比卡鲁胺联合紫杉醇对雄激素受体阳性三阴性乳腺癌MDA-MB-231细胞的增殖抑制作用

doi:10.3877/cma.j.issn.1674-0807.2018.03.002

目的

探讨比卡鲁胺(BIC)联合化疗药物紫杉醇(PTX)对雄激素受体(AR)阳性的三阴性乳腺癌MDA-MB-231细胞的增殖抑制作用及可能的作用机制。

方法

采用CCK-8试剂盒观察不同浓度的BIC (0.1、1.0、10.0 μmol/L)和PTX(0.1、1.0、10.0、100.0、1 000.0、10 000.0 nmol/L)以单药及不同联合给药方式处理后，对MDA-MB-231细胞增殖的抑制作用。细胞增殖抑制率比较采用单因素方差分析。组间两两比较采用LSD法。选取10 nmol/L PTX及10 nmol/L DMSO分别处理MDA-MB-231细胞样品(各3个)72 h，采用生物信息学方法分析样品的相关基因表达芯片数据，采用校正t检验筛选出差异基因。

结果

使用不同浓度的BIC分别处理MDA-MB-231细胞24、48、72 h后，各组MDA-MB-231细胞增殖抑制率在不同时间点差异均有统计学意义(F＝4.124、8.189、4.139, P＝0.037、0.004、0.032)。BIC 10.0 μmol/L组MDA-MB-231细胞增殖抑制率在48 h最高，为(12.9 ± 5.5)%。不同浓度的PTX分别处理MDA-MB-231细胞24、48、72 h后，不同浓度组MDA-MB-231细胞增殖抑制率在不同时间点差异均有统计学意义(F＝8.407、47.432、14.907, P均<0.001)。PTX在48 h时对MDA-MB-231细胞的半数抑制浓度(IC₅₀)为5 380.0 nmol/L。5 000.0 nmol/L PTX单药或联合不同浓度(0.1、1.0、10.0 μmol/L)的BIC同时处理MDA-MB-231细胞48 h后，5 000.0 nmol/L PTX单药处理组与3个实验组中细胞增殖抑制率分别为(53.2±2.7)%、(53.2±3.1)%、(51.7±3.4)%、(51.0±2.3)%，组间差异无统计学意义(F＝0.831，P＝0.492)。采用5 000.0 nmol/L PTX和10.0 μmol/L BIC以不同的序贯方式联合给药处理MDA-MB-231细胞(PTX 24 h ＋BIC 24 h组、BIC 24 h ＋PTX 24 h组、PTX 48 h ＋BIC 24 h组、BIC 48 h＋PTX 24 h组)，并用5 000.0 nmol/L PTX(PTX 48 h组)和10.0 μmol/L BIC(BIC 48 h组)单药处理及同时联合给药(PTX 48 h＋ BIC 48 h组)分别处理MDA-MB-231细胞后，各组间细胞增殖抑制率差异有统计学意义(F＝241.466，P<0.001)。其中，两两比较结果显示，PTX 24 h ＋BIC 24 h组细胞增殖抑制率为(72.9±1.9)%，高于BIC 24 h ＋PTX 24 h组的(42.9±1.7)%(P<0.001)，PTX 48 h组的(60.9±3.7)%(P<0.001)和PTX 48 h ＋BIC 48 h组的(60.3±4.1)%(P<0.001)。PTX处理组中有EGR1、FST、FOS、IL8、IL6、RPL27A及CA2 7个基因的表达量与DMSO处理组比较，差异均有统计学意义(t＝18.647、10.336、10.098、9.683、9.408、9.050、8.001，P均<0.050)。

结论

通过先PTX再BIC的序贯联合给药方式较单药及其他联合给药方式能够更有效抑制AR阳性三阴性乳腺癌细胞MDA-MB-231的增殖，两者间可能存在协同作用。

关键词: 乳腺肿瘤, 雄激素受体拮抗剂, 化学疗法, 联合药物疗法, 早期生长反应蛋白质1

Objective

To investigate the inhibitory effect of bicalutamide (BIC) combined with paclitaxel (PTX) on the proliferation of androgen receptor (AR) -positive triple negative breast cancer MDA-MB-231 cells and its possible mechanism.

Methods

The CCK-8 kit was used to determine the effect of BIC (0.1, 1.0, 10.0 μmol/L) and PTX at different concentrations (0.1, 1.0, 10.0, 100.0, 1000.0, 10 000.0 nmol/L)in monotherapy or in sequential combination of both on the proliferation of MDA-MB-231 cells. Inhibition rate was compared using one-way analysis of variance. The pairwise comparison was performed using the LSD method. MDA-MB-231 cells were treated with 10 nmol/L PTX and 10 nmol/L DMSO respectively for 72 h. Three cell samples were taken in each group to analyze the relevant gene expression profiling in array using a bioinformatic method. The adjusted t test was used to screen out differential genes.

