Bcl-2反义寡核苷酸对人小细胞肺癌NCI-H69化疗敏感性的影响
|
摘要
目的:研究Bcl-2反义寡核苷酸对人小细胞肺癌细胞NCI-H69化疗药物敏感性的促进作用。
方法:将NCI-H69细胞培养传代,细胞共分4组,反义寡核苷酸组、正义寡核苷酸组、无义寡核苷酸组和空白对照组,通过脂质体将不同寡核苷酸导人细胞中,Western-Blot法检测Bcl-2蛋白的表达。化疗药物依托泊甙(vp-16)分别以不同浓度加入4组细胞。流式细胞仪的检测细胞凋亡率,MTT法测定细胞存活分数。
结果:4组细胞中,与空白对照组相比较,反义寡核苷酸组的Bcl-2蛋白表达明显受到抑制,而正义寡核苷酸组和无义寡核苷酸组细胞的Bcl-2蛋白表达则与空白对照组差别不明显。细胞中加入vp-16后,反义寡核苷酸组细胞在Vp-16浓度0.25、0.5、1.0、2.0、4.0ug/ml的凋亡率明显高于空白对照组,统计结果差异具有显著性(P<0.05),正义寡核苷酸组和无义寡核苷酸组与空白对照组相比则无统计学差异。四组细胞接受vp-16后,转染反义寡核苷酸组在不加人Vp-16和加入vp-16不同剂量后的存活分数均低于空白组,且有统计学差异(P<0.01),而转染正义链和无义链组与空白组比较则无统计学差异。
结论:Bcl-2反义寡核苷酸能有效封闭Bcl-2基因的表达,增强小细胞肺癌细胞NCI-H69对化疗药物的敏感性。
Objective: To study the effect of bcl-2 antisense oligodexynucleotides on chemotherapy of human small cell lung cancer cell line NCI-H69. Methods: Cultured cells were devided into 4 groups: antisense oligodexynucleotides(ASODN) sense oligodexynucleotides(SODN) nonsense oligodexynucleotides(NSODN) and control. The different bcl-2 oligodexynucleotides was transfected into corresponding cells using Oligofectamine. The expression of bcl-2 was examined by Westem-Blot. The different group cells were treated by etopside(Vp-16) for four concentrations, and men the apoptotic cells were detected with flowcytometry and cell growth was assessed by MTT test after irradiation. Results: The bcl-2 expression in ASODN group was significantly inhibited compared to the control group, but it was not obviously inhibited in SODN and NSODN groups. The apoptotic rate of ASODN group was significantly higher than that of the control group, and the survival fraction of ASODN group was significantly lower than that of the control group.Conclusions: The bcl-2 ASODN could enhance the sensitivity of chemotherapy of small cell lung cancer cell line NCI-H69 by blocking bcl-2 gene effectively.
方法:将NCI-H69细胞培养传代,细胞共分4组,反义寡核苷酸组、正义寡核苷酸组、无义寡核苷酸组和空白对照组,通过脂质体将不同寡核苷酸导人细胞中,Western-Blot法检测Bcl-2蛋白的表达。化疗药物依托泊甙(vp-16)分别以不同浓度加入4组细胞。流式细胞仪的检测细胞凋亡率,MTT法测定细胞存活分数。
结果:4组细胞中,与空白对照组相比较,反义寡核苷酸组的Bcl-2蛋白表达明显受到抑制,而正义寡核苷酸组和无义寡核苷酸组细胞的Bcl-2蛋白表达则与空白对照组差别不明显。细胞中加入vp-16后,反义寡核苷酸组细胞在Vp-16浓度0.25、0.5、1.0、2.0、4.0ug/ml的凋亡率明显高于空白对照组,统计结果差异具有显著性(P<0.05),正义寡核苷酸组和无义寡核苷酸组与空白对照组相比则无统计学差异。四组细胞接受vp-16后,转染反义寡核苷酸组在不加人Vp-16和加入vp-16不同剂量后的存活分数均低于空白组,且有统计学差异(P<0.01),而转染正义链和无义链组与空白组比较则无统计学差异。
结论:Bcl-2反义寡核苷酸能有效封闭Bcl-2基因的表达,增强小细胞肺癌细胞NCI-H69对化疗药物的敏感性。
Objective: To study the effect of bcl-2 antisense oligodexynucleotides on chemotherapy of human small cell lung cancer cell line NCI-H69. Methods: Cultured cells were devided into 4 groups: antisense oligodexynucleotides(ASODN) sense oligodexynucleotides(SODN) nonsense oligodexynucleotides(NSODN) and control. The different bcl-2 oligodexynucleotides was transfected into corresponding cells using Oligofectamine. The expression of bcl-2 was examined by Westem-Blot. The different group cells were treated by etopside(Vp-16) for four concentrations, and men the apoptotic cells were detected with flowcytometry and cell growth was assessed by MTT test after irradiation. Results: The bcl-2 expression in ASODN group was significantly inhibited compared to the control group, but it was not obviously inhibited in SODN and NSODN groups. The apoptotic rate of ASODN group was significantly higher than that of the control group, and the survival fraction of ASODN group was significantly lower than that of the control group.Conclusions: The bcl-2 ASODN could enhance the sensitivity of chemotherapy of small cell lung cancer cell line NCI-H69 by blocking bcl-2 gene effectively.
