The Chemotaxis For Colon Cancer Cell HCT-116by Platelet Proteinase-activated Receptor-1activation

Objective:To observe the effect of chemotaxis for colon cancer cell HCT-116afterplatelet proteinase-activated receptor1（PAR1）activation.Methods:1HCT-116was treated with a concentration gradient of PAR1agonistTFLLR-NH2(1μM,3μM,5μM,7μM,9μM,11μM,13μM,15μM,17μM,19μM), the blank control group only added culture medium.At24h, MTTwas used to detect the effect of proliferation.2The platelets were activated by a concentration gradient of TFLLR-NH2(0.5μM,1μM,3μM,10μM,30μM),and then FCM was used to detect theCD62P expression of platelet. The control group hadn’t added TFLLR-NH2.3The platelets were activated by a concentration gradient of TFLLR-NH2（1μM、3μM、5μM、7μM、9μM）.Transwell cell migration assays wereperformed to observe the effect of chemotaxis in HCT-116induced by plateletsupernate. At the same time,we setted up the blank group and non-activatedplatelet group. The blank group only added culture medium supplementedwith10%FBS.47μM TFLLR-NH2was used to activate platelet,and then plateletsupernate was extracted to treat HCT-116.At24h,FCM was performed todetect the CXCR4expression of HCT-116.Results:1The result of MTT assay showed that there were no statisticallysignificant difference between the OD values of1μM-17μM TFLLR-NH2groups and the blank control group（P＞0.05）.However, The OD valueofTFLLR-NH2group increased when compared with the control group(0.682 ±0.014vs0.647±0.291,P＜0.05).So the TFLLR-NH2concentration shouldbe lower than19μM in the following experiment.2The CD62P expressions of platelets activated by differentconcentration of TFLLR-NH2(0.5μM,1μM,3μM,10μM,30μM)were(33.82±17.96)%,(39.6±19.53)%,(60.99±12.42)%,(55.78±10.49)%,(57.58±11.77)%, respectively.It was increased when compared with the controlgroup (14.97±6.28)%. There were statistically significant difference(P<0.01).3The result of Transwell cell migration assays showed that the numberof migrated HCT-116cells induced by platelet supernate extracted from theplatelet activated by different concentration of TFLLR-NH2(1μM,3μM,5μM,7μM,9μM)were18.15±1.62,19.60±3.05,19.50±2.35,17.65±0.96,17.85±2.79,respectively,while the number of blank control groupwas13.50±1.91.The number of each platelet supernate group increased whencompared with the blank control group（P＜0.05）.But it hadn’t effect in theplatelet supernate group without adding TFLLR-NH2（16.65±1.19vs13.50±1.91，P＞0.05）.At the same time,1-9μM TFLLR-NH2agonist groups weresimilar to the blank control group.47μM TFLLR-NH2was used to activate platelets,and thenplateletsupernate was extracted.HCT-116cells were treated with the plateletsupernate.At24h，the CXCR4expression of HCT-116cells increased whencompared with the blank control（[40.89±6.74vs3.47±1.40）%，P＜0.01）].Conclusions:1The activation of platelet PAR1had a chemotactic effect on HCT-116cell.2The activation of platelet PAR1could upregulate CXCR4expressionof HCT-116cell.