Results

After MDA-MB-231 cells were treated with different concentrations of BIC for 24, 48 and 72 h, respectively, the inhibition rates of MDA-MB-231 cells were statistically different at different time points (F=4.124, 8.189, 4.139, P=0.037, 0.004, 0.032). The inhibition rate of MDA-MB-231 cells reached the highest [(12.9 ± 5.5)%] at 48 h after the treatment of 10.0 μmol/L BIC. The inhibition rates of MDA-MB-231 cells were significantly different at different time points (F=8.407, 47.432, 14.907, P<0.001) after the treatment of PTX at different concentrations. The half inhibitory concentration (IC₅₀) of PTX in MDA-MB-231 cells at 48 h was 5 380.0 nmol/L. After 48 h treatment of 5 000.0 nmol/L PTX alone or combined with 0.1, 1.0, 10.0 μmol/L BIC, the inhibition rate of MDA-MB-231 cells was (53.2 ± 2.7)%, (53.2 ± 3.1)%, (51.7 ± 3.4)%, (51.0 ± 2.3)% in PTX monotherapy group and three experimental groups, respectively, indicating no significant difference (F=0.831, P=0.492). MDA-MB-231 cells were treated with sequential combination of 5 000.0 nmol/L PTX and 10.0 μmol/L BIC (PTX 24 h+ BIC 24 h group, BIC 24 h+ PTX 24 h group, PTX 48 h+ BIC 24 h group, BIC 48h+ PTX 24 h group), the monotherapy with 5 000.0 nmol/L PTX (PTX 48 h group) or 10.0 μmo/L BIC (BIC 48 h group) and the synchronous combined therapy of PTX and BIC(PTX 48 h+ BIC 48 h group), respectively. The result showed that there was a statistically significant difference in inhibition rate (F=241.466, P<0.001). The result of pairwise comparison showed that the inhibition rate in PTX 24 h + BIC 24 h group was (72.9 ± 1.9)%, significantly higher than (42.9 ± 1.7)% in BIC 24 h + PTX 24 h group (P<0.001), (60.9 ± 3.7)% in PTX 48 h group(P<0.001) and (60.3 ± 4.1)% in PTX 48 h + BIC 48 h group (P<0.001). There was a significant difference in the expression of seven genes (EGR1, FST, FOS, IL8, IL6, RPL27A and CA2) between PTX-treated group and DMSO-treated group (t=18.647, 10.336, 10.098, 9.683, 9.408, 9.050, 8.001, all P<0.050)

Conclusions

Sequential administration of PTX and BIC can inhibit the proliferation of AR-positive triple negative breast cancer MDA-MB-231 cells more effectively compared with the monotherapy and other combination methods. The two drugs may have the synergistic effect.

Key words: Breast neoplasms, Androgen receptor antagonists, Chemotherapy, Combination modality therapy, Early growth response protein 1

表1 不同浓度比卡鲁胺作用不同时间后各组MDA-MB-231细胞增殖抑制率比较(%，±s)

组别	实验次数	作用时间
组别	实验次数	24 h	48 h	72 h
0.1 μmol/L组	3	3.9±4.1	2.4±3.1	－0.9±4.8
1.0 μmol/L组	3	3.9±1.8	8.0±4.5^a	1.8±5.5
10.0 μmol/L组	3	8.1±2.3^ab	12.9±5.5^a	7.5±5.4^a
F值	?	4.124	8.189	4.391
P值	?	0.037	0.004	0.032

表1 不同浓度比卡鲁胺作用不同时间后各组MDA-MB-231细胞增殖抑制率比较(%，±s)

组别	实验次数	作用时间
组别	实验次数	24 h	48 h	72 h
0.1 μmol/L组	3	3.9±4.1	2.4±3.1	－0.9±4.8
1.0 μmol/L组	3	3.9±1.8	8.0±4.5^a	1.8±5.5
10.0 μmol/L组	3	8.1±2.3^ab	12.9±5.5^a	7.5±5.4^a
F值	?	4.124	8.189	4.391
P值	?	0.037	0.004	0.032

表2 不同浓度紫杉醇作用不同时间后各组MDA-MB-231细胞增殖抑制率比较(%，±s)

