他莫西芬与β-榄香烯或吉非替尼联合治疗对乳腺癌MCF-7 细胞的影响

doi:10.3877/cma. j. issn.1674-0807.2015.02.002

中华乳腺病杂志(电子版) ›› 2015, Vol. 09 ›› Issue (02) : 78 -84. doi: 10.3877/cma. j. issn.1674-0807.2015.02.002

论著

他莫西芬与β-榄香烯或吉非替尼联合治疗对乳腺癌MCF-7 细胞的影响

张斌¹^,², 张霞³, 张改容², 刘越坚⁴, 张阳⁵^,^†(

)

1.116011 大连医科大学附属第一医院肿瘤科
2.116044 大连医科大学研究生院
3.250017 济南,山东省肿瘤医院肿瘤内三科
4.116011 大连医科大学附属第一医院实验中心
5.116027 大连医科大学附属第二医院肿瘤内科

收稿日期:2014-03-20 出版日期:2015-04-01
通信作者: 张阳
基金资助:
山东省自然科学基金(ZR2013C03050)山东省医学科学院青年基金(2013-35)大连医科大学附属第一医院青年基金(2014QN004)资助项目

Effect of combined use of tamoxifen with β-elemene or gefitinib in breast cancer MCF-7 cells

Bin Zhang¹^,², Xia Zhang³, Gairong Zhang², Yuejian Liu⁴, Yang Zhang⁵^,^†()

1.Department of Oncology, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
2.Graduate School,Dalian Medical University, Dalian 116044, China
3.Department of Medical Oncology, Shandong Provincial Tumor Hospital, Jinan 250017, China
4.Experimental Center, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
5.Department of Medical Oncology, Second Affiliated Hospital of Dalian Medical University, Dalian 116044, China

Received:2014-03-20 Published:2015-04-01
Corresponding author: Yang Zhang

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引用本文：

张斌, 张霞, 张改容, 刘越坚, 张阳. 他莫西芬与β-榄香烯或吉非替尼联合治疗对乳腺癌MCF-7 细胞的影响[J/OL]. 中华乳腺病杂志(电子版), 2015, 09(02): 78-84.

Bin Zhang, Xia Zhang, Gairong Zhang, Yuejian Liu, Yang Zhang. Effect of combined use of tamoxifen with β-elemene or gefitinib in breast cancer MCF-7 cells[J/OL]. Chinese Journal of Breast Disease(Electronic Edition), 2015, 09(02): 78-84.

目的

探讨内分泌药物与中药或化疗药物的联合应用对乳腺癌MCF-7 细胞的影响。

方法

分别应用1.5×10^-6 mol/L 或0.15×10^-6 mol/L 他莫西芬(tamoxifen,TAM)与梯度浓度(5、10、20、40、80 μg/ml )的β-榄香烯(β-elemene,β-ELE)或吉非替尼(gefitinib)序贯或同时作用于乳腺癌MCF-7 细胞。应用MTT 法检测不同用药方案对MCF-7 细胞的增殖抑制作用,计算药物联合指数(combine index,CI)来评价疗效。流式细胞仪检测不同方案对MCF-7 细胞周期的影响。细胞增殖抑制率采用重复测量方差分析进行比较,细胞周期数据采用方差分析。

结果

与β-ELE 单独作用组相比,随着β-ELE 浓度的增加,TAM 与β-ELE 联合用药组的细胞增殖抑制率显著升高(分组:F=34809.538,P=0.000;β-ELE浓度:F=118.540,P=0.000, 交互作用:F=312.912,P=0.000)。与gefitinib 单独作用组相比,随者gefitinib 浓度的增加,TAM 与gefitinib 联合用药组的细胞增殖抑制率显著升高,差异具有统计学意义(分组:F=21186.430, P=0.000;gefitinib 浓度:F=415.842,P=0.000;交互作用:F=134.464,P=0.000)。TAM 浓度为1.5×10^-6 mol/L 时,β-ELE-TAM、TAM-β-ELE 两种方案的CI 值(0.8627±0.0088、0.8388±0.0072)低于TAM+β-ELE 方案(0.9464±0.0038)(P 均＜0.050)。 gefitinib-TAM、TAM-Gefitinib 两种方案的CI 值(0.3383±0.0024、0.4481±0.0029)低于TAM+gefitinib 方案(0.5319±0.0015)(P 均＜0.050)。流式细胞仪检测显示,20 μg/ml 及40 μg/ml β-ELE 作用于MCF-7 细胞后,G₀/G₁ 期细胞比例由49.26%提高到64.04%和63.88%;10 μg/ml 及20 μg/ml gefitinib 作用于MCF-7 细胞后,G₀/G₁ 期细胞比例由49.26%提高到54.89%,68.35%。

结论

β-ELE 及gefitinib 与TAM 序贯联用具有协同作用,同时应用具有叠加作用,均可使MCF-7 细胞阻滞于G₀/G₁ 期并抑制细胞增殖。

关键词: 乳腺肿瘤, 化学疗法, 辅助, 他莫西芬, β-榄香烯, 吉非替尼

Objective

To investigate the effect of combined use of endocrine drug with traditional Chinese medicine or chemotherapy drug in breast cancer MCF-7 cells.

