5-杂氮-2’-脱氧胞苷诱导裸鼠HepG2种植瘤细胞表达T-cadherin及对种植瘤的抑制作用
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摘要
目的:本研究旨在探讨去甲基化药物5-杂氮-2’-脱氧胞苷(5-Aza-CdR)能否在裸鼠体内诱导HepG2细胞的T-cadherin表达,并对HepG2种植瘤生长产生抑制作用.
方法:皮下接种法建立HepG2细胞裸鼠种植瘤模型,随机分为实验组(5-Aza-CdR组)和对照两组,实验组11只,对照组10只。4周后处死裸鼠,观察种植瘤的生长情况;用RT-PCR技术及免疫组织化学技术检测T-cadherin mRNA及蛋白的表达。
结果:用药4周后,实验组肿瘤组织的T-cadherin mRNA的表达水平显著高于对照组(T-cadherin基因PCR产物吸光度值与β-actin PCR产物吸光度值比值分别为0.545±0.119,0.256±0.079,t=6.613, P<0.05 );对照组HepG2细胞几乎检测不到T-cadherin蛋白表达,而实验组裸鼠种植瘤细胞膜上可检测到T-cadherin蛋白表达;实验组肿瘤的平均体积明显小于对照组肿瘤平均体积(分别为1005.78±419.69 mm3 , 1423.78±645.54mm3, t=2.337,P=0.025)。
结论:5-Aza-CdR能诱导裸鼠皮下种植HepG2瘤细胞的T-cadherin表达,抑制HepG2种植瘤的生长。其机制可能与5-Aza-CdR使甲基化的T-cadherin启动子去甲基化恢复了T-cadherin的表达,从而抑制HepG2种植瘤生长.
AIM: This study was to observe whether the demethylation agent 5-Aza-2’–deoxycytidine ( 5-Aza-CdR) can induce the HepG2 cell to express T-cadherin gene, and inhibit the proliferation of the cancer cells in nude mice.
METHODS : The HepG2-derived tumor model in nude mice was generated by subcutaneous inoculation of HepG2 cells. 21 Nude mice were randomly divided into two groups, the experiment group with 11 mice (5-Aza-CdR group) and the control group with 10 mice. The mice of the experiment group were given 5-Aza-CdR at 1mg/kg(in 200μl PBS) by intraperitoneal injection, three times per week( at an interval of 2-3 days) for 4 weeks, and the mice of the control group were only given the same volume of PBS. The mice were then sacrificed at the end of the fourth week. The tumor growth in nude mice was observed,and the T-cadherin mRNA and protein expressions of the tumors were detected by reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemistry.
RESULTS: At the end of the fourth week, the T-cadherin mRNA expression in the tumors of the experiment group was significantly higher than that in the tumors of the control group (t=6.613, P<0.05); Immunohistochemistry further showed that the protein expression of T-cadherin in the HepG2-derived tumor cells of the control group was not found, however, the T-cadherin expression appeared on the membranes of the HepG2-derived tumor cells of the experiment group. After 4 weeks treatment with 5-Aza-CdR, the average volume of HepG2-derived tumors in the experiment group was significantly smaller than that in the control group(1005.78±419.69 vs 1423.78±645.54mm3 ) , there was a significant difference in tumor volume between the two groups(t=2.337,P=0.025<0.05).
CONCLUSION:5-Aza-CdR induces HepG2 cells to re-express T-cadherin, and inhibits the growth of HepG2-derived tumors in nude mice. The mechanism may correlate with the demethylation of the methylated T-cadherin promoter induced by 5-Aza-CdR,and the T-cadherin reexpression inhibits the growth of the HepG2-derived tumors in nude mice.
