E-selectin在食管癌转移中的作用及cimetidine抑制血管内皮细胞E-selectin表达的实验研究
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摘要
目的 了解E-selectin及其配体sLe A/X在食管癌细胞和血管内皮细胞早期粘附中的作用。方法 用细胞粘附试验检测E-selectin及其配体sLe A和sLe X在食管癌细胞株EC9706与血管内皮细胞株ECV304早期粘附。结果 1、激活内皮细胞组、E-selectin单抗1:200、1:400、1:800组相对粘附率分别为5.2900±0.790、1.8818±0.8289、1.9436±0.3935、3.1730±0.4887,差别具有统计学意义(F值28.789,p值<0.001)。随着E-selectin单抗浓度增加,相对粘附率逐渐降低,E-selectin单抗阻断食管癌细胞和内皮细胞间粘附。2、sLe A、sLe X单抗阻断组相对粘附率分别为2.5436±0.6789、3.1286±0.8306,均较未处理(6.5170±0.9293)低,差别具显著统计学差异(p<0.01)。结论1、E-selectin介导食管癌细胞株EC9706和内皮细胞株ECV304早期粘附,从体外实验看E-selectin可能与食管癌转移有关。2、sLe A、sLe X介导食管癌细胞株EC9706和内皮细胞株ECV304粘附,提示sLe A、sLe X与食管癌转移相关。
Objective1 To study the Role of E-selectin and its ligands sLe A/X on the early adhesion of EC9706 and vascular endothelial cell line ECV304 activated by LPS2 To learn expression of E-selectin on vascular endothelial cell of liver of nude mice induced by EC9706;3 To study the mechanism of cimtidine inhibiting expression of E-selectin.Methods1 To learn the role of E-selectin and its ligands sLeA/X and cimetidine on EC9706 adhesion on ECV304 by cell adhesion;2 To study expression of E-selectin on vascular endothelial cell of liver of nude mice induced by EC9706 with RT-PCR and IHC;3 To study the expression of protein of E-selectin on ECV304 activated by LPS inhibited by cimetidine with cell ELISA and flow cytometry ;4 To study expression of E-selectin mRNA on vascular endothelial cell inhibited by cimetidine with RT-PCR;5 To investigate the influence of cimetidine on metastasis with nude mice model of liver metastasis of EC9706.
Results1 , ECV304 were stimulated with lug/ml LPS for 5 hours in a 96-well plate, EC9706 cells were added onto a semiconfluent monplayer culture of ECV304. The augment of EC9706 cell adhesion on ECV304 was augmented by 5.2900 + 0.790-fold by LPS 1 u g/ml. When ECV304 were pretreated by mAb against E-selectin half an hour before adding EC9706 cells, the extent of augment of EC9706 cells induction decreased to 1.8818 ± 0.8289-, 1.9436 + 0.3935- and 3.1730 + 0.4887-fold, at mAb against E-selectin concentrations of 1:200, 1:400 and 1:800, respectively. The reduction of augment of cell adhesion was statisticly significant (p< 0.001). 2> The augment of cell adhesion in group of stimulated ECV304, mAb against sLe A(l:100), mAb against sLe X (1:100) were 6.5170 + 0.9293-, 2.5436 + 0.6789- and 3.1286 + 0.8306-fold, statisticly significant difference from group with mAb and without mAb (p< 0.01).3> EC9706 5 X 106 cells were injected beneath the splenic capsule, 8 hours later, E-selectin mRNA of liver was detected by RT-PCR and protein of E-selectin on liver vascular endothelial cell were found by IHC in group of injected EC9706, but none with PBS.4, Adhesive EC9706 cells were increased when ECV304 were stimulated by LPS, but reduced by cimetidine. The augnent of adhesion cells of group of activated ECV304> cimetidine 10"4M> 10~6M> 10"8M were 7.384 + 1.565-fold, 2.397 + 0.459-fold, 3.779 + 0.968-fold. 4.767 + 0.723-fold. The difference was statisticly significant (pO.OOl).5. Effects of cimetidine on E-selectin expression on ECV304 stimulated by LPS, the E-selectin level on ECV304 was augmented by 6.42 + 1.637-fold
