Effect Of Anthraquinone Derivative C10 On Colon Cancer Cell Proliferation Through Jak/Stat3 Signaling Pathway

The Janus kinase(Jak)/signal transducer and transcription activator(Stat)is a signaling pathway related to inflammatory mediators that regulates many physiological processes,such as cell growth and differentiation,immunity,inflammation,and tumorigenesis.Anthraquinone is an active ingredient of traditional Chinese medicines such as aloe,rhubarb,cassia,et al.It has various pharmacological activities such as anti-inflammatory,anti-hypertensive,anti-bacterial,anti-angiogenesis,anti-tumor,which have attracted great attention of researchers in recent years.Drugs that have been marketed clinically include mitoxantrone,doxorubicin,arubicin,and epirubicin.However,anthraquinones have poor solubility and low yields due to natural extraction and separation.Therefore,researchers have focused on artificial synthesis and modification to improve the biological activity of compounds.In the early stage,our team synthesized 1-nitro-2-acylanthraquinone-glycine(C10)using 2-methylanthraqui-none as a raw material through a series of chemical modifications.It has been measured that it has good biological activity on colon cancer cells HCT116 and HT29 cells.Therefore,we conduct in-depth research on this basis.The main findings are as follows:1.C10 inhibits the proliferation of colon cancer cells HCT116 and HT29 cells,but does not affect the proliferation of normal intestinal epithelial cells,and induces these two cell cycle arrests,cell migration and colony-forming capabilities.We first tested HCT116 and HT29 with different concentrations of C10 and normal intestinal epithelial cells by MTT for 24,48,and 72 h.It was found that C10 can inhibit the proliferation of these two colon cancer cells in dose-dependent manner.But C10 does not affect the viability of normal intestinal epithelial cells.At the same time,the morphological observation of cells showed that with the increase of C10 concentration,the number of cells gradually decreased,the distance between cells became larger,and the cell morphology became smaller and rounder.Nuclei condense,chromatin is unevenly distributed,and nucleus morphology becomes uneven.Flow cytometry,western blot,and q PCR detection revealed that the expression of the cycle-associated protein Cyclin B and its downstream signal molecule Cdk1 protein and gene gradually decreased with increasing concentration.These results indicate that C10 blocks the cell cycle at G2/M period.It was found through the scratch test that the increase in scratch distance becomes significantly smaller with time and concentration compared with the control group,which indicates that C10 can inhibit cell migration.Subsequently,we tested the effect of C10 on the colony-forming ability of these two cells through clone formation experiments.The results showed that the number of clones formed gradually decreased with the increase of the concentration of C10,indicating that C10 can inhibit the colony-forming ability of these two cells.2.C10 has strong affinity with Jak family protein Jak2.We have studied the affinity of C10 with Jak1,Jak2,and Jak3 through molecular docking prediction.The results show that C10 forms two hydrogen bonds with Asn1008 of Jak1 and one hydrogen bond with Arg1007.C10 is close to the β-sheet region of its active site.C10 forms a hydrogen bond with Lys882 of Jak2,forms two hydrogen bonds with Asn859,and binds to the α-helix position of the active site of Jak2.C10 forms a hydrogen bond with Lys885 and Leu905 of Jak3 and binds to the β-sheet region structure of the secondary structure of Jak3 protein.The docking scores of C10 and these three proteins were 4.2043,5.3796,and 4.5155,respectively.This shows that C10 and Jak2 have a higher binding affinity.Therefore,in the next step,when we study the effect of C10 on the Jak/Stat3 signaling pathway,we will examine the effect of C10 on the expression level of Jak2.3.C10 regulates the Jak / Stat3 signaling pathway and inhibits the proliferation of HCT116 and HT29 cells.Phosphorylation levels of Jak/Stat3 signaling pathway-related signaling molecules Jak2,PI3 K,Akt,m TOR,and Stat3 were detected by Western blot.The results showed that the phosphorylation level of signaling pathway-related signaling molecules gradually decreased with the increase of the concentration of C10.Subsequently,the expression of these five signal molecule genes was detected by q PCR.The results showed that the expression of genes gradually decreased with the increase of C10 concentration.Subsequently,the fluorescence intensity of phosphorylated Stat3 was detected by immunofluorescence.It was found that the fluorescence intensity of phosphorylated Stat3 gradually decreased with the increase of the concentration of C10,which was consistent with the results of western blot.Previous literature reported that PIAS3 is an upstream signal molecule of Stat3,which can regulate the phosphorylation expression of Stat3.We detected PIAS-3 expression by western blot and q PCR.The results showed that the expression of PIAS-3 protein and gene gradually increased with the increase of C10 action concentration,which indicated that C10 further inhibited the phosphorylation of Stat3 by up-regulating the expression of PIAS-3.First stimulated with a certain concentration of IL-6 and then treated with C10.The two cells were tested for the expression of phosphorylated Jak2 and phosphorylated Stat3 genes by western blot and q PCR.The results showed that the expression levels of phosphorylation of the two signal molecules after IL-6 treatment were increased compared with the control group.After IL-6 stimulation,80μg/m L C10 effected the phosphorylation level of these two signal molecules.The gene had the same trend as the protein.This indicates that C10 can reverse the expression of genes and proteins of Jak2 and Stat3 stimulated by IL-6.The effect of C10 on the proliferation of HCT116 and HT29 cells was detected by RNA interference Jak2 gene.The results showed that compared with the control group,the cell viability gradually decreased with the increase of the action concentration.However,compared with the effect of C10 treated,its effect on cell proliferation activity is not very obvious.In conclusion,C10 regulates colon cancer cell HCT116 and HT29 cell proliferation and cycle arrest by mediating the Jak/Stat3 signaling pathway.It provides new prospects for the study of new anthraquinone derivatives.