中国普外基础与临床杂志

中国普外基础与临床杂志

曲妥珠单抗对 SW-620 人结肠癌细胞增殖凋亡的影响及其与奥沙利铂协同作用的研究

查看全文

目的 观察不同浓度曲妥珠单抗单独或联合奥沙利铂对 SW-620 人结肠癌细胞增殖、凋亡及细胞周期的影响,并探讨其作用机制。 方法 体外培养 SW-620 人结肠癌细胞。① 细胞增殖实验:将细胞分为 2 个大组:曲妥珠单抗组和曲妥珠单抗+奥沙利铂组,每个大组内设 8 个浓度小组(每个小组设 5 个复孔),曲妥珠单抗组的浓度依次为 0、0.001、0.01、0.1、1、10、100 及 1 000 μg/mL,对应的曲妥珠单抗+奥沙利铂组曲妥珠单抗的浓度与前相同,不同之处在于均加入了奥沙利铂(10 μmol/L)。采用 CCK-8 法检测各组细胞的吸光度(A)值。② 细胞凋亡实验:组别设置同增殖实验,只是曲妥珠单抗的浓度仅包括 0、0.1、1 及 10 μg/mL。采用流式细胞仪检测各组细胞的细胞凋亡率及细胞周期分布。③ 人表皮生长因子受体 2(her-2)蛋白表达检测:分别以 0、100 及 1 000 μg/mL 曲妥珠单抗,以及 0、1 及 10 μg/mL 曲妥珠单抗联合奥沙利铂(10 μmol/L)处理 SW-620 细胞,采用 Western blot 法检测各组细胞中 her-2 蛋白的表达。 结果 ① 细胞增殖实验:同种浓度下曲妥珠单抗+奥沙利铂组的 A 值均低于曲妥珠单抗组(P<0.05),且随着曲妥珠单抗浓度的增加,A 值在逐渐降低。② 细胞凋亡实验:同种曲妥珠单抗浓度下,曲妥珠单抗组+奥沙利铂组的细胞凋亡率均较曲妥珠单抗组高(P<0.05)。流式细胞检测:不同浓度曲妥珠单抗+奥沙利铂处理后,随浓度增加,G1 期细胞比例呈下降趋势,S 期细胞比例呈上升趋势,且呈剂量依赖性;至 1 μg/mL 和 10 μg/mL 时,与曲妥珠单抗组比较,曲妥珠单抗+奥沙利铂组的 G1 期细胞比例较低,S 期细胞比例较高(P<0.05);在 0.1、1 和 10 μg/mL 浓度的曲妥珠单抗条件下,曲妥珠单抗联合奥沙利铂组的 G2 期细胞比例较曲妥珠单抗组升高(P<0.01)。③ her-2 蛋白的表达水平:1、100 及 1 000 μg/mL 曲妥珠单抗组细胞中 her-2 蛋白的表达水平逐渐降低,3 组间两两比较差异均有统计学意义(P<0.05);0、1 及 10 μg/mL 曲妥珠单抗+奥沙利铂组中 her-2 蛋白的表达水平也逐渐降低,3 组间两两比较差异也有统计学意义(P<0.05)。 结论 高浓度曲妥珠单抗可抑制 SW-620 人结肠癌细胞的增殖,诱导其凋亡。曲妥珠单抗和奥沙利铂具有协同抑制细胞增殖、促进细胞凋亡的作用。

