Class III beta-tubulin expression and in vitro resistance to microtubule targeting agents

Abstract

Background: Class III beta-tubulin overexpression is a marker of resistance to microtubule disruptors in vitro, in vivo and in the clinic for many cancers, including breast cancer. The aims of this study were to develop a new model of class III beta-tubulin expression, avoiding the toxicity associated with chronic overexpression of class III beta-tubulin, and study the efficacy of a panel of clinical and pre-clinical drugs in this model.

Methods: MCF-7 (ER+ve) and MDA-MB-231 (ER-ve) were either transfected with pALTER-TUBB3 or siRNA-tubb3 and 24 h later exposed to test compounds for a further 96 h for proliferation studies. RT-PCR and immunoblotting were used to monitor the changes in class III beta-tubulin mRNA and protein expression.

Results: The model allowed for subtle changes in class III beta-tubulin expression to be achieved, which had no direct effect on the viability of the cells. Class III beta-tubulin overexpression conferred resistance to paclitaxel and vinorelbine, whereas downregulation of class III beta-tubulin rendered cells more sensitive to these two drugs. The efficacy of the colchicine-site binding agents, 2-