方法:皮下接种法建立HepG2细胞裸鼠种植瘤模型,随机分为实验组(5-Aza-CdR组)和对照两组,实验组11只,对照组10只。4周后处死裸鼠,观察种植瘤的生长情况;用RT-PCR技术及免疫组织化学技术检测T-cadherin mRNA及蛋白的表达。
结果:用药4周后,实验组肿瘤组织的T-cadherin mRNA的表达水平显著高于对照组(T-cadherin基因PCR产物吸光度值与β-actin PCR产物吸光度值比值分别为0.545±0.119,0.256±0.079,t=6.613, P<0.05 );对照组HepG2细胞几乎检测不到T-cadherin蛋白表达,而实验组裸鼠种植瘤细胞膜上可检测到T-cadherin蛋白表达;实验组肿瘤的平均体积明显小于对照组肿瘤平均体积(分别为1005.78±419.69 mm3 , 1423.78±645.54mm3, t=2.337,P=0.025)。
结论:5-Aza-CdR能诱导裸鼠皮下种植HepG2瘤细胞的T-cadherin表达,抑制HepG2种植瘤的生长。其机制可能与5-Aza-CdR使甲基化的T-cadherin启动子去甲基化恢复了T-cadherin的表达,从而抑制HepG2种植瘤生长.
AIM: This study was to observe whether the demethylation agent 5-Aza-2’–deoxycytidine ( 5-Aza-CdR) can induce the HepG2 cell to express T-cadherin gene, and inhibit the proliferation of the cancer cells in nude mice.
METHODS : The HepG2-derived tumor model in nude mice was generated by subcutaneous inoculation of HepG2 cells. 21 Nude mice were randomly divided into two groups, the experiment group with 11 mice (5-Aza-CdR group) and the control group with 10 mice. The mice of the experiment group were given 5-Aza-CdR at 1mg/kg(in 200μl PBS) by intraperitoneal injection, three times per week( at an interval of 2-3 days) for 4 weeks, and the mice of the control group were only given the same volume of PBS. The mice were then sacrificed at the end of the fourth week. The tumor growth in nude mice was observed,and the T-cadherin mRNA and protein expressions of the tumors were detected by reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemistry.
RESULTS: At the end of the fourth week, the T-cadherin mRNA expression in the tumors of the experiment group was significantly higher than that in the tumors of the control group (t=6.613, P<0.05); Immunohistochemistry further showed that the protein expression of T-cadherin in the HepG2-derived tumor cells of the control group was not found, however, the T-cadherin expression appeared on the membranes of the HepG2-derived tumor cells of the experiment group. After 4 weeks treatment with 5-Aza-CdR, the average volume of HepG2-derived tumors in the experiment group was significantly smaller than that in the control group(1005.78±419.69 vs 1423.78±645.54mm3 ) , there was a significant difference in tumor volume between the two groups(t=2.337,P=0.025<0.05).
CONCLUSION:5-Aza-CdR induces HepG2 cells to re-express T-cadherin, and inhibits the growth of HepG2-derived tumors in nude mice. The mechanism may correlate with the demethylation of the methylated T-cadherin promoter induced by 5-Aza-CdR,and the T-cadherin reexpression inhibits the growth of the HepG2-derived tumors in nude mice.
引文
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3. Bender CM, Pao MM, Jones PA. Inhibition of DNA methylation by 5-aza-2’- deoxycytidine suppresses the growth of human tumor cell lines.Cancer Res,1998,58(1):95-101.
4. Lantry LE, Zhang Z, Crist KA, Wang Y, Kelloff GJ, Lubet RA, You M.5-Aza-2,-deoxycytidine is chemopreventive in a 4- (methylnitrosamino)- 1- (3-pyridy1)- 1-butanone-induced primary mouse lung tumor model [J]. Carcinogenesis,1999,20(2):343—346.
5. Huang ZY, Wu Y, Hedrick N, Gutmann DH. T- cadherin-Mediated Cell Growth Regulation Involves G2 Phase Arrest and requires p21CIP1/WAF1 Expression. Mol Cell Biol, 2003, 23: 566–578.
6. Qun Yan , Zhi-fa Zhang, Xiao-ping Chen, David H Gutmann, Min Xiong Zhen-yu Xiao, Zhi-yong Huang. Reduced T-cadherin expression and promoter methylation are associated with the development and progression of hepatocellular carcinoma.IntJ Oncol, 2008, 32:1057-1063.
