NDGA对HT-29结肠癌细胞诱导凋亡、凋亡机制及对其转移影响的研究
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摘要
背景 结肠癌是常见的消化道肿瘤。近年来,随着生活水平的提高,饮食中脂肪含量增高等原因,使结肠癌在我国有上升趋势,而且结肠癌发病年龄逐渐年轻化,早期容易发生转移。对于结肠癌的治疗,除手术切除外,化疗和放疗也是重要的治疗方法。传统的化疗药物如5-氟尿嘧啶等药物效果差,副作用较多,病人的耐受性较差。积极寻找一种新的治疗结肠癌有效药物,受到学者们的普遍重视。
有关肿瘤细胞花生四烯酸(AA)的代谢途径是国内外近年来研究的热点。花生四烯酸的代谢分为环氧化酶(COX)和脂氧合酶(LOX)途径。COX是环氧化酶途径的关键酶,有COX-1和COX-2两种异构体。抑制COX途径能够诱导结肠癌细胞凋亡,抑制癌细胞转移,对这一点国内外很多学者已达成共识。而对于抑制LOX途径能否抑制HT-29结肠癌细胞生长、诱导其凋亡、抑制转移,目前国内外鲜有报道。
目的 探讨脂氧合酶抑制剂NDGA在体外对结肠癌HT-29细胞株能否诱导凋亡;凋亡机制探讨,以及对其转移方面的影响。
材料与方法 结肠癌细胞株HT-29培养于含10%小牛血清的RPMI1640培养基中,5%CO_2,37℃恒温暖箱中常规培养。然后加入小同浓度NDGA处理结肠癌细胞。分别应用(1)MTT法绘制生长曲线。(2)倒置相差显微镜观察细胞形态变化,扫描电镜观察细胞的
Background: Colon cancer is a common kind of malignant tumor. Due to the factors like the improvement of people's living standard , the increasing of fat content in people's everyday diet, the incidence rate of this diease is on the rise in recent years. The episode age is gradually declining and metastasis is easily seen in early stage. Apart from surgical operation, treatments of it include chemotherapy and radiotherapy. However, the effects of traditional chemotherapy drugs such as 5-FU are unsatisfying, with too many side-effects and poor tolerance of patients. Therefore, scientists are now vigorously engaged in seeking an new effective drug to treat colon cancer.The focus of research, domestic or abroad, is on the metabolism pathway of arachidonic acid in tumor cell. Arachidonic acid can be classified into pathways of cyclooxygenase (COX) and lipoxygenase(LOX). COX is the key factor in the COX pathway and consists of COX-1 and COX-2. Many scholars agree that inhibiting the pathway of COX can induce tumor cell apoptosis and affect its metastasis. But there is little agreement and few reports on whether
inhibiting the pathway of LOX can inhibit colon cancer's growth, induce its apotosis and suppress its metastasis.Objective: To find out the effect of NDGA, the lipoxygenase inhibitor, on colon cancer cell line HT-29 in vitro from the aspects of cell apoptosis, apoptosis mechanism, and metastasis.Materials and Methods: The colon cancer cell line HT-29 was cultivated in RPMI1640 medium, which contained 10% calf blood serum and was placed in a constant temperature oven of 37°C and 5% CO_2. Then NDGA of different concentrations was used to dispose the cancer cell. We applied respectively. a.) MTT to draw the growth curve. b.) inverted phase contrast microscope to observe morphologic change of cells as well as scanning electron microscope to observe changes of cell's ultra-microstructure and apoptotic body. c.) flow cytometry to examine the apoptosis and periodic changes of cells. d.) Study of cell's apoptosis mechanisms: RT-PCR to detect the expression of 5-LOX messenger ribonucleic acid (5-LOXmRNA) and that of messenger ribonucleic acid about human telomerase reverse transcriptase (hTERTmRNA) respectively; flow cytometry to detect the expression of Bcl-2; confocal laser scanning electron microscope to examine freeing calcium of cell. e.) influence of cell metastasis: RT-PCR to detect VEGF, scanning electron microscope to observe cell's microvilli, flow cytometry to detect expression of CD44v6, confocal laser scanning electron microscope detect cytoskeleton change.The data was statistically processed with the software SPSS12.0,with the value of P smaller than 0.05 being considered statistically significant.Results: a.)Cancer cell's growth apoptosis examination: We used different concentration of NDGA to dispose cancer cell independently. Accompanying the rise in drug's concentration,
