Study On The Efficacy And Mechanism Of Pantoprazole Enhances Osimertinib Activity By Inhibiting Autophagy In Human Lung Cancer Cells And Patient Derived Xenograft Models Of Lung Cancer

BackgroundAt present,lung cancer has the highest morbidity and mortality rate in the world.Only a small number of patients have the opportunity of surgery,most patients who can not be treated by surgery need to receive chemotherapy-based anti-cancer drugs.At present,the third representative of EGFR-TKI,osimertinib,has been included in the clinical first-line treatment.Although the use of osimertinib in the treatment of non-small cell lung cancer reverses the clinical resistance of the first generation of gefitinib and erlotinib,new acquired resistance has generally emerged one year after the use of osimertinib.Current studies have confirmed that autophagy can affect the effect of targeted drugs on cancer cells.It has been found that besides the resistance to EGFR-TKI caused by gene mutation,autophagy can also cause resistance to EGFR-TKI and affect its treatment.Pantoprazole is a commonly used proton pump inhibitor(PPI)in clinic.It has been proved that pantoprazole can inhibit autophagy.Therefore,we studied whether pantoprazole could affect the effect and mechanism of osimertinib in the treatment of lung cancer by inhibiting autophagy.Objective1.To detect the effects of pantoprazole combined with osimertinib on autophagy and growth inhibition of lung cancer cell lines in non-small cell lines,and to analyze the relationship between autophagy level and drug action.2.Establish the xenograft(PDX)models of human lung cancer,and screen the available and representative xenograft(PDX)models of human lung cancer for in vivo experiment.3.Human lung cancer xenograft(PDX)models was used to evaluate the feasibility,efficacy,safety and anti-tumor mechanism of pantoprazole and osimertinib in vivo.Methods.1.The effects of OSI ± pantoprazole were studied in four different wild-type and EGFR-mutant non-small-cell lung cancer(NSCLC)cell lines,along with two human xenograft models.The effects of OSI± pantoprazole on autophagy were evaluated by quantifying LC3-?,LC3-?,ATG-5and p62 protein expression by western blot,and by fluorescence microscopy of cells transfected with RFP-GFP-LC3.Drug effects were quantified by reactive oxygen species(ROS)and apoptosis in vivo,which was detected by TUNEL and caspase-3.CCK-8 and flow cytometry were used to detect the effects in vitro.Alterations in PI3K/Akt/mTOR pathway activation were evaluated by immunohistochemistry and western blot.2.Establish the the PDX models of human lung cancer,screen two PDX models,and test the usability of the models by routine pathology,immunohistochemistry and gene sequencing.Results1.Pantoprazole had mild inhibitory effects on A549,NCI-H1975,HCC827,and PC9 lung cancer cell lines,but there was no statistically significant compared with the control group.OSI had significant inhibitory effects on the four lung cancer cell lines,and there was no statistically significant compared with the control group(p<0.01),but the inhibitory effect of OSI in wild-type A549 cell lines was slightly lower than that of other cells.The combination of pantoprazole and OSI had significant inhibitory effects on four lung cancer lines,and there were significant differences compared with pantoprazole alone or OSI alone(p<0.01).2.Combination of pantoprazole and OSI produced the highest ROS and the highest apoptotic rate in A549,NCI-H197 lung cancer cell lines.After transfection of ATG5-SiRNA,the apoptotic rate of lung cancer cell line A549 and NCI-H197 treated with drugs was the highest,which was higher than that of untransfected group,and the ROS of combined drugs was the highest,which was higher than that of untransfected group.The apoptotic rate and the increasing trend of ROS induced by transfection of ATG5-SiRNA+oxetinib were similar to pantoprazole+OSI.3.Autophagy test results:The expression of LC3-? in A549 cells treated with OSI alone was higher than that in the control group,while the expression of P62 was lower than that in the control group;the expression of LC3-? in lung cancer cells treated with pantoprazole alone was lower than that in the control group,while the expression of P62 was higher than that in the control group;the expression of LC3-?in cells was lower than that in the control group after the combination of the two drugs.The expression of LC3-? in control group and OSI group was significantly higher than that in control group and oxitinib group(p<0.05).A549,NCI-H197 were treated with fluorescent labeling LC3.The red fluorescence of OSI group was higher than that of control group,while the yellow fluorescence was lower than that of control group.Pantoprazole alone or in combination with pantoprazole showed that the red fluorescence representing the binding of autophage and lysosome was less than that of the control group,while the yellow fluorescence representing the normal autophage was more than that of the control group,the difference was statistically significant(p<0.05).The expression of LC3-? in the cells transfected with ATG5-SiRNA was lower than that in the control group,while the expression of P62 was higher than that in the control group.The expression of LC3-? was slightly increased and P62 was slightly decreased in lung cancer cells transfected with OSI alone compared with the control group;the expression of LC3-? was slightly decreased in lung cancer cells transfected with pantoprazole alone,while the expression of P62 was slightly increased in lung cancer cells compared with the control group;the expression of LC3-? in cells could be observed in combination with the two drugs.The expression of LC3-? in OSI group was lower than that in control group and OSI group,while the expression of P62 was higher than that in control group and OSI group,but there was no statistical difference.In the two cells transfected with ATG5-SiRNA,the red fluorescence of OSI group was higher than that of control group,while the yellow fluorescence was lower than that of control group.Pantoprazole alone or combination of two drugs can be observed that red fluorescence representing autophagy and lysosome binding is less than that of the control group,while yellow fluorescence representing ordinary autophage is more than that of the control group,but there is no