The Study On The Discovery Of STAT3 Inhibitors In Jatamansi’s Epoxy-iridoid-Ester And Underlying Its Anti-cancer Activity

Objective To discover novel inhibitors of STAT3 from herbs jatamansi,and investigate the potential anti-cancer activities as well as underlying molecular mechansims of epoxy iridoid ester(EIE)class Valtrate in pancreatic cancer cells.To provide a scientific basis for rational designing the leading compounds of anti-pancreatic cancer.Methods1.To use of the STAT3 luciferase reporter gene technology screening the five compounds in purified from jatamansi and detecting whether STAT3 transcription inhibitory activity.2.To research EIE class of STAT3 inhibitors on human pancreatic cancer cell line inhibition by MTT assay and to investigate the relations of pancreatic cancer cell growth inhibition with the exposure time and concentration.3.Annexin V/FITC-PI staining assay and flow cytometry were used to study EIE class of STAT3 inhibitors’s apoptosis on PANC-1 cell(human pancreatic cancer).4.To use of the RT-PCR assay detected Valtrate on expression of apoptosis related genes Bcl-2,Bax and other target genes in STAT3 signaling pathway in PANC-1 cell.5.Western blot assay was performed to investigate the expressions of apoptosis related proteins Bcl-2,Bax and the expression of proteins in STAT3 signaling pathway.6.Western blot assay was performed to investigate the expressions of PANC-1 cell proteins in STAT3 signaling pathway by antioxidants NAC intervention Valtrate on it.7.UPLC-ESI-MS were used to study EIE class of STAT3 inhibitors activity of binding with GSH,to preliminary study on its molecular mechanism.Results1.STAT3 Luciferase reporter assay results showed that the five compounds in purified from jatamansi we have been selected to epoxy iridoid ester(EIE)compounds Valtrate and 1-Homoisovaltrate capable of inhibiting STAT3 transcriptional activity.2,MTT assay results showed that EIE class of STAT3 inhibitor Valtrate and 1-Homoisovaltrate have significant antiproliferative effect on a panel of three human pancreatic cancer cells in vitro,such as PANC-1,BXPC-3 and ASPC-1.The cells with Valtrate for 48h,IC50 was 1.21,2.52,1.07μg/mL,the cells with 1-Homoisovaltrate for 48h,IC50 was 17.17,1.34,0.80μg/mL.Both of the two compounds could inhibit the pancreatic cancer cells based on a time-and concentration-dependent manner.3.Annexin V/FITC-PI staining assay and flow cytometry results showed that Valtrate can induce human pancreatic cancer cell PANC-1 apoptosis.The cells were treated with different concentrations(5,10,20μg/mL)of Valtrate for 48h,the percentage of apoptotic cells rate were 28.33±3.63%,39.71±3.01%and 54.62±6.22%.The cells were treated with different time by the highest dose of Valtrate(20μg/mL),the percentage of apoptotic cells rate were 20.04±0.99%,27.12±1.44%and 40.05±2.74%.Both of them have based on a time-and concentration-dependent manner.4.RT-PCR assay results showed that Valtrate can up-regulation of proapoptotic gene Bax expression in PANC-1 cells,down-regulation the expression of anti-apoptotic gene Bcl-2 and other STAT3 signaling pathway genes such as STAT3,c-Myc,Cyclin D1.Both of them have based on a time-and concentration-dependent manner.5.Western blot assay results showed that Valtrate can up-regulation of proapoptotic protein Bax expression in PANC-1 cells,down-regulation the expression of anti-apoptotic protein Bcl-2 and other STAT3 signaling pathway proteins such as STAT3,c-Myc,Cyclin D1,inhibition of phosphorylation levels of STAT3.Both of them have based on a time-and concentration-dependent manner.6.NAC inversion results showed that thiol heterosexual antioxidants NAC could reverse that Valtrate down-regulation the expression of proteins such as c-Myc,Cyclin D1 and phosphorylation levels of STAT3 in PANC-1 cells.7.UPLC-ESI-MS detection results showed that EIE class of STAT3 inhibitors Valtrate and 1-Homoisovaltrate have activity to bind with GSH.Conclusion1.It have benn screened two new EIE class of STAT3 inhibitors from jatamansi that were Valtrate and 1-Homoisovaltrate.2.The EIE class of STAT3 inhibitors’s mechanism of anti-pancreatic cancer activity maybe are Valtrate induced the formation of inactive STAT3 dimers then by induced degradation STAT3 dimers to down-regulation the expression of the total STAT3 while could inhibit the phosphorylation levels of STAT3.On the basis of influence on STAT3 signaling pathway Valtrate could regulate the target-proteins intervente by STAT3 such Bcl-2,STAT3,c-Myc,Cyclin D1 which inducing PANC-1 cell apoptosis and inhibiting the pancreatic cancer cells ultimately.3.Valtrate,the EIE class of STAT3 inhibitor’s molecular mechanism of inhibiting transcriptional activity of STAT3 maybe are its structure of "Three oxygen ring" can bind to critical cysteine residue on the specific domains of STAT3 proteins and alkylate the modified protein;Valtrate can induce the formation of inactive STAT3 dimers to down-regulation the expression of the total STAT3.After Valtrate alkylate the modified STAT3 protein also can inhibit the phosphorylation levels of STAT3 to inhibite transcriptional activity of STAT3 ultimately.