Establishment Of Paclitaxel-resistant Colon Cancer Cell Line LoVo/Taxol And Studies On Its Resistance Mechanisms

Subjective To establish a paclitaxel resistant cell line Lo Vo/Taxol and to explore the potential molecular mechanism of paclitaxel resistance in colon cancer.Methods The cell line LoVo was cultured by gradually increasing dose of Taxol to generate its resistant cell line LoVo/Taxol.Drug resistance of LoVo and LoVo/Taxol was determined using MTT assay,the morphological features were observed by inverted microscope and transmission electron microscope,the distribution of their cell cycles and apoptosis rate were detected by flow cytometry,the mRNA levels of Mdr1、Mrp1、GST-π、LRP、TopoⅡ、TUBB3、Cdk1、Tau、Bcl-2、TP53 were assessed by Real-time PCR,the protein expression level of MRP1、Bcl-2,TUBB3、Cdk1 and Tau were detected by Western blotting.Results(1)A paclitaxel-resistant human colon cancer cell line,LoVo/Taxol,was successfully established.Compared to the parent cell line,it was 42.32-fold resistant to paclitaxel,it also exhibited high-level cross-resistance to Doxorubicin(ADM)and 5-fluorouracil(5-FU).(2)It was observed that the cell morphology and structure had also changed,and the adhesion between the resistant cells was significantly less than that of the parent cells.The cytoplasmic ratio was slightly smaller and the mitochondria were significantly increased.(3)Moreover,the proportion of G2/M phase decreased significantly(P < 0.05),meanwhile the apoptosis rate of LoVo/Taxol cells was also decreased significantly(P < 0.05)than that of LoVo cells after treated with 1.5umol/L Taxol for 24 h.(4)The levels of transcription and translation of Multidrug resistance protein(MRP1),microtubule-associated protein(MAP-Tau)and cyclin-dependent kinase 1(CDK1)gene in drug-resistant cell lines significantly increased that detected by q RT-PCR and Western blot.Meanwhile,the levels of transcription and translation of β-tubulin-3 and B-lymphoblastoma-2(Bcl-2)genes were significantly decreased.Conclusion A paclitaxel-resistant human colon cancer cell line,LoVo/Taxol,has been successfully established,which might play a role for mechanism study of paclitaxel-resistance.Overexpression of the multidrug resistance-associated protein MRP1 、 microtubule-associated protein(MAP)-Tau as well as Cyclin-dependent kinase 1(CDK1)might contribute to the acquired paclitaxel resistance in colon cancer cell.