The Study Of Human Islet Amyloid Polypeptide Regulating Gemcitabine Resistance In Pancreatic Cancer

BACKGROUND:Pancreatic cancer is a malignant tumor of the digestive system,and difficult to diagnose and treat.The ductal adenocarcinoma which originating from the ductal gland epithelium accounts for about 90% among all the pancreatic tumor,and its morbidity and mortality have increased significantly in recent years.The5-year survival rate is <1%,which leads the pancreatic ductal adenocarcinoma(PDAC)to one of the worst malignant tumors.The early diagnosis rate of PDAC is not high,but the cure rate is very low.PDAC has a high degree of malignancy,a low surgical resection rate and a poor prognosis.Although surgery is still the primary treatment,PDAC is often found too late to lose the chance for R0 resection,so comprehensive treatment for PDAC is highly needed.There is still no comprehensive treatment for PDAC which is fully applicable to help it.The current comprehensive treatment is still based on surgical treatment,supplemented by radiotherapy and chemotherapy.Chemotherapy can be performed on pancreatic cancer that cannot be surgically removed,or performed to prevent postoperative recurrence of pancreatic cancer,the main manifestation of chemotherapy failure is the recurrence and metastasis of pancreatic cancer.Glycolysis has a close relationship with drug resistance in the pathogenesis of tumor formation,development,and recurrence.Gemcitabine has an irreplaceable role in the chemotherapy of pancreatic cancer,and in the process of gemcitabine resistance,the glycolysis effect shows a significant enhancement.In normal cells,glucose maintains an equilibrium state.In the absence of oxygen,glucose converts to pyruvate and turns it into lactic acid.When the oxygen content is normal,pyruvic acid enters the tricarboxylic acid(TCA)cycle.A common feature of tumor cells is that even in the case of normal oxygen levels,the amount of glucose uptake and lactic acid accumulation will gradually increase,using glycolysis as a source of major energy metabolism,we call this phenomenon as the Warburg effect.In recent years,we tried to treat tumors by inhibiting the activity of key enzymes in the tumor glycolytic pathway.Human islet amyloid polypeptide(hIAPP)is a hormone formed in islet β cells and secreted with insulin by a certain proportion,this reveals that hIAPP has a physiological role in regulating sugar metabolism.Pramlintide is a synthetic analog of amyloidoid peptide,which was approved by the FDA in March2005 for adjuvant therapy of type 1 and type 2 diabetes.A recent study published by nature showed that hIAPP or pramlintide enters cells via the CalR and RAMP3 channels and reduces the formation of G-6-P by inhibiting hexokinase,suggesting that hIAPP has a very close relationship with glycolysis.However,its relationship with gemcitabine resistance has not been reported.Therefore,this study means to improve the pancreatic cancer gemcitabine resistance by inhibiting glycolysis by using exogenous hIAPP,and providing a new research idea for the treatment of pancreatic cancer.METHODS: 1.Human pancreatic cancer cell line PANC-1 was cultured by gemcitabine to establish Gem-R with gemcitabine resistance.IC50 was determined by MTS assay,and cells were cultured by gemcitabine until the IC50 concentration increased to 10 times(ie 316 n M),Gem-R cells were stimulated periodically with gemcitabine(316 n M)to maintain their resistance.After the drug induction,the Gem-R cells were cultured in the culture solution containing no gemcitabine for 2weeks,and used for the experiment.Gem-R was stably transfected with lentivirus,and a novel cell line with puromycin resistance was established.After screening with puromycin 2μg/ml,the stable transgenic cell line hIAPP-Gem-R1 overexpressing hIAPP was screened and only with control.Group cell line CON-Gem-R;q RT-PCR method was used to detect the expression of hIAPP gene in hIAPP-Gem-R cells and CON-Gem-R cells;thiosulfate S stained hIAPP-Gem-R cells and CON After-Gem-R cells,the expression of hIAPP was detected by inverted fluorescence microscopy;after thiosulfurin S stained hIAPP-Gem-R cells and CON-Gem-R cells,the expression of hIAPP was detected by chemiluminescence Fluorescence intensity;the proliferation rate of hIAPP-Gem-R cells and CON-Gem-R cells in gemcitabine at 1μM was detected by MTS assay,and hIAPP-Gem-R cells and CON-Gem-R cells were detected by colony formation assay.The proliferation ability of gemcitabine at 1 μM;the apoptosis of hIAPP-Gem-R cells and CON-Gem-R cells in gemcitabine at 1 μM was detected by Annexin V-FITC/PI method.2.Gem-R gemcitabine-resistant cell line Gem-R was cultured using a concentration of puromycin-resistant cell line Gem-R,and Gem-R cells were detected in gemcitabine 1μM using pramlintide(10 mg/L)concentration.The difference in the effect of pramlintide(10 mg/L)versus pramlintide was used to determine whether pramlintide could improve chemosin chemotherapy resistance in pancreatic cancer.Cloning experiments were performed to detect the effect of pramlintide(10 mg/L)and no pramlintide on the proliferative capacity of Gem-R cells at 1 μM of gemcitabine.The condition of Gem-R cells in gemcitabine at 1 μM was detected by EdU assay.The effect of pramlintide(10 mg/L)and pramlintide on proliferation was observed.Using Annexin V-FITC/PI method,Gem-R cells were detected using pramlintide(10 mg/L)in gemcitabine at 1 μM.And the effect of not using pramlintide on apoptosis.3.The expression of CalR or RAMP3 protein in Gem-R gem-resistant cell line after transfection of CalR-siRNA plasmid or RAMP3-siRNA plasmid was detected by Western blot.Gem-R,a gemcitabine-resistant cell line,was cultured at a concentration of pramlintide(10 mg/L)to detect the difference in the effect of whether the CalR