The Role Of E-selectin In Metastasis Of Esophageal Carcinoma And Expression Of E-selectin On Vascular Endothelial Cell Inhibited By Cimetidine

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-foldby 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 intrasplenicallyinjection 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.