引文
1 Parkin DM, et al. Eur J cancer 2001;37csapp(8): S4-S66
2 Greenlee Rt, et al. CA cancer J clin 2000;50: 7-33
3 殷蔚伯,谷铣之主编。肿瘤放射治疗学。第三版。北京,中国协和医科大学出版社。2002,1-15。
4 Nair CKK, Parida DK and Nomura T. Radioprotectors in radiotherapy. J Radiat. Res. 2001;42: 21-37.
5 Nicholson DW, Thornberry NA. Apoptosis: Life and death decisions. [J] Science, 2003, 299 (6504): 214-215.
6 Yang E ,Korsmeyer S. Molecular Thanatopsis: A discourse on the BCL-2 family and cell death [J]. Blood, 1996, 88(2): 388-401.
7 Vaux D, Cory S, Fulco M, et al. Effects of Bcl-2 gene in the tumour cell culture in vitro[J]. Nature, 1998, 335(6): 440.
8 姚和瑞,欧阳能太,李锦梅。癌基因Bcl-2蛋白在肺癌组织中的表达。癌症[J]1997,16(5):348-349。
9 刘慧,孔庆兖,吴永平,王绪。SCLC与NSCLC基因调控的差异—凋亡调控基因p53、Bcl-2表达的差异及意义。徐州医学院学报,2003,23(2)115-118。
10 Stefanaki K, Rontogiannis D, Vamvouka C, et al. Immunohistochemical detection of bcl-2, p53, mdm2 and p21 wafl proteins in small-cell lung carcinomas[J]. Anticancer Res, 1998, 18(3A): 1689-1695.
11 Whitesell L, Geselowitz DC, Havang C, et al. Stability, clearance and disposition of intraventricularly administered oligodeoxynucleotides: implications for therapeutic application within the central nervous systerm[J]. Proc Natl Acad Sci USA, 1993, 90: 4665-4669.
12 欧阳能太,冉丕鑫,邱志辉等,BCL-2反义寡核苷酸对顺铂诱导肺癌细胞凋亡的促进作用。中华结核和呼吸杂志,2000,23 (12):722-724。
13 Ziegler A, Luedke GH, Fabbro D, et al. Induction of apoptosis in small-cell lung cancer cell by an antisense oligodeoxynucleotide targeting the bcl-2 coding sequence[J]. J Natil Cancer Institute, 1997, 89(14): 1027-1036.
14 ZangemeisterWU, Schenker T, Luedke GH, et al. Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etotopside, doxorubicin, and cisplatin on small-cell lung cancer lines[J] . Br J Cancer, 1998, 78 (8): 1035-1044.
15 Rudin CM, Otterson GA, et a]. A pilot trial of G3139, a bcl-2 antisense oligonucleotide, and paclitaxel in patients with chemorefractory small-cell lung cancer. Ann Oncol. 2002 Apr;13(4): 539-45.
16 Rudin CM, Kozloff M, Hoffman PC, et al. Phase I study of G3139, a bcl-2 antisense oligonucleotide, combined with carboplatin and etoposide in patients with small-cell lung cancer. J Clin Oncol. 2004 Mar 15;22(6): 1110-7.
