碧萝芷对乳腺癌MCF-7细胞增殖、迁移和转移的影响

doi:10.3877/cma.j.issn.1674-0807.2019.03.006

目的

探讨抗氧化剂碧萝芷对乳腺癌MCF-7细胞的抑制作用，并对观察碧萝芷对MCF-7细胞的转移和迁移功能的影响。

方法

用10、20、40 μg/ml的碧萝芷处理乳腺癌MCF-7细胞作为实验组，空白对照组只加入DMEM培养基(无血清), MTT法在处理细胞后24、48、72 h于波长490 nm处测定各组吸光度值；用流式细胞仪检测细胞凋亡率；用β-半乳糖苷酶染色法检测MCF-7细胞衰老率；用Transwell小室法分别检测各浓度碧萝芷对乳腺癌MCF-7细胞迁移和侵袭能力的影响；用Western blot法分别检测乳腺癌MCF-7细胞衰老相关蛋白P53、P21、P16、P27、E2F1以及基质金属蛋白酶(matrix metalloproteinase, MMP)-2、MMP-9的表达。细胞迁移、侵袭数目及各种蛋白的表达水平比较采用单因素方差分析，吸光度值比较采用重复测量的方差分析，两两比较采用LSD法。

结果

MTT结果显示，在处理乳腺癌MCF-7细胞24、48、72 h后，各组吸光度值比较，差异有统计学意义(F＝149.439, P<0.001)，在不同时间点之间比较，差异有统计学意义(F＝27.922，P<0.001)；分组与时间点之间存在交互作用(F＝18.466, P<0.001)。流式细胞仪检测结果显示：空白对照组及10、20、40 μg/ml碧萝芷组的细胞凋亡率分别为(2.36±0.27)%、(6.44±1.43)%、(7.52±2.09)%和(11.68±1.65)%，差异有统计学意义(F＝19.143, P<0.001)。β-半乳糖苷酶染色法结果显示：空白对照组和10、20、40 μg/ml碧萝芷组的细胞衰老率分别为(5.35±1.32)%、(20.08±2.14)%、(40.55±4.61)%和(59.26±4.10)%，差异有统计学意义(F＝150.150, P<0.001)。Transwell小室结果显示：空白对照组及10、20、40 μg/ml碧萝芷组的迁移细胞数目分别为(41±5)、(27±2)、(19±1)、(11±2)个，差异有统计学意义(F＝59.330, P<0.001)；侵袭转移的细胞数目分别为(24±4)、(17±1)、(12±2)、(7±2)个，差异也有统计学意义(F＝26.230, P<0.001)。Western blot结果显示：碧萝芷可上调P53、P21、P16、P27蛋白表达(F＝263.905、424.937、217.515、391.115，P均<0.001)，下调E2F1蛋白的表达(F＝64.003，P<0.001)；同时，各组MCF-7细胞中的MMP-2及MMP-9蛋白表达水平明显降低(F＝44.104、45.594, P均<0.001)。

结论

碧萝芷可能是以促衰老的方式抑制乳腺癌MCF-7细胞的生长，且能抑制细胞的迁移和转移。

关键词: 乳腺肿瘤, 细胞衰老, 迁移, 转移, 碧萝芷

Objective

To investigate the inhibitory effect of the antioxidant pycnogenol on human breast cancer MCF-7 cells, and to observe the effect of pycnogenol on the metastasis and migration of MCF-7 cells.

Methods

MCF-7 cells were treated with 10, 20, 40 μg/ml pycnogenol, respectively, as experimental groups. The control group was only treated with blank DMEM medium (without fetal bovine serum). The optical density of MCF-7 cells at the wavelength of 490 nm was determined by the MTT method at 24, 48 and 72 h after treatment. The apoptosis rate of MCF-7 cells was measured by the flow cytometry. The senescence rate of MCF-7 cells was measured by β-galactosidase staining. The effect of pycnogenol at different concentrations on the migration and invasion of MCF-7 cells were detected by Transwell chamber assay. The expression of senescence-associated proteins P53, P21, P16, P27 and E2F1 in MCF-7 cells was determined by Western blot analysis. The expression of matrix metalloproteinase (MMP)-2 and MMP-9 was also determined by Western blot analysis. The number of migrated cells and metastatic cells, and the protein expression among four groups were compared by one-factor analysis of variance. The optical density was compared by repeated measurement analysis of variance and pairwise comparison was performed by LSD method.