apoptosis rate of cells rose gradually with difference of notable significance, b.) Morphologic change of cells: In the control group, cancer cells showed pararound shape, some of them showing fusiform due to extension. After the drug's disposing, morphologic of cells became round, the volume became small and they abscised from the inner surface of the bottle. With the passage of time, the trend became increasingly apparent. Apoptotic body could be found through scanning electron microscope after NDGA (100M-mol/L) had been used for 48 hours, c.) Using flow cytometry to examine cell's apoptosis, we found that the rate of cancer cell apoptosis rise following the drug's concentration change and the growth of cells was blocked on the stage of Go/Gi. In contrast to the control section, the difference was obvious, d.) Mechanism of cell apoptosis: colon cancer cell lines HT-29 showed positive expression of5-LOXmRNA.This expression became weaker following the rising of the drug's concentration and so were hTERTmRNA and bcl-2. Freeing calcium of cell increased following the rise of cell's apoptosis. e.) Cell's metastasis affected by NDGA: The expression of CD44v6 went down following the rising of drug concentration, and so was VEGF detected with RT-PCR. Through scanning electron microscope, it was found that cell's microvilli curled to microball after the drug ( lOP-mol/L) had been used for 48 hours. Confocal laser scanning electron microscope was used to detect cytoskeleton change. It was also observed that after the cells had been disposed with NDGA for 36 hours, the actin showed depolymerization, the intensity of fluorescence became weaker and the annular structure around cell nuclei disappeared.Conclusions: NDGA can inhibit colon cancer cell line HT-29 growth and induce its apoptosis, the rate of which rises following the
有关肿瘤细胞花生四烯酸(AA)的代谢途径是国内外近年来研究的热点。花生四烯酸的代谢分为环氧化酶(COX)和脂氧合酶(LOX)途径。COX是环氧化酶途径的关键酶,有COX-1和COX-2两种异构体。抑制COX途径能够诱导结肠癌细胞凋亡,抑制癌细胞转移,对这一点国内外很多学者已达成共识。而对于抑制LOX途径能否抑制HT-29结肠癌细胞生长、诱导其凋亡、抑制转移,目前国内外鲜有报道。
目的 探讨脂氧合酶抑制剂NDGA在体外对结肠癌HT-29细胞株能否诱导凋亡;凋亡机制探讨,以及对其转移方面的影响。
材料与方法 结肠癌细胞株HT-29培养于含10%小牛血清的RPMI1640培养基中,5%CO_2,37℃恒温暖箱中常规培养。然后加入小同浓度NDGA处理结肠癌细胞。分别应用(1)MTT法绘制生长曲线。(2)倒置相差显微镜观察细胞形态变化,扫描电镜观察细胞的
Background: Colon cancer is a common kind of malignant tumor. Due to the factors like the improvement of people's living standard , the increasing of fat content in people's everyday diet, the incidence rate of this diease is on the rise in recent years. The episode age is gradually declining and metastasis is easily seen in early stage. Apart from surgical operation, treatments of it include chemotherapy and radiotherapy. However, the effects of traditional chemotherapy drugs such as 5-FU are unsatisfying, with too many side-effects and poor tolerance of patients. Therefore, scientists are now vigorously engaged in seeking an new effective drug to treat colon cancer.The focus of research, domestic or abroad, is on the metabolism pathway of arachidonic acid in tumor cell. Arachidonic acid can be classified into pathways of cyclooxygenase (COX) and lipoxygenase(LOX). COX is the key factor in the COX pathway and consists of COX-1 and COX-2. Many scholars agree that inhibiting the pathway of COX can induce tumor cell apoptosis and affect its metastasis. But there is little agreement and few reports on whether