17 Tsujmoto Y, Gorham J, Croce CM, et al. The t(14, 18)chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining [J]. Science, 1985, 229(4720): 1390-1393.
18 Borner C. The Bcl-2 protein family: sensors and check points for life or death decisions. [J] Mol Immunol, 2003, 39(11): 615 647.
19 Ashe PC, Berry MD. Apoptotic signaling cascades[J]. Prog Neuropsychopharmacol Biol Psychiatry, 2003, 27(2): 199-214.
20 Xu J, Xu Z, Jiang Y,et al. Cryptorchidism induces mouse testicular germ cell apoptosis and changes in bcl-2 and bax protein expression [J]. J-Environ -pathol-Toxicol-oncol, 2000, 19(1-2): 25-33.
21 Bernard D. Anticancer drug resistance and inhibition of apoptosis [J]. Anticancer Res, 1994, 14(6A): 2291-2294.
22 陈协群,黄高升,王文亮等。非霍奇金淋巴中抗凋零蛋白Bcl-2的表达与分布。第四军医大学学报,1996;17(6):461。
23 Sarbia M, Bittinger F, Porschen R, et al. bcl-2 expression and prognosis in squamous cell carcinoma of the esophagus[J]. Int J Cancer, 1996, 69(4): 324 328.
24 Ohbu M, Saegusa M, Kobayashi N, et al. Expression of bcl-2 protein in esophagus squamous cell carcinomas and its association with lymph node metastasis[J]. Cancer, 1997, 79(7): 1287 1293.
25 黄莺,王康敏,何长武等,bcl-2和p16蛋白在食管鳞癌组织中的表达及其意义。[J]肿瘤防治研究2003,30(4):259-261。
26 Mc Donnell TJ, Deane N, Platt FM, et al. bcl-2 immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lympho proliferation[J]. Cell, 1989, 57(1): 79-88.
27 Klatka J, Rolinski J, Kupisz K, et al. Expression of bcl-2 protein in lymphocytes of patients with laryngeal carcinoma[J]. Eur Arch Otorhino]aryngol, 1999, 256(6): 299.
28 Pruneri G, Pignataro L, Carboni N, et al. Clinical relevance of p53 and bcl-2 protein over-expression in laryngeal squamous-cell carcinoma[J]. Int J Cancer, 1998, 79(3): 263.
29 张明生。袁宏银等。大肠肿瘤中Bcl-2表达的免疫组化研究。湖北预防医学杂志[J]2002,13(2):21-22。
30 刘海峰,刘为纹,房殿春等,胃癌和胃癌前病变bcl-2基因表达及其对细胞凋亡的调控作用.第三军医大学学报[J] 2004,26(7):580-583。
31 Morales Ducret CRJ, vander Rijn M, Le Brun DP, et al. bcl-2 expression in primary malignancies of the skin. Arch Dermatol, 1995, 131: 909.
32 张士发,路平,岳辉等。恶性黑素瘤和良性黑素细胞肿瘤bcl-2蛋白表达。中国皮肤性病学杂志[J],1999,13(3):133-134。
33 Ramon Y, Cajal S, Suster S, et al. Induction of different morphologic features of malignant melanoma and pigmented lesions after transformation of murine melanocytes with b-FGF Cdna and H-ras, myc, neu and Ela oncogens. Am J Pathol, 1991, 138: 349.
34 Husain Z, Fitz Gerald GB, Wick MM. Comparison of cellular proto oncogen activity of human melanocytes and metastatic melanoma. J Invest Dermatol, 1990, 95: 571.
35 Charlotte F, Hemine A, Martin N, et al. Immunohistochemical detection of bcl-2 protein in normal and pathological human liver[J]. AM-J-pathol, 1994, 144(3): 460-465.
36 秦军,李敏,孙丽娟等,蛋白在肝胆癌及慢性肝病组织中的表达,实用医药杂志,2002,19 (4):265。
37 王万忠,王宗耀,杜德利。Bcl-2基因在肝癌中的表达[J]。中华医学杂志,1996,76(6):470-473。
38 Kassim SK, Ali HS, Sallam MM, et al. Increased bcl-2 expression is associated with primary resistance to chemotherapy, in human epithelial ovarian cancer. Clin Biochem, 1999, 32: 333-338.
