| Background and Purpose:Pancreatic cancer(PC)is one of the common nausea tumors of the digestive tract.Its onset is hidden,the course of the disease progresses rapidly,and the prognosis is extremely poor.For decades,the clinical progress of PC therapy is still limited.Gemcitabine(Gem)-based treatment is still the first choice for the treatment of locally advanced and metastatic PC patients.However,due to the low efficiency of Gem chemotherapy,many patients are receiving After chemotherapy,the development of acquired drug resistance leads to disease resistance or even worsening.Therefore,new methods are urgently needed to improve the chemotherapy sensitivity of Gem.Cell cycle checkpoint kinase 1(Chk1)is a key factor that maintains cell proliferation,survival and cell cycle integrity,especially in the repair of DNA double-strand breaks(DSBs).Clinical studies have found that increased Chk1 expression levels are positively correlated with disease recurrence,treatment resistance,and shorter overall survival.When Gem blocks DNA synthesis in cancer cells and induces DNA damage in cancer cells,tumor cells start DNA Injury response,by activating Chkl related signaling pathways to repair damaged DNA double strands to protect cancer cells from the genotoxicity of conventional chemotherapeutics.Based on this,clinically,Chkl inhibitors can be combined to enhance the sensitivity of genotoxic chemotherapeutics.This study aimed to explore the effects of the five-ring triterpene compound Celastrol(Celastrol,Cel)and its carboxymethylated product Pristimerin(Pristimerin,Pri)on the Chkl protein in human pancreatic cancer cells,and to explore the effect of the two drugs The structure-activity relationship and the molecular mechanism of Pri regulating DNA damage repair through the Chk1/53BP1 pathway to enhance the chemosensitivity of Gem to pancreatic cancer cells provide a basis for the development of new natural Chkl inhibitors as tumor radiosensitizers.Methods:The thiazolyl(MTT)method was used to detect the cytotoxic effects of Pri,Cel and Gem alone on human pancreatic cancer cell lines AsPC-1,BxPC-3 and PANC-1,and the structure-activity relationship analysis between Pri and Cel;discussion of drug interaction analysis Whether there is a synergistic effect between Pri and Gem;clone colony formation test to detect the effect of single Pri,Gem and combination drugs on the long-term inhibition of human pancreatic cancer cells;EdU proliferation test quantitative analysis of single Pri,Gem and combination drugs on human pancreatic cancer cells The effect of proliferation;flow cytometry analysis of the effects of Pri,Gem,and combination of drugs on the cell cycle distribution of human pancreatic cancer cells;Western Blot(Western Blot)detection of Pri,Gem,and the combination of the two drugs in cells-Chk1,p-Chk1 protein;DNA damage protein γH2aX,PARs,DNA damage repair related protein 53BP1,RAD51,and apoptosis related protein PARP1,Bax and Bcl2 protein expression;immunofluorescence test detects Pri,Gem and combination alone After treatment,the expression of DNA damage protein and repair protein in human pancreatic cancer cells and their distribution in the cells;comet electrophoresis test(single gel electrophoresis experiment)to detect the DNA in human pancreatic cancer cells after Pri and Gem alone and after combined treatment The damage effect;Results:1.The results of the MTT experiment show that compared with the control group,Pri and Cel have a significant inhibitory effect on human pancreatic cancer cells after 24,48,and 72 h.The cytotoxic effect of Pri is stronger than that of Cel at the same concentration.Among them,BxPC-3 cells are the most sensitive to Pri and Cel.2.Western Blot experiment results show that both Pri and Cel can effectively inhibit the expression of Chkl total protein in PC cells;both Pri and Cel can effectively inhibit the expression of the phosphorylation level of Chkl protein,and present time and dose dependence;at the same time;It was found that Pri’s ability to inhibit the phosphorylation of Chkl was stronger than Cel.3.Through computer simulation drug interaction analysis,it is found that Pri and Gem have a good synergistic effect at their respective sub-IC50 concentrations.4.Clonal colony formation experiments show that Pri and Gem have long-term and continuous inhibition of tumor proliferation,and the combination of the two can further inhibit the clonal formation of PC cells.5.Flow cytometry analysis of the cell cycle showed that Pri could block the G1 phase of the cell cycle and Gem could block the G1/S phase of the cell cycle,and the combination of the two could synergistically block the cell cycle in the S prophase.6.Westem Blot results showed that Pri could reverse Gem-induced Chkl activation,DNA damage marker yH2AX and Cleaved PARP1 protein were significantly increased.7.The results of comet experiment showed that Pri could aggravate the Gem-induced DNA damage;8.Immunofluorescence assay results showed that after Pri combined with Gem,the recruitment of DNA damage protein yH2AX in the nucleus was increased,while the recruitment of DNA repair protein 53BP1 was decreased.In order to further explore the molecular biological events in the process of DNA replication fork arrest and DNA damage caused by the combined treatment of Pri and Gem in PC cells,we tested the downstream oxidative stress and DNA repair functions,and found that Pri can be silenced.The non-homologous recombination downstream of Chkl repairs the expression of the protein 53BP1.In addition,the combination of the two drugs ultimately promotes the apoptosis of pancreatic cancer cells.Conclusion:Cel and Pri can inhibit the survival rate of PC cells,reduce the viability of PC cells,and inhibit the proliferation and growth of PC cells;through the comparison of the efficacy of the two drugs,it is found that Pri has a stronger ability to inhibit the proliferation of PC cells than Cel;Pri is through lysozyme The somatic hydrolysis pathway degrades the expression of Chkl protein in cells while inhibiting its phosphorylation expression level;Pri combined with Gem can aggravate the DNA damage of pancreatic cancer cells,and Pri inhibits DNA damage repair by blocking the Chk1/53BP1 signaling pathway,thereby enhancing Gem’s effect on PC cells Chemosensitivity. |