inhibiting the pathway of LOX can inhibit colon cancer's growth, induce its apotosis and suppress its metastasis.Objective: To find out the effect of NDGA, the lipoxygenase inhibitor, on colon cancer cell line HT-29 in vitro from the aspects of cell apoptosis, apoptosis mechanism, and metastasis.Materials and Methods: The colon cancer cell line HT-29 was cultivated in RPMI1640 medium, which contained 10% calf blood serum and was placed in a constant temperature oven of 37°C and 5% CO_2. Then NDGA of different concentrations was used to dispose the cancer cell. We applied respectively. a.) MTT to draw the growth curve. b.) inverted phase contrast microscope to observe morphologic change of cells as well as scanning electron microscope to observe changes of cell's ultra-microstructure and apoptotic body. c.) flow cytometry to examine the apoptosis and periodic changes of cells. d.) Study of cell's apoptosis mechanisms: RT-PCR to detect the expression of 5-LOX messenger ribonucleic acid (5-LOXmRNA) and that of messenger ribonucleic acid about human telomerase reverse transcriptase (hTERTmRNA) respectively; flow cytometry to detect the expression of Bcl-2; confocal laser scanning electron microscope to examine freeing calcium of cell. e.) influence of cell metastasis: RT-PCR to detect VEGF, scanning electron microscope to observe cell's microvilli, flow cytometry to detect expression of CD44v6, confocal laser scanning electron microscope detect cytoskeleton change.The data was statistically processed with the software SPSS12.0,with the value of P smaller than 0.05 being considered statistically significant.Results: a.)Cancer cell's growth apoptosis examination: We used different concentration of NDGA to dispose cancer cell independently. Accompanying the rise in drug's concentration,
apoptosis rate of cells rose gradually with difference of notable significance, b.) Morphologic change of cells: In the control group, cancer cells showed pararound shape, some of them showing fusiform due to extension. After the drug's disposing, morphologic of cells became round, the volume became small and they abscised from the inner surface of the bottle. With the passage of time, the trend became increasingly apparent. Apoptotic body could be found through scanning electron microscope after NDGA (100M-mol/L) had been used for 48 hours, c.) Using flow cytometry to examine cell's apoptosis, we found that the rate of cancer cell apoptosis rise following the drug's concentration change and the growth of cells was blocked on the stage of Go/Gi. In contrast to the control section, the difference was obvious, d.) Mechanism of cell apoptosis: colon cancer cell lines HT-29 showed positive expression of5-LOXmRNA.This expression became weaker following the rising of the drug's concentration and so were hTERTmRNA and bcl-2. Freeing calcium of cell increased following the rise of cell's apoptosis. e.) Cell's metastasis affected by NDGA: The expression of CD44v6 went down following the rising of drug concentration, and so was VEGF detected with RT-PCR. Through scanning electron microscope, it was found that cell's microvilli curled to microball after the drug ( lOP-mol/L) had been used for 48 hours. Confocal laser scanning electron microscope was used to detect cytoskeleton change. It was also observed that after the cells had been disposed with NDGA for 36 hours, the actin showed depolymerization, the intensity of fluorescence became weaker and the annular structure around cell nuclei disappeared.Conclusions: NDGA can inhibit colon cancer cell line HT-29 growth and induce its apoptosis, the rate of which rises following the
引文
1.陈灏珠主编.实用内科学.北京:人民卫生出版社,1998:1619-1621
2.伍治平,蒋永新.非甾体类抗炎药对大肠癌的化学预防及机理探讨.肿瘤研究与临床;2001;13(2)142-143
3. Vane JR, Botting RM. The mode of action of anti-inflammatory drugs[J]. postgrad MedJ,1990,66(Supp 14):S 2-17.
4. Ford Hutclinson AW, Bray MA, Doig MV, et al. EukotrieneB, a potent chemokinetic and a gregating substance relased from polymorphonuclear leukocytes[J]. Nature, 1980, 286(5770): 264-265.
5. Vernon E. Stele, Cathy A. Holmes, Ernest T. et al. Lipoxygenase Inhibitors as Potential Cancer Chemopreventives. Cancer Epidemiology Biomarkers & Prevention 1999, 5(8): 467-483