Methods

MCF-cells were divided into several groups respectively receiving the sequential or combined treatment of tamoxifen (TAM, 1.5×10^-6 mol/L and 0.15×10^-6 mol/L) and β-elemene(β-ELE) or gefitinib at gradient concentrations of 5,10,20,40,80 μg/ml.After the different drug treatments, the proliferation inhibition rates of MCF-7 cells were assayed by MTT, the efficacy of the drug combination was evaluated by the combine index (CI), and the cell cycle was detected by flow cytometry. The inhibition rates were compared by repeated measurement analysis of variance, and the dataon cell cycle were analyzed by variance analysis.

Results

Compared with β-ELE group, with the increase of β-ELE concentration, the proliferation inhibition rates of MCF-7 cells after the different regimen of β-ELE plus TAM were significantly increased (groups:F=34809.538,P=0.000;β-ELE concentration:F=118.540,P=0.000;interaction:F=312.912,P=0.000). Compared with gefitinib group, with the increase of gefitinib concentration, the proliferation inhibition rates of MCF-7 cells after the different regimen of gefitinib plus TAM were significantly increased (groups:F=21186.430, P=0.000;gefitinib concentration:F= 415.842,P =0.000;interaction:F=134.464,P=0.000). With TAM at the concentration of 1.5 × 10^-6 mol/L, CI value of β-ELE-TAM or TAM-β-ELE was lower than that of TAM + β-ELE (0.8627 ± 0.0088/0.8388 ± 0.0072 vs 0.9464 ± 0.0038 , both P＜0.050); CI value of gefitinib-TAM or TAM- gefitinib was lower than that of TAM+ gefitinib (0.3383±0.0024/0.4481±0.0029 vs 0.5319±0.0015, both P ＜0.050). The results of flow cytometry showed that the proportion of MCF-7 cells at phase of G₀/G₁ was increased from 49.26% to 64.04%and 63.88% after the treatment of 20, 40 μg/ml β-ELE, from 49.26% to 54.89% and 68.35% after the treatment of 10,20 μg/ml gefitinib.

Conclusions

The sequential treatment of TAM and β-ELE or gefitinib has synergistic effect and the combined treatment has addictive effect. It can cause MCF-7 cells arrested in G₀/G₁ phase and inhibit cell proliferation.

Key words: Breast neoplasms, Chemotherapy, adjuvant, Tamoxifen, β-elemene, Gefitinib

表1 不同用药方案处理后MCF-7 细胞的增殖抑制率比较(±s,%)

分组	β-ELE(μg/ml)
分组	5	10	20	40	80
β-ELE	0.01±0.01	2.81±0.25	15.80±1.33	47.09±2.53	95.30±1.86
β-ELE-TAM(1.5)	33.98±1.86	36.59±1.80	56.33±2.47	80.22±1.56	98.00±0.89
TAM(1.5)-β-ELE	25.25±1.88	33.44±2.07	65.89±2.55	76.95±1.82	97.63±1.88
β-ELE+TAM(1.5)	12.40±0.90	17.99±2.02	31.27±2.29	61.86±3.09	95.71±1.81
β-ELE-TAM(0.15)	26.38±2.05	31.38±1.55	40.35±1.45	74.39±1.95	98.04±0.92
TAM(0.15)-β-ELE	9.62±0.89	22.81±1.84	54.36±2.18	61.32±2.10	96.64±2.07

表1 不同用药方案处理后MCF-7 细胞的增殖抑制率比较(±s,%)

分组	β-ELE(μg/ml)
分组	5	10	20	40	80
β-ELE	0.01±0.01	2.81±0.25	15.80±1.33	47.09±2.53	95.30±1.86
β-ELE-TAM(1.5)	33.98±1.86	36.59±1.80	56.33±2.47	80.22±1.56	98.00±0.89
TAM(1.5)-β-ELE	25.25±1.88	33.44±2.07	65.89±2.55	76.95±1.82	97.63±1.88
β-ELE+TAM(1.5)	12.40±0.90	17.99±2.02	31.27±2.29	61.86±3.09	95.71±1.81
β-ELE-TAM(0.15)	26.38±2.05	31.38±1.55	40.35±1.45	74.39±1.95	98.04±0.92
TAM(0.15)-β-ELE	9.62±0.89	22.81±1.84	54.36±2.18	61.32±2.10	96.64±2.07