组别	实验次数	作用时间
组别	实验次数	24 h	48 h	72 h
0.1 nmol/L组	3	10.0±6.2	29.0±2.1	59.5±5.5
1.0 nmol/L组	3	17.5±6.9	23.7±5.7	54.5±4.4
10.0 nmol/L组	3	22.9±7.9	29.2±4.1	58.5±4.4
100.0 nmol/L组	3	28.0±6.9	40.0±2.8	64.4±4.2
1 000.0 nmol/L组	3	30.5±4.3	42.4±1.6	66.0±3.6
10 000.0 nmol/L组	3	28.6±7.2	48.0±2.0	72.9±1.8
F值	?	8.407	47.432	14.907
P值	?	<0.001	<0.001	<0.001

表2 不同浓度紫杉醇作用不同时间后各组MDA-MB-231细胞增殖抑制率比较(%，±s)

组别	实验次数	作用时间
组别	实验次数	24 h	48 h	72 h
0.1 nmol/L组	3	10.0±6.2	29.0±2.1	59.5±5.5
1.0 nmol/L组	3	17.5±6.9	23.7±5.7	54.5±4.4
10.0 nmol/L组	3	22.9±7.9	29.2±4.1	58.5±4.4
100.0 nmol/L组	3	28.0±6.9	40.0±2.8	64.4±4.2
1 000.0 nmol/L组	3	30.5±4.3	42.4±1.6	66.0±3.6
10 000.0 nmol/L组	3	28.6±7.2	48.0±2.0	72.9±1.8
F值	?	8.407	47.432	14.907
P值	?	<0.001	<0.001	<0.001

表3 比卡鲁胺与紫杉醇不同给药方式处理后MDA-MB-231细胞增殖抑制率比较(%，±s)

组别	实验次数	增殖抑制率
PTX 48 h组	3	60.9±3.7
PTX 48 h ＋BIC 48 h组	3	60.3±4.1
PTX 24 h ＋BIC 24 h组	3	72.9±1.9
PTX 48 h ＋BIC 24 h组	3	75.8±1.8
BIC 24 h ＋PTX 24 h组	3	42.9±1.7
BIC 48 h ＋PTX 24 h组	3	47.0±3.1
BIC 48 h组	3	25.0±0.8
F值	?	241.466
P值	?	<0.001

表3 比卡鲁胺与紫杉醇不同给药方式处理后MDA-MB-231细胞增殖抑制率比较(%，±s)

组别	实验次数	增殖抑制率
PTX 48 h组	3	60.9±3.7
PTX 48 h ＋BIC 48 h组	3	60.3±4.1
PTX 24 h ＋BIC 24 h组	3	72.9±1.9
PTX 48 h ＋BIC 24 h组	3	75.8±1.8
BIC 24 h ＋PTX 24 h组	3	42.9±1.7
BIC 48 h ＋PTX 24 h组	3	47.0±3.1
BIC 48 h组	3	25.0±0.8
F值	?	241.466
P值	?	<0.001

图1 7个差异基因在紫杉醇和DMSO处理后的MDA-MB-231细胞中的表达量热图

表4 紫杉醇和DMSO处理后MDA-MB-231细胞中差异基因表达比较(±s)

组别	样本数	差异基因
组别	样本数	EGR1	FST	FOS	IL8	IL6	RPL27A	CA2
DMSO处理组	3	5.798±0.103	8.267±0.088	5.984±0.073	4.703±0.276	5.653±0.031	5.060±0.045	3.772±0.095
紫杉醇处理组	3	7.160±0.031	9.027±0.065	6.681±0.050	5.910±0.059	6.261±0.053	5.687±0.078	4.428±0.109
t值	?	18.647	10.336	10.098	9.683	9.408	9.050	8.001
P值	?	<0.001	0.023	0.023	0.024	0.024	0.028	0.048

表4 紫杉醇和DMSO处理后MDA-MB-231细胞中差异基因表达比较(±s)

组别	样本数	差异基因
组别	样本数	EGR1	FST	FOS	IL8	IL6	RPL27A	CA2
DMSO处理组	3	5.798±0.103	8.267±0.088	5.984±0.073	4.703±0.276	5.653±0.031	5.060±0.045	3.772±0.095
紫杉醇处理组	3	7.160±0.031	9.027±0.065	6.681±0.050	5.910±0.059	6.261±0.053	5.687±0.078	4.428±0.109
t值	?	18.647	10.336	10.098	9.683	9.408	9.050	8.001
P值	?	<0.001	0.023	0.023	0.024	0.024	0.028	0.048

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Inhibitory effect of bicalutamide and paclitaxel on proliferation of androgen receptor-positive triple negative breast cancer MDA-MB-231 cells

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Inhibitory effect of bicalutamide and paclitaxel on proliferation of androgen receptor-positive triple negative breast cancer MDA-MB-231 cells