6. Cortese J. F., Spannhake E. W., Eisinger W., et al. The 5-lipoxygenase pathway in cultured human intestinal epithelial cells. Prostaglandins, 1995, 49: 155-166
7. Sjolander A., Schippert A., Hammarstrom S. A human epithelial cell line, intestine-407, can produce 5-hydroxyeicosatetraenoic acid and leukotriene B_4. Prostaglandins, 1993, 45: 85-96
8.司徒镇强,吴军正主编.细胞培养.西安:世界图书出版公司,1996
9.尹格平.流式细胞术检测化疗前后卵巢癌细胞凋亡的临床意义.癌症.2000;19(4):383
10. Hong SH, Avis I, Vos MD, et al. Relationship of arachidonic acid metabolizing enzyme expression in epithelial cancer cell lines to the growth effect of selective biochemical inhibitors. Cancer Res. 1999 May 1; 59(9): 2223-8
11. Gupta S, Srivastava M, Ahmad N, et al. Lipoxygenase-5 is
??overexpressd in prostate adenocarcinoma[J]. Cancer,2001,91(4): 73 7-743
12. Avis I. M, Jett M, Boyle T, Vos M. D, Moody T., et al. Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling. J. Clin. Invest, 1996 ,97: 806-813
13.Ghosh J,Myers CE. Inhibition of arachidonate 5- lipoxygenase triggers massive apoptosis in human prostate cancer cells[J].Proc Natl Acad Sci USA,1998,95(22):13 182-13 187
14. Anderson K. M., Seed T., Ondrey F., Harris J. E. The selective 5-lipoxygenase inhibitor A63162 reduces PC3 proliferation and initiates morphologic changes consistent with secretion. Anticancer Res, 1994., 14: 1951-1960
15. Kort W. J., Bijma A. M., van Dam J. J., et al. Eicosanoids in breast cancer patients before and after mastectomy. Prostaglandins Leukotrienes Essent. Fatty Acids, 1992, 45: 319-327
16. McCormick D. L., Spicer A. M. Nordihydroguaiaretic acid suppression of rat mammary carcinogenesis induced by N-methyl-N-nitrosourea. Cancer Lett. 1987, 37: 139-146
17.Avis I,Hong SH,Martinez A, et al. Five-lipoxygenase inhibitors can mediate apoptosis in human breast cancer cell lines through complex eicosanoid interactions[J].FASEB J,2001,15(11):2007-2009
18. Krieg P., Kinzig A., Ress-Loschke M., et al. 12-Lipoxygenase isoenzymes in mouse skin tumor development. Mol. Carcinog., 1995 ( 14) : 118-129,
19.Dittmann KH, Mayer C, Rodemann HP, et al. MK886,a leukotriene biosynthesis inhibitor,induces antiproliferative effects and apoptosis in HL-60 cells[J]. Leuk Res,1998,22(1):
49-53
20. Hennig R,Ding XZ,Tong WG, et al. 5-Lipoxygenase and Leukotriene B(4) receptor are expressed in human pancreatic cancers but not in pancreatic ducts in normal tissue[J]. Am J Pathol, 2002, 161(2): 421-428
21. Seufferlein T, Seckl MJ, Schwarz E, et al. Mechanisms of nordihydroguaiaretic acid-induced growth inhibition and apoptosis in human cancer cells[J]. Br J Cancer, 2002, 86(7): 1188-1196
22.樊晓明.脂质加氧酶抑制剂NDGA诱导胃癌细胞凋亡的研究.胃肠病学和肝病学杂志 2001,10(3):212-216
23. Dias V. C., Wallace J. L., Parsons H. G. Modulation of cellular phospholipid fatty acids and leukotriene B4 synthesis in the human intestinal cell (CaCo-2). Gut, 1992, 33: 622-627
24.翟中和,王善忠,丁明孝主编.细胞生物学.北京:高等教育出版社 2000,454-467
25.权华.5-脂氧合酶代谢通路与肿瘤细胞增值及凋亡的关系.国外医学肿瘤学分册 2003,30(1):57-59
26.张向阳,李爱英.流式细胞仪的原理及其在医学研究中的应用.邯郸医学高等专科学校学报 2002;15(4):470-471
27.牛朝诗,傅先明,汪业汉.反义IGF-IR基因片断诱导胶质瘤细胞凋亡的流式细胞仪分析和电镜观察.现代神经疾病杂志 2003,3(1):26-29
28.邵世和,李咏梅,陈曦,等.流式细胞仪法检测4HPR诱导Hela细胞凋亡的百分率.吉林医学院学报 1998,18(2):16-17
29. Huang L, Ouyang Q, Wei D, et al. The growth inhibition of colorectal adenoma cells by sulindac and its mechanisms. Hua Xi Yi Ke Da Xue Xue Bao. 2002; 33(1): 101-3.
30. Zhu Q, Deng Apoptosis of lymphocytes induced by Fas/FasL of
2.伍治平,蒋永新.非甾体类抗炎药对大肠癌的化学预防及机理探讨.肿瘤研究与临床;2001;13(2)142-143
3. Vane JR, Botting RM. The mode of action of anti-inflammatory drugs[J]. postgrad MedJ,1990,66(Supp 14):S 2-17.
4. Ford Hutclinson AW, Bray MA, Doig MV, et al. EukotrieneB, a potent chemokinetic and a gregating substance relased from polymorphonuclear leukocytes[J]. Nature, 1980, 286(5770): 264-265.
