整合素αvβ3单克隆抗体对乳腺癌MDA-MB-435S细胞增殖、侵袭和凋亡的影响
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摘要
目的:检测整合素αvβ3在乳腺癌MDA-MB-435细胞中的表达情况,探讨整合素αvβ3单克隆抗体对乳腺癌MDA-MB-435细胞增殖、侵袭和凋亡的作用.
方法:细胞免疫组化(SP)检测乳腺癌MDA-MB-435细胞表面整合素αvβ3的表达。MTT法测定整合素αvβ3单克隆抗体对乳腺癌MDA-MB-435细胞增殖的影响。Transwell侵袭实验测单抗对乳腺癌MDA-MB-435细胞侵袭力的影响。Hoechst 33258检测整合素αvβ3单克隆抗体作用48 h后细胞凋亡情况。流式细胞仪检测药物作用24 h后的细胞凋亡率。
结果:乳腺癌MDA-MB-435细胞表面整合素αvβ3阳性表达。整合素αvβ3单克隆抗体抑制乳腺癌MDA-MB-435S细胞增殖呈时效和量效关系,且在时间和浓度中,时间起主要作用。Transwell侵袭实验表明单抗处理对乳腺癌MDA-MB-435S细胞的侵袭能力有抑制效应。Hoechst 33258检测紫杉醇作用48 h后可见凋亡细胞,镜下观察凋亡细胞数随药物浓度增加而增多。流式定量分析,10μg/ml的单克隆抗体对乳腺癌细胞凋亡的影响与对照组相比有显著差异(P<0.05)。
结论:乳腺癌MDA-MB-435S细胞表面整合素αvβ3阳性表达,使用单克隆抗体封闭抗原,通过影响其下游的信号转导,可抑制乳腺癌细胞增殖、侵袭并促进其凋亡。
Objectives:To investigate the effects of monoclonal antibody to integrinαvβ3 on cell proliferation and apoptosis of MDA-MB-435 cells .
Methods:immunohistochemistry staining (SP)was used to examine the expression of integrinαvβ3 receptor in breast cancer cell lines MDA-MB-435s.Cell growth inhibition of monoclonal antibody to integrinαvβ3 on MDA-MB-435 cells was analyzed by MTT assay.Cell migration assays were performed in 24-transwell chambers with a polystyrene membrane (6.5-mm diameter, 10-μm thickness, and 8-μm pore size).Cell apoptosis was detected by Hoechst 33258 staining after monoclonal antibody to integrinαvβ3 treatment for 48 hours;apoptotic rate were analyzed by cytometry.
Results:Integrinαvβ3 expression in cellular membrane was high in MDA-MB-435s cell lines.monoclonal antibody to integrinαvβ3 could inhibit the proliferation of MDA-MB-435 cells in a time-and dose-dependant manner. monoclonal antibody to integrinαvβ3 could inhibit cell invasion.Hoechst 33258 staining indicated that apoptosis was induced by monoclonal antibody to integrinαvβ3 After treated for 48 hours, And apoptosis rate increased in dose-dependant manner.Cell apoptosis rate of monoclonal antibody to integrinαvβ3 groups were significantly higher than that of control group(P<0.01).
Conclusions:monoclonal antibody to integrinαvβ3 has not only targeted neovascularization but also direct tumor-killing effects. integrinαvβ3 antagonist is an ideal therapeutic agent for potential tumor targeting therapy.
方法:细胞免疫组化(SP)检测乳腺癌MDA-MB-435细胞表面整合素αvβ3的表达。MTT法测定整合素αvβ3单克隆抗体对乳腺癌MDA-MB-435细胞增殖的影响。Transwell侵袭实验测单抗对乳腺癌MDA-MB-435细胞侵袭力的影响。Hoechst 33258检测整合素αvβ3单克隆抗体作用48 h后细胞凋亡情况。流式细胞仪检测药物作用24 h后的细胞凋亡率。
结果:乳腺癌MDA-MB-435细胞表面整合素αvβ3阳性表达。整合素αvβ3单克隆抗体抑制乳腺癌MDA-MB-435S细胞增殖呈时效和量效关系,且在时间和浓度中,时间起主要作用。Transwell侵袭实验表明单抗处理对乳腺癌MDA-MB-435S细胞的侵袭能力有抑制效应。Hoechst 33258检测紫杉醇作用48 h后可见凋亡细胞,镜下观察凋亡细胞数随药物浓度增加而增多。流式定量分析,10μg/ml的单克隆抗体对乳腺癌细胞凋亡的影响与对照组相比有显著差异(P<0.05)。
结论:乳腺癌MDA-MB-435S细胞表面整合素αvβ3阳性表达,使用单克隆抗体封闭抗原,通过影响其下游的信号转导,可抑制乳腺癌细胞增殖、侵袭并促进其凋亡。
Objectives:To investigate the effects of monoclonal antibody to integrinαvβ3 on cell proliferation and apoptosis of MDA-MB-435 cells .
