Chrysin Directly Disrupts Hsp90/Cdc37 Complex And Inhibits Pancreatic Cancer Cell Growth

Pancreatic cancer is a lethal digestive system carcinoma,with the overall 5-year survival of 4%.The underlying mechanism is rather complex.The treatment regimens for pancreatic cancer have no substantial improvement over the past few decades.Thus,novel agents for treatment of pancreatic cancer are highly desired.Due to the critical role of molecular chaperone Hsp90(90 kDa heat shock protein)in regulating the stability,activity and sorting of its client proteins involved in multiple oncogenic progress,Hsp90 inhibitors are promising therapeutic agents for cancer treatment.The co-chaperone Cdc37(cell division cycle protein 37)is an important partner for Hsp90,assisting in molecular chaperone activities with regard to the regulation of protein kinases,many of which are oncogenic proteins in cancer cells.The crucial role of the Hsp90/Cdc37 complex in regulation of protein kinase has made it a novel target for cancer treatment.Therefore,screening small molecule inhibitors targeting Hsp90/Cdc37 interaction might be a promising strategy for developing novel cancer therapeutics.In our previous study,we utilized a newly developed method split synthetic Renilla luciferase protein-fragment-assisted complementation(SRL-PFAC)to study Hsp90/Cdc37 interaction in HEK293 T cells.We also proved that SRL-PFAC is sensitive and specific to quantitatively monitor Hsp90/Cdc37 interactions in HEK293 T cells,thus providing a rationale to screen Hsp90/Cdc37 inhibitors.In this study,we selected 9 flavonoids with reference to spatial structure of binding site of Hsp90/Cdc37,then screened these candidate compounds using SRL-PFAC bioluminescence method.We hypothesized there were potential specific inhibitors of Hsp90/Cdc37 among these candidate compounds.To verify our hypothesis,we co-transfected equal molar of pcDNA3.1(+)-NRL-Hsp90α and pcDNA3.1(+)-Cdc37-CRL plasmids into HEK293 T cells,then treated the cells with candidate compounds for 24 h respectively.As a result,we found that natural flavonoid chrysin reduced the complemented NRL-Hsp90α/Cdc37-CRL activity in a concentration dependent manner.In addition,the false positive assay data showed that chrysin did not show any inhibitory effect on full length Renilla luciferase’s catalytic activity compared to non-treated group,indicating that chrysin directly disrupted Hsp90/Cdc37 complex.And then,we further utilized SRL-PFAC bioluminescence method to further verify if chrysin is indeed specific inhibitors of Hsp90/Cdc37.In order to verify the inhibitory effect of chrysin on pancreatic cell lines,we treated AsPc-1,Bx Pc-3,CFPAC-1,HPAC,MIAPaCa-2 and PANC-1 with chrysin for 72 h respectively.The MTS assay data showed that chrysin inhibited the growth of these pancreatic cell lines.Taken together,we verified 9 flavonoids as potential Hsp90/Cdc37 inhibitors on the basis of SRL-PFAC method,and finally found that chrysin directly disrupted Hsp90/Cdc37 complex.Then we proved that chrysin also inhibited the growth of 6 pancreatic cell lines.This study may provide proof to the development of novel Hsp90/Cdc37 inhibitors.