Drug Sensitivity Studies Of HEK-293-Twist ,a Inducible Twist Expression Cell Line

bjective: To further explore the drug resistance promoting mechanism of Twist gene by observing the chemosensitivity of trichostatin A （TSA）, 5-fluorouracil （5Fu） and ,paclitaxel （Taxol） in HEK-293-Twist cell lines ,and analysing the differential expression of Twist,E-Cadherin and several drug resistance associated proteins,including multidrug resistance-associated protein（MRP）,p-glycoprotein（p-gp）,lung resistance-related protein（LRP）,TopoisomeraseII（TOPOII） in HEK-293-Twist and HEK-293-vector .Methods:（1）HEK-293 cells with a Flp-In-T-REx system were transfected with the pcDNA5/FRT/TO empty vector and the vector containing the full-length hTWIST cDNA, respectively. Cells were cultured in medium containing 5μg/ml blasticidin and 200μg/ml hygromycin B for 10 days. The surviving cells were expanded and treated with 0.1μg/ml of DOX to examine inducible expression of F-TWIST. The cells with empty vector were treated in the same way to serve as a control. Proteins of calculated HEK-293-Twist and HEK-293-vector cells were extracted after tetracycline treating （ 2h,4h,6h,8h,10h and 12h respectively） , and then analyzed by western blot to detect the expression levels of Twist.（2） Cell Counting Kit-8（CCK-8） assay was used to observe the differential drug sensitivity of trichostatin A （TSA）, 5-fluorouracil （5Fu） and ,paclitaxel （Taxol） in HEK-293-Twist and HEK-293-vector cell lines.Cultured HEK-293-Twist and HEK-293-vector cells were divided into control group,taxol group,TSA group and 5Fu group, each group had 3 parallel wells. All cells were cultured with DMEM contining 10% fetal serum in 5% CO₂ at 37℃for 2days,then the medium in test groups was exchanged by medium containing taxol,TSA and 5Fu respectively in different concentrations . After two day’s culture,CCK-8 were added to every groups and incubated for 4 hours in dark. O.D. value at 450nm was detected by Microplate Reader .（3） The differential expression of Twist,TOPOII,MRP,LRP and P-gp genes in HEK-293-Twist and HEK-293-vector was investigated by RT-PCR. Total RNA of calculated HEK-293-Twist and HEK-293-vector cells were extracted by using total RNA extraction kit. The concentration and purity of total RNA were calculated according to the values of OD260/OD280. The total RNA was transcribed reversely into cDNA by reverse transcriptase kit with random primers,and then the Twist gene was selectively amplified in vitro by RT-PCR with its specific primers which were fund in PrimerBank and GeneBand.The standardization was calibrated by GAPDH gene as an internal control. The data were compared and analyzed by gel imaging systems, Quantity One software and fluorescense and chemiluminescence’s analyzer.（4） Correlation of Twist and TOPOII, MRP, LRP,P-gp, E-Cadherin expression in HEK-293-Twist was investigated by Immunohistochemical method. HEK-293-Twist and HEK-293-vector cells were cultured with DMEM contining 10% fetal serum in 5% CO₂ at 37℃for 3days,and then detected by MRP,p-gp,LRp,TOPOIIand E-Cadherin antibodies respecitively following SABC method .（5）The correlation of Twist and E-Cadherin protein expression in HEK-293-Twist was investigated by Western Blot. HEK-293-Twist and HEK-293-vector cells were cultured with DMEM contining 10% fetal serum in 5% CO₂ at 37℃for 3days.Proteins of calculated cells were extracted by using total protein extraction kit, then detected by WB. Results:（1） WB resulte showed that Twist was successfully transfected and expressed stably in HEK-293-Twist cells.After Inducing for 6h,the expression level of Twist come to peak （2）CCK-8 assay result indicated that the growth inhibition of HEK-293-Twist cells by Taxol and 5Fu was weaker than that of HEK-293-vector cells（p<0.05）, suggesting that HEK-293-Twist cells was relatively insensitivity to Taxol and 5Fu compared with HEK-293-vector .（3） RT-PCR result indicated that Twist、TOPOII、MRP、LRP、P-gp and E-Cadherin genes expressed in both of HEK-293-Twist and HEK-293-vector cells, but the expression level of Twist、LRP and P-gp in HEK-293-Twist cells was higher than that in HEK-293-vector cells（p<0.05）.（4）Immunostaining results indicated that Twist、LRP and P-gp proteins expression were higher in HEK-293-Twist cells than that in HEK-293-vector cells ,but the expression of E-Cadherin protein in HEK-293-vector cells was higher than that in HEK-293-Twist cells .（5）Western blot result showed that the expression level of E-Cadherin protein in HEK-293-vector cells was higher than that in HEK-293-Twist cells.Conclutions:（1）A inducible Twist expression cell line, HEK-293-TWIST was constructed successfully.（2）HEK-293-Twist cells with high expression level of Twist accqures drug-resistance to Taxol and 5FU to some extant.（2）The high expression level of Twist is consistent with drug resistance-related protein including LRP and P-gp proteins ,which may provide a possible mechanism of drug resistance promoting function of Twist.（3）The expression level of Twist is negative correlation to E-Cadherin in HEK-293-Twist,suggesting that Twist can promates durg resistance and metastasis synchronously.