| BackgroundGastric cancer is one of the most common malignant tumors,and it is also the top three malignant tumors in tumor mortality.The proportion of patients with gastric cancer in China has been increasing in recent years and accounts for more than 40%of the total number of gastric cancer patients worldwide,which is significantly higher than that of other countries.In addition,because of the lack of early clinical diagnosis of gastric cancer and the neglect of the patient’s own stomach disease,the diagnosis of gastric cancer is generally in the middle and late stages,and the 5-year survival rate is below 5%.Therefore,it is important to explore the mechanisms that reveal the mechanism of gastric carcinoma and to seek new drug targets with clinical value.Helicobacter pylori(H.pylori)mainly results in gastric atrophic gastritis,intestinal type gastric cancer and non-atrophic gastritis,which gradually develops into diffuse gastric cancer.Helicobacter pylori stimulates the accumulation of neutrophils in epithelial cells,producing inflammatory mediators,reactive oxygen species and nitrogen species,which helps disrupt gastric epithelial function and DNA damage.Neutrophil infiltration is also associated with downregulation of E-cadherin,cell proliferation and gastric carcinogenesis.Epigenetic regulation includes DNA modifications,histone modifications,non-coding RNA regulation,chromatin remodeling,and so on.Abnormalities in epigenetic regulation are closely related to human diseases,including inflammatory diseases,metabolic disorders,neurological diseases,and cancers.The related pathogenic mechanisms are constantly being revealed.At present,the research on small molecule inhibitors of epigenomic regulators is more and more extensive,and clinically,histone deacetylase inhibitors and DNA methyltransferase inhibitors have been approved for treatment.More prodrugs that regulate epigenetics are in clinical trials.RBP2 is a molecule belonging to the histone demethylase KDM5 family which contains a Jmj C domain.Demethylation of dimethylation and trimethylation of Histone H3 lysine 4 is produced by the dioxygenase reaction of the cofactors Fe(II)and a-ketoglutarate(α-KG).Studies have shown that RBP2 is involved in tumor cell proliferation,invasion,migration,apoptosis and autophagy,which plays an important role in the pathogenesis of gastric cancer,breast cancer,non-small cell lung cancer,leukemia,etc.High expression of RBP2 and poor prognosis of tumor patients is also closely related.Our team has demonstrated that histone demethylase RBP2 can promote Helicobacter pylori-mediated malignant transformation and invasion and metastasis of gastric mucosal epithelial cells.In summary,RBP2 was found the key regulatory molecule in the malignant transformation of gastritis to gastric cancer mediated by H.pylori.It can provide more optionsfor the treatment of diseases caused by RBP2 to design and synthesis of small molecule compounds that can specifically inhibit the activity of histone demethylase RBP2,which might be the prodrugs for the diseases,and perform relevant biological function verification in gastric cancer models.ObjectiveTaking the pharmacophore hexamethylene formyl fragment as the core,it is screened and synthesized for small molecule compounds that inhibit the activity of histone demethylase RBP2,and detect the function in the gastric cancer model,which can provide more relevant clinical therapeutic prodrugs.Methods and Results1.Compound WXSA-055B can inhibit the expression of RBP2 to promote the level of H3K4me3 and regulate the downstream target genes expression of RBP2 in gastric cancer cell BGC-823.Using Western-blot,the compound WXSA-055B was tested for the regulation of RBP2 expression,histone H3K4me3 level,and the expression of downstream target genes p21,p27 and CCND1 in human gastric cancer cell line BGC-823.The results showed that WXSA-055B could inhibit the expression of RBP2 in BGC-823,and inhibit the activity of RBP2 histone demethylase activity,which could promote the level of H3K4me3 and regulate the downstream target genes expression of RIBP2.2.Compound WXSA-059A down regulated the RBP2 protein level and regulated the expression of downstream genes of RBP2 that inhibited the proliferation and migration of gastric cancer cells.The gastric cancer cell BGC-823 was treated with compound WXSA-059A,and the changes of RBP2,H3K4me3,p21 and CCND1 expression were detected by Western-blot.The proliferation of the cells was detected by EdU test.The results showed that the corresponding proteins were regulated and the proliferation ability of BGC-823 cells was significantly down-regulate in the cells treated with WXSA-059A.3.Compound WXSA-051B inhibited the expression of RBP2,promoted the level of H3K4me3 and the expression of BGC-823 apoptosis-related proteins,which inhibited cell proliferation and migration.After the treatment of the compound WXSA-051B were performed on the gastric cancer cell BGC-823,the corresponding changes of proteins expression and cells functions were detected.The results showed that the expression of RBP2 in BGC-823 cells decreased with the increase of H3K4me3 level and Caspase-3 activity.At the same time,the treated cells were checked for cells proliferation by EdU assay,flow analysis and Transwell chamber testing.After treatment with compound WXSA-051B,the abilities of cells proliferation and migration were inhibited,while the apoptosis was unchanged.4.Compound WXSA-082B could inhibit the expression of RBP2 and thus increased the level of H3K4me3 and promoted the expression of apoptosis-related proteins,which inhibited cells proliferation and migration.After the gastric cancer cell BGC-823 was treated by the compound WXSA-082B,the corresponding proteins and cells functions changes were detected.The results showed that after treatment with WXSA-082B,the expression of RBP2 in BGC-823 cells decreased,and the expression of H3K4me3,Caspase-3 and PARP1 increased.Then the EdU assay,flow analysis and Transwell chamber testing were detected.The results showed that compound WXSA-082B could inhibit the proliferation and migration of BGC-823 cells while the apoptosis ability was unchanged.5.Compound WXSA-072A inhibited the expression of RBP2 and promoted the level of H3K4me3 and apoptosis-related proteins,which inhibited cell proliferation,migration and apoptosis of gastric cancer cells.After the gastric cancer cell BGC-823 was treated with the compound WXSA-072A,the corresponding proteins and cells functions changes were detected.The results showed that after treatment with WXSA-072A,the expression of RBP2 in BGC-823 cells decreased,and the expression of H3K4me3 and Caspase-3 increased.At the same time,EdU assay,flow analysis and Transwell chamber testing were detected.The results showed that the proliferation and migration ability of BGC-823 cells were inhibited and apoptosis was significantly increased after the treatment of WXSA-072A.Conclusions1.WXSA-055B only affected the RBP2 expression and enzyme substrate level which regulated the downstream target molecules of RBP2 in gastric cancer cells.It had no further effect on the changes of cells functions.2.WXSA-059A affected RBP2 expression,which regulated the downstream target molecules of RBP2 at the protein level that could inhibit the proliferation of cells.But it did not play a role on RBP2 demethylase function.3.Both WXSA-051B and WXSA-082B had the effect on the RBP2 expression and the enzyme substrate level which could affect the downstream target molecules of RBP2 in gastric cancer cells,inhibiting the proliferation and migration of gastric can-cer cells.4.WXSA-072A affected the RBP2 expression,enzyme substrate level and the downstream target molecules of RBP2 at the protein level in gastric cancer cells,which functionally inhibited the proliferation and migration of gastric cancer cells,and promoted apoptotic process. |
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