Results

The result of MTT assay showed that at 24, 48, and 72 h after treatment, the optical density of breast cancer MCF-7 cells was significantly different among four groups (F=149.439, P<0.001) and at different time points (F=27.922, P<0.001); there was an interaction between grouping and time points (F=18.466, P<0.001). The result of flow cytometry showed that the apoptosis rate of MCF-7 cells was (2.36±0.27)%, (6.44±1.43)%, (7.52±2.09)% and (11.68±1.65)% in the blank control group, 10, 20 and 40 μg/ml pycnogenol group, respectively, indicating a significant difference (F=19.143, P<0.001). The result of β-galactosidase staining showed that the senescence rate of MCF-7 cells was (5.35±1.32)%, (20.08±2.14)%, (40.55±4.61)% and (59.26±4.10)% in the blank control group, 10, 20 and 40 μg/ml pycnogenol group, respectively, indicating a significant difference (F=150.150, P<0.001). The result of Transwell chamber assay showed that the number of migrated cells was 41±5, 27±2, 19±1 and 11±2 in the blank control group, 10, 20 and 40 μg/ml pycnogenol group, respectively, indicating a significant difference (F=59.330, P<0.001); the number of metastatic cells was 24±4, 17±1, 12±2 and 7±2, respectively in the blank control group, 10, 20 and 40 μg/ml pycnogenol group, respectively, indicating a significant difference (F=26.230, P<0.001). Western blot results showed that pycnogenol up-regulated the expression of P53, P21, P16 and P27 (F=263.905, 424.937, 217.515, 391.115, all P<0.001), and down-regulated the expression of E2F1 (F=64.003, P<0.001); the expression levels of MMP-2 and MMP-9 in MCF-7 cells were significantly decreased (F=44.104, 45.594, both P<0.001).

Conclusion

Pycnogenol may inhibit the growth of breast cancer MCF-7 cells by promoting the cellular senescence and it can also inhibit the cell migration and metastasis.

Key words: Breast neoplasms, Cellular senescence, Migration, Metastasis, Pycnogenol

表1 采用MTT法检测不同时间点各组乳腺癌MCF-7细胞的吸光度值(±s)

组别	24 h	48 h	72 h
空白对照组	0.918±0.028	1.005±0.043	1.122±0.142
10 μg/ml碧萝芷组	0.887±0.038	0.770±0.015^a	0.650±0.013^a
20 μg/ml碧萝芷组	0.712±0.031^ab	0.652±0.020^ab	0.510±0.030^ab
40 μg/ml碧萝芷组	0.721±0.052^ab	0.556±0.032^abc	0.334±0.033^abc

表1 采用MTT法检测不同时间点各组乳腺癌MCF-7细胞的吸光度值(±s)

组别	24 h	48 h	72 h
空白对照组	0.918±0.028	1.005±0.043	1.122±0.142
10 μg/ml碧萝芷组	0.887±0.038	0.770±0.015^a	0.650±0.013^a
20 μg/ml碧萝芷组	0.712±0.031^ab	0.652±0.020^ab	0.510±0.030^ab
40 μg/ml碧萝芷组	0.721±0.052^ab	0.556±0.032^abc	0.334±0.033^abc

图1 采用流式细胞仪检测各组乳腺癌MCF-7细胞凋亡情况　a～d图分别所示空白对照组及10、20、40 μg/ml碧萝芷组的细胞凋亡检测结果

图2 采用β-半乳糖苷酶染色法观察碧萝芷处理24 h后各组乳腺癌MCF-7细胞形态学特征　a～d图分别所示空白对照组及10、20、40 μg/ml碧萝芷组细胞的β-半乳糖苷酶染色结果(×200)

图3 采用Transwell小室实验检测各组乳腺癌MCF-7细胞的迁移和侵袭能力　a～d图分别所示空白对照组及10、20、40 μg/ml碧萝芷组迁移实验结果(结晶紫　×200)；e～h图分别所示空白对照组及10、20、40 μg/ml碧萝芷组侵袭实验结果(结晶紫　×200)

