| Objectives: 1.To study the effects of ?-Sitosterol and combined with gemcitabine on the proliferation,apoptosis,cell cycle,metastasis,invasion and epithelial-mesenchymal transition of the MIA-Pa Ca-2 and BXPC-3 human pancreatic cancer cells,and to explore its mechanism of action.2.To observe the effects of BS combined with GEM on proliferation,apoptosis and epithelial-mesenchymal transformation of subcutaneous tumors in nude mice,and to explore its mechanism of action.Methods: 1.The effects of BS on the proliferation,apoptosis and epithelial-mesenchymal transition of human pancreatic cancer cells: the human pancreatic cancer cells were treated with different concentrations of BS for 24,48 and72 hours,the growth inhibition of human pancreatic cancer cells was detected by MTT,Flow cytometry was used to analyze the apoptosis and cell cycle,Hoechst33258 staining was used to observe the apoptosis,Transwell chamber was used to detect the effect on cell migration and invasion.Western blot was used to detected the expression of Apoptotic signal proteins NF-?B,p-NF-?B,Bax and Bcl-2 after treat of BS and BAY(NF-?B receptor inhibitor).The expression of epithelial-mesenchymal transition signal proteins AKT,p-AKT,GSK-3?,p-GSK-3?,Snail,Vimentin and E-cadherin were detected after treated with of BS and PER(AKT receptor inhibitor)and Li CL(GSK-3? receptor inhibitor).2.The effects of combined BS with GEM on the proliferation,apoptosis and epithelial-mesenchymal transition of human pancreatic cancer cells: the human pancreatic cancer cells were treated with BS combined with GEM,The cell growth inhibition was detected by MTT,The best way to combine the two drugs was determined by Calcusyn 2.1 software.Flow cytometry was used to analyze the effect of the cell apoptosis and the cell cycle.Hoechst 33258 staining was used to observe the apoptosis.Transwell chamber was used to detect the effect on cell migration and invasion.Western blot was used to detected the expression of Apoptotic signal proteins NF-?B,p-NF-?B,Bax,Bcl-2 and epithelial-mesenchymal transition signalproteins AKT,p-AKT,GSK-3?,p-GSK-3?,Snail,Vimentin and E-cadherin by the combination of BS and GEM.3.Effects of BS combined with GEM on proliferation,apoptosis and EMT of subcutaneous tumors in nude mice: the subcutaneous transplantation model of human pancreatic cancer cell line BXPC-3 was established in nude mice.The experimental group consisted of control group,BS group,GEM group and BS+GEM group.The body weight and tumor volume of nude mice were measured every two days.The mice were killed after four weeks administration.The organ index was calculated and the tumour weight was measured.The expressions of Ki-67,p-NF-?B,Bax,Bcl-2,p-AKT,p-GSK-3?,Snail,Vimentin and E-cadherin were detected by immunohistochemistry.Apoptosis was observed by Tunel staining.Results: 1.The MTT assay showed that BS inhibited the proliferation of human pancreatic cancer cells with the increased concentration and time.Flow cytometry showed that the Apoptotic cells and the percentage of cells in G0/G1 Phase were increased significantly.Hoechst 33258 staining showed that the apoptosis cells were increased.Transwell migration and invasion experiments showed that the number of transmembrane cells were decreased significantly.Compared with the control group,the Western blot results of treatment of BS and BAY showed that there was no significant change in the expression of NF-?B protein,the p-NF-?B and Bcl-2 protein were decreased,the Bax protein was increased.After the treatment of BS and PER,the protein expression of AKT,GSK-3? protein did not change significantly,the protein expression of p-AKT,p-GSK-3?,Snail,Vimentin protein decreased,while the expression of E-cadherin protein increased.After the treatment of Li CL,the expression of GSK-3? protein did not change significantly.The expression of p-GSK-3?,Snail and Vimentin protein increased,while the expression of E-cadherin protein decreased.2.Compared with control group,BS,GEM and BS+GEM could significantly inhibit the proliferation of human pancreatic cancer cells.Calcusyn 2.1 software showed that 250 ?mol/L BS combined with 50 ?mol/L GEM have the strongest inhibitory effect.Flow cytometry showed that BS+GEM could increase the cells of apoptosis and G0/G1 Phase.Hoechst 33258 staining showed that BS+GEM could increase Apoptotic cells.Transwell migration and invasion experiments showed that BS+GEM could significantly reduce the number of transmembrane cells.Compared with control group,BS and GEM,the Western blot results showed that the expressionof NF-?B and AKT protein did not change significantly after the treatment of BS+GEM.The expression of p-NF-?B,Bcl-2,p-AKT,p-GSK-3?,Snail and Vimentin protein decreased,while the expression of Bax and E-cadherin protein increased.3.The results showed that there were no significant changes in body weight and organ index of nude mice in control group,BS group,GEM group and BS + GEM group.Compared with control group,BS and GEM,BS + GEM group had the smallest tumor volume and weight.Compared with control group,BS and GEM,the results of BS+GEM group showed that the expression of protein Ki67,p-NF-?B,Bcl-2,p-AKT,p-GSK-3?,Snail and Vimentin was the lowest,while the expression of protein Bax and E-cadherin was the highest.Tunel staining showed that the BS+GEM group have the most number cells of the Apoptotic cells.Conclusions: 1.BS can inhibit the cell proliferation,induce the cell apoptosis,inhibit the cell migration and invasion of human pancreatic cancer,and inhibit the epithelial-mesenchymal transition through AKT/GSK-3? signaling Pathway.2.The combination of BS with GEM is superior to single drug,Which showed in inhibiting proliferation,inducing apoptosis,inhibiting migration and invasion of human pancreatic cancer cells,and inhibiting epithelial-mesenchymal transition through AKT/GSK-3? signaling Pathway.3.The inhibitory effect of BS combined with GEM on subcutaneous transplantation of human pancreatic cancer cells in nude mice was better than the single drug. |
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