Objective To observe the effects of different concentrations of trastuzumab alone or in combination with oxaliplatin on proliferation, apoptosis, and cell cycle of SW-620 human colon cancer cell, and to explore its mechanism. Methods SW-620 human colon cancer cells were culturedin vitro. ① Cell proliferation experiment: the cells were divided into two large groups: trastuzumab group and trastuzumab combined with oxaliplatin group. There were eight concentration groups in each large group (five holes for each group). The concentration of the trastuzumab group was 0, 0.001, 0.01, 0.1, 1, 10, 100, and 1 000 μg/mL, corresponding to the trastuzumab combined with oxaliplatin group. The concentration of the antibiotic was the same as before, except that oxaliplatin (10 μmol/L) was added. The absorbance (A) value of each group was measured by CCK-8 method. ② Apoptosis experiment: the same proliferation experiment was performed in the group, except that the concentrations of trastuzumab only included 0, 0.1, 1 and 10 μg/mL. Flow cytometry was used to detect the proportion of apoptotic cells and cell cycle distribution in each group. ③ Determination of human epidermalgrowth factor receptor-2 (her-2). The SW-620 cells were divided into two large groups, the concentration of trastuzumab group concluded 0, 100, and 1 000 μg/mL, as well as the concentration of trastuzumab in the trastuzumab combined with oxaliplatin group concluded 0, 1, and 10 μg/mL. Expressions of her-2 protein in SW-620 cells were detected by Western blot method. Results ① Cell proliferation assay: the A values at100 μg/mL and 1 000 μg/mL were significantly lower than that at 0 μg/mL (P<0.05). At the same concentration, theA value of the trastuzumab combined with oxaliplatin group was lower than that of the trastuzumab group (P<0.05 ), and theA value gradually decreased with the increase of the concentration of trastuzumab. ② Apoptosis experiment: the proportion of apoptotic cells in the trastuzumab combined with oxaliplatin group was higher than that in the trastuzumab group (P<0.05) at the same concentration of trastuzumab. Flow cytometry: after treatment with different concentrations of trastuzumab combined with oxaliplatin, cells in G1 phase showed a downward trend, and cells in S phase showed an upward trend in a dose-dependent manner. At 1 and 10 μg/mL concentration of trastuzumab, the trastuzumab combined with oxaliplatin group significantly reduced the proportion of cells in the G1 phase of SW-620 cell cycle compared with the trastuzumab group, but S phase ratio was higher (P<0.05). The proportion of G2 phase cells was significantly higher in the trastuzumab combined with oxaliplatin group than the trastuzumab group at 0.1, 1 and 10 μg/mL concentrations of trastuzumab (P<0.01). ③ Expressions of her-2 protein: the expression level of her-2 protein gradually decreased at 1, 100, and 1 000 μg/mL trastuzumab group (P<0.05). The expression levels of her-2 protein in 0, 1 and 10 μg/mL trastuzumab combined with oxaliplatin group also gradually decreased (P<0.01). Conclusions High concentration of trastuzumab can inhibit the proliferation of SW-620 human colon cancer cells and induce apoptosis. Trastuzumab combined with oxaliplatin has synergistic effect on inhibiting cell proliferation and promoting apoptosis.

关键词: 结肠癌; 曲妥珠单抗; 奥沙利铂; 人表皮生长因子受体-2; SW-620 细胞; 体外实验

Key words: colon cancer; trastuzumab; oxaliplatin; human epidermalgrowth factor receptor-2; SW-620 cell; experiment in vitro

引用本文: 王建, 安华松, 宋正霞, 何跃君, 郭贞. 曲妥珠单抗对 SW-620 人结肠癌细胞增殖凋亡的影响及其与奥沙利铂协同作用的研究. 中国普外基础与临床杂志, 2018, 25(12): 1433-1438. doi: 10.7507/1007-9424.201808044 复制

