| BackgroudAccording to the American Cancer Statistics survey,There is about 1,685,210 new cases are diagnosed as malignant tumors in 2016,which is equivalent to more than 4,600 new cases diagnosed each day.In many types of malignant tumor,pancreatic cancer mortality is the highest,which overall survival rate was 8%,and 5-year survival rate was only 2%,mainly due to more than one-half of cases are diagnosed at a distant metastasis.The the most effective treatment of pancreatic cancer is operation,but even after treated actively,the 5-year survival rate is only 25% ~ 35%.In recent years,it has been found that ALDHs(aldehyde dehydrogenases)is higher expressed in pancreatic cancer,and is closely related to the characteristics of pancreatic cancer stem cells.In addition,ALDHs can not only as a surface marker to identify and isolate pancreatic cancer stem cells,also have certain relations with drug resistance,cancer metastasis,recurrence and prognosis in pancreatic cancer.Metformin is currently the most widely used antidiabetic oral drug all over the world.It can reduce blood sugar and insulin concentrations by inhibiting liver gluconeogenesis and increasing insulin sensitivity in peripheral tissues.A retrospective study shows that metformin can reduce the cancer incidence and mortality in diabetic patients.In addition,it has been reported that metformin not only inhibits the proliferation of pancreatic cancer cells through the mTOR pathway,but also inhibits the expression of ALDH1 in pancreatic cancer animal models,but its mechanism is unclear yet.In this experiment,we will observe the effect of metformin and rapamycin on the expression of ALDH1 in pancreatic cancer cell line and pancreatic cancer stem cells by blocking the activation of mTOR signal using mTOR inhibitor--rapamycin,and explore the relationship between the expression of ALDH1 and mTOR in pancreatic cancer.ObjectiveTo investigate the effect of metformin on the expression of ALDH1 in pancreatic cancer PANC-1 cell line and pancreatic cancer stem cell,and explore its mechanism.MethodsPancreatic cancer cell line PANC-1 cells were used to detect the inhibition rate and the expression levels of ALDH1 and p-mTOR by CCK-8 and Western Blot after treated with metformin of different concentrations.Western Blot,Immunofluorescence and Quantitative real-time PCR(qRT-PCR)were used to detect the protein expression and mRNA level of ALDH1、4EBP1、p-mTOR among metformin group,rapamycin group and combination group with mammalian Target Of Rapamycin(mTOR)inhibitor--Rapamycin;Using ultra low adhesion culture plate to culture PANC-1 cell and observe the effect of spheroid body cells forming capacity with different drugs;Using Western Blot to detect the expression of ALDH1 protein level in pancreatic cancer stem cells.The tumorigenicity between pancreatic cancer cells and spheroid body cells were compared by establishing mouse transplantation model of pancreatic cancer.Results1.CCK-8 results showed that: Pancreatic cancer cell PANC-1 cells were treated with different concentrations of metformin,such as 1,2,5,10,15 and 20 mmol/L,the proliferation inhibition rate of 24 h was respectively:(5.23±2.03)% 、(10.23±4.19)%、(16.89±4.19)%、(20.60±5.24)%、(29.40±2.05)%、(35.38±1.70)%;The proliferation inhibition rate of 48 h was respectively:(8.30±3.48)% 、(14.20±3.76)%、(22.10±6.10)%、(31.20±2.03)%、(42.33±2.17)%、(49.58±1.84);The proliferation inhibition rate of 72 h was respectively:(19.02±2.67)% 、(22.13±6.11)%、(25.83±2.69)%、(36.65±6.30)%、(49.67±3.80)%、(53.70±3.13)%.Those results shows that,compared with the control group,metformin could significantly inhibit the proliferation of PANC-1,along with the increase of treatment time and metformin concentrations.The optimal respond time and concentration of metformin were 48 hours and 20 mmol/L.Statistical analysis showed that the difference was statistically significant in each experimental group compared with the control group(P<0.05).2.Western Blot results in pancreatic cancer showed that: Different concentrations of metformin(2,5,10,15,20 mmol/L)could inhibit the expressions of ALDH1 and p-mTOR in pancreatic cancer cells,and with the increasing of concentrations of metformin,the expression of ALDH1 and p-mTOR are gradually reduced,which is dose-dependent.The results in the mechanism of metformin is that: Metformin and rapamycin could inhibit the expression of 4EBP1 and p-mTOR significantly,but the effect of combination group is stronger.Compared with rapamycin group,the expression of ALDH1 in metformin group was significantly reduced,the difference is statistically significant(P<0.05);The expressions of 4EBP1 and p-mTOR in metformin group were not changed.3.The results of RT-PCR showed that: Compared with control group,the mRNA level of ALDH1,4EBP1 and p–mTOR in metformin group and rapamycin group were reduced;But the effcet of metformin is stronger than rapamycin on the mRNA transcription level ALDH1,the difference is statistically significant(P<0.05).4.The results of Immunofluorescence showed that: Compared with control group,metformin could inhibit the expression of ALDH1,and so does Rapamycin.5.The results of spheroid body experiments showed that: The number of spheroid body cells in control group was 85.00 ± 5.12,the number of spheroid body cells was 43.00 ± 3.66 in metformin group,the rapamycin group was 55.00 ± 3.21.To investigate the effect of metformin on the expression of ALDH1 in pancreatic spheroid body cells by Western Blot and found that: Compared with pancreatic cancer cells,the expression of ALDH1 in pancreatic cancer spheroid body cells was increased significantly.After the intervation in pancreatic cancer spheroid body cells,the expression of ALDH1 protein in metformin group,rapamycin group were significantly lower than control group,the difference was statistically significant(P<0.05).6.The comparison of tumorigenic ability showed that :Pancreatic cancer PANC-1 cells had lower tumorigenesis ability(7/10)than pancreatic cancer spheroid body cells(10/10).In addition,The tumor volume of pancreatic cancer spheroid body cells was significantly larger than that in pancreatic cancer PANC-1 cells,and the difference was significant(P<0.05).Conclusion1.Metformin could inhibit the proliferation of PANC–1 and the expression of ALDH1 significantly,along with the prolongation of intervention time and concentrations.2.Metformin and rapamycin could inhibit the expression of ALDH1 in pancreatic cancer PANC-1 cells,the mechanism may be via the mTOR pathway.3.Metformin and rapamycin could inhibit the formation of pancreatic cancer spheroid body cells and the expression of ALDH1 in pancreatic spheroid body cells,the mechanism may be through the mTOR pathway. |