表2 各组乳腺癌MCF-7细胞迁移和侵袭能力的比较

组别	实验次数	细胞数目(个，±s)
组别	实验次数	迁移	侵袭
空白对照组	3	41±5	24±4
10 μg/ml碧萝芷组	3	27±2^a	17±1^a
20 μg/ml碧萝芷组	3	19±1^ab	12±2^ab
40 μg/ml碧萝芷组	3	11±2^abc	7±2^abc
F值	?	59.330	26.230
P值	?	<0.001	<0.001

表2 各组乳腺癌MCF-7细胞迁移和侵袭能力的比较

组别	实验次数	细胞数目(个，±s)
组别	实验次数	迁移	侵袭
空白对照组	3	41±5	24±4
10 μg/ml碧萝芷组	3	27±2^a	17±1^a
20 μg/ml碧萝芷组	3	19±1^ab	12±2^ab
40 μg/ml碧萝芷组	3	11±2^abc	7±2^abc
F值	?	59.330	26.230
P值	?	<0.001	<0.001

图4 采用Western blot检测各组乳腺癌MCF-7细胞中细胞衰老通路重要效应蛋白的表达

表3 各组乳腺癌MCF-7细胞中细胞衰老通路重要效应蛋白的表达

组别	实验次数	P53	P21	P16	P27	E2F1
空白对照组	3	1.000±0.000	1.000±0.000	1.000±0.000	1.000±0.000	1.000±0.000
10 μg/ml碧萝芷组	3	1.252±0.045^a	1.309±0.045^a	1.244±0.024^a	1.309±0.036^a	0.821±0.051^a
20 μg/ml碧萝芷组	3	1.618±0.036^ab	1.796±0.059^ab	1.422±0.048^ab	1.825±0.042^ab	0.688±0.033^ab
40 μg/ml碧萝芷组	3	1.941±0.067^abc	2.021±0.022^abc	1.782±0.056^abc	1.954±0.055^abc	0.532±0.061^abc
F值	?	263.905	424.937	217.515	391.115	64.003
P值	?	<0.001	<0.001	<0.001	<0.001	<0.001

表3 各组乳腺癌MCF-7细胞中细胞衰老通路重要效应蛋白的表达

组别	实验次数	P53	P21	P16	P27	E2F1
空白对照组	3	1.000±0.000	1.000±0.000	1.000±0.000	1.000±0.000	1.000±0.000
10 μg/ml碧萝芷组	3	1.252±0.045^a	1.309±0.045^a	1.244±0.024^a	1.309±0.036^a	0.821±0.051^a
20 μg/ml碧萝芷组	3	1.618±0.036^ab	1.796±0.059^ab	1.422±0.048^ab	1.825±0.042^ab	0.688±0.033^ab
40 μg/ml碧萝芷组	3	1.941±0.067^abc	2.021±0.022^abc	1.782±0.056^abc	1.954±0.055^abc	0.532±0.061^abc
F值	?	263.905	424.937	217.515	391.115	64.003
P值	?	<0.001	<0.001	<0.001	<0.001	<0.001

图5 采用Western blot检测各组乳腺癌MCF-7细胞中MMP-2及MMP-9的表达

表4 各组乳腺癌MCF-7细胞中MMP-2及MMP-9的表达

组别	实验次数	MMP-2	MMP-9
空白对照组	3	1.000±0.000	1.000±0.000
10 μg/ml碧萝芷组	3	0.953±0.048	0.801±0.055^a
20 μg/ml碧萝芷组	3	0.762±0.052^ab	0.727±0.041^a
40 μg/ml碧萝芷组	3	0.563±0.078^abc	0.564±0.063^abc
F值	?	44.104	45.594
P值	?	<0.001	<0.001

表4 各组乳腺癌MCF-7细胞中MMP-2及MMP-9的表达

组别	实验次数	MMP-2	MMP-9
空白对照组	3	1.000±0.000	1.000±0.000
10 μg/ml碧萝芷组	3	0.953±0.048	0.801±0.055^a
20 μg/ml碧萝芷组	3	0.762±0.052^ab	0.727±0.041^a
40 μg/ml碧萝芷组	3	0.563±0.078^abc	0.564±0.063^abc
F值	?	44.104	45.594
P值	?	<0.001	<0.001

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Effect of pycnogenol on proliferation, migration and metastasis of human breast cancer MCF-7 cells

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Effect of pycnogenol on proliferation, migration and metastasis of human breast cancer MCF-7 cells