39 陈勇,郑新民,李世文等。前列腺癌细胞凋亡与bcl-2蛋白表达及其意义。临床泌尿外科杂志,2000,15(5):218-219。
40 生秀杰,宋和存,何秀琴。抗凋亡基因bcl-2在子宫内膜癌的表达及意义,中华 肿瘤杂志,1998,20(5):365。
41 Bhargava V, Kell DL, Van-de-Rijin M, et al. Bcl-2 immuno reactivity in breast carcinoma correlates with hormone receptor positivity. Am J Pathol, 1994, 145: 5356.
42 Joensun H, Pylkkanen L, Toikkanen S. Bc1-2 protein expression and long-term survival in breast cancer. Am J Pathol, 1994, 145: 1191.
43 徐云峰,毕铁男,尚贤文。bcl-2蛋白在乳腺癌中的表达及临床意义。实用肿瘤学杂志[J]2004,18(2):143-144。
44 郭庆明,王照明,张建民等。乳腺癌中bel-2的表达与激素受体及预后的关系。南京铁道医学院学报 1997;16(3):182～184。
45 Bargou RC, Daniel PT, Mapara MY, et al . Expression of the bcl-2 gene family in normal and malignant breast tissue: low bax-alpha expression in tumor cells correlates with resistance towards apoptosis. Int J Cancer, 1995 Mar 16: 60(6): 854-9.
46 陈鑫基,胡建达,战榕等。反义bcl-2硫代磷酸寡脱氧核苷酸对小细胞肺癌细胞株NCI-H446的抑制作用及诱导细胞凋亡的研究。中国病理生理杂志,2003,19(3):374-378。
47 林芸秀,吕联煌,林振兴等。联合新型BCL-2反义核酸与足叶乙甙抑制人小细胞肺癌细胞的增殖。中国药理学通报,2004 Jan;20(1):58-60.
48 Jaworska A, Szumiel I, DeAngelis P, et al. Evaluation of ionizing radiation sensitivity markers in a panel of lymphoid cell lines. Int J Radiat Biol. 2001 Mar77(3): 269-80.
49 Chaumont CG, Scksek O, Grzybowska J, et al. Delivery system for antisense oligonucleotides[J]. Pharmacol Ther, 2000, 87: 255-277.
50 Hara T, Omura-Minamisawa M, Chao C, et al. Bcl-2 inhibitors potentiate the cytotoxic 'effects of radiation in Bcl-2 overexpressing radioresistant tumor cell, Int J Radiat Oncol Phy. 2005 Feb 1;61(2): 517-28.
2 Greenlee Rt, et al. CA cancer J clin 2000;50: 7-33
3 殷蔚伯,谷铣之主编。肿瘤放射治疗学。第三版。北京,中国协和医科大学出版社。2002,1-15。
4 Nair CKK, Parida DK and Nomura T. Radioprotectors in radiotherapy. J Radiat. Res. 2001;42: 21-37.
5 Nicholson DW, Thornberry NA. Apoptosis: Life and death decisions. [J] Science, 2003, 299 (6504): 214-215.
6 Yang E ,Korsmeyer S. Molecular Thanatopsis: A discourse on the BCL-2 family and cell death [J]. Blood, 1996, 88(2): 388-401.
7 Vaux D, Cory S, Fulco M, et al. Effects of Bcl-2 gene in the tumour cell culture in vitro[J]. Nature, 1998, 335(6): 440.
8 姚和瑞,欧阳能太,李锦梅。癌基因Bcl-2蛋白在肺癌组织中的表达。癌症[J]1997,16(5):348-349。
9 刘慧,孔庆兖,吴永平,王绪。SCLC与NSCLC基因调控的差异—凋亡调控基因p53、Bcl-2表达的差异及意义。徐州医学院学报,2003,23(2)115-118。
10 Stefanaki K, Rontogiannis D, Vamvouka C, et al. Immunohistochemical detection of bcl-2, p53, mdm2 and p21 wafl proteins in small-cell lung carcinomas[J]. Anticancer Res, 1998, 18(3A): 1689-1695.
