| Cancer has become a serious disease that threatens human health and is a public health problem that needs to be solved.The 2018 China Cancer Registry Annual Report shows that the new cancer cases in China account for about one-fourth of the world's total;of these,the number of new cases of gastric cancer is the second and the number of deaths is the third.Therefore,the development of an effective cancer prevention and control strategy is of great significance for safeguarding people's health and promoting economic and social development.Most cancer patients are already at the advanced stage of diagnosis,and palliative chemotherapy is the main treatment for advanced cancer patients.At present,the platinum compounds represented by cisplatin(DDP)are still used in the treatment of many tumors,especially some solid tumors,such as lung cancer,gastric cancer,etc.Cisplatin mainly induces DNA double-strand damage and promotes apoptosis.Although cisplatin has toxic side effects on the body,it is still clinically used due to its extensive and effective killing effect on tumors.The intrinsic resistance and acquired resistance to cisplatin severely affect its efficacy,limiting its clinical application.Cisplatin-resistant tumor cells also produce cross-resistance to other drugs.The molecular mechanisms of cisplatin resistance are complex and diverse,including the activation of drug transporters,which enhancing intracellular drug detoxification;enhanced DNA repair;inhibition of apoptosis and cell cycle arrest;altered drug metabolism.After DNA damage induced by platinum drugs,the activation of DNA repair pathway is one of the key factors to promote the tolerance of tumor cells to platinum drugs.Therefore,elucidating the abnormally expressed or modified proteins in tumor drug-resistant cells and developing of the inhibitors,agonists,or combined with other target mechanisms for multi-target interventions aimed at the newly discovered targets is an urgent task to overcome tumor resistance.JWA,also known as ARL6IP5,encodes a protein located in the endoplasmic reticulum and is a microtubule binding protein.Our group has focused on the structure and function of JWA gene for a long time.JWA is an active environmental response gene that repairs DNA damage caused by oxidative stress.When JWA proximal-promoter-76G/C polymorphism is mutated,the cells cannot respond to oxidative stress,which aggravates DNA damage and genomic instability caused by environmental toxicants,and the susceptibility to various malignancies such as gastric cancer and esophageal cancer increased significantly.Our previous study found that the level of JWA in gastric cancer tissues was lower than that in adjacent tissues and was significantly negatively correlated with the prognosis of patients.The level of JWA was related to the overall survival rate caused by platinum-based chemotherapy.Increasing JWA expression in tumor cells can be independent or synergistic with chemotherapeutic drugs to inhibit multiple malignant phenotypes.Compared with parental cells,JWA level in cisplatin-resistant gastric cancer cells decreased significantly,while the DNA double-strand damage repair signaling pathway represented by XRCC1 was overactivated.In cisplatin-resistant gastric cancer cells,JWA inhibited XRCC1 518S/519T/523T site phosphorylation by inhibiting CK2 and promoted ubiquitination and degradation of XRCC1,thereby inhibiting XRCC1 over-activation and super strong DNA repair ability,promote cisplatin-induced apoptosis.In addition to the DNA repair signal network abnormalities,the apoptosis signal pathways of cisplatin-resistant tumor cells are disordered,showing dynamic changes one after another.Our previous study found that acquired gastric cancer cells are sensitive to TRAIL.TRAIL functions to induce apoptosis by binding to its receptor.Although we have found that cisplatin-resistant cells are sensitive to TRAIL,the exact molecular mechanism needs to be further studied.Previous studies have found that JWA protects oxidative stress-induced single-strand DNA damage by modulating PARP1 activity(PAR).When DNA damage occurs,PARP1 activates and recruits other DNA repair proteins such as XRCC1 to participate in DNA repair.More and more studies have reported that PARP1 inhibitors act as sensitizers to increase the chemotherapeutic effect of various tumors.However,whether PARP1 