登录后 ,请手动点击刷新查看全文内容。 没有账号,
登录后 ,请手动点击刷新查看图表内容。 没有账号,
1. Ferlay J, Shin H, Bray F, et al. GLOBOCAN 2008, cancer incidence and mortality worldwide: IARC CancerBase No. 10. Lyon, France. 2010-08-01. http://globocan.iarc.fr.
2. Carethers JM, Jung BH. Genetics and genetic biomarkers in sporadic colorectal cancer. Gastroenterology, 2015, 149(5): 1177-1190.
3. Ahmad M, Attoub S, Singh MN, et al. Gamma-synuclein and the progression of cancer. FASEB J, 2007, 21(13): 3419-3430.
4. De Mattia E, Cecchin E, Toffoli G. Pharmacogenomics of intrinsic and acquired pharmacoresistance in colorectal cancer: toward targeted personalized therapy. Drug Resist Updat, 2015, 20(4): 39-70.
5. Birkman EM, Avoranta T, Ålgars A, et al. EGFR gene copy number decreases during anti-EGFR antibody therapy in colorectal cancer. Hum Pathol, 2018, [Epub ahead of print].
6. 王珊珊, 梅金红. HER2 与肿瘤浸润转移. 生命的化学, 2008, 28(5): 566-568.
7. Pandya K, Wyatt D, Gallagher B, et al. PKCα attenuates jagged-1-mediated notch signaling in ErbB-2-positive breast cancer to reverse trastuzumab resistance. Clin Cancer Res, 2016, 22(1): 175-186.
8. Lin Z, Lu Y, Meng Q, et al. miR372 promotes progression of liver cancer cells by upregulating erbB-2 through enhancement of YB-1. Mol Ther Nucleic Acids, 2018, 11: 494-507.
9. Testa U, Pelosi E, Castelli G. Colorectal cancer: genetic abnormalities, tumor progression, tumor heterogeneity, clonal evolution and tumor-initiating cells. Med Sci (Basel), 2018, 6(2): 31-41.
10. 李春莲, 周旭婕, 娄培培, 等. RNA 结合蛋白 38 通过增加人表皮生长因子受体 2 的表达诱导乳腺癌 BT474 细胞对曲妥珠单抗的敏感性. 中华肿瘤杂志, 2016, 38(3): 172-178.
11. 许惠玉, 陈志伟, 郭洪英. 岩大戟内酯 B 诱导乳腺癌 MCF7 细胞凋亡实验研究. 中国医学创新, 2012, 9(13): 1-3.
12. Pan G, Li D, Li X, et al. SPECT/CT imaging of HER2 expression in colon cancer-bearing nude mice using. Biochem Biophys Res Commun, 2018, 504(4): 765-770.
13. Takegawa N, Yonesaka K. HER2 as an emerging oncotarget for colorectal cancer treatment after failure of anti-epidermal growth factor receptor therapy. Clin Colorectal Cancer, 2017, 16(4): 247-251.
14. Ménard S, Casalini P, Campiglio M, et al. HER2 overexpression in various tumor types, focussing on its relationship to the development of invasive breast cancer. Ann Oncol, 2001, 12(Suppl 1): S15-S19.
15. Lee JH, Kim HM, Koo JS. Differential expression of cancer-associated fibroblast-related proteins in ductal carcinoma in situ according to molecular subtype and stromal histology. Pathobiology, 2018, 17(6): 1-11.
16. 梁霞, 张勇晶, 刘冰, 等. HER-2 基因 Ile655Val 多态性与结直肠癌易感性分析. 中华医学遗传学杂志, 2009, 26(3): 302-305.
17. Buhmeida A, Assidi M, Al-Maghrabi J, et al. Membranous or cytoplasmic HER2 expression in colorectal carcinoma: evaluation of prognostic value using both IHC – BDISH. Cancer Invest, 2018, 36(2): 129-140.
18. 李东升, 王巍, 李晴, 等. 结直肠癌组织中 Her-2 和 Claudin-1 的表达及意义. 广东医学, 2012, 33(2): 237-239.
19. Zhu H, Zhang G, Wang Y, et al. Inhibition of ErbB2 by Herceptin reduces survivin expression via the ErbB2-beta-catenin/TCF4-survivin pathway in ErbB2-overexpressed breast cancer cells. Cancer Sci, 2010, 101(5): 1156-1162.
20. Rebischung C, Barnoud R, Stéfani L, et al. The effectiveness of trastuzumab (Herceptin) combined with chemotherapy for gastric carcinoma with overexpression of the c-erbB-2 protein. Gastric Cancer, 2005, 8(4): 249-252.
21. Liu D, Li X, Chen C, et al. Target-specific delivery of oxaliplatin to HER2-positive gastric cancer cells. Oncol Lett, 2018, 15(5): 8079-8087.
22. Huang R, Sun Y, Zhang XY, et al. Biological evaluation of a novel Herceptin-platinum (Ⅱ) conjugate for efficient and cancer cell specific delivery. Biomed Pharmacother, 2015, 73: 116-122.
23. Carter WB, Niu G, Ward MD, et al. Mechanisms of HER2-induced endothelial cell retraction. Ann Surg Oncol, 2007, 14(10): 2971-2978.
24. Hudziak RM, Lewis GD, Shalaby MR, et al. Amplified expression of the HER2/ERBB2 oncogene induces resistance to tumor necrosis factor alpha in NIH 3T3 cells. Proc Natl Acad Sci U S A, 1988, 85(14): 5102-5106.
25. 张丽华, 李静. 曲妥珠单抗联合紫杉醇诱导过表达 HER-2 的乳腺癌裸鼠皮下移植瘤乳腺癌细胞凋亡的作用机制和效果研究. 临床和实验医学杂志, 2016, 15(14): 1373-1377.