11 Whitesell L, Geselowitz DC, Havang C, et al. Stability, clearance and disposition of intraventricularly administered oligodeoxynucleotides: implications for therapeutic application within the central nervous systerm[J]. Proc Natl Acad Sci USA, 1993, 90: 4665-4669.
12 欧阳能太,冉丕鑫,邱志辉等,BCL-2反义寡核苷酸对顺铂诱导肺癌细胞凋亡的促进作用。中华结核和呼吸杂志,2000,23 (12):722-724。
13 Ziegler A, Luedke GH, Fabbro D, et al. Induction of apoptosis in small-cell lung cancer cell by an antisense oligodeoxynucleotide targeting the bcl-2 coding sequence[J]. J Natil Cancer Institute, 1997, 89(14): 1027-1036.
14 ZangemeisterWU, Schenker T, Luedke GH, et al. Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etotopside, doxorubicin, and cisplatin on small-cell lung cancer lines[J] . Br J Cancer, 1998, 78 (8): 1035-1044.
15 Rudin CM, Otterson GA, et a]. A pilot trial of G3139, a bcl-2 antisense oligonucleotide, and paclitaxel in patients with chemorefractory small-cell lung cancer. Ann Oncol. 2002 Apr;13(4): 539-45.
16 Rudin CM, Kozloff M, Hoffman PC, et al. Phase I study of G3139, a bcl-2 antisense oligonucleotide, combined with carboplatin and etoposide in patients with small-cell lung cancer. J Clin Oncol. 2004 Mar 15;22(6): 1110-7.
17 Tsujmoto Y, Gorham J, Croce CM, et al. The t(14, 18)chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining [J]. Science, 1985, 229(4720): 1390-1393.
18 Borner C. The Bcl-2 protein family: sensors and check points for life or death decisions. [J] Mol Immunol, 2003, 39(11): 615 647.
19 Ashe PC, Berry MD. Apoptotic signaling cascades[J]. Prog Neuropsychopharmacol Biol Psychiatry, 2003, 27(2): 199-214.
20 Xu J, Xu Z, Jiang Y,et al. Cryptorchidism induces mouse testicular germ cell apoptosis and changes in bcl-2 and bax protein expression [J]. J-Environ -pathol-Toxicol-oncol, 2000, 19(1-2): 25-33.
21 Bernard D. Anticancer drug resistance and inhibition of apoptosis [J]. Anticancer Res, 1994, 14(6A): 2291-2294.
22 陈协群,黄高升,王文亮等。非霍奇金淋巴中抗凋零蛋白Bcl-2的表达与分布。第四军医大学学报,1996;17(6):461。
23 Sarbia M, Bittinger F, Porschen R, et al. bcl-2 expression and prognosis in squamous cell carcinoma of the esophagus[J]. Int J Cancer, 1996, 69(4): 324 328.
24 Ohbu M, Saegusa M, Kobayashi N, et al. Expression of bcl-2 protein in esophagus squamous cell carcinomas and its association with lymph node metastasis[J]. Cancer, 1997, 79(7): 1287 1293.
25 黄莺,王康敏,何长武等,bcl-2和p16蛋白在食管鳞癌组织中的表达及其意义。[J]肿瘤防治研究2003,30(4):259-261。
26 Mc Donnell TJ, Deane N, Platt FM, et al. bcl-2 immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lympho proliferation[J]. Cell, 1989, 57(1): 79-88.
27 Klatka J, Rolinski J, Kupisz K, et al. Expression of bcl-2 protein in lymphocytes of patients with laryngeal carcinoma[J]. Eur Arch Otorhino]aryngol, 1999, 256(6): 299.
28 Pruneri G, Pignataro L, Carboni N, et al. Clinical relevance of p53 and bcl-2 protein over-expression in laryngeal squamous-cell carcinoma[J]. Int J Cancer, 1998, 79(3): 263.
29 张明生。袁宏银等。大肠肿瘤中Bcl-2表达的免疫组化研究。湖北预防医学杂志[J]2002,13(2):21-22。
30 刘海峰,刘为纹,房殿春等,胃癌和胃癌前病变bcl-2基因表达及其对细胞凋亡的调控作用.第三军医大学学报[J] 2004,26(7):580-583。
31 Morales Ducret CRJ, vander Rijn M, Le Brun DP, et al. bcl-2 expression in primary malignancies of the skin. Arch Dermatol, 1995, 131: 909.
