Effects Of Cucurmosin On Proliferation Inhibition And Apoptosis In Human Pancreatic Cancer Cell Line SW1990

Objective:To investigate the effect of cucurmosin(CUS) on proliferation inhibition and apoptosis in human pancreatic cancer cell line SW1990 in vitro.To establish NOD-SCID mice orthotopic transplantation model,and estimate the proliferation inhibition effect of CUS on human pancreatic cancer cell line SW1990 in vivo.To clarify the possible mechanism of proliferation inhibition and apoptosis,then provide some theoretical foundation and experiment basis for the possible anticancer use of CUS.Methods:1.MTT assay was used to detect the proliferation inhibition of CUS on the pancreatic cancer cell SW1990 in vitro.2.To establish NOD-SCID mice orthotopic transplantation model,and estimate the proliferation inhibition effect of CUS on human pancreatic cancer cell line SW1990 in vivo.3.Flow cytometric analysis was used to examine the pancreatic cancer SW1990 cell cycle change after exposure to CUS.4.Transmission electron microscopy was used to observe the ultrastructure change of SW1990 cell after CUS intervention.5.Annexin V-FITC Apoptosis Detection Kit was used to analyze the apoptosis rate after CUS treatment.6.Western blot was used to determine the protein level of Caspase-3,Bcl-2 and Bax after CUS treatment. Results:1.Exposure to CUS at 1.25,2.50,5.00,10.00,20.00,40.00 and 80.00 mg/Lfor 24,48 and 72h,the proliferation inhibition effect was elevated gradually with the in crease of concentration and the extension of time.At 24,48 and 72h after CUS treatment, the half maximal inhibitory concentration(IC50) was(49.12±2.35) mg/L,(19.33±1.13) mg/L,(6.47±0.59) mg/L,which implies that CUS inhibited SW1990 cells growth in a time and dose dependent manner.2.After establishing NOD-SCID mice orthotopic transplantation model successfully, we injected the mice with 0.50,1.00,2.00 mg/kg CUS for 5 weeks.The tumour proliferation inhibition rate were 33.58%,43.75%,58.36%,compared with the NS control group,which implies CUS can inhibit SW1990 cells growth in a dose dependent manner in vivo.3.Under the treatment of CUS at 0,2.50,10.00,40.00 mg/L for 72h, the proportion of SW1990 cell in G0/G1 phase increased gradually as ( 40.79±4.05 ),(45.39±3.63)%,(52.37±4.76)%,(66.46±5.02)%,and the proportion in S phase decreased gradually as(49.87±3.98)%,(47.06±3.12)%,(41.19±2.85)%,(22.76±1.91)%, which implies CUS might inhibit the SW1990 cell growth in the way of reducing the ratio of G0/G1 phase cells to S phase cells,and then delaying the cell-cycle progression from G0/G1 phase into S phase.4.Cultivating the SW1990 cells in the medium for 72h ,we observed that the nuclei became larger and hyperchromatic, the nuclei and the nuclear membrane ratio imbalanced,and double nucleoli emerged.The typical apoptosis changes were observed after CUS treatment for 72h,including that the membrane structure was still intact, the cytoplasm arranged loosely , the nucleus located mainly in the nuclear membrane,the nuclear membrane and nucleolus broke,the nucleoplasm concentrated,and the apoptosis body appeared.5.After exposure to CUS at 0,2.50,10.00,40.00mg/L for 72h, the apoptosis rate was increased gradually as (0.30±0.11)%,(18.93±1.06)%,(28.00±2.07)% and (49.93±3.25)%,which implies that CUS could effectively induce the pancreatic cancer cell SW1990 apoptosis in a dose dependent manner.6.Under the treatment of CUS at 0,2.50,10.00,40.00 mg/L for 72h,the expression of Caspase-3 and Bax protein was elevated gradully ,whereas the expression of Bcl-2 protein was lessened gradually in a dose dependent manner.Conclution:1.CUS can inhibit human pancreatic SW1990 cells growth in vitro and in vivo.2.CUS may inhibit the SW1990 cells growth in the way of reducing the ratio of G0/G1 phase cells to S phase cells,and then delaying the cell-cycle progression from G0/G1 phase into S phase.3.CUS may induce the apoptosis of SW1990 cells and finally inhibit the cells growth through up-regulating the expression of Caspase-3 and Bax,down-regulating the expression of Bcl-2,reducing the ratio of Bcl-2 expression to Bax expression,and then activating the expression of Caspase-3 in downstream levels.