| [Objective]Gastric cancer is the fourth leading cause of cancer related death worldwide.There are no obvious specific symptoms of early gastric cancer,and most patients are in the advanced stage when they are diagnosed.Chemotherapy is the mainstay of treatment for patients with advanced gastric cancer.5-fluorouracil(5-FU)is a commonly used first-line basic chemotherapy drug in the treatment of gastric cancer,often in combination with other drugs.However,gastric cancer is highly heterogeneous,and the current clinical targeted drugs are limited.The research group found that the new derivative of ACP-196(Acalabrutinib,ACP-196),HZ-A-018,has good antigastric cancer activity and can enhance the efficacy of 5-FU in gastric cancer.In this project,the effect and mechanism of HZ-A-018 sensitizing 5-FU against gastric cancer were preliminarily studied.[Methods]1.The inhibitory effect of ACP-196 or HZ-A-018 at different concentrations on the proliferation of HGC-27 and BGC-823 human gastric cancer cells in vitro after 72 h treatment was determined by CCK-8 assay.2.The inhibitory effect of 5-FU,ACP-196 or HZ-A-018 and their combined treatment for 72 h on the proliferation of HGC-27 and BGC-823 human gastric cancer cells in vitro was determined by CCK-8 assay.The inhibitory effect of 5-FU and HZ-A-018 single treatment and their combination on the colony forming ability of HGC-27 and BGC-823 human gastric cancer cells in vitro was determined by clone formation assay.3.The in vitro apoptosis effects of 5-FU,ACP-196 or HZ-A-018 and their combined treatment for 72 h were detected by flow cytometry.4.The protein levels of HGC-27 and BGC-823 human gastric cancer cells after single treatment of 5-FU,ACP-196/HZ-A-018 and combined treatment for 24 h were detected by Western Blot analysis.5.The model of HGC-27 xenotransplantation in nude mice was established and treated with 5-FU,HZ-A-018 and combined therapy.The tumor diameter and tumor size of nude mice were measured,and the anti-gastric cancer effect of drugs on nude mice was evaluated.The hepatorenal toxicity of the drug in nude mice was evaluated by the results of immunohistochemical section of liver and kidney in nude mice.6.The key proteins affecting the combination of HZ-A-018 and 5-FU were Screened by proteomic analysis and bioinformatics analysis.7.The expression of key proteins in the combination was verified by Western Blotting,immunofluorescence and immunohistochemistry.The relationship between drugs and key proteins was studied by giving protein synthesis inhibitors and proteasome degraders.8.Knockdown and overexpression transfection techniques were used to study the effects of key proteins on the malignant degree and drug sensitivity of gastric cancer cells.[Results]1.The results of CCK-8 assay showed that the IC50 values of ACP-196 in HGC-27 and BGC823 human gastric cancer cell lines after 72 h were 35.6±2.4μM and 44.9±5.2 μM,respectively.The IC50 values in HGC-27 and BGC-823 human gastric cancer cell lines after 72 h were 17.9±0.7 μM and 17.1±3.6 μM,respectively.2.The results of CCK-8 assay showed that in HGC-27 gastric cancer cells,5-FU(5 μM)combined with HZ-A-018(10 μM)had a synergistic anti-proliferative effect with an inhibition rate of 77.8%±6.0%and the combination index was about 0.68.In BGC-823 gastric cancer cells,5-FU(0.5μM)combined with HZ-A-018(10 μM)also had a synergistic anti-proliferative effect with an inhibition rate of 58.7%±7.7%and the combination index was about 0.83.In contrast,there was no significant effect when 5-FU was combined with ACP-196 on both cell lines.The results of clone formation assay showed that in HGC-27 cells,the inhibition rates of clone formation were 32.4%± 7.44%and 25.2%± 25.7%for 5-FU(2.5 μM)and HZ-A-018(10 μM)alone,respectively,and 93.8%± 3.0%for the combination of the two drugs(p<0.001 vs 5-FU,p<0.05 vs HZ-A-018).In BGC-823 gastric cancer cells,the inhibition rate of clone formation were 40.2%±18.3%and 42.7%±19.6%for 5-FU(0.25μM)and HZ-A-018(10 μM)alone,respectively,and 87.0%±8.3%for the combination of the two drug.The difference was statistically significant(p<0.05 vs 5-FU,p<0.05 vs HZ-A-018).3.The results of flow cytometry showed that the apoptosis rates of 5-FU(5 μM)and HZ-A018(10 μM)in HGC-27 gastric cancer cells were 12.8%±7.1%,13.6%±5.0%and 44.9%±9.9%,respectively(p<0.05 