| Objective The aim of this study is to investigate the anti-tumor effect of pPeOp and its effect on cell viability,metastasis,apoptosis,cell cycle and JAK/STAT3 signal pathway,and to explore the relationship between the antitumor effect of pPeOp and JAK/STAT3 signal pathway,so as to provide theoretical and experimental basis for utilization of pPeOp.Methods(1)Experimental cell lines and animals: human colon cancer SW620 cells,human gastric cancer MC-4 cells,human gastric cancer SGC-7901 cells and human gastric mucosal normal cells MC-1 used as experimental cell lines.Sixty female nude mice(five-week-old)were used as experimental animals.(2)Experimental grouping and drug intervention:(1)The colon cancer SW620 cells in logarithmic growth phase were randomly divided into 6 groups: the blank control group,the negative control group and 4 pPeOp intervention groups with different concentrations.The blank control group was treated with cell culture medium;the negative control group was treated with 120 μg/m L PVP extraction buffer;the pPeOp intervention groups were treated with 30 μg/m L,60 μg/m L,90 μg/m L,and 120 μg /m L pPeOp,respectively.After drug intervention,each group was incubated at 37 ℃ for 24 h.(2)The gastric cancer MC-4,SGC-7901 cells and gastric mucosal normal cells MC-1 in the logarithmic growth phase were randomly divided into 6 groups: the blank control group,the pPeOp-treated group,the agonist group,the combination therapy group with agonist and pPeOp,the inhibitor group,combined treatment group with inhibitor and pPeOp.The blank control group was treated with culture medium;the pPeOp-treated group was treated with 60 μg/m L pPeOp;the agonist group was treated with 100 ng/m L IL-6;the combination therapy group treated with 100 ng/m L IL-6 and 60 μg/m L pPeOp;the inhibitor group was treated with 100 ng/m L of NSC74859(the optimal concentration of NSC74859 in SGC-7901 cells was 50 ng/m L);the combined treatment group treated with NSC74859 and pPeOp.After drug intervention,each group was incubated at 37 ℃ for 24 h.(3)In this study,lentiviral(LV)-STAT3-green fluorescent protein(GFP)was constructed and transfected into MC-4 gastric cancer cells.There were three types of MC-4 gastric cancer cells: LV-STAT3-GFP type cells,LV-GFP type cells,and wild-type MC-4 cells.Sixty nude mice were divided into three large groups which formed tumor with the different type of MC-4 cells.Each large group of nude mice was randomly divided into two groups(10 mice in each group),one of which were regarded as the control group and the other were treated with pPeOp(10 u L/g)through caudal vein.(3)Indicator detection(1)After treated with pPeOp for 24 h,the MTS method used to detect the effect of pPeOp on cell viability of SW620;the Ed U method used to detect the effect of pPeOp on cell proliferation of SW620;the Hoechest33258 was used to detect the effect of pPeOp on the nuclear morphology of SW620 cells;the flow cytometry was used to detect the effect of pPeOp on apoptosis of SW620 cells;the Western blotting was used to detect the effect of pPeOp on apoptosis-related protein expression.(2)After MC-4、SGC-7901 and MC-1 cells treated with pPeOp for 24 h,the MTS method used to detect the effect of IL-6 and NSC74859 on the cell viability regulayed by pPeOp;the wound healing method used to detect the effect of IL-6 and NSC74859 on metastasis regulated by pPeOp;the flow cytometry used to detect the effect of IL-6 and NSC74859 on apoptosis and cell cycle regulated by pPeOp;the immunofluorescence fluorescence used to detect the effect of IL-6 and NSC74859 on the STAT3 protein regulated by pPeOp.(3)After the tumor formation in nude mice for seven days,pPeOp(10 u L/g)was injected through the caudal vein every 5 days.After injection for 5 times,the nude mice were killed and dissected to detect the effect of pPeOp protein on tumor volume and tumor weight.The tumor inhibition rate was calculated by VD25/VD1.Tumors from different groups of nude mice were taken for protein extraction.The protein expression of gp130,JAK1,STAT3,p-STAT3,Ee F2 and SOCS1 in the JAK/STAT3 signal pathway were detected by Western blotting.