5. Vernon E. Stele, Cathy A. Holmes, Ernest T. et al. Lipoxygenase Inhibitors as Potential Cancer Chemopreventives. Cancer Epidemiology Biomarkers & Prevention 1999, 5(8): 467-483
6. Cortese J. F., Spannhake E. W., Eisinger W., et al. The 5-lipoxygenase pathway in cultured human intestinal epithelial cells. Prostaglandins, 1995, 49: 155-166
7. Sjolander A., Schippert A., Hammarstrom S. A human epithelial cell line, intestine-407, can produce 5-hydroxyeicosatetraenoic acid and leukotriene B_4. Prostaglandins, 1993, 45: 85-96
8.司徒镇强,吴军正主编.细胞培养.西安:世界图书出版公司,1996
9.尹格平.流式细胞术检测化疗前后卵巢癌细胞凋亡的临床意义.癌症.2000;19(4):383
10. Hong SH, Avis I, Vos MD, et al. Relationship of arachidonic acid metabolizing enzyme expression in epithelial cancer cell lines to the growth effect of selective biochemical inhibitors. Cancer Res. 1999 May 1; 59(9): 2223-8
11. Gupta S, Srivastava M, Ahmad N, et al. Lipoxygenase-5 is
??overexpressd in prostate adenocarcinoma[J]. Cancer,2001,91(4): 73 7-743
12. Avis I. M, Jett M, Boyle T, Vos M. D, Moody T., et al. Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling. J. Clin. Invest, 1996 ,97: 806-813
13.Ghosh J,Myers CE. Inhibition of arachidonate 5- lipoxygenase triggers massive apoptosis in human prostate cancer cells[J].Proc Natl Acad Sci USA,1998,95(22):13 182-13 187
14. Anderson K. M., Seed T., Ondrey F., Harris J. E. The selective 5-lipoxygenase inhibitor A63162 reduces PC3 proliferation and initiates morphologic changes consistent with secretion. Anticancer Res, 1994., 14: 1951-1960
15. Kort W. J., Bijma A. M., van Dam J. J., et al. Eicosanoids in breast cancer patients before and after mastectomy. Prostaglandins Leukotrienes Essent. Fatty Acids, 1992, 45: 319-327
16. McCormick D. L., Spicer A. M. Nordihydroguaiaretic acid suppression of rat mammary carcinogenesis induced by N-methyl-N-nitrosourea. Cancer Lett. 1987, 37: 139-146
17.Avis I,Hong SH,Martinez A, et al. Five-lipoxygenase inhibitors can mediate apoptosis in human breast cancer cell lines through complex eicosanoid interactions[J].FASEB J,2001,15(11):2007-2009
18. Krieg P., Kinzig A., Ress-Loschke M., et al. 12-Lipoxygenase isoenzymes in mouse skin tumor development. Mol. Carcinog., 1995 ( 14) : 118-129,
19.Dittmann KH, Mayer C, Rodemann HP, et al. MK886,a leukotriene biosynthesis inhibitor,induces antiproliferative effects and apoptosis in HL-60 cells[J]. Leuk Res,1998,22(1):
49-53
20. Hennig R,Ding XZ,Tong WG, et al. 5-Lipoxygenase and Leukotriene B(4) receptor are expressed in human pancreatic cancers but not in pancreatic ducts in normal tissue[J]. Am J Pathol, 2002, 161(2): 421-428
21. Seufferlein T, Seckl MJ, Schwarz E, et al. Mechanisms of nordihydroguaiaretic acid-induced growth inhibition and apoptosis in human cancer cells[J]. Br J Cancer, 2002, 86(7): 1188-1196
22.樊晓明.脂质加氧酶抑制剂NDGA诱导胃癌细胞凋亡的研究.胃肠病学和肝病学杂志 2001,10(3):212-216
23. Dias V. C., Wallace J. L., Parsons H. G. Modulation of cellular phospholipid fatty acids and leukotriene B4 synthesis in the human intestinal cell (CaCo-2). Gut, 1992, 33: 622-627
24.翟中和,王善忠,丁明孝主编.细胞生物学.北京:高等教育出版社 2000,454-467
25.权华.5-脂氧合酶代谢通路与肿瘤细胞增值及凋亡的关系.国外医学肿瘤学分册 2003,30(1):57-59
26.张向阳,李爱英.流式细胞仪的原理及其在医学研究中的应用.邯郸医学高等专科学校学报 2002;15(4):470-471
27.牛朝诗,傅先明,汪业汉.反义IGF-IR基因片断诱导胶质瘤细胞凋亡的流式细胞仪分析和电镜观察.现代神经疾病杂志 2003,3(1):26-29
28.邵世和,李咏梅,陈曦,等.流式细胞仪法检测4HPR诱导Hela细胞凋亡的百分率.吉林医学院学报 1998,18(2):16-17
29. Huang L, Ouyang Q, Wei D, et al. The growth inhibition of colorectal adenoma cells by sulindac and its mechanisms. Hua Xi Yi Ke Da Xue Xue Bao. 2002; 33(1): 101-3.
30. Zhu Q, Deng Apoptosis of lymphocytes induced by Fas/FasL of
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