Screening And Anti-tumor Mechanism Of Targeted JAK2 Inhibitor

JAK-STAT signaling pathway plays a critical role in mediating cytokine and growth factor response.Downstream genes of JAK-STAT signaling pathway such as C-myc,Cyclin D1,Survivin,Mcl-1,etc.are essential in the regulation of physiological processes such as cell survival,proliferation and apoptosis and so on.Delicate regulation of this pathway is very important for the maintenance of normal cell physiological function.Dysregulation of components in JAK-STAT signaling are closely related to pathogenesis and progression of many cancers.In JAK-STAT3 signaling dependent solid tumors,JAK2 is necessary for the activation of STAT3.Hyperactivated JAK2 mutations have been extensively reported in patients with hematopoietic proliferative diseases such as polycythemia vera,idiopathic thrombocythemia,primary myelofibrosis and some leukemia cases.JAK2 inhibitors have been used in the treatment of myeloproliferative neoplasm in clinical,and JAK2 inhibitor in the treatment of solid tumors is under clinical trial.However,some defects of JAK2 inhibitors are observed in clinical,including drug resistance,side effects and so on.With the development of the applications of combined therapy in cancer treatment,different drug combinations may provide stronger efficacy but also may lead to greater side effects.The principle of drug screening is to restore a certain amount of lead compounds,and then select the best.Therefore,the development of new JAK2 inhibitors will provide more choices for clinical application.In this study,bioinformatics methods as well as molecular and cell biology experimental methods were integrated,and computer-aided virtual screening for JAK2 inhibitors was performed.A novel natural product inhibitor of JAK2 Dehydrocrenatidine was discovered,and the inhibitory effects of Dehydrocrenatidine on JAK-STAT signaling pathway in solid tumor and leukemia cells were studied at cellular and molecular levels.In this study,we screened 1062608 drug-like compounds from ZINC database and 2080 natural products from small molecule compound library from National Compound Resource Center(Shanghai,China)using UCSF DOCK software.Then we re-screened top ranking 10,000 compounds by Autodock software.Biological activity of the four highest ranking small molecules was studied.Western blotting results showed that one of the small molecule compounds from natural products library Dehydrocrenatidine inhibited JAK2 phosphorylation level in JAK-STAT3 persistently activated DU145 cells.To further elucidate the inhibition mechanism of Dehydrocrenatidine on JAK-STAT3 signaling pathway,first we studied the selective inhibition role of Dehydrocrenatidine on JAK-STAT3 signaling dependent cells.We chose JAK-STAT3 signaling dependent prostate cancer cells DU 145 and breast cancer cells MDA-MB-468,JAK-STAT3 signaling independent normal cells hTERT-BJ and MCF 10A were used as control.Cell viability was tested by MTT assay.We found that Dehydrocrenatidine exhibited similar characteristics with known JAK2 inhibitors AG490 and AZD1480,and selectively inhibited survival of DU 145 and MDA-MB-468 cells,but did not show significant inhibitory effects on normal cells hTERT-BJ and MCF 10A.At the molecular level,we used Western blot method to investigate the inhibitory role of Dehydrocrenatidine on JAK-STAT3 signaling pathway constitutively activated DU 145 and MDA-MB-468 cells,and cytokine induced JAK-STAT activity on HeLa,HepG2 and HEK293T cells,in which basal level of JAK-STAT3 signaling activity is relatively low.The results showed that Dehydrocrenatidine inhibited persistent JAK2 and STAT3 activity in DU 145 and MDA-MB-468 cells in a dose-and time-dependent manner.Dehydrocrenatidine also inhibited cytokines IL-6-and IFNs-induced JAKs(JAK1,JAK2 and TYK2)and STATs(STAT1,STAT2 and STAT3)activity.In order to investigate whether Dehydrocrenatidine could inhibit other signaling pathways,we examined the activity of two other signaling pathways that are closely related with tumor pathogenesis:PI3K-AKT and NF-?B.We found that Dehydrocrenatidine did not reduce the activity of key components of these two pathways,AKT and p65,whereas their positive inhibitors significantly inhibited their activity respectively.JAK-STAT signaling pathway has a regulation role on downstream gene transcription.Therefore we examined whether Dehydrocrenatidine has an inhibitory role on JAK-STAT signaling pathway downstream gene transcription.RT-qPCR results showed that in HeLa and HepG2 cells which possess low STAT3 activity,Dehydrocrenatidine inhibited IL-6-induced STAT3 specific downstream gene socs3 transcription.Moreover,Dehydrocrenatidine inhibited the transcription of IFN-a induced STAT1 specific downstream gene in HeLa cells.In order to study the anti-tumor mechanism of Dehydrocrenatidine,we tested whether Dehydrocrenatidine induces apoptosis of DU145 and MDA-MB-468 cells.Hochest staining and Annexin V-PI double staining flow cytometry results showed that Dehydrocrenatidine significantly induced apoptosis of JAK-STAT3 signaling dependent solid tumor cells DU 145 and MDA-MB-468.In order to verily whether Dehydrocrenatidine directly inhibits the JAK kinase activity,we constructed JAKs-JH1 overexpressing cell lines.The results showed that Dehydrocrenatidine inhibited JAK tyrosine kinase domain overexpression induced total tyrosine phosphorylation as well as STAT3 and STAT1 phosphorylation.However,Dehydrocrenatidine had little inhibitory effect on EGF-induced EGFR and STAT3 activity and Src overexpression induced Src and STAT3 activity.In Vitro kinase assays demonstrated that Dehydrocrenatidine directly inhibited the catalytic activity of JAK2-JH1 kinase domain.In addition to the study of solid tumor cells,we primarily explored the inhibitory effect of Dehydrocrenatidine on JAK2-dependent leukemia cells.In HEL cell line that contains activating mutation JAK2V617F,Dehydrocrenatidine significantly inhibited the sustained activation of JAK2,STAT3 and STAT5 and inhibited the survival of HEL cells and promoted apoptosis.This study first reported that Dehydrocrenatidine is a novel JAK inhibitor.Our data proved that Dehydrocrenatidine selectively inhibited JAK-STAT signaling pathway at the cellular and molecular level and provided a theoretical basis for Dehydrocrenatidine to be developed as an anti-cancer drug.Pharmacological studies have shown that bitter wood extract has anti-bacterial,anti-inflammatory and anti-tumor effects.This study suggested the possible molecular mechanism of the anti-tumor effect of bitter wood extract and offered new knowledge of the medicinal value of bitter wood plant resources.