表2 不同用药方案处理后MCF-7 细胞的增殖抑制率比较(±s,%)

分组	gefitinib(μg/ml)
分组	5	10	20	40	80
gefitinib	0.01±0.01	1.23±0.24	24.32±1.91	39.48±1.64	50.46±2.14
gefitinib-TAM(1.5)	38.32±0.76	50.27±1.83	63.12±1.84	83.36±1.22	92.82±0.91
TAM(1.5)-gefitinib	21.97±1.19	44.86±1.31	55.42±1.80	81.48±1.33	96.41±0.81
gefitinib+TAM(1.5)	25.46±2.03	36.96±1.17	46.33±2.17	70.39±1.90	86.11±1.80
gefitinib-TAM(0.15)	27.39±1.02	35.03±0.98	58.90±1.34	71.46±1.32	91.53±1.06
TAM(0.15)-gefitinib	4.74±0.25	22.00±1.05	40.45±1.43	48.73±1.80	59.30±3.22

表2 不同用药方案处理后MCF-7 细胞的增殖抑制率比较(±s,%)

分组	gefitinib(μg/ml)
分组	5	10	20	40	80
gefitinib	0.01±0.01	1.23±0.24	24.32±1.91	39.48±1.64	50.46±2.14
gefitinib-TAM(1.5)	38.32±0.76	50.27±1.83	63.12±1.84	83.36±1.22	92.82±0.91
TAM(1.5)-gefitinib	21.97±1.19	44.86±1.31	55.42±1.80	81.48±1.33	96.41±0.81
gefitinib+TAM(1.5)	25.46±2.03	36.96±1.17	46.33±2.17	70.39±1.90	86.11±1.80
gefitinib-TAM(0.15)	27.39±1.02	35.03±0.98	58.90±1.34	71.46±1.32	91.53±1.06
TAM(0.15)-gefitinib	4.74±0.25	22.00±1.05	40.45±1.43	48.73±1.80	59.30±3.22

图1 β-榄香烯或吉非替尼作用后MCF-7 细胞周期分布注:β-ELE 为β-榄香烯;gefitinib 为吉非替尼;a 为MCF-7 对照组;b 为20 μg/ml β-ELE 作用组;c 为40 μg/ml β-ELE 作用组; d 为10 μg/ml gefitinib 作用组;e 为20 μg/ml gefitinib 作用组

表3 不同用药方案作用后MCF-7 细胞周期的变化(±s,%)

分组	凋亡率	G₀/G₁	G₂/M	S
对照组	25.76±2.08	49.26±1.74	7.76±0.48	42.98±1.41
20 μg/ml β-ELE	34.19±2.26^a	64.04±1.39^a	6.71±1.33	29.25±0.54^a
40 μg/ml β-ELE	29.60±1.02^ab	63.88±1.81^a	4.71±0.67^ab	31.41±1.18^a
10 μg/ml gefitinib	28.37±1.89^b	54.89±1.31^abc	2.95±0.55^abc	42.16±0.76^bc
20 μg/ml gefitinib	24.26±1.55^bcd	68.35±2.54^abcd	2.14±0.67^abc	29.51±1.90^ad
检验值	13.313	55.479	26.903	91.440
P值	0.001	0.000	0.000	0.000

表3 不同用药方案作用后MCF-7 细胞周期的变化(±s,%)

分组	凋亡率	G₀/G₁	G₂/M	S
对照组	25.76±2.08	49.26±1.74	7.76±0.48	42.98±1.41
20 μg/ml β-ELE	34.19±2.26^a	64.04±1.39^a	6.71±1.33	29.25±0.54^a
40 μg/ml β-ELE	29.60±1.02^ab	63.88±1.81^a	4.71±0.67^ab	31.41±1.18^a
10 μg/ml gefitinib	28.37±1.89^b	54.89±1.31^abc	2.95±0.55^abc	42.16±0.76^bc
20 μg/ml gefitinib	24.26±1.55^bcd	68.35±2.54^abcd	2.14±0.67^abc	29.51±1.90^ad
检验值	13.313	55.479	26.903	91.440
P值	0.001	0.000	0.000	0.000

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Effect of combined use of tamoxifen with β-elemene or gefitinib in breast cancer MCF-7 cells

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Effect of combined use of tamoxifen with β-elemene or gefitinib in breast cancer MCF-7 cells