vs 5-FU).In BGC-823 gastric cancer cells,the apoptosis rates of 5-FU(0.5 μM)and HZ-A-018(10 μM)were 17.3%±0.4%,6.0%±1.7%and 47.1%±5.4%,respectively(p<0.001 vs 5-FU).However,when 5-FU and ACP-196 were used together,there was no significant synergistic effect on inducing apoptosis in HGC-27 and BGC-823 gastric cancer cell lines.4.The results of Western Blot assay showed that HZ-A-018(10 μM)treatment downregulated the expression of p-AKTSer473,p-S6Ser240/244,p-S6Ser235/236 in HGC-27 and BGC-823 gastric cancer cells,and 5-FU(5 μM in HGC-27 cells and 0.5μM in BGC-823 cells)combined with HZ-A-018(10 μM)further inhibited the activation of S6.5.The results of tumor diameter results showed that the tumor suppression rates after 5-FU(40 mg/kg)monotherapy,HZ-A-018(100 mg/kg)monotherapy,and combination therapy were 39.8%± 12.6%,32.0%± 29.5%,and 70.0%± 8.7%,respectively,compared with the control group,and the differences in the results were statistically significant(p<0.05 vs 5FU,p<0.05 vs HZ-A-018).The tumor weight results showed that the tumor weights of the control,5-FU alone,HZ-A-018 alone and combined groups were 0.90 ± 0.32 g,0.72 ± 0.31 g,0.61 ± 0.18 g and 0.29 ± 0.14 g,respectively,and the differences in the results were statistically significant(p<0.05 vs 5-FU,p<0.05 vs HZ-A-018).The immunohistochemical results of liver and kidney of nude mice showed no significant toxicity of 5-FU(40 mg/kg)and HZ-A-018(100 mg/kg)monotherapy and combination treatment compared with the control group.6.The results of proteomic analysis showed that RRM2 was one of the more significantly down-regulated proteins in the combination group of 5-FU and HZ-A-018 compared with the group of 5-FU alone in HGC-27 gastric cancer cells.Among them,RRM2 is one of the proteins with significant down-regulation.The results of bioinformatics analysis showed that RRM2 was highly expressed in gastric cancer,and the expression in gastric cancer was higher than that in matched tissues.7.The results of Western Blot showed that the combination of 5-FU(5μM)and HZ-A-018(10μM)could down-regulate the expression level of RRM2 protein in vivo and in vitro.The results of immunofluorescence showed that the average fluorescence intensity of the 5-FU and HZ-A-018 combined group was lower than that of the 5-FU and HZ-A-018 alone group,and the difference was statistically significant(p<0.05 vs 5-FU,p<0.05 vs HZ-A-018).The results of immunohistochemistry showed that the combination of 5-FU and HZ-A-018 could reduce the expression of RRM2 to some extent.By administering protein synthesis inhibitors and proteasome degradation agents,it was found that the combination of 5-FU and HZ-A-018 may inhibit the protein synthesis of RRM2.8.The results of RRM2 knockdown experiments showed that the growth inhibition rate of cells after knockdown of RRM2 in HGC-27 cells was 57.6%±2.5%,and the difference of the results was statistically significant compared to Scr control group(p<0.01).the sensitivity of cells to drugs was enhanced after RRM2 knockdown.In the Scr control and RRM2-KD groups,the survival rates of HGC-27 cells were 75.4%±0.7%,39.8%±0.8%(p<0.01),61.3%±2.7%,32.2%±2.3%(p<0.01),after 5-FU(5 μM)alone,HZ-A-018(10μM)alone and in combination,respectively.16.2%±0.3%,7.8%±0.8%(p<0.05).the proliferation rate of cells after RRM2 overexpression was 1.6 times higher than that of the null group.In the EV group,the survival rates of cells in the 5-FU(5 μM),HZ-A-018(10μM)alone and in the combination group were 42.4%±2.2%,73.4%±1.9%and 29.0%±1.9%;in the RRM2 overexpression group,the survival rates of cells in the 5-FU(5 μM),HZ-A-018(10 μM)alone and in the combination group were 69.8%±2.3%,79.9%±3.6%and 48.1%±1.7%.The drug sensitivity of cells in the overexpression group to 5-FU(5 μM)and HZ-A-018(10 μM)alone and in combination became weaker compared with the EV group.Western Blot results showed that the apoptosis-related protein expression of cl-PARP and cl-Caspase 3 became weaker after RRM2 overexpression group administration compared with the EV group.[Conclusion]The combination of HZ-A-018 and 5-FU has a synergistic effect on gastric cancer in vitro and in vivo,and its mechanism is related to the inhibition of AKT/S6 pathway and the decrease of RRM2 expression. |
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