Eastablishment Of A Taxol Resistant Human Mammary Adenocarcinoma Cell Subline And Correlation Analysis Of Drug Resistance And Twist Expression

Abstract:objective:To establish taxol-resistant human mammary adenocarcinoma cell subline(MCF-7/Taxol) and investigate the relationship between twist gene expression and taxol resistance dynamically. Methods:(1)A taxol-resistant human breast cancer cell subline (MCF-7/Taxol) was established by repeatedly exposing human mammary adenocarcinoma cell subline (MCF-7) to high concentrations of taxol (up to 15μg/ml). Inhibiting growth curve of MCF-7 and MCF-7/Taxol,50% inhibiting concentration (IC50) and taxol resistance index(RI) were got based on MTT assay. The morphological featuers of MCF-7 and MCF-7/Taxol were observed under light microscopy.The dynamic analysis for 4 different stages of taxol resistance MCF-7(named MCF-7/Taxol I,MCF-7/Taxol II,MCF-7/TaxolⅢ,MCF-7/TaxolIV respectively) were carried out. (2)Real-time PCR for twist gene was established as following steps:twist gene was amplified by RT-PCR and subcloned into PMD18T vector which was transformed into E.coil, then the positive recombinant plasmid was used as quantitative template to generate standard curve. GAPDH as the internal standard and Rotor-Gene 3000 series sofeware was performed to detect the level of Twist in the MCF-7. Results:(1) Taxol IC50 of MCF-7,MCF-7/TaxolⅠ, MCF-7/TaxolⅡ,MCF-7/TaxolⅢand MCF-7/TaxolIV were 3.83±0.04μg/ml,3.76±0.07μg/ml (P>0.05),10.41±0.27μg/ml (P<0.05),24.82±0.32μg/ml (P<0.01),107.05±1.79μg/ml (P<0.01) respectively. Index number of drug resistance (RI) to taxol were 0.94±0.04 (MCF-7/TaxolⅠ),2.36±0.33 (MCF-7/TaxolⅡ,P<0.05),7.51±1.23 (MCF-7/TaxolⅢ,P<0.01),28.83±1.05 ((MCF-7/TaxolⅣ,P<0.01), indicating MCF-7/TaxolⅡ,MCF-7/TaxolⅢand MCF-7/TaxolⅣshowed stable resistance to taxol. (2)The vinblastine IC50 of MCF-7 and MCF-7/TaxolⅣwere 12.86±0.34μg/ml,22.06±1.12μg/ml(P<0.05) respectively,suggesting MCF-7/TaxolIV also showed tolenrance to vinblastine. (3)The standard curve of Real-time PCR for twist indicated the linear relationship between CT(cycle threshold) and template concentration. The lowest value of copy for SYBR GREEN I for Twist was 1.The melting cure showed a peak of Twist, and the coefficient correlation of GAPDH and Twist were 0.99897and 0.99927 respectively.(4)Quantitative analysis of twist gene by Real-time PCR showed that Twist expression were 0.84±0.05 (MCF-7/TaxolⅠ, P>0.05),1.63±0.30 (MCF-7/TaxolⅡ, P<0.05),3.38±0.14 (MCF-7/TaxolⅢ,P<0.01),65.28±0.26 (MCF-7/TaxolⅣ, P<0.01) respectively. The correlation coefficient of RI and Twist expression was 0.999,P<0.05.Conclusions:(1)MCF-7/Taxol cell sublines was established successfully by 4-step taxol shock method lasting 4 months.It is an ideal model for screening drug-resistant gene and drug-reversal agent. MCF-7/Taxol was tolerant to vinblastine while showing stable resistance to taxol suggesting it had the characteristic of multidrug resistance.The expression of Twist in MCF-7 was getting higher following the tendency of acquired taxol resistance. There was significant correlation between drug resistant and Twist expression during the establishment of MCF-7/Taxol, indicating high expression of Twist is of significance for taxol resistance in MCF-7.