Experimental Study Of GANT61 On Apoptosis And Proliferation Of Hela Cells In Cervical Cancer

ObjectTo study the effect of Hedgehog(Hh)signaling pathway antagonist GANT61 on Hela cell apoptosis in vitro and in vivo,and to observe the effect of GANT61 on the proliferation of transplanted cervical cancer in nude mice in order to provide experimental basis for targeted therapy of cervical cancer.MethodPart I The effect of GANT61 on apoptosis of Hela cells and xenografts of cervical cancer in nude mice1.He La cells as materials,GANT61 was added to the experimental group and DMSO was added to the control group.2.Western-blot and RT-PCR were used to detect the expression of Gli1 protein and m RNA in Hela cells.3.TUNEL method was used to detect the apoptotic rate of He La cells in two groups.4.Cells of the two groups were inoculated subcutaneously in nude mice to observe the growth of subcutaneous transplanted tumors in the two groups.5.HE staining was used to observe the morphology of transplanted tumors in nude mice.6.Immunohistochemistry was used to detect the expression of Caspase-3 and Survivin in transplanted tumors of the two groups.HE staining to observe the pathological morphology of heart,liver,spleen,lung and kidney in nude micePart II:The effect of GANT61 on the proliferation of transplanted cervical cancer Hela cells in nude mice1.Nude mice were subcutaneously injected with Hela cells and divided into groups after tumorigenesis.The experimental group was injected with GANT61 and the control group was injected with solvent.2.After removing the tumors,weigh them and compare the weight of transplanted tumors between the two groups.3.HE staining was used to observe the visceral morphology of nude mice in the two groups.Western-blot and RT-PCR were used to detect the expression of Gli1 in the transplanted tumors of the two groups.4.The expressions of Fox M1,Cyclin D1 and Ki-67 in transplanted tumors were detected by immunohistochemistry.Result:Part I The effect of GANT61 on apoptosis of Hela cells and xenografts of cervical cancer in nude mice(1)In vitro experiment:The expression levels of Gli1 in He La cells of experimental group were(0.81+0.47)and(0.15+0.07),respectively,lower than those of control group(2.37+1.61)and(0.44+0.07),(P<0.05).The apoptotic rate of He La cells in the experimental group was(27.1+11.4)%,which was higher than that in the control group(6.9+3.1)%(P<0.05).(2)In vivo experiment:28 days after inoculation,the volume of transplanted tumors in nude mice of experimental group was(217+78)mm3,and that in nude mice of control group was(677+177)mm3(P<0.05).Under light microscope,the apoptosis of transplanted tumors in experimental group was more obvious than that in control group.The IOD values of Caspase-3 and Survivin protein in experimental group were29 335.26+1512.23 and 123.23+1078.56,respectively,while those in control group were 11 530.32+1012.13 and 1.56,respectively.The difference between the two groups was significant(P<0.05).Part II:The effect of GANT61 on proliferation of Hela cells and xenografts of cervical cancer in nude miceAfter 28 days of treatment,the volume and weight of transplanted tumors in nude mice in the experimental group were(222+9)mm~3and(0.33+0.07)g,respectively,lower than those in the control group(1223+284)mm3 and(2.06+0.32)g,with significant difference(P<0.05).3.The expression levels of Gli1 protein and m RNA were(0.087+0.052),(0.52+0.074),lower than those of the control group(0.34+0.12),(1.091+0.13),the difference was statistically significant(P<0.05).4.The values of Fox M1,Cyclin D1 and Ki-67 IOD in the transplanted tumors of the experimental group were 18600+900,13100+1100 and 16200+300,lower than those of the control group 25300+500,28800+700 and 34700+190.5.Under light microscopy,there was no significant difference in the morphology of heart,liver,spleen,lung and kidney between the two groups.Conclusion:GANT61 can inhibit Gli1 expression in He La cells in vitro and in vivo.2.GANT61can inhibit the growth of cervical cancer xenografts in nude mice.3.The mechanism of GANT61 inhibiting the growth of cervical cancer xenografts in nude mice may be related to promoting apoptosis and inhibiting proliferation,and the mechanism of promoting apoptosis may be related to up-regulation of Caspase-3 and down-regulation of Survivin,and the mechanism of inhibiting proliferation may be related to down-regulation of Fox M1,Cyclin D1 and Ki-67.