statistical difference.4.The effect of drug treatment on ATG5 before and after transfection of ATG5-SiRNA.In the non-transfection group,the expression of ATG-5 in lung cancer cells treated with OSI alone was higher than that in the control group,while the expression of ATG-5 in lung cancer cells treated with pantoprazole alone was lower than that in the control group,and the expression of ATG-5 in lung cancer cells treated with pantoprazole alone was lower than that in the control group.In the transfection group,the expression of ATG-5 in lung cancer cells treated with OSI alone was not significantly different from that in the untransfected group;the expression of ATG-5 in lung cancer cells treated with pantoprazole alone was lower than that in the control group;the expression of ATG-5 in cells treated with OSI and pantoprazole alone was not significantly different from that in the control group.5.We selected 2 PDXS to detect HE and IHC in primary and G3 generations,and found no significant difference in the expressions of Ki67,HER1,HER2 and ALK between GO and G3.We compared the gene mutations of 51 lung cancer-related genes in NEJM2016 with Panel.Among them,36 genes did not detect mutations in two models.The consistency rates of GO and G3 gene expression were PDX1 92.1%(47/51)and PDX2 92.1%(47/51)respectively.The EGFR-T790M mutation was detected by PDX 2.We compared the mutation frequencies of GO 7.3%,G3.7%,and no Statistical differences were found.6.In both PDX models,OSI combined with pantoprazole could significantly inhibit the growth of tumors,which was significantly different from that of OSI alone(p<0.05).Compared with pantoprazole alone,the volume of tumors in PBS group was also inhibited,but there was no statistical significance.There was significant difference between OSI alone and pantoprazole alone(p<0.05).In PDX 2 with EGFR mutation,the inhibitory effect of OSI combined with pantoprazole was more obvious.The average volume of tumors was significantly lower than that of OSI group and pantoprazole group(p<0.01).7.IHC was used to detect the expression of P62 to evaluate the level of autophagy in tumors of different treatment groups.It can be seen that the expression of P62 in OSI group was significantly lower than that in pantoprazole group(p<0.01),while the expression of P62 in pantoprazole group was high,but there was no statistical difference.P62 in combination group was higher than that in OSI group(p<0.01).There was statistical difference(p<0.05);P62 in the two drug combination group was lower than that in pantoprazole group(p<0.01).8.The expression of Ki67 and Caspase-3 in tumors of different treatment groups was detected by IHC,and the apoptotic cells in tumors were detected by TUNEL assay to evaluate the apoptotic level in tumors.The results showed that in two PDX models,the expression of Ki-67 in OSI combined pantoprazole group was significantly lower than that in OSI alone group and pantoprazole alone group(p<0.01).The expression of Caspase-3 in OSI combined pantoprazole group was significantly higher than that in OSI alone group and pantoprazole alone group(p<0.01).The expression of Caspase-3 in OSI combined pantoprazole group was significantly higher than that in OSI alone group and pantoprazole alone group(p<0.01).)The number of apoptotic cells in combination group was significantly higher than that in OSI group and pantoprazole group(p<0.01).In PDX1,there was no significant difference in Ki67 and Caspase-3 expression between OSI group and pantoprazole group.There was significant difference in apoptotic cell number in TUNEL test(p<0.05).The expression of Ki67 and Caspase-3 and the number of apoptotic cells in control group were affected by pantoprazole alone,but there was no significant difference.In PDX2,the expression of KI67,caspase-3 and apoptotic cells in TUNEL assay were significantly different between oxetini group and pantoprazole group(p<0.01).Pantoprazole alone had a certain effect on the number of Ki67,caspase-3 and apoptotic cells compared with the control group,but there was no statistical difference.9.We used IHC to detect the expression of PI3K p-AKT and p-mTOR in tumors of different treatment groups.The results showed that in Model 1,the expression of p-AKT and PI3K was affected by the combination of two drugs,which was significantly different from that of OSI alone(p<0.05).The expression of p-mTOR was affected by the combination of two drugs,but there was no significant difference compared with that of OSI alone.The expression of PI3K p-AKT and p-mTOR was inhibited by OSI,which was significantly different from that of pantoprazole alone(p<0.01).In Model 2,the expression of p-AKT,PI3K and p-mTOR was affected by the combination of the two drugs,which was significantly different from that of OSI alone(p<0.05)and pantoprazole alone(p<0.01);OSI inhibited the expression of PI3K p-AKT and p-mTOR,which was significantly different from pantoprazole alone(p<0.01).10.There was no significant difference in the weight of mice between the two PDXs.The weight of mice in the combined drug group decreased slightly but did not reach statistical significance.Several biochemical indexes of liver and kidney function showed that OSI combined with pantoprazole had no significant effect on the function of various organs in mice.Conclusions1.In all lung cancer cell lines PDXs of human lung cancer,OSI increased the level of autophagy.Pantoprazole inhibited the autophagy induced by OSI.2.Pantoprazole activated the PI3K/Akt/mTOR pathway inhibited by OSI in various lung cancer cell lines and human lung cancer xenotransplantation models.3.Pantoprazole enhanced the anti-tumor activity of OSI in lung cancer cell lines and human lung cancer xenotransplantation models,especially in lung cancer cell lines with EGFR gene mutation and human lung cancer PDX models.4.Pantoprazole enhances OSI-induced apoptosis by ROS in cell lines.5.Pantoprazole enhanced OSI-induced apoptosis in PDX models of human lung cancer,especially in the PDX model of human lung cancer with EGFR mutation.6.Successfully screened two suitable PDX models,which proved that the first and third generation models could maintain the characteristics of primary tumors at the level of histomorphology,immunohistochemical markers and gene molecules.7.The combination of OSI and pantoprazole is feasible and safe.