The Study Of Berberine And Olaparib Induced Synthetic Lethal Effect And Its Underlying Mechanism In Pancreatic Cancer Cells

Pancreatic cancer is one of the most common gastrointestinal malignancies.Due to its high degree of malignancy and poor prognosis,the five-year survival rate is only 9.3%.Pancreatic cancer is expected to become the second most deadly malignancy by 2030.Poly(ADP-ribose)polymerase(PARP)inhibitors,as a new type of anti-tumor targeted drugs,have therapeutic effects on BRCA-deficient breast cancer and ovarian cancer patients."Synthetic lethality" is the theory that two genetic mutations that occur independently have no effect on a cell,but when they occur together in the same cell can cause cell death.The dual inhibition of PARP and BRCA has become a research hotspot in relevant fields.In late2019,the PARP inhibitor olaparib received its first approval for the treatment of pancreatic cancer patients with embryogenic BRCA(GBRCA)mutations,becoming the first targeted treatment for pancreatic cancer.In clinical study,Olaparib reduced the risk of death in patients with pancreatic cancer with g BRCA mutations by approximately 47%.However,globally,with approximately 460000 newly diagnosed cases of pancreatic cancer,patients with g BRCA mutations only account for 5-7%of all cases.So,it is still urgent to seek more effective and more widely applicable treatment methods and strategies.Berberine has a variety of biological activities and can inhibit a variety of human tumor cell lines.It has been reported that in ovarian cancer cells,berberine can inhibit homologous recombination repair and induce DNA oxidative damage,produces an effect similar to that of a BRCA defect,and enhancing the sensitivity of ovarian cancer cells to PARP inhibitors.In pancreatic cancer cells,berberine can induce ROS production to cause DNA oxidative damage,and PARP1 plays an important role in DNA oxidative damage repair,which means berberine may become a sensitizer of PARP inhibitor in pancreatic cancer cells.This study was designed to investigate the synthetic lethal effect of berberine and PARP inhibitor olaparib on pancreatic cancer cells,and to preliminarily explore the molecular mechanism.Objective: To investigate the synthetic lethal effect of berberine combined with PARP inhibitor Olaparib on pancreatic cancer cells and its molecular mechanism.Methods: Pancreatic cancer cells(Panc-1,Miapaca-2,Aspc-1 and Capan-2)were dealt with different concentration of berberine and olaparib alone and in combination.CCK8 assay were used to observe the cell vialilities.Synergistic effect of berberine and olaparib was analyzed by Kim’s formula.Colony formation assay was used to investigate the proliferation of cells.Cell activity was detected by combination of apoptosis inhibitors and necrotizing apoptosis inhibitors respectively.Cell apoptosis rate was detected by flow cytometry with Annexin V-FITC/PI.Real-time fluorescence quantitative PCR(q-PCR)was used to detect the expression of m RNA relative apoptosis and DNA damage repair.Results: Berberine inhibited the activity of pancreatic cancer cells in a dose-dependent way.The IC50 value of miapaca-2 cell treated with berberine at 48 h was 48.55μM.The IC50 value of Panc-1,Aspc-1 and Capan-2 cells was not reached when the maximum dose of berberine was200μM.Olaparib inhibited Panc-1 and Aspc-1 cells activity in a dose-dependent manner,but was insensitive to Miapaca-2 and Capan-2cells.The IC50 value of Asp C-1 cell was 34.41μM at 48 h after olaparib treatment.The IC50 value of Panc-1,Miapaca-2 and Capan-2 cells was not reached when olaparib treated with the maximum dose of 40μM.5μM berberine was combined with olaparib(5μM,10μM,20μM)to treat four pancreatic cancer cells,respectively.According to Kim’s formula,berberine and olaparib combined to treat four pancreatic cancer cells showed synergistic effect.Compared with berberine and olaparib alone,the survival rates of Panc-1,Miapaca-2,Aspc-1,and Capan-2 of pancreatic cancer cells were significantly decreased in the combination of berberine and olaparib.In Panc-1,Miapaca-2 and Aspc-1 cells,the apoptosis inhibitor z-VADFMK or the necrotizing apoptosis inhibitor Nec-1 can reverse the synergistic effect of berberine and olaparib.Compared with berberine or olaparib alone,cell proliferation was significantly inhibited in expression of Bcl-xl was down-regulated and the expressions of Bid,Bak,Bax,P21 and TP53 were up-regulated in the combination group.Conclusion: Berberine combined with olaparib may exert synthetic lethality on pancreatic cancer cells by down-regulating the expression of RAD51 and PARP,and induce cell apoptosis and enhance the sensitivity of pancreatic cancer cells to olaparib by down-regulating the expression of Bcl-xl and up-regulating the expressions of Bid,Bak,Bax,P21 and TP53.