inhibitors promote cisplatin-induced apoptosis in gastric cancer-resistant cells still requires systematic studies.Based on our previous study,this study intends to explore the regulation of abnormal expression proteins in cisplatin-resistant gastric cancer from the perspective of DNA damage repair and death receptor signaling pathways;and to conduct interventions against newly discovered targets and proposed new methods to reverse cisplatin resistance in gastric cancer.Objective:This present study was based on the role of DNA damage repair and death receptor signaling pathways in cisplatin resistance,and explored the molecular mechanisms and intervention of cisplatin resistance in gastric cancer cells.Methods:1.In this study,gastric tumor-bearing model was first used to verify the sensitivity of JWA to cisplatin and to observe the effect of JWA knockdown on tumor growth and cisplatin sensitivity in tumor cells.Then,the tumor-bearing model was used to analyze the effect of JWA-targeted peptide alone or in combination with cisplatin on the growth of gastric cancer.2.Western blot,flow cytometric analysis,immunofluorescence and other methods were used to analyze the effect of JWA-MARCH8-DR4 signaling axis on the sensitization of TRAIL-induced cisplatin-resistant gastric cancer cells.3.CCK8,Western blot,immunofluorescence,plate colony formation assay,Tunel,DNA damage detection system and other methods were used to analyze the role and molecular mechanism of PARP1 inhibitors in cisplatin resistance in gastric cancer cells.Results:1.HGC-27 and NCI-N87 cells were primary cisplatin-resistant gastric cancer cells.CCK8 results showed that the IC50 values of cisplatin on HGC-27 and NCI-N87 were 5.163 ?g/ml and 10.809 ?g/ml,respectively;while the IC50 values of BGC823 and SGC7901 were 0.811 ?g/ml and 0.408 ?g/ml,respectively.Tunel staining showed that the apoptosis rates of BGC823 and SGC7901 were 35.01±0.91%and 43.89±1.26%,respectively;while the apoptosis rates of HGC-27 and NCI-N87 were 9.27±1.64%and 4.57±1.07%,respectively.The difference was statistically significant(P<0.001)2.JWA promotes cisplatin sensitivity in gastric cancer cells.The western blot results showed that the level of JWA protein expression in primary and acquired cisplatin-resistant gastric cancer cells was both decreased compared with the cisplatin-sensitive cells.In vivo,after knocking down JWA level in gastric cancer cells,it promoted the growth of gastric cancer cells and the tolerance of cisplatin.The inhibition rate of cisplatin on si-con group was 55.8%(P<0.05),and the inhibition rate in si-JWA group was 23.9%(P>0.05)3.JWA-targeting peptide alone and synergistically inhibits the growth of gastric cancer.The results of tumor-bearing model showed that the cisplatin group(inhibition rate of 56.9%,P<0.01)and JWA polypeptide group(inhibition rate of 39.4%,P<0.05)effectively inhibited the growth of gastric cancer,and the combined group had the most significant inhibitory effect(inhibition rate was 71.8%,P<0.001).Western blot analysis of gastric cancer tissue showed that the expression of MMP2 in JWA-targeting peptide group and the combination group was decreased by 0.74 and 0.78 times compared with the control group,respectively,suggesting that the JWA targeting peptide targets the inhibition of MMP2 expression.The expression of ?H2AX in JWA peptide group and cisplatin treatment group was 2.17 times and 2.28 times as much as that in control group.The expression of yH2AX in combined group was 3.26 times as much as that in control group.The expression of XRCC1 in JWA peptide group and the combination group was decreased by 0.57 and 0.63 fold,respectively,compared with the control group,suggesting that the JWA-targeting peptide inhibited the expression of XRCC1 and increased the sensitivity of cisplatin chemotherapy.4.DR4 upregulation promotes TRAIL-sensitive cells in acquired cisplatin-resistant gastric cancer cells.Western blot results showed that DR4 expression was increased in acquired cisplatin-resistant gastric cancer cells.Flow cytometry showed that the DR4 content in the acquired gastric cancer cisplatin-resistant cell membrane was significantly higher than that in the corresponding sensitive cells,and the fold increase was 7.17±0.35 and 5.74±0.55,respectively.In cisplatin-resistant cells,TRAIL-induced reduction in caspase3 splicing decreased after