The Mechanism Research On HER2-positive Esophagus Cancer Cell OE19 Resistance To Hsp90 Inhibitor 17-AAG

Objective : To identify target proteins associated with the development of 17-AAG-acquired resistance,we cultivated 17-AAG-acqui-red resistance OE19(OE19R)cells,investigated the signal trans-duction difference between parental OE19 and OE19 R cells accord-ing to protein microarray and hoped to catch the strategy of ov-ercoming 17-AAG and similar Hsp90 inhibitor resistance.Methods:(1)MTT assay was used to assess IC50 value of 17-AA in HER2-positive esophagus cancer cell OE19.The 17-AAG-resistant OE19 cell line,OE19 R,was established by long-term exposure of cells to 17-AAG.(2).Western blot was applied to delineate the contrast of proliferous signaling pathway-PI3K/AKT/m TOR and Ras/Raf/ERK in two previously mentioned cells.(3)Immunohistochemistry was used to test the P-AKT and P-ERK expression of xenograft tumor in nude mice.(4)The expression difference of Receptor Tyrosine Kinase(RTK)was tested in Human RTK Phosphorylation Antibody Array.(5)Western Blot and q RT-PCR was applied to verified the result of protein microarray.(6)Target protein selective inhibitor was choosed to demonstrate its impact on drug resistance.(7)Lipofectamine 3000 Reagent and the plasmid of target gene was used to overexpress the target protein of OE19 for proving its functions.Results:(1)17-AAG-acquired resistance cell(OE19R)was established with IC50 twenty-eight higher than parental OE19.(2)Western Blot detected that the expression of P-AKT and P-ERK was higher in OER.(3)Immunohistochemistry result revealed the expression P-AKT and P-ERK was up-regulation in xenograft tumor of nude mice.(4)The expression variation of RTKs was scaned by protein microarray,and the FYN was seleced to follow-up study.(5)The level of FYN was verified to express higher in OER by Western Blot and q RT-PCR.(6)Based on the speculation about the relationship among FYN,P-AKT and P-ERK,the selected inhibitor of FYN,PP2,was applied to OER,and which decreased the P-AKT and P-ERK remarkably.Then PP2 suppressed the proliferation of OER with 17-AAG in synernetic manner which restored the sensibility of OE19 to 17-AAG to a degree.(7)Overexpressed FYN could turn OE19 into the characteristic of OE19 R obvioiusly.Conclusion: The mechanism of FYN inducing 17-AAG-acquired resistance was preliminary found by protein microarray.In addition,we further discovered that OE19 not only resisited to 17-AAG but also to other similar Hsp90 inhibitor to some extent,such as STA-9090(Ganetespib),which implied the universality in the mechanism of Hsp90 inhibitor resistance.In a word,this research could provided a new strategy for reduceing 17-AAG and similar Hsp90 inhibitor resistance in the basic research and clinical application.