| ObjectivesTo study the effect of somatosatin analogue octreotide at different concentrations on proliferation and apoptosis in colonic cancer cell line SW480. To explore the effect of octreotide on expression of GSK-3β,GSK-3β(Phospho-Ser9) and GSK-3β(Phospho-Tyr216) proteins in Wnt/β-Cateninsignal transduction pathway in colonic cancer cell line. to elucidate the anti-tumor mechanism of somatostatin and the targets somatostatin targeted and to elucidate furtherly the anti-tumor mechanism of somatostatin.Methods1. Cultivate human colon cancer SW480 cell in vitro to investigate.2. The inhibitory action of octreotide to the growth of olonic cancer cell line SW480.2.1 MTT assay was used to analyse the effect of octreotide at different concentrations on the proliferation of SW480 cell. The best inhibiting concentration was screened which had the maxlmum potency.2.2 Colony formation assay on Plate was used to test inhibition of clone formation in SW480 cells by octreotide.3. The effectiveness that octreotide induced apoptosis in SW480 cells.3.1 Fluorescence after AO/EB staining was used to observed the morphologic changes of apoptosis induced by octreotide(OCT) in SW480 cell line.3.2 DNA agarose gel electrophoresis was used to test apotosis induced by octreotide in SW480 cell line.4. The effectiveness that octreotide regulate the phosphorylation level in SW480 cell line.4.1 Western blot was used to analyze expression of GSK-3βproteinin in SW480 cells treated by octreotide at different concentrations. 4.2 Western blot was used to analyze expression of GSK-3β(Phospho-Tyr216) proteinin in SW480 cells treated by octreotide at different concentrations.4.3 Western blot was used to analyze expression of GSK-3β(Phospho-Ser9) proteinin in SW480 cells treated by octreotide at different concentrations.Results1.The result that octreotide inhibited the growth and proliferation in SW480 cells.1.1 The MTT assay showed that different concentrations of OCT can differently inhibited the proliferation of SW480 cells, the inhibition ratio of SW480 cells was 2.36%,15.12%,34.80%,35.16%(P<0.05)respectively after treatment with OCT at the concentrations of 10-12M,10-10 M -10-8M,10-6M(mol/L),and the inhibitory effect of OCT at 10(-8M was the most significant.1.2 Colony formation assay on Plate showed that OCT significantly inhibited the anchorage dependent growth of SW480 cells in a concentration-dependent manner, the inhibition ratio of SW480 cells was 19.67%,34.43%,37.70%(P<0.05)respectively after treatment with OCT at the concentrations of 10-10 M,-10-8M,10-6M(mol/L).2. The result that octreotide induce apoptosis in SW480 cells.2.1 Typical morphologic changes of apoptosis in SW480 cell could be observed after treatment with OCT by fluorescence microscope using AO/EB fluorescence staining, the apoptotic index of SW480 cells was 21.8%±6.4%,34.9%±9.6%,41.5%±10.8%(P<0.05) respectively after treatment with OCT at the concentrations of 10(-10 M,10-8M,10-6M(mol/L).2.2 DNA agarose gel electrophoresis shown that DNA ladder bands could appear after treatment with OCT(10-8 to 10-6M) for 48h.3. The result that octreotide regulate the phosphorylation level in SW480 cell line.3.1 Otreotide increased the amount of GSK-3βprotein expression in SW480 cell in a dose-dependent manner. Western-blot analysis indicated that exposure to OCT at10-10M,10-8M,10-6M for 24h,the protein level of GSK-3βwas up-regulated by 9.21±1.62%,24.93±3.49%,32.52±3.82%(P<0.05)respectively in comparision with the control group.3.2 Otreotide increased the amount of GSK-3β(Phospho-Tyr216) protein expression in SW480 cell in a dose-dependent manner. Western-blot analysis indicated that exposure to OCT at10-10M,10-8M,10-6M for 24h,the protein level of GSK-3β(Phospho-Tyr216) was up-regulated by 9.23±1.02%,21.36±3.34%,102.27±7.37%(P<0.05)respectively in comparision with the control group.3.3 Otreotide decreased the amount of GSK-3β(Phospho-Ser9) protein expression in SW480 cell in a dose-dependent manner. Western-blot analysis indicated that exposure to OCT at10-10M,10-8M,10-6M for 24h,the protein level of GSK-3β(Phospho-Ser9) was down-regulated by 35.12±4.72%,58.63±6.43%,67.84±7.87%(P<0.05) respectively in comparision with the control group.Conclusion1. OCT caused inhibition of colonic cancer cell line SW480 growth and proliferation,and the effect was dose dependent in a certain degree.2. OCT can significantly induce apoptosis of colonic cancer cell line SW480,and the effect was dose dependent.3. OCT can increased the amount of GSK-3βprotein and GSK-3β(Phospho-Tyr216) protein expression in SW480 cell, and decreased the amount of GSK-3β(Phospho-Ser9) protein expression in SW480 cell.indicated that OCT can encourag the activation of GSK-3β, accordingly inhibit the Wnt signal transduction pathway in SW480 cells4. The inhibition of growth and induction of apoptosis of SW480 cells by OCT are associated with the reinforcement of activation of GSK-3βin Wnt signal transduction pathway.
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