7.张志发,严群,黄志勇.黏附分子cadherin与肝癌生物学特性的关系.世界华人消化杂志, 2006年3月8日;14(7):697-701.
8. Levenberg S, Yarden A, Kam Z, Geiger B. P27 is involved in N-cadherin mediated contact inhibition of cell growth and S-phase entry. Oncogene, 1999, 18: 869–876.
9. Ivanov D B, Philippova MP, Tkachuk VA.Structure and Functions of Classical Cadherins,Biochemistry (Moscow), Vol. 66, No. 10, 2001.
10. Angst BD, Marcozzi C, Magee AI.The cadherin superfamily: diversity in form andfunction. Journal of Cell Science, 2001, 114(Pt4): 629-641.
11. Philippova M, Ivanov D, Tkachuk V, Erne P, Resink TJ. Polarisation of T-cadherin to the leading edge of migrating vascular cells in vitro: a function in vascular cell motility? Histochemistry and Cell Biology, 2003, 120:353-360.
12. Sato M, Mori Y, Sakurada A, Fujimura S, Horii A. The H-cadherin (CDH13) gene is inactivated in human lung cancer. Hum Genet, 1998, 103:96–101.
13. Sakai M, Hibi K, Koshikawa K, Inoue S, Takeda S, Kaneko T, Nakao A. Frequent promoter methylation and gene silencing of CDH13 in pancreatic cancer. Cancer Sci, 2004, 95: 588–591.
14. Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC, Baylin SB, Sidransky D. 5’CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med, 1995, 1: 686–692.
15. Dammann R, Schagdarsurengin U, Liu L, Otto N, Gimm O, Dralle H, Boehm BO, Pfeifer GP, Hoang-Vu C. Frequent RASSF1A promoter hypermethylation and K-ras mutations in pancreatic carcinoma. Oncogene, 2003, 22:3806–3812.
16. Fukushima N, Sato N, Ueki T, Rosty C, Walter KM, Wilentz RE, Yeo CJ, Hruban RH, Goggins M. Aberrant methylation of preproenkephalin and p16 genes in pancreatic intraepithelial neoplasia and pancreatic ductal adenocarcinoma. Am J Pathol,2002, 160: 1573–1581.
17. Santini V, Kantarjian HM, Issa JP,Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann Intern Med. 2001 Apr 3; 134 (7):573-86.
18. Plumb JA,Strathdee G,Sludden J,et a1.Reversal of drug resistance in human tumor xenografts by 2’-deoxy-5-azacytidine induced demethylation of the hMLH1 gene promoter [J]. Cancer Res,2000,60(21):6039—6044(可删除,正文相应删除)
19. Wijermans PW, Krulder JW, Huijgens PC, NeveP. Continuous infusion of low-dose 5-Aza-2'-deoxycytidine in elderly patients with high-risk myelodysplastic syndrome. Leukemia 1997; 11: 1-5.
20. Kantarjian H, Oki Y, Garcia-Manero G, Huang X, O'Brien S, Cortes J, Faderl S, Bueso-Ramos C, Ravandi F, Estrov Z, Ferrajoli A, Wierda W, Shan J, Davis J, Giles F, Saba HI, Issa JP. Results of a randomized study of 3 schedules of low-dose decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic leukemia.Blood 2007; 109: 52-57.
21. Kanai Y, Ushijima S, Hui AM, Ochiai A, Tsuda H, Sakamoto M, Hirohashi S. The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas, Int. J. Cancer: 71, 355–359.
22. Lee YY, Kang SH, Seo JY, Jung CW, Lee KU, Choe KJ, Kim BK, Kim NK, Koeffler HP, Bang YJ. Alterations of p16INK4A and p15INK4B genes in gastric carcinomas. Cancer, 80, 1889–1896.
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2. Kiyomi O. Toyooka, Shinichi Toyooka, Arvind K. Virmani, Ubaradka G. Sathyanarayana, David M. Euhus, Michael Gilcrease, John D. Minna, and Adi F. Gazdar.Loss of Expression and Aberrant Methylation of the CDH13 (H-Cadherin) Gene in Breast and Lung Carcinomas. Cancer Res 61, 4556–4560, June 1, 2001.