32 张士发,路平,岳辉等。恶性黑素瘤和良性黑素细胞肿瘤bcl-2蛋白表达。中国皮肤性病学杂志[J],1999,13(3):133-134。
33 Ramon Y, Cajal S, Suster S, et al. Induction of different morphologic features of malignant melanoma and pigmented lesions after transformation of murine melanocytes with b-FGF Cdna and H-ras, myc, neu and Ela oncogens. Am J Pathol, 1991, 138: 349.
34 Husain Z, Fitz Gerald GB, Wick MM. Comparison of cellular proto oncogen activity of human melanocytes and metastatic melanoma. J Invest Dermatol, 1990, 95: 571.
35 Charlotte F, Hemine A, Martin N, et al. Immunohistochemical detection of bcl-2 protein in normal and pathological human liver[J]. AM-J-pathol, 1994, 144(3): 460-465.
36 秦军,李敏,孙丽娟等,蛋白在肝胆癌及慢性肝病组织中的表达,实用医药杂志,2002,19 (4):265。
37 王万忠,王宗耀,杜德利。Bcl-2基因在肝癌中的表达[J]。中华医学杂志,1996,76(6):470-473。
38 Kassim SK, Ali HS, Sallam MM, et al. Increased bcl-2 expression is associated with primary resistance to chemotherapy, in human epithelial ovarian cancer. Clin Biochem, 1999, 32: 333-338.
39 陈勇,郑新民,李世文等。前列腺癌细胞凋亡与bcl-2蛋白表达及其意义。临床泌尿外科杂志,2000,15(5):218-219。
40 生秀杰,宋和存,何秀琴。抗凋亡基因bcl-2在子宫内膜癌的表达及意义,中华 肿瘤杂志,1998,20(5):365。
41 Bhargava V, Kell DL, Van-de-Rijin M, et al. Bcl-2 immuno reactivity in breast carcinoma correlates with hormone receptor positivity. Am J Pathol, 1994, 145: 5356.
42 Joensun H, Pylkkanen L, Toikkanen S. Bc1-2 protein expression and long-term survival in breast cancer. Am J Pathol, 1994, 145: 1191.
43 徐云峰,毕铁男,尚贤文。bcl-2蛋白在乳腺癌中的表达及临床意义。实用肿瘤学杂志[J]2004,18(2):143-144。
44 郭庆明,王照明,张建民等。乳腺癌中bel-2的表达与激素受体及预后的关系。南京铁道医学院学报 1997;16(3):182～184。
45 Bargou RC, Daniel PT, Mapara MY, et al . Expression of the bcl-2 gene family in normal and malignant breast tissue: low bax-alpha expression in tumor cells correlates with resistance towards apoptosis. Int J Cancer, 1995 Mar 16: 60(6): 854-9.
46 陈鑫基,胡建达,战榕等。反义bcl-2硫代磷酸寡脱氧核苷酸对小细胞肺癌细胞株NCI-H446的抑制作用及诱导细胞凋亡的研究。中国病理生理杂志,2003,19(3):374-378。
47 林芸秀,吕联煌,林振兴等。联合新型BCL-2反义核酸与足叶乙甙抑制人小细胞肺癌细胞的增殖。中国药理学通报,2004 Jan;20(1):58-60.
48 Jaworska A, Szumiel I, DeAngelis P, et al. Evaluation of ionizing radiation sensitivity markers in a panel of lymphoid cell lines. Int J Radiat Biol. 2001 Mar77(3): 269-80.
49 Chaumont CG, Scksek O, Grzybowska J, et al. Delivery system for antisense oligonucleotides[J]. Pharmacol Ther, 2000, 87: 255-277.
50 Hara T, Omura-Minamisawa M, Chao C, et al. Bcl-2 inhibitors potentiate the cytotoxic 'effects of radiation in Bcl-2 overexpressing radioresistant tumor cell, Int J Radiat Oncol Phy. 2005 Feb 1;61(2): 517-28.