(4)Expression microarray assay: After treated with 90 μg/mL pPe Op,the total RNA of MC-4 cells was extracted,and was reversed transcribed to c RNA according to manufacturer instruction.The quantitative of c RNA was hybridized to Human Experiment 12x135K microarrays in hybridization oven.The significant differential gene expression was screened with a > 4-fold differential expression and a P<0.05 cut-off in statistical tests.The differential expression signal pathways were enriched by KEGG.Result(1)Compared with the control group,pPeOp at different concentrations could significantly inhibit the cell viability,arrest the cell proliferation and induce apoptosis in human colon cancer cells SW620,whereas after treatment with PVP in the negative control group didn’t cause any significant effect on cell viability,proliferation and apoptosis in SW620 cells.(2)The KEGG signaling pathway enrichment and GO annotation used to assay differentially expressed genes screened,the results suggested that numerous signal pathway were regulated by pPeOp,of which the JAK/STAT signal pathway was the most significant regulated pathway.(3)MTS viability method and real-time fluorescence quantitative PCR method were used to screen the most suitable concentration of agonists and inhibitors in JAK/STAT3 signal pathway.The most suitable concentration of SGC-7901 was 100 ng/m L IL-6 and 100 ng/m L NSC74859.The most suitable concentration of MC-4 was 100 ng/m L IL-6 and 50 ng/m L NSC74859.The most suitable concentration of MC-1 was 100 ng/m L IL-6 and 100 ng/m L NSC74859.(4)The MTS method,the wound-healing,the flow cytometry and immunofluorescence method were used to detect the effects of IL-6 and NSC74859 on cell viability,metastasis,apoptosis,cell cycle and STAT3 protein expression,respectively,regulated by pPeOp.The results suggested that pPeOp significantly inhibit cell viability and metastasis,induce apoptosis,arrest the cell cycle in gastric cancer cells,whereas had no significant effect in normal gastric mucosal MC-1 cells.In addition,NSC74859 significantly reduce the inhibitory effect of pPeOp,while IL-6 significantly enhances the anti-cancer effect pPeOp.(5)Empty vector MC-4 cells and STAT3 knockdown MC-4 cells were successfully transfected by lentivirus.The STAT3 expression was detected by Western blotting and real-time fluorescence quantitative PCR.Compared with empty vector MC-4 cells,the knockdown rate was 70.6% in STAT3 knockdown MC-4 cells.(6)In vivo experiments showed that pPeOp significantly inhibited the growth of tumor in nude mice,and the tumor inhibition rates of the blank control group,the negative control group,and the knockdown group were 26.9%,27.8% and 36.4%,respectively.(7)The protein expression of key nodes in JAK/STAT3 signal pathway in nude mice was detected by Western blotting.The results showed that pPeOp significant down-regulated the expression of gp130,JAK1,Ee F2,STAT3,and p-STAT3,whereas significant up-regulated SOCS1 protein expression.Conclusion pPeOp significantly inhibited cell viability,triggered apoptosis,inhibited metastasis and arrest cell cycle in human gastrointestinal cancer cells,whereas did not cause toxicity to normal gastric cells.The animal experiments suggest that pPeOp also showed the significantly anti-tumor effect in nude mice.The JAK/STAT3 signal pathway was significantly regulated by pPeOp.According to the results of in virto and in vivo,pPeOp significantly down-regulated the protein levels of gp130、JAK1、Ee F2、STAT3、p-STAT3,while SOCS1,the negative feedback adjustment factor of JAK/STAT3,was significantly up-regulated.The anti-gastrointestinal cancer effect of pPeOp protein may be related to its regulation of JAK / STAT3 signal pathway. |
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