Effect Of ATR Inhibitor On Proliferation Of Pancreatic Cancer Cells And Its Mechanism

BackgroundCancer is the main cause of death in all countries,and it is also a major challenge to prolong life expectancy.According to statistics,pancreatic cancer ranks eighth in the incidence rate of all cancers,but among all tumors,pancreatic cancer ranks third in the cause of cancer death.In the past 50 years,with the development and progress of medical treatment,the 5-year survival rate of most cancer species has reached more than 50%,but the 5-year survival rate of pancreatic cancer is only 11%,which is the lowest of all tumors and a major challenge for people to overcome diseases.The incidence of pancreatic cancer is affected by genetic variation(gene mutation)and environment(such as diabetes,smoking,obesity and heavy drinking).The pathological classification of pancreatic cancer is 90%ductal adenocarcinoma.The rare types are mucinous cystadenocarcinoma,acinar cell carcinoma and squamous adenocarcinoma.At present,the treatment of pancreatic cancer is mainly surgery,radiotherapy and chemotherapy.Surgical treatment is relatively effective for medium-term pancreatic cancer,but the overall recurrence rate after surgery is higher than other cancers.Most pancreatic cancer is insidious with no prominent early symptoms,and there is no sensitive,accurate and efficient early diagnosis method.Most patients were in the advanced stage of pancreatic cancer when they were found,and lost the opportunity of surgery.Moreover,the clinical effects of radiotherapy and chemotherapy were poor,and the prognosis of patients was poor.Therefore,it is of great significance for patients with pancreatic cancer to explore a new and more effective treatment scheme for pancreatic cancer.The clinical treatment plan for pancreatic cancer recommended according to NCCN guidelines.Gemcitabine alone;Gemcitabine-based combination drugs,such as gemcitabine combined with albumin-binding paclitaxel,teggio,erlotinib,fluorouracil and cisplatin;Capecitabine combined with 5-fluorouracil;GTX scheme(gemcitabine,docetaxel,capecitabine);FOLFIRINOX scheme(oxaliplatin,irinotecan,calcium folinate,5-fluorouracil),of which FOLFIRINOX4 drug combination scheme has a good effect.The side effects of multi drug combination are large,and many patients with pancreatic cancer cannot tolerate it.Although the side effects of the single drug regimen are much lower,the efficacy is difficult to guarantee.NCCN guidelines recommend clinical trials for the vast majority of pancreatic cancer patients.It is conceivable that the current scheme is difficult to achieve satisfactory results.At present,ATR kinase inhibitors targeting DNA damage response(DDR)repair pathway have shown good effects in most cancer species.Charlie J et al.screened the first efficient and highly selective ATR inhibitor VE-821 through high-throughput screening,and found that VE-821 can significantly enhance the sensitivity of human colon cancer cell HCT116 to gemcitabine,and this effect will not affect normal cells.Then Prevo R et al found that VE-821 was combined with radiotherapy and gemcitabine to act on pancreatic cancer cells,and found that VE-821 significantly enhanced the sensitivity of pancreatic cancer cells to radiation and gemcitabine.VE-822,a highly selective derivative of VE-821,has shown that VE-822 combined with cisplatin or PI3K inhibitors has a strong anti-tumor effect on esophageal cancer.Moreover,VE-822 combined with gemcitabine in the treatment of ovarian cancer patients,VE-822 combined with topotecan or gemcitabine in the treatment of non-small cell lung cancer patients and other clinical trials have achieved good results.Another selective inhibitor AZD-6738 also has good efficacy,such as AZD-6738 combined with camptothecin analogues,topoisomerase I inhibitors to inhibit the proliferation of ovarian cancer cells,AZD-6738 combined with gemcitabine to inhibit the proliferation of pancreatic cancer cells;The clinical trials of AZD-6738 combined with PARP inhibitor Olapali in the treatment of ovarian cancer patients and AZD-6738 combined with Duvalizumab in the treatment of gastric cancer patients have sustained and good results.The mechanism of ATR inhibitor against pancreatic cancer cells remains to be further studied.In this topic,we aim to explore the effect and mechanism of ATR inhibitor on the growth and proliferation of pancreatic cancer cells,and provide new ideas for the treatment of pancreatic cancer patients.Methods1.Detect the sensitivity of ATR inhibitor AZD-6738 and VE-822 to pancreatic cancer cell lines 6606PDA,MIA Paca-2 and SUIT2 through in vitro experiments(1)The drug sensitivity of pancreatic cancer cell lines 6606PDA,MIA Paca-2 and SUIT2 to ATR inhibitors AZD-6738 and VE-822 was detected by CCK-8 experiment,and IC50 values were calculated by GraphPad Prism8.0.