Anti-cancer Mechanism Of Plant Cytokinin In Acute Myeloid Leukemia

Background: Previous studies on cytokinins have been mainly focused on regulating plant growth and development,and their effects on animal cells have not been taken seriously.In recent years,the anti-tumor activity of adenosine cytokinins has gradually attracted the attention of nucleoside drug researchers.It has been found that adenosine cytokinins can inhibit the proliferation of cancer cells and induce apoptosis and differentiation of cancer cells through adenosine transporters.This suggests that natural nucleoside cytokinins have great clinical and pharmaceutical value.However,the mechanism of their action in cancer cells is still in the preliminary stage of research.The study found that epigenetic modification plays a non-negligible role in the development of tumor cells.Adenosine DNA methyltransferase inhibitors in epigenetic treatment of tumors are a hot topic in current research.However,the role and mechanism of nucleoside cytokinins in tumor epigenetics has not been reported.Research purposes: In this study,AML cell line was used as the research vector,and nucleoside cytokinin was used as the research object to investigate its anti-tumor effect on AML cells and its effect on epigenetic modified tumor suppressor pathway.Research methods:(1)To investigate the inhibitory effect of cytokinin on the proliferation of AML cells: the inhibition of proliferation of AML by cytokinin was detected by CCK-8,Giemsa staining and flow cytometry analysis;(2)Explore the cytokinin pair Mechanism of differentiation of AML cells: Western Blot,PCR and flow cytometry were used to detect changes in protein levels of differentiation-related proteins and mRNA levels of differentiation-related genes;(3)oTR with the strongest inhibitory effect on proliferation of AML cell lines Molecular docking analysis of bioinformatics with DNMT1 was performed by libdock and cdocker molecules,comparing the degree of chimerism of DNMT1 natural substrate SAH and oTR with DNMT1 structure,and comparing the scoring function;(4)In vitro test for inhibition of DNMT1 activity by oTR: The DNMT activity/inhibition assay kit was used to further detect the inhibitory activity of oTR on DNMT1;(5)to explore the demethylation of oTR on hypermethylated miR-34 a tumor cells and to miR-34a-SIRT1-p53 Effect of tumor suppressor pathway: detection of miR-34 a expression and methylation status by Northern blot.Results :(1)It was found by CCK-8 that cytokinin inhibited the proliferation of AML cells in a concentration-dependent manner;Giemsa staining and flow cytometry analysis showed that cytokinin can induce differentiation of AML cells into myeloid cells,CD11 b The expression of myeloid differentiation-related protein was detected by Western Blot.The expression of p-JAK and p-STAT3 was down-regulated,while the expression of p-STAT1 was up-regulated.The expression of mRNA of myeloid differentiation-related genes was detected by PCR.The mRNA levels of PU.1 and CXCL-10 were increased.(3)Molecular docking simulation by bioinformatics showed that oTR was embedded in the active pocket of DNMT1 crystal model compared with SAH,and oTR and SAH The scores differed slightly,and the spatial interaction between protein and small molecule showed that 85% of the amino acid residues between oTR and DNMT1 were the same as the natural substrate,suggesting that oTR may affect the activity of DNMT1;(4)utilization DNMT activity/inhibition assay kit found that oTR significantly inhibited the activity of total DNMT and DNMT1 in a concentration-dependent manner;(5)Northern blot found that in p53 wild-type AML cells,miR-34 a The expression level of AML cells was significantly lower than that of p53 mutants.The expression of miR-34 a was up-regulated after oTR treatment,and the expression of miR-34 a target gene SIRT1 was found to be oTR concentration-dependent after oTR-acting THP-1 cell line.Decreased,p53 acetylation levels were significantly upregulated.Conclusion: Nucleoside cytokinins inhibit the proliferation of AML cells and induce their differentiation into myeloid cells;oTR can restore the expression of hypermethylated miR-34 a by inhibiting DNMT1 activity and restore miR-34a-SIRT1-p53 tumor suppressor pathway.