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据国际癌症研究机构(IARC,https://www.iarc.who.int/)提供的数据显示,2020年女性乳腺癌现已成为全球最常见的癌症之一[1]。其中有15%~25%的乳腺肿瘤存在人类表皮生长因子受体2(HER2)过表达。HER2阳性的乳腺癌浸润性强、无病生存期短、预后差,对化疗敏感性差,且易复发[2]。曲妥珠单抗是一株靶向HER2的人源化单克隆抗体。虽然曲妥珠单抗单药对乳腺癌治疗起到了很好的改善的效果, 但其疗效还有所不足,如对HER2过表达的乳腺癌患者初次治疗有效率仅在30%左右[3]。EGCG是绿茶中主要的多酚[4],有抑制肿瘤细胞生长、增殖、转移和血管生成,诱导细胞凋亡和增强抗肿瘤免疫等多种抗肿瘤作用[5-9]。据报道,EGCG在乳腺癌、胃癌、白血病、膀胱癌治疗中均显示有抗肿瘤作用[10-13]。本实验旨在探讨曲妥珠单抗与EGCG对HER2过表达细胞株是否具有协同增殖抑制作用,为HER2高表达乳腺癌的治疗提供新的思路。
The effect and mechanism of epigallocatechol gallate combined with trastuzumab on the proliferation of HER2 overexpressing breast cancer cells
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摘要:
目的 研究表没食子儿茶素没食子酸酯(EGCG)联合曲妥珠单抗对人表皮生长因子受体2(HER2)过表达乳腺癌细胞增殖的影响及其作用机制。 方法 表达纯化曲妥珠单抗;用CCK-8 细胞增殖检测试剂盒(CCK8)检测不同浓度EGCG、曲妥珠单抗及两药联用对HER2过表达乳腺癌细胞BT474、SK-BR-3的增殖抑制作用;用Western blot法检测EGCG、曲妥珠单抗及两药联用对BT474乳腺癌细胞中HER2,表皮生长因子受体(EGFR),丝裂原激活的蛋白激酶(MAPK)和蛋白激酶B(Akt)及它们的磷酸化蛋白的表达水平的影响。 结果 细胞增殖试验结果显示,EGCG、曲妥珠单抗以及二者联用均能有效抑制BT474和SK-BR-3细胞的增殖,且在一定浓度范围内,EGCG与曲妥珠单抗联用显示出协同增殖抑制作用。Western blot结果显示EGCG、曲妥珠单抗以及二者联合均能抑制BT474细胞中Akt,MAPK,EGFR,HER2的磷酸化蛋白表达,与单药相比,二者联合抑制作用显著增强,其差异具有统计学意义(P<0.05)。 结论 EGCG联合曲妥珠单抗能协同抑制HER2过表达乳腺癌细胞的增殖,其机制可能与Akt、MAPK信号通路有关。 -
关键词:
- 表没食子儿茶素没食子酸酯 /
- 曲妥珠单抗 /
- 人表皮生长因子受体2过表达 /
- 乳腺癌 /
- 协同 /
- 增殖抑制
Abstract:Objective To study the effect and mechanism of epigallocatechol gallate (EGCG) combined with trastuzu-mab on the proliferation of human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer cells. Methods Trastuzumab was expressed and purified. The cell proliferation of HER2 overexpressing breast cancer cells BT474 and SK-BR-3 treated with trastuzumab, EGCG, or trastuzumab plus EGCG was evaluated by CCK8 assay. The effects of EGCG and trastuzumab on the expression of HER2, epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), protein kinase B (Akt), and their phosphorylated proteins in BT474 breast cancer cells were detected by Western blot. Results The results of cell proliferation assay indicated that EGCG and trastuzumab, alone or in combination, effectively inhibited the proliferation of BT-474 and SK-BR-3 cells. And within a certain concentration range, EGCG and trastuzumab showed a synergistic proliferation inhibitory effect on HER2 overexpressing breast cancer cells. Consistent with these results, Western blot results showed that trastuzumab and EGCG, alone or in combination significantly reduced the phosphorylation levels of Akt, MAPK, EGFR, and HER2 in BT474 cells. Moreover, the inhibition effect of EGCG plus trastuzumab was significantly more potent than either EGCG or trastuzumab. Conclusion EGCG and trastuzumab could synergistically inhibit the proliferation of HER2 overexpressing breast cancer cells, which may be related to the regulation of Akt and MAPK signaling pathways. -
Key words:
- EGCG /
- trastuzumab /
- HER2 overexpression /
- breast cancer /
- synergy /
- proliferation inhibition
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图 3 EGCG单药组、曲妥珠单抗单药组及两者联用组对BT474细胞增殖的影响
注:A.EGCG对BT474细胞增殖的影响;B.曲妥珠单抗对BT474细胞增殖的影响;C.16.67 nmol/L曲妥珠单抗、16.67 nmol/L IgG 曲妥珠单抗同型对照、45 μmol/L EGCG、16.67 nmol/L曲妥珠单抗与45 μmol/L EGCG联用对BT474细胞增殖的影响;D.16.67 nmol/L曲妥珠单抗联合不同浓度EGCG对BT474细胞增殖的影响,计算联合指数(CI),Fa表示效应值;*P<0.05,****P<0.0001,与空白组比较;△△△△P<0.0001,与IgG组比较;####P<0.0001,与曲妥珠单抗组比较
图 4 EGCG单药组、曲妥珠单抗单药组及两者联用组对SK-BR-3细胞增殖的影响
注:A.EGCG对SK-BR-3细胞增殖的影响;B. 曲妥珠单抗对SK-BR-3细胞增殖的影响;C. 16.67 nmol/L 曲妥珠单抗、16.67 nmol/L IgG 曲妥珠单抗同型对照、15 μmol/L EGCG、16.67 nmol/L曲妥珠单抗与15 μmol/L EGCG联用对SK-BR-3细胞增殖的影响;D. 16.67 nmol/L曲妥珠单抗联合不同浓度EGCG对SK-BR-3细胞增殖的影响,计算联合指数(CI),Fa表示效应值;**P<0.01,***P<0.001,与空白组比较;△△△P<0.001,与IgG组比较;####P<0.0001,与曲妥珠单抗组比较
图 5 EGCG单药组、曲妥珠单抗单药组及两者联用组对BT474细胞中MAPK、Akt、EGFR、HER2及其磷酸化蛋白的表达的影响
注:A. p-Akt、Akt的蛋白表达水平;B. p-Akt、Akt的相对蛋白表达量;C. p-MAPK、MAPK的蛋白表达水平;D. p-MAPK、MAPK的相对蛋白表达量;E. p-EGFR、EGFR的蛋白表达水平;F. p-EGFR、EGFR的相对蛋白表达量;G. p-HER2、HER2的蛋白表达水平;H. p-HER2、HER2的相对蛋白表达量;****P<0.0001,与对照组比较;##P<0.01、###P<0.001、 ####P<0.0001,与曲妥珠单抗组比较
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