(2)The drug sensitivity of pancreatic cancer cell lines 6606PDA,MIA Paca-2 and SUIT2 to ATR inhibitor was detected by clone formation test,live dead cell staining and trypan blue staining.2.Detect the sensitivity of ATR inhibitor VE-822 combined with FOLFIRINOX protocol to pancreatic cancer cell lines 6606PDA,MIA Paca-2 and SUIT2 through in vitro experiments(1)The drug sensitivity of pancreatic cancer cell lines 6606PDA,MIA Paca-2 and SUIT2 to FOLFIRINOX protocol and ATR inhibitor VE-822 combined with FOLFIRINOX protocol were tested by CCK-8 experiment,and the effect of the combination of the two drugs was judged by Kim Jong Jun formula.(2)The drug sensitivity of pancreatic cancer cell lines 6606PDA,MIA Paca-2 and SUIT2 to ATR inhibitor VE-822 combined with FOLFIRINOX protocol was detected by clone formation test,live dead cell staining and trypan blue staining.3.To explore the mechanism of ATR inhibitor enhancing cell death and the mechanism of ATR inhibitor enhancing drug sensitivity of pancreatic cancer cells through molecular cell biology experiments(1)Western blot assay was used to detect the effects of autophagy and pyroptosis in death related pathways in 6606PDA cells of pancreatic cancer cells.(2)According to the second generation sequencing,we screened out the changes of ABC transporter related protein expression in pancreatic cancer cell 6606PDA after the influence of VE-822.Using Western blot,we detected the influence of the expression of CFTR in ABC transporter related pathway in pancreatic cancer cell 6606PDA.Results1.ATR inhibitor inhibits the proliferation of pancreatic cancer cells and promotes the death of pancreatic cancer cells.In the experiment of ATR inhibitor on the proliferation and death of pancreatic cancer cell lines 6606-PDA,MIA Paca-2 and SUIT2,we found that ATR inhibitor has a strong inhibitory effect on pancreatic cancer cells.ATR inhibitor reduces cell proliferation and promotes cell death in a dose-dependent manner.In pancreatic cancer cells,the ATR inhibitor VE-822 has a stronger effect than AZD-6738.2.ATR inhibitor VE-822 combined with FOLFIRINOX regimen inhibited the proliferation of pancreatic cancer cells.In the ATR inhibitor VE-822 combined with FOLFIRINOX scheme,we found that the combination of VE-822 combined with FOLFIRINOX scheme has a stronger effect on inhibiting cell enhancement and promoting cell death under the same single drug dosage compared with the cells treated with the control group or with VE-822 alone or FOLFIRINOX alone,and through the Kim Jong Un formula(Q=E(a+b)/(Ea+Eb Ea × Eb))The calculated Q value of the combined medication is greater than 1.15,and VE-822 combined with FOLFIRINOX has synergistic effect.3.ATR inhibitors promote cell death by enhancing autophagy.In order to understand how ATR inhibitor VE-822 can promote cell death,we tried to find out the related proteins such as pyrolysis,iron death and autophagy in cell death.Finally,we found that ATR inhibitor significantly enhanced the expression of autophagy related proteins P62 and LC3B,thus confirming that VE-822 mainly enhances the autophagy of cells and thus promotes cell death.4.ATR inhibitor VE-822 inhibits the proliferation of pancreatic cancer cells by inhibiting the expression of ABC transporter and sensitizing FOLFIRINOX.According to the results of previous sequencing,we tried ABC transporter-associated protein,and found that VE-822 significantly reduced the expression of ABC transporter-associated protein CFTR.Conclusions1.ATR inhibitor VE-822 has significant inhibitory effect on pancreatic cancer cells.2.ATR inhibitor VE-822 combined with FOLFIRINOX regimen has synergistic inhibitory effect on pancreatic cancer cells.3.ATR inhibitor VE-822 can enhance autophagy and promote cell death of pancreatic cancer cells.ATR inhibitor can inhibit the expression of ABC transporter related proteins,reduce drug resistance of pancreatic cancer cells,and thus enhance drug sensitivity.