| Objectives:FTY720, a novel immunosuppressive agent, is a synthetic compound produced by modification of a metabolite from Isaria sinclairil. FTY720has inhibitory effects on various cancer growth and metastasis. In this study we investigated the effect of FTY720on proliferation inhibition and apoptosis-inducing of human pancreatic cancer Miopac2cells and its mechanism. There result may be used to establish the experimental basis for its clinical application.Methods:1.Cultured human pancreatic cancer Miopac2cells were divided into the control group (the negative control group), FTY720-treated groups(1.25,2.5,5,10,20μg/ml) and ADM(2μg/ml) group(the positive control group).2. The growth curve of the human pancreatic cancer Miopac2cells was obstained by MTT assay.3. The inhibitory rate and50%inhibiting concentration (IC5o) values were evaluated by MTT assay after treatment with FTY720for12h,24h,48h and72h.4. After being treated with FTY720for24h, morphological changes were observed with microscope.5. After being treated with FTY720for24h, morphological changes of apoptosis were observed with fluorescance microscope after Rhodamine123(Rh123) staining.6. After being treated with FTY720for24h, DNA ladder was examined by DNA agarosegel electrophoresis in1,2,4μg/ml FTY720-treated groups.7. After being treated with FTY720for24h, the effect of FTY720on cell cycle and apoptotic rate were detected by flow cytometry.8. After being treated with FTY720for24h, the mitochondrial membrane potential were detected after Rh123staining in1,2,4,8μg/ml FTY720-treated groups..9. After being treated with FTY720for24h, the expression of intracellular bcl-2, were detected by Western Blot in2,4,8μg/ml FTY720-treated groups. 10. After being treated with FTY720for24h, the expression of intracellular caspase-9and caspase-3were detected by spectrophotometric method in2,4,8p.g/ml FTY720-treated groups.Results:1. Logarithmic growth phase of Miopac2cells appeared on the second to the sixth day after being cultured by MTT assay.2. FTY720displayed strong proliferation inhibitory effect against Miopac2cells in a dose-and-time-dependent manner. The inhibitory rate which was treated with1.25,2.5,5,10,20μg/ml FTY720and2μg/ml ADM for12h was (2.124±0.044)%,(10.796±0.030)%,(42.832±0.085)%,(69.735±0.021)%,(72.920±0.027)%and (58.938±0.051)%, respectively. After treatment for24h was (4.839±0.021)%,(15.249±0.029)%,(59.971±0.050)%,(80.938±0.009)%,(81.672±0.015)%and (75.513±0.041)%, respectively.After treatment for48h was (4.332±0.072)%,(14.851±0.087)%,(50.128±0.116)%,(87.005±0.006)%,(87.376±0.005)%and (86.633±0.019)%, respectively. After treatment for72h was (13.953±0.040)%,(22.272±0.065)%,(50.629±0.202)%,(90.878±0.012)%,(91.771±0.008)%and (90.877±0.005)%, respectively. The inhibitory rate was much higher in FTY720groups and ADM group than that of control group(P<0.001). Besides, after treatment with FTY720for12,24,48and72h, the IC50value was6.39,4.88,4.79and3.78μg/ml, respectively.3. There were obvious changes of morphology, biochemistry. We found morphologic changes by microscopy and DNA ladder on agarosegel electrophoresis, as seen in typical apoptotic cells. No changes were observed in control group. The result of flow cytometry demonstrated that FTY720induced Miopac2cells apoptosis with dose-dependent.4. The mitochondrial membrane potential of Miopac2was decreased. After being treated with T,2,4,8μg/ml FTY720for24h, the fluorescanc potent of Rhl23was2.218±0.112,2.009±0.055,1.827±0.153andl.405±0.022respectively. There was significant difference in FTY720groups compared with control group (3.716±0.051, P<0.01).5. The bcl-2protein expression was down-regulated, while the caspase-9and caspase-3were increased in FTY720groups. After being treated with2,4,8μg/ml FTY720for24h, the ratio of bcl-2to GAPDH was0.472±0.034,0.259±0.045and0.218±0.042respectively. There was significant difference in FTY720groups compared with control group (0.616±0.021, P<0.01). After being treated with2,4,8μg/ml FTY720for24h, the OD value of caspase-9was0.398±0.068,0.524±0.034and0.640±0.031respectively. There was significant difference in FTY720groups compared with control group (0.224±0.040, P<0.01). After being treated with2,4,8μg/ml FTY720for24h, the OD value of caspase-3wasl.029±0.026,1.532±0.034and2.108±0.031respectively. There was significant difference in FTY720groups compared with control group (0.809±0.015, P<0.01).Conclusion:1. FTY720has strong proliferation inhibitory effect in a dose-and-time-dependent manner against Miopac2cells.2. FTY720induced Miopac2cells arrest in G1phase. FTY720induce the apoptosis of Miopac2cells and the rate of apoptosisis dose-dependent.3. The effect of FTY720on inducing apoptosis of Miopac2cells may be related to the passage way of mitochondrosome, that bcl-2and caspase-9may be the regulating-target for FTY720. |
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