Methods:immunohistochemistry staining (SP)was used to examine the expression of integrinαvβ3 receptor in breast cancer cell lines MDA-MB-435s.Cell growth inhibition of monoclonal antibody to integrinαvβ3 on MDA-MB-435 cells was analyzed by MTT assay.Cell migration assays were performed in 24-transwell chambers with a polystyrene membrane (6.5-mm diameter, 10-μm thickness, and 8-μm pore size).Cell apoptosis was detected by Hoechst 33258 staining after monoclonal antibody to integrinαvβ3 treatment for 48 hours;apoptotic rate were analyzed by cytometry.
Results:Integrinαvβ3 expression in cellular membrane was high in MDA-MB-435s cell lines.monoclonal antibody to integrinαvβ3 could inhibit the proliferation of MDA-MB-435 cells in a time-and dose-dependant manner. monoclonal antibody to integrinαvβ3 could inhibit cell invasion.Hoechst 33258 staining indicated that apoptosis was induced by monoclonal antibody to integrinαvβ3 After treated for 48 hours, And apoptosis rate increased in dose-dependant manner.Cell apoptosis rate of monoclonal antibody to integrinαvβ3 groups were significantly higher than that of control group(P<0.01).
Conclusions:monoclonal antibody to integrinαvβ3 has not only targeted neovascularization but also direct tumor-killing effects. integrinαvβ3 antagonist is an ideal therapeutic agent for potential tumor targeting therapy.
引文
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[2] glass ag, lacey jv jr, carreon d, hoover rn. breast cancer incidence, 1980--2006: combined roles of menopausal hormone therapy, screening mammography, and estrogen receptor status. j natl cancer inst 2007;99:1152—61.
[3] Ries LAG, Melbert D, Krapcho M, Stinchcomb DG, Howlader N, Horner MJ, Mariotto A, Miller BA, Feuer EJ, Altekruse SF, Lewis DR, Clegg L, Eisner MP, Reichman M, Edwards BK (eds). SEER Cancer Statistics Review, 1975-2005,National Cancer Institute. Bethesda, MD, http://seer.cancer.gov/csr/1975_2005/, based on November 2007 SEER data submission, posted to the SEER web site, 2008.
[4] Ratnikov BI,Rozanov DV,Postnova TI,et al.An alternative processing of integrin al-pha(v) subunit in tumor cells by mem-brane type-1 matrix metalloproteinase[J].J Biol Chem,2002,277(9)∶7377-7385.
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[6] LiapisH, Flath A, Kitazawa S,et al. Integrin alpha Vbeta 3 expres-sion by bone-residing breast eancer metastases[J]. Diagn Mol Pathol,1996,5(2):127-135.
[7] Brooks PC,Clark RA, Cheresh DA: Requirement of vascular integrin alpha v beta 3 for angiogenesis . Science 1994;264(5158):569-571.
[8] BrookS PC,Montgomery AMp,Rosenfeld M et al Integrin αvβ3antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels .Cell1 994;79:1157-1164.
[9] Zitzmann S, Ehemann V, Schwab M. Arginine-glycine-aspartic acid (RGD)-peptide binds to both tumor and tumor-endothelial cells in vivo.Cancer Res. 2002; 62 (18):5139-43.
[10] Haubner R, Wester HJ, Weber WA, et al. Noninvasive imaging of alph(v)beta3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography[J]. Cancer Res, 2001, 61(5):1781-1785.
[11] Chatterjee S, Matsumura A, Schradermeier, et al. Human malignant glioma therapy using anti-αvβ33 integrin agents [J]. J Neurooncol, 2000, 46(2): 135-144.
[12] 司徒镇强, 吴军正. 细胞培养[M]. 西安: 世界图书出版公司, 1996: 118-227.
[13] MATTERN J, KOOMAGI R, VOLM M. Biological charac-terization of subgroups of squamous cell lung carcinomas[J].Clin Cancer Res, 1999,5(6):1459-1463.
[14] Fujishita T,Doi Y,Sonoshita M,et al.Development ofspontaneous tumours and intestinal lesions in Fhit gene knockout mice[J].Br J Cancer,2004,91(8):1571-1574.
[15] Chaudhuri AR,Khan IA,Prasad V,et al.The tumor suppressor protein Fhit:a novel interaction with tubulin[J].J Biol Chem,1999,274:24378 ～ 24382.
[16] Gopalakrishnan VK,Banerjee AG,Vishwanatha JK,et al. Effect of FHIT gene replacement on growth,cell cycle and apoptosis in pancreatic cancer cells[J].Pancreatology,2003,3(4):293-302.
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