadherin, ezrin and p-ezrin in four groups were detected by Western blot. The growth inhibition rates of cells were compared using variance analysis of factorial design. The mean values between two groups were compared using t test, the mean values among multiple groups were compared using single factor analysis of variance. Pairwise comparison was performed using LSD method.

Results

After 2-week treatment of XCL1, the inhibitory effect of epirubicin on MCF-7 cells was decreased (group comparison: F=23.780,P＜0.001;different concentrations of epirubicin:F=160.602,P ＜0.001; no interaction between grouping and concentration of epirubicin: F=1.565, P=0.208). The clone formation rates of MCF-7 NC, MCF-7 XCL1, MCF-7 E and MCF-7 XCL1 + E groups were(15.63±0.61)%, (19.47±1.94)%, (7.77±0.71)% and (12.37±1.55)%, respectively, indicating a significant difference (F=41.925, P＜0.001). The number of cells penetrating the membrane in the MCF-7 XCL1 group was significantly higher than that in the MCF-7 NC group (migration: 180.00±20.42 vs 88.00±7.00, t = -7.382, P = 0.002; invasion: 176.33±8.02 vs 90.33 ±12.90, t = -9808, P = 0.001).Immunofluorescence assay showed that the expression of p-ezrin protein was significantly increased in MCF-7 cells after XCL1 stimulation (MCF-7 XCL1:1.08±0.12, MCF-7 NC:0.65 ± 0.11, t=-4.528, P=0.011).Western blot analysis showed that the expressions of E-cadherin and p-ezrin among four groups were significantly different (F=6.317,48.517, both P＜0.050), while there was no significant difference in ezrin expression (F=0.868, P=0.497). Compared with the control group(1.10 ± 0.09), the expression of E-cadherin in MCF-7 E group, MCF-7 XCL1 group and MCF-7 XCL1 + E group was decreased (0.65±0.22,0.67±0.14,0.71±0.11, all P＜0.05). The expression of p-ezrin in MCF-7 XCL1 group and MCF-7 XCL1+E group (0.93±0.10,1.28±0.15) was increased compared with control group (0.37±0.07, both P＜0.050).

Conclusion

XCL1 can reduce the epirubicin sensitivity of human breast cancer MCF-7 cells and increase the ir invasion and metastasis ability of cells.

Key words: Breast neoplasms, Lymphokines, Epirubicin, Drug resistance, neoplasm, Ezrin

表1 不同浓度表柔比星处理后MCF-7 细胞的生长抑制率(%)

组别	试验次数	0.125μg/ml	0.250μg/ml	0.500μg/ml	0.750μg/ml	1.000μg/ml	1.500μg/ml
MCF-7 NC	3	22.21±1.11	32.15±3.11	51.57±3.40	60.83±1.81	61.64±2.05	62.26±5.19
MCF-7 XCL1	3	21.39±2.42	27.12±2.66	40.93±2.66	54.59±3.23	56.41±2.43	58.96±5.48

表1 不同浓度表柔比星处理后MCF-7 细胞的生长抑制率(%)

组别	试验次数	0.125μg/ml	0.250μg/ml	0.500μg/ml	0.750μg/ml	1.000μg/ml	1.500μg/ml
MCF-7 NC	3	22.21±1.11	32.15±3.11	51.57±3.40	60.83±1.81	61.64±2.05	62.26±5.19
MCF-7 XCL1	3	21.39±2.42	27.12±2.66	40.93±2.66	54.59±3.23	56.41±2.43	58.96±5.48

图1 平板克隆实验检测各组MCF-7 细胞的克隆形成能力注:图a ～d 分别为MCF-7 NC、MCF-7 XCL1、MCF-7 E 和MCF-7 XCL1+E 组

图2 平板克隆实验检测MCF-7 细胞克隆形成率注:4 组比较,F=41.925,P＜0.001;与MCF-7 NC 组比较,^aP＜0.050;^b 与MCF-7 E 组比较,P＜0.050

图3 MCF-7 NC 组和MCF-7 XCL1 组细胞Transwell 迁移和侵袭实验(结晶紫 ×100) 注:a、b 图分别为MCF-7 NC 组和MCF-7 XCL1 组的迁移实验结果;c、d 图分别为MCF-7 NC 组和MCF-7 XCL1 组的侵袭实验结果

图4 MCF-7 NC 组和MCF-7 XCL1 组Transwell 迁移和侵袭实验的穿膜细胞数注:^aP＜0.050;a 图为迁移实验结果;b 图为侵袭实验结果

图5 免疫荧光检测乳腺癌MCF-7 细胞中ezrin 蛋白表达(DAPI ×200) 注: a、b 图为MCF-7 NC 组和MCF-7 XCL1 组ezrin 蛋白表达; c、d 图为MCF-7 NC 组和MCF-7 XCL1 组p-ezrin 蛋白表达

图6 Western bolt 检测各组MCF-7 细胞中E-cadherin、ezrin 及p-ezrin 的蛋白表达

表2 各组MCF-7 细胞中E-cadherin、ezrin 及p-ezrin 的蛋白表达

组别	试验次数	E-cadherin	p-ezrin	ezrin
MCF-7 NC	3	1.10±0.09	0.37±0.07	0.88±0.11
MCF-7 E	3	0.65±0.22^a	0.54±0.06^b	0.96±0.09
MCF-7 XCL1	3	0.67±0.14^a	0.93±0.10^ab	1.00±0.19
MCF-7 XCL1+E	3	0.71±0.11^a	1.28±0.15^a	1.00±0.06
F值		6.317	48.517	0.868
P值		0.017	＜0.001	0.497

表2 各组MCF-7 细胞中E-cadherin、ezrin 及p-ezrin 的蛋白表达

组别	试验次数	E-cadherin	p-ezrin	ezrin
MCF-7 NC	3	1.10±0.09	0.37±0.07	0.88±0.11
MCF-7 E	3	0.65±0.22^a	0.54±0.06^b	0.96±0.09
MCF-7 XCL1	3	0.67±0.14^a	0.93±0.10^ab	1.00±0.19
MCF-7 XCL1+E	3	0.71±0.11^a	1.28±0.15^a	1.00±0.06
F值		6.317	48.517	0.868
P值		0.017	＜0.001	0.497

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Effect of lymphotactin on epirubicin sensitivity and invasion and metastasis ability of human breast cancer MCF-7 cells

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Effect of lymphotactin on epirubicin sensitivity and invasion and metastasis ability of human breast cancer MCF-7 cells