Preliminary Study Of Twist On Multidrug Resistance And It’s Possible Mechanism

Objective:To preliminary explore the effects of Twist on the cell multidrug resistance and it’s possible molecular mechanism by observing the chemosensitivity of HEK-293-Twist and HEK-293-Vector cell lines on trichostatin A (TSA),5-fluorouracil (5-Fu) and paclitaxel (Taxol) treatment and differential expression of Twist, NF-Kb, Akt and FHIT in these two cell lines using CCk-8, RT-PCR and Western blot. Methods:(1) Cell scarification test:HEK-293-Twist cells and HEK-293-vector cells were made into5×105/ml cells suspensions with DMEM containing10%fetal serum, cells were then vaccinated into6-well plate.1-2traces were made on cell monolayer in every well by sterile transferpettor after cell density reaching90%. Cells scarifications repair was observed0,7h,24h later respectively to compare the scarifications repairing speeds of different cells.(2) Cell Counting Kit-8(CCK-8) assay:Cultured HEK-293-Twist and HEK-293-vector cells were divided into control group, blank group, Taxol group, TSA group and5-Fu group, each group had6parallel wells. All cells had been cultured for24h, then the medium in test groups was exchanged by medium containing Taxol, TSA and5-Fu respectively in different concentrations. After two day’s culture, CCK-8was added to every groups and incubated for3hours in dark. IOD. value at450nm was detected by Microplate Reader.(3) RT-PCR was used to detect the expressions of Twist and FHIT gene in HEK-293-Twist and HEK-293-vector cell lines. Total RNA of calculated HEK-293-Twist and HEK-293-vector cells were extracted by using total RNA extraction kit. The total RNA was transcribed reversely into cDNA by reverse transcriptase kit with random primers, and then the Twist and FHIT gene were selectively amplified in vitro by PCR with their specific primers which were designed by Primer5.0and Olig6.0. Quantity One software was used to analyze the gray value of DNA bands.(4) total proteins in HEK-293-Twist and HEK-293-vector cell lines which were treated or untreated by taxol, TSA and5-Fu respectively for24h were extracted by using total protein extraction kit, then the expressions of Twist and NF-κB, Akt and FHIT were detected by Western Blot. The gradation values of interest proteins were analyzed by Quantity one software. Results:(1) On cell scarification test, it was found that with the culture time prolonged, both cell lines migrated to the scarification obviously, but the migratory speeds were different. The immigration of two cell lines were not different after7h cultivation, however, scarification of HEK-293-Twist was nearly closed while the scarification of HEK-293-vector cells was not obviously changed after24h cultivation compared with those after7h cultivation.(2) In CCK-8assay, we found that the growth of HEK-293-Twist and HEK-293-vector cells lines was inhibited by TSA、Taxol and5-Fu (p<0.05), but HEK-293-Twist cell lines were relatively insensitive to TSA、Taxol and5-Fu compared with HEK-293-vector cell lines. RT-PCR results indicated that the expression of Twist gene in HEK-293-Twist cell lines was significantly higher than that of HEK-293-vector cell lines (p<0.05); FHIT gene was expressed in both cell lines, but the expression level of FHIT in HEK-293-Twist cell lines was lower than that in HEK-293-vector cell lines (p<0.05). WB results indicated that the expression of Twist protein in HEK-293-Twist cell lines was high, while it was not detected in HEK-293-vector cell lines; the expressions of NF-κB, Akt protein in HEK-293-Twist were higher than those in HEK-293-vector simultaneousl; while the expression of FHIT protein in HEK-293-Twist cells was lower than that of HEK-293-vector cells. The expressions of Twist, NF-κB, Akt and FHIT in HEK-293-Twist cell lines treated by TSA, Taxol and5-Fu respectively were all higher than those of HEK-293-vector cell lines. With increasing drug concentration, positive correlation was obtained between the expressions of Twist and NF-κB, Akt (p<0.05), while negative correlation was gained between the expression of Twist and FHIT (p<0.05). Conclusions:1. Twist can enhance cell migration and decrease the chemosensitive to TSA, Taxol and5-Fu in HEK-293.2. In HEK-293-Twist, Twist might positively regulates the expression of NF-κB and Akt, while negatively regulate the expression of FHIT.3. Those conclusions suggested that NF-κB, Akt and FHIT may involved in the mechanism of multidrug resistance promotion of Twist.