or RAMP3 protein was inhibited or not inhibited in Gem-R cells at1 μM of gemcitabine.To determine whether pramlintide is a mechanism for entry into cells via CalR or RAMP3 proteins to improve chemoresistance in pancreatic cancer gemcitabine.Using a colony formation assay to detect whether glu-R cells are inhibited or not inhibited by calci or RAMP3 protein in gemcitabine at 1 μM,whether pramlintide is a mechanism for entry of cells via CalR or RAMP3 protein to improve gemcitabine chemotherapy resistance in pancreatic cancer Using Annexin V-FITC/PI method to detect whether pramlintide is a mechanism for entering cells through CalR or RAMP3 protein when Gem-R cells are inhibited or not inhibited by calcibine at 1μM.Improve chemotherapy resistance to gemcitabine in pancreatic cancer;Results: 1.PANC-1 cells were cultured with gemcitabine 0.01 μM,and the concentration of gemcitabine was increased by 20 n M,until 10 times of PC50(Fig.1-1A,B)concentration,316 n M,no death of gemcitabine cultured cells.Gem-R-PANC-1 cell line with gemcitabine resistance,culture period of 6 months.Gem-R cells were stably transfected with lentivirus and screened for 12 days with puromycin 2μg/ml to establish a new cell line with puromycin resistance,CON-Gem-R and hIAPP-Gem-R cell lines,which were successfully screened after 14 days.set up.The fluorescence intensity of hIAPP-Gem-R cells was significantly enhanced compared with CON-Gem-R cells by thiosulfate S staining.The expression of hIAPP in CON-Gem-R cells and hIAPP-Gem-R cells was detected by q RT-PCR.The results showed that the expression of hIAPP in hIAPP-Gem-R cells was significantly higher than that of CON-Gem-R cells.Up.The P-gp protein was detected by Western Blot,and the P-gp protein of the drug-resistant strain was found to be significantly expressed,while the P-gp of wild-type PANC-1 cells was not expressed.The cell proliferation activity of each group was determined by MTS method.It was found that the cell proliferation activity of overexpressing hIAPP resistant cell line was significantly decreased,and overexpression of hIAPP could significantly improve drug resistance.Annexin V-FITC/PI double staining cell apoptosis assay showed that the apoptosis of overexpressing hIAPP resistant cell lines was significantly increased,and overexpression of hIAPP could improve drug resistance.The proliferation of cells in each group was determined by colony formation assay.It was found that the cell proliferation ability of overexpressing hIAPP resistant cell lines was significantly decreased,and overexpression of hIAPP could improve drug resistance.2.The cells of the resistant strains were cultured with gemcitabine(1 μM)plus pramlintide(10 mg/L)or gemcitabine alone(1 μM)for 14 days.The proliferation of each group was determined by colony formation method.The pramlintide(10 mg/L)was observed.The cell proliferation ability of the drug-resistant cell lines was significantly down-regulated,and pramlintide could improve the drug resistance.Cloning assays were performed to detect the inhibition of exogenous hIAPP(pramlintide)on the proliferation of pancreatic cancer resistant cell lines.The drug-resistant cells were cultured with gemcitabine(1 μM)plus pramlintide(10mg/L)or gemcitabine alone(1 μM),and the proliferation of each group was determined by EdU method.The drug-resistant cells of pramlintide(10 mg/L)group were observed.The cell proliferation ability of the strain was significantly down-regulated,and pramlintide could improve the drug resistance.Gemcitabine(1 μM)plus pramlintide(10 mg / L)or gemcitabine alone(1 μM)were used to culture the drug-resistant cells,and Annexin V-FITC/PI double-stained cells were used to determine the apoptosis of each group.The apoptosis of the drug-resistant cell line of the peptide(10mg/L)group was significantly increased,and the exogenous hIAPP(pramlintide)could improve the drug resistance.After using CalR-siRNA and RAMP3-siRNA in Gem-R cells,the expression of CalR and RAMP3 was detected by Western Blot,and the expression of both was inhibited to some extent.After using CalR-siRNA and RAMP3-siRNA for Gem-R cells,gemcitabine(1 μM)plus pramlintide(10 mg/L)was used to culture the drug-resistant cell group and siRNA transfection group,and Annexin V-FITC/PI double.The apoptosis rate of siRNA transfection group was significantly decreased by staining cell apoptosis.After using CalR-siRNA and RAMP3-siRNA for Gem-R cells,gemcitabine(1 μM)plus pramlintide(10 mg/L)was used to culture the drug-resistant cell group and siRNA transfected group,respectively.Proliferation showed that the proliferation ability of siRNA transfection group was higher than that of the control group.Conclusion: Gem-R,a gemcitabine resistance model,has been successfully established.The hIAPP-Gem-R cell line overexpressing hIAPP has been successfully established,and the proliferation ability of hIAPP-Gem-R cell model at 1 μM concentration of gemcitabine is significantly decreased;its apoptosis has been restored,and hIAPP has been verified to improve pancreatic cancer.Gemcitabine resistance has important significance and role.At the concentration of gemcitabine at 1 μM,the proliferative ability of gemcitabine-resistant cells cultured under pramlintide(10 mg/L)was significantly decreased compared with the non-planin-resistant cells,and the apoptosis was restored.Pramlintide plays an important role in improving gemcitabine resistance in pancreatic cancer;gemcitabine-resistant cells cultured at a concentration of 1 μM in gemcitabine and pramlintide(10 mg/L),if CalR or RAMP3 is inhibited,its proliferative capacity Recovery and decreased apoptosis have fully demonstrated that pramlintide,an exogenous hIAPP,enters pancreatic cancer cells through CalR or RAMP3 to improve the resistance of pancreatic cancer to gemcitabine.