knockdown of DR4 expression,whereas TRAIL-induced caspase3 splicing showed no significant change after knockdown of DR5 expression by western blot assay.5.JWA negatively regulates DR4 through the lysosomal pathway.The western blot results showed that si-JWA was transfected into the sensitive gastric cancer cells and DR4 expression was increased.The expression of DR4 was decreased in flag-JWA transfecting cisplatin-resistant gastric cancer cells.The flow cytometry results showed that the expression of DR4 on the membrane surface was significantly up-regulated after si-JWA transfection into BGC823 and SGC7901,and the expression of DR4 protein was increased by 3.69 ± 0.48 and 2.66 ±0.06 folds,respectively.After transfection of the flag-JWA plasmid into BGC823/DDP and SGC7901/DDP to restore JWA expression,the DR4 protein level on the cell membrane surface was significantly decreased by 0.46±0.05 and 0.59± 0.04 fold,respectively.Western blot results showed that lysosomal inhibitors recover the expression of DR4,which was decreased because of high JWA expression.Immunofluorescence results showed that the expression of DR4 on the cell membrane was decreased after increased JWA expression,and the expression in cytoplasm was increased and co-localized with lysosomal marker LAMP2.6.JWA activates MAPK signaling pathway,and promotes DR4 ubiquitination by positive regulation of MARCH8.Western blot results showed that over-expression of JWA in drug-resistant cells promoted ubiquitin degradation of flag-DR4(wild-type)but failed to promote the degradation of flag-DR4(mutant K273R).Further analysis of the Western blot results revealed that JWA promotes DR4 ubiquitination by positively modulating MARCH8.After transfection of flag-JWA in drug-resistant cells,pERK was activated,MARCH8 expression was up-regulated,DR4 expression was decreased,but after MEK inhibitor treatment,pERK was blocked,and JWA lost its regulation of MARCH8 and DR4.7.The protein levels of JWA and DR4 were negatively correlated in gastric cancer tissues.Western blot results showed that the expression of JWA and DR4 protein in 21 gastric cancer tissues was negative correlation,R2=0.3908,the difference was statistically significant(P<0.01).8.Primary cisplatin-resistant gastric cancer cells were resistant to TRAIL.CCK8 results showed that both HGC-27 and NCI-N87 cells were insensitive to TRAIL(IC50=413 ng/ml and 1632 ng/ml,respectively).Western blot results showed that DR4 expression in primary cisplatin-resistant cells was significantly lower than acquired cisplatin-resistant cells.9.PARP1 inhibitors promote cisplatin-induced apoptosis in cisplatin-resistant gastric cancer cells.Western blot results showed that PARP1 activity(PAR)expression was increased in cisplatin-resistant gastric cancer cells.PARP1 inhibitor AG 14361 alone could effectively inhibit PAR and not promote apoptosis in cisplatin-resistant cells,but significantly promoted cisplatin-induced caspase3 cleavage in the resistant cells by western blot assay.Tunel staining also showed that the apoptosis rate in the cisplatin group was 12.11±0.54%,the apoptosis rate was 19.53± 0.16%and 14.43± 0.46%in combined group(AG14361+cisplatin or BYK204165+cisplatin),respectively.The difference was statistically significant.CCK8 results showed that the survival rate of cisplatin group cells in HGC-27 cells was 82.38±3.36%,and the cell survival rate in the combined group(AG14361+cisplatin or BYK204165+cisplatin)was 66.83±2.50%and 71.09±1.83%,respectively;The survival rate of NCI-N87 cells in cisplatin group was 94.47±2.05%,and the cell survival rate in combined group(AG14361+cisplatin or BYK204165+cisplatin)was 77.08±4.37%and 71.72±0.89%,respectively,and the difference in HGC-27 and NCI-N87 cells was both statistically significant.Clone formation experiments showed that the two PARP1 inhibitors AG 1461 and BYK204165 alone could not inhibit the proliferation of drug-resistant cells(P>0.05);but significantly increase chemotherapy sensitivity of cisplatin.Compared with the cisplatin group,the number of clones in BYK204165+cisplatin or AG14361+cisplatin group was reduced by 21.67±0.48 and 19.20 ± 0.76 in HGC-27 cells,respectively.In NCI-N87 cells,the number of clones in the combination group(BYK204165+ cisplatin or AG14361+cisplatin)was reduced by 48.34±8.18 and 51.33±1.70,respectively,compared with the cisplatin group.In BGC823/DDP cells,the number of clones in the combination group(BYK204165+cisplatin or AG14361+cisplatin)decreased by 121.22±10.61 and 115.13±10.18,respectively,compared with the cisplatin group,and all the differences were statistically significant(P<0.001).10.PARP1 inhibitors promote cisplatin-induced DNA damage and G0/G1 arrest in cisplatin-resistant cells.Immunofluorescence staining showed that PARP1 inhibitor AG 14361 combined with cisplatin significantly increased cisplatin-induced the number of ?