by LPS 1 u g/ml. When ECV304 were pretreated by cimetidine 2-hours before LPS stimulation, the extent of E-selectin induction decreased to 2.772±0.50-fold> 4.004+0.630-fold and 4.963 + 0.658-fold at cimetidine concentrations of 10~4M. 10~6M and 10~8M, respectively(p<0.001). 6> Quantitation of induced ECV304 cell surface E-selectin expression blockde by cimetidine also detected by flow cytometry. Fraction of ECV304 cells with high-level E-selectin expression stimulated by LPS was increased to (35.6 + 3.347)%. When ECV304 were pretreated by cimetidine 2-hours before LPS stimulation, the extent of E-selectin induction decreased to (14.65 + 4.11)% > (20.2 + 4.80)% > (27.8 + 2.19)% at cimetidine concentrations of 10~\ 10"6 and 10~8M, respectively(p<0.001). 7> Effects of cimetidine on E-selectin gene expression on ECV304 induced by LPS with half-quantitation RT-PCR. The proportionality of E-selectin mRNA/PAPDH was 0.7533 + 0.0744, when stimulated by LPS. When ECV304 were pretreated by cimetidine 2-hours before LPS stimulation , the proportion were 0.6844 + 0.0427, 0.7642 + 0.083 and 0.7153 + 0.075 at cimetidine concentrations of 10 M ^ 10 M and 10 M, respectively(p<0.001). No statistic significance (p value 0.249). 8> Effects of cimetidine on serum E-selectin level of nude mice induced by EC9706 5X106 cell were detected by ELISA. Nude mice were injected intraspenically with EC9706 5X106 cell. Blood was drawn at point of 2^ 4-n 8-^ 16- and 24- hours. The serum E-selectin level was 0.238 + 0.075ng/mk 9.59 + 0.789 ng/mk 16.65 + 0.659 ng/mk 15.85 + 0.85 ng/mk 4.99 + 0.238 ng/ml, respectively. Cimetidine 200mg/kg was intraperitoneally administrated once a day for 5 days before intrasplenically
injection of EC9706. The serum E-selectin levels of nude mice induced by EC9706 5 X 106 cell were decreased to 0.235 + 0.077 ng/mk 4.576 + 0.768 ng/mh 8.615+ 0.92 ng/mk 7.38 + 0.69 ng/mh 3.458 + 0.96 ng/ml at point of2-x 4-> 8-> 16-and 24-hours later, respectively. There was stastisticly significance at moment of 4-> 8- and 16-hours (p<0.001), but no stastistic significance at point of 2- and 24-hours(p>0.05).9> Suppression of liver metastasis in balb/c mice by cimetidine. To induced hepatic metastasis, EC9706 5X106cell were injected intrasplenically and spleenctomy was taken 1 hour later. Mice were treated with 200mg/kg cimetidine or saline (control) i.p for 10 consecutive days (5 day before and 5 day after EC9706 cell injection). Subsequently cimetidine of saline( control) was administered every other day for an additional 8 weeks and 4 days. Six mice had liver metastasis in saline group (6/8) and one in cimetidine group (1/8), There was stastistically significance (p value 0.041).Conclusions1 ■? E-selectin >sLeA and sLeX are important molecular in the early adhesionof human esophageal carcinoma cell line EC9706 and vascular endothelialcell line ECV304.2> Human esophageal carcinoma cell line EC9706 can induce balb/c miceliver vascular endothelial cell E-selectin expression when intrasplenecally.3> Cimetidine can inhibit human esophageal carcinoma cell line EC9706cell adhesion on endothelial cell line ECV304 by blocking E-selectinexpression.4> Cimetidine did not influence vascular endothelial cell E-selectin mRNAtranscription induced by LPS.
5^ Cimetidine can prevent liver metastasis of human esophageal carcinoma with blocking E-selectin expression.