3. Bender CM, Pao MM, Jones PA. Inhibition of DNA methylation by 5-aza-2’- deoxycytidine suppresses the growth of human tumor cell lines.Cancer Res,1998,58(1):95-101.
4. Lantry LE, Zhang Z, Crist KA, Wang Y, Kelloff GJ, Lubet RA, You M.5-Aza-2,-deoxycytidine is chemopreventive in a 4- (methylnitrosamino)- 1- (3-pyridy1)- 1-butanone-induced primary mouse lung tumor model [J]. Carcinogenesis,1999,20(2):343—346.
5. Huang ZY, Wu Y, Hedrick N, Gutmann DH. T- cadherin-Mediated Cell Growth Regulation Involves G2 Phase Arrest and requires p21CIP1/WAF1 Expression. Mol Cell Biol, 2003, 23: 566–578.
6. Qun Yan , Zhi-fa Zhang, Xiao-ping Chen, David H Gutmann, Min Xiong Zhen-yu Xiao, Zhi-yong Huang. Reduced T-cadherin expression and promoter methylation are associated with the development and progression of hepatocellular carcinoma.IntJ Oncol, 2008, 32:1057-1063.
7.张志发,严群,黄志勇.黏附分子cadherin与肝癌生物学特性的关系.世界华人消化杂志, 2006年3月8日;14(7):697-701.
8. Levenberg S, Yarden A, Kam Z, Geiger B. P27 is involved in N-cadherin mediated contact inhibition of cell growth and S-phase entry. Oncogene, 1999, 18: 869–876.
9. Ivanov D B, Philippova MP, Tkachuk VA.Structure and Functions of Classical Cadherins,Biochemistry (Moscow), Vol. 66, No. 10, 2001.
10. Angst BD, Marcozzi C, Magee AI.The cadherin superfamily: diversity in form andfunction. Journal of Cell Science, 2001, 114(Pt4): 629-641.
11. Philippova M, Ivanov D, Tkachuk V, Erne P, Resink TJ. Polarisation of T-cadherin to the leading edge of migrating vascular cells in vitro: a function in vascular cell motility? Histochemistry and Cell Biology, 2003, 120:353-360.
12. Sato M, Mori Y, Sakurada A, Fujimura S, Horii A. The H-cadherin (CDH13) gene is inactivated in human lung cancer. Hum Genet, 1998, 103:96–101.
13. Sakai M, Hibi K, Koshikawa K, Inoue S, Takeda S, Kaneko T, Nakao A. Frequent promoter methylation and gene silencing of CDH13 in pancreatic cancer. Cancer Sci, 2004, 95: 588–591.
14. Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC, Baylin SB, Sidransky D. 5’CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med, 1995, 1: 686–692.
15. Dammann R, Schagdarsurengin U, Liu L, Otto N, Gimm O, Dralle H, Boehm BO, Pfeifer GP, Hoang-Vu C. Frequent RASSF1A promoter hypermethylation and K-ras mutations in pancreatic carcinoma. Oncogene, 2003, 22:3806–3812.
16. Fukushima N, Sato N, Ueki T, Rosty C, Walter KM, Wilentz RE, Yeo CJ, Hruban RH, Goggins M. Aberrant methylation of preproenkephalin and p16 genes in pancreatic intraepithelial neoplasia and pancreatic ductal adenocarcinoma. Am J Pathol,2002, 160: 1573–1581.
17. Santini V, Kantarjian HM, Issa JP,Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann Intern Med. 2001 Apr 3; 134 (7):573-86.
18. Plumb JA,Strathdee G,Sludden J,et a1.Reversal of drug resistance in human tumor xenografts by 2’-deoxy-5-azacytidine induced demethylation of the hMLH1 gene promoter [J]. Cancer Res,2000,60(21):6039—6044(可删除,正文相应删除)
19. Wijermans PW, Krulder JW, Huijgens PC, NeveP. Continuous infusion of low-dose 5-Aza-2'-deoxycytidine in elderly patients with high-risk myelodysplastic syndrome. Leukemia 1997; 11: 1-5.
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