-H2AX-positive cells,which increased by 30.40± 8.12%and 29.82 ± 6.07%,respectively,in HGC-27 and NCI-N87 cells.The differences were statistically significant(P<0.01).Western blot results showed that the ?H2AX level in the combined group(BYK204165+cisplatin or AG 14361+cisplatin)was increased compared with cisplatin group,showing a time effect and dose effect in HGC-27 cells.Flow cytometry results showed that the cell cycle did not change significantly after treatment of HGC-27 and NCI-N87 cells with AG14361 alone;however,after treatment with cisplatin alone,G0/G1 phase arrest was observed.In the combination group,G0/G1 phase arrest was further increased compared with the cisplatin group,in which HGC-27 increased by 10.22 ± 0.73%and NCI-N87 increased by 7.93 ± 1.23%,and the differences were statistically significant.11.PARP1 inhibitors block the activation of NHEJ pathway.CCK8 results showed that the IC50 values of BGC823 and HGC-27 cells against MX were 3.190 mg/ml and 3.069 mg/ml,respectively,and there was no statistical difference between the two groups.After MX and cisplatin treatment of HGC-27 cells,CCK8 results showed that MX could not reduce cisplatin-induced cell survival,and the difference was not statistically significant(P>0.05).The western blot results also showed that MX could not increase cisplatin-induced yH2AX expression levels.CCK8 results showed that the IC50 values of curcumin in BGC823 and HGC-27 cells were 18.212 and 9.794 ?g/ml respectively,and the difference was statistically significant(P<0.001).CCK8 results showed that the survival rate of curcumin and cisplatin combination group was 11.30 ± 0.87%lower than that of cisplatin group,and the difference was statistically significant(P<0.05).Western blot results also showed that curcumin can increase cisplatin-induced yH2AX expression levels.The HR and NHEJ detection report system analysis showed that the number of GFP-positive cells in PARP1 inhibitor group was reduced by 1.26±0.34%,and the difference was statistically significant(P<0.001),but could not effectively inhibit the number of GFP-positive cells in HR pathway,which has no statistical difference(P>0.05)12.PARP1 inhibitors block the expression and activation of DNA-PKcs and XRCC1.Western blot results showed that the expression of DNA-PKcs and XRCC1 in primary and acquired gastric cancer cisplatin-resistant cells was higher than that in the sensitive cells.The DNA-PKcs inhibitor NU7441 effectively promoted the apoptosis of HGC-27 cells induced by cisplatin;compared with the cisplatin group,the cell survival rate of the combination group decreased by 8.35±1.16%,and the difference was statistically significant(P<0.01).Immunofluorescence,western blot and co-immunoprecipitation assays showed that PARP1 inhibitors inhibitted DNA-PKcs and XRCC1 expression and activation,and blocked their interactions,thereby inhibiting the NHEJ repair pathway,and promoting DNA damage in cisplatin-induced resistant cells.Conclusion:(1)JWA promotes the chemosensitivity of cisplatin,and JWA-targeting peptide can effectively promote cisplatin-induced apoptosis in gastric cancer in vivo.(2)Acquired cisplatin-resistant gastric cancer cells are sensitive to TRAIL because of up-regulaged DR4 in the resistant cells.Its molecular mechanism is the decline of JWA expression in cisplatin-resistant cells,inhibition of activation of MEK-ERK signaling pathway,which in turn down-regulates MARCH8,and blocks ubiquitin degradation of DR4 from cell membrane to lysosomes.(3)PARP1 inhibitors promote cisplatin-induced DNA damage and apoptosis in cisplatin-resistant gastric cancer cells by inhibiting PAR.The specific mechanism is that PARP1 inhibitors block the expression and activation of DNA-PKcs and XRCC1,the key molecules in NHEJ repair pathway,to prevent DNA repair and promote cisplatin induced cell death. |
PDF Full Text Request