Objective1 To study the Role of E-selectin and its ligands sLe A/X on the early adhesion of EC9706 and vascular endothelial cell line ECV304 activated by LPS2 To learn expression of E-selectin on vascular endothelial cell of liver of nude mice induced by EC9706;3 To study the mechanism of cimtidine inhibiting expression of E-selectin.Methods1 To learn the role of E-selectin and its ligands sLeA/X and cimetidine on EC9706 adhesion on ECV304 by cell adhesion;2 To study expression of E-selectin on vascular endothelial cell of liver of nude mice induced by EC9706 with RT-PCR and IHC;3 To study the expression of protein of E-selectin on ECV304 activated by LPS inhibited by cimetidine with cell ELISA and flow cytometry ;4 To study expression of E-selectin mRNA on vascular endothelial cell inhibited by cimetidine with RT-PCR;5 To investigate the influence of cimetidine on metastasis with nude mice model of liver metastasis of EC9706.
Results1 , ECV304 were stimulated with lug/ml LPS for 5 hours in a 96-well plate, EC9706 cells were added onto a semiconfluent monplayer culture of ECV304. The augment of EC9706 cell adhesion on ECV304 was augmented by 5.2900 + 0.790-fold by LPS 1 u g/ml. When ECV304 were pretreated by mAb against E-selectin half an hour before adding EC9706 cells, the extent of augment of EC9706 cells induction decreased to 1.8818 ± 0.8289-, 1.9436 + 0.3935- and 3.1730 + 0.4887-fold, at mAb against E-selectin concentrations of 1:200, 1:400 and 1:800, respectively. The reduction of augment of cell adhesion was statisticly significant (p< 0.001). 2> The augment of cell adhesion in group of stimulated ECV304, mAb against sLe A(l:100), mAb against sLe X (1:100) were 6.5170 + 0.9293-, 2.5436 + 0.6789- and 3.1286 + 0.8306-fold, statisticly significant difference from group with mAb and without mAb (p< 0.01).3> EC9706 5 X 106 cells were injected beneath the splenic capsule, 8 hours later, E-selectin mRNA of liver was detected by RT-PCR and protein of E-selectin on liver vascular endothelial cell were found by IHC in group of injected EC9706, but none with PBS.4, Adhesive EC9706 cells were increased when ECV304 were stimulated by LPS, but reduced by cimetidine. The augnent of adhesion cells of group of activated ECV304> cimetidine 10"4M> 10~6M> 10"8M were 7.384 + 1.565-fold, 2.397 + 0.459-fold, 3.779 + 0.968-fold. 4.767 + 0.723-fold. The difference was statisticly significant (pO.OOl).5. Effects of cimetidine on E-selectin expression on ECV304 stimulated by LPS, the E-selectin level on ECV304 was augmented by 6.42 + 1.637-fold
by LPS 1 u g/ml. When ECV304 were pretreated by cimetidine 2-hours before LPS stimulation, the extent of E-selectin induction decreased to 2.772±0.50-fold> 4.004+0.630-fold and 4.963 + 0.658-fold at cimetidine concentrations of 10~4M. 10~6M and 10~8M, respectively(p<0.001). 6> Quantitation of induced ECV304 cell surface E-selectin expression blockde by cimetidine also detected by flow cytometry. Fraction of ECV304 cells with high-level E-selectin expression stimulated by LPS was increased to (35.6 + 3.347)%. When ECV304 were pretreated by cimetidine 2-hours before LPS stimulation, the extent of E-selectin induction decreased to (14.65 + 4.11)% > (20.2 + 4.80)% > (27.8 + 2.19)% at cimetidine concentrations of 10~\ 10"6 and 10~8M, respectively(p<0.001). 7> Effects of cimetidine on E-selectin gene expression on ECV304 induced by LPS with half-quantitation RT-PCR. The proportionality of E-selectin mRNA/PAPDH was 0.7533 + 0.0744, when stimulated by LPS. When ECV304 were pretreated by cimetidine 2-hours before LPS stimulation , the proportion were 0.6844 + 0.0427, 0.7642 + 0.083 and 0.7153 + 0.075 at cimetidine concentrations of 10 M ^ 10 M and 10 M, respectively(p<0.001). No statistic significance (p value 0.249). 8> Effects of cimetidine on serum E-selectin level of nude mice induced by EC9706 5X106 cell were detected by ELISA. Nude mice were injected intraspenically with EC9706 5X106 cell. Blood was drawn at point of 2^ 4-n 8-^ 16- and 24- hours. The serum E-selectin level was 0.238 + 0.075ng/mk 9.59 + 0.789 ng/mk 16.65 + 0.659 ng/mk 15.85 + 0.85 ng/mk 4.99 + 0.238 ng/ml, respectively. Cimetidine 200mg/kg was intraperitoneally administrated once a day for 5 days before intrasplenically
injection of EC9706. The serum E-selectin levels of nude mice induced by EC9706 5 X 106 cell were decreased to 0.235 + 0.077 ng/mk 4.576 + 0.768 ng/mh 8.615+ 0.92 ng/mk 7.38 + 0.69 ng/mh 3.458 + 0.96 ng/ml at point of2-x 4-> 8-> 16-and 24-hours later, respectively. There was stastisticly significance at moment of 4-> 8- and 16-hours (p<0.001), but no stastistic significance at point of 2- and 24-hours(p>0.05).9> Suppression of liver metastasis in balb/c mice by cimetidine. To induced hepatic metastasis, EC9706 5X106cell were injected intrasplenically and spleenctomy was taken 1 hour later. Mice were treated with 200mg/kg cimetidine or saline (control) i.p for 10 consecutive days (5 day before and 5 day after EC9706 cell injection). Subsequently cimetidine of saline( control) was administered every other day for an additional 8 weeks and 4 days. Six mice had liver metastasis in saline group (6/8) and one in cimetidine group (1/8), There was stastistically significance (p value 0.041).Conclusions1 ■? E-selectin >sLeA and sLeX are important molecular in the early adhesionof human esophageal carcinoma cell line EC9706 and vascular endothelialcell line ECV304.2> Human esophageal carcinoma cell line EC9706 can induce balb/c miceliver vascular endothelial cell E-selectin expression when intrasplenecally.3> Cimetidine can inhibit human esophageal carcinoma cell line EC9706cell adhesion on endothelial cell line ECV304 by blocking E-selectinexpression.4> Cimetidine did not influence vascular endothelial cell E-selectin mRNAtranscription induced by LPS.
5^ Cimetidine can prevent liver metastasis of human esophageal carcinoma with blocking E-selectin expression.
引文
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7.陈秉燮,卢诗杰,李绍余等.食管癌切除术后随访至死亡100例分析.癌症,1992,11(5):394～395.
8.郑立新,石应康.选择素与器官移植免疫的研究进展.中华实验外科杂志,2000,17(6):592～593.
9. Krause T, Turner GA. Are selectins involved in metastasis? Clin Exp Metastasis, 1999, 17(3): 183
10. D'Amato M, Flugy AM, Alaimo G, et al. Role of calcium in E-selectin induced phenotype of T84 colon carcinoma cells. Biochem Biophys Res Commun, 2003, 301(4): 907～914.
11. Kawakami-Kimura N, Narita T, Ohmori K et al. Involement of hepatocyte growth factor in increased integrin expression on HepG2 cells triggered by adhesion to endothelial cells. Br J Cancer, 1997, 75(1): 47～53.
12. Taisuke Sawada, Masayuki Yoshida, Yukio Yasukouchi, et al. Colon Cancer Cell Adhesion to Endothelial E-selectin Inhibits Detachment of endothelial cells through activation of β_1-integrin. Biochem Biophys Res Commun, 2001, 286(1): 20～27.
13. Laferriere J, Houle F, Taher M, et al. Transendothelial migration of colon carcinoma cells requires expression of E-selectin by endothelial cells and activation of stress-activation protein kinase-2 (SAPK2/p38) in the tumor cells. J Biol Chem, 2001, 276(36): 33762～33772.
14. Tei K, Kawakami-Kimura N, Taguchi O, et al. Roles of cell adhesion molecules in tumor angiogenesis induced by cotransplantation of cancer and endothelial cells to nude rats. Cancer Res, 2002, 62(21): 6289～6296.
15.阎齐,赵雍凡.采用组织芯片技术检测人食管癌组织选择素P及其配体表达(硕士论文).2002,4月:26～29.
16. Khatib AM, Fallavollita L, Wancewicz EV, et al. Intibition of hepatic endothelial e-selectin expression by c-raf antisense oligonucleotides blocks colorectal carcinoma liver metastasis. Cancer Res, 2002, 62(19): 5393～5398.
17. Kobayashi K, Matsumoto S, Morishima T, et al. Cimetidine inhibits cancer cell ashesion to endothelial cell and prevents metastasis by blocking e-selectin expression. Cancer Res, 2000, 60(14): 78～3984
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19. Tozawa K, Sakurada S, Kohri K, et al. Effects of anti-nuclear factor kB reagents in blocking adhesion of human cancer cells to vascular endothelial cells. Cancer Res, 1995, 55(18): 4162～4167.
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22. Collins T, Williams A, Johnston G, et al. Structure and chromosomal location of the gene for endothelial-leukocyte adhesion molecule 1. J Bio Chem, 1991, 266(4): 2466-2473.
23. Johnston GI, Cook RG, McEver RP. Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. Cell, 1989, 56(6): 1033-1044.
24. Gallatin WM, Weissman IL, Butcher EC. A cell-surface molecule involved in organ-specific homing of lymphocytes. Nature, 1983, 304(5921): 30-34.
25. Tedder TF, Isaacs CM, Ernst TJ, et al. Isolation and chromosomal localization of cDNAs encoding a novel human lymphocyte cell surface molecule, LAM-1,homology with the mouse lymphocyte honing receptor and other human adhesion proteins. J Exp Med, 1989,170(l):123~133.
26. Geng JG, Heawvener GA, Mmcever RP. Lectin domain peptides from selectins interact with both cell surface ligands and Ca2+ ions. J Biol Chem, 1992,267(28): 19846-19853.
27. Jutila MA, Watts G, Walcheck B,et al. Characterization of a functionally important and evolutionarily well-conserved epitope mapped to the short consensus repeats of E-selectin and L-selectin JExp Med, 1992, 175(6): 1565-1573.
28. Crockett-Torabi E, Sullenbarger B, Smith CW, et al. Activation of human neutrophils through L-selectin and Mac-1 molecules. J Immunol, 1995,154(5): 2291-2302.
29. Springer TA. Adhesion receptors of the immune system. Nature, 1990 , 346(6283): 425-434.
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2. Morita M, Kuwano H, Ohno S, et al. Characteristics and sequence of the recurrent patterns after curative esophagectomy for squamous cell carcinoma. Surgery, 1994, 116(1): 1～7.
3. Sugimachi K, Inokuchi K, Kuwano H, et al. Patterns of recurrence after curative resection for carcinoma of the thoracic part of the esophagus. Surg Gynecol Obstet, 1983, 157(6): 537～540.
4. Osugi H, Takemura M, Higashino M, et al. Causes of death and pattern of recurrence after esophagectomy and extended lymphadenectomy for squamous cell carcinoma of the thoracic esophagus. Oncol Rep, 2003, 10(1): 81～87.
5. Chen J, Sang M, Chen Y. Recurrence pattern and prognosis of esophageal cancer following tumor resection. Zhonghua Zhong Liu Za Zhi, 1998, 20(4): 293～295.
6. Law SY, Fok M, Wong J. Pattern of recurrence after oesophageal resection for cancer: clinical implications. Br J Surg, 1996, 83(1): 107～111.
7.陈秉燮,卢诗杰,李绍余等.食管癌切除术后随访至死亡100例分析.癌症,1992,11(5):394～395.
8.郑立新,石应康.选择素与器官移植免疫的研究进展.中华实验外科杂志,2000,17(6):592～593.
9. Krause T, Turner GA. Are selectins involved in metastasis? Clin Exp Metastasis, 1999, 17(3): 183
10. D'Amato M, Flugy AM, Alaimo G, et al. Role of calcium in E-selectin induced phenotype of T84 colon carcinoma cells. Biochem Biophys Res Commun, 2003, 301(4): 907～914.
11. Kawakami-Kimura N, Narita T, Ohmori K et al. Involement of hepatocyte growth factor in increased integrin expression on HepG2 cells triggered by adhesion to endothelial cells. Br J Cancer, 1997, 75(1): 47～53.
12. Taisuke Sawada, Masayuki Yoshida, Yukio Yasukouchi, et al. Colon Cancer Cell Adhesion to Endothelial E-selectin Inhibits Detachment of endothelial cells through activation of β_1-integrin. Biochem Biophys Res Commun, 2001, 286(1): 20～27.
13. Laferriere J, Houle F, Taher M, et al. Transendothelial migration of colon carcinoma cells requires expression of E-selectin by endothelial cells and activation of stress-activation protein kinase-2 (SAPK2/p38) in the tumor cells. J Biol Chem, 2001, 276(36): 33762～33772.
14. Tei K, Kawakami-Kimura N, Taguchi O, et al. Roles of cell adhesion molecules in tumor angiogenesis induced by cotransplantation of cancer and endothelial cells to nude rats. Cancer Res, 2002, 62(21): 6289～6296.
15.阎齐,赵雍凡.采用组织芯片技术检测人食管癌组织选择素P及其配体表达(硕士论文).2002,4月:26～29.
16. Khatib AM, Fallavollita L, Wancewicz EV, et al. Intibition of hepatic endothelial e-selectin expression by c-raf antisense oligonucleotides blocks colorectal carcinoma liver metastasis. Cancer Res, 2002, 62(19): 5393～5398.
17. Kobayashi K, Matsumoto S, Morishima T, et al. Cimetidine inhibits cancer cell ashesion to endothelial cell and prevents metastasis by blocking e-selectin expression. Cancer Res, 2000, 60(14): 78～3984
18. Tang NH, Chen YL, Wang XQ, et al. Cooperative inhibitory effects of antisense oligonucleotide of cell adhesion molecules and cimetidine on cancer cell adhesion. World J Gastroenterol, 2004, 10(1): 62～66.
19. Tozawa K, Sakurada S, Kohri K, et al. Effects of anti-nuclear factor kB reagents in blocking adhesion of human cancer cells to vascular endothelial cells. Cancer Res, 1995, 55(18): 4162～4167.
20. Bevilacqua MP, Pober JS, Mendrick DL, et al. Identification of a inducible endothelial leukocyte adhesion molecules ELAM-1. Proc Natl Acad Sci USA, 1987, 84(24): 9238～9242.
21. Bevilacqua MP, Stengelin S, Gimbrone MA, et al. Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science, 1989, 243(4895): 1160～1165.
22. Collins T, Williams A, Johnston G, et al. Structure and chromosomal location of the gene for endothelial-leukocyte adhesion molecule 1. J Bio Chem, 1991, 266(4): 2466-2473.
23. Johnston GI, Cook RG, McEver RP. Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. Cell, 1989, 56(6): 1033-1044.
24. Gallatin WM, Weissman IL, Butcher EC. A cell-surface molecule involved in organ-specific homing of lymphocytes. Nature, 1983, 304(5921): 30-34.
25. Tedder TF, Isaacs CM, Ernst TJ, et al. Isolation and chromosomal localization of cDNAs encoding a novel human lymphocyte cell surface molecule, LAM-1,homology with the mouse lymphocyte honing receptor and other human adhesion proteins. J Exp Med, 1989,170(l):123~133.
26. Geng JG, Heawvener GA, Mmcever RP. Lectin domain peptides from selectins interact with both cell surface ligands and Ca2+ ions. J Biol Chem, 1992,267(28): 19846-19853.
27. Jutila MA, Watts G, Walcheck B,et al. Characterization of a functionally important and evolutionarily well-conserved epitope mapped to the short consensus repeats of E-selectin and L-selectin JExp Med, 1992, 175(6): 1565-1573.
28. Crockett-Torabi E, Sullenbarger B, Smith CW, et al. Activation of human neutrophils through L-selectin and Mac-1 molecules. J Immunol, 1995,154(5): 2291-2302.
29. Springer TA. Adhesion receptors of the immune system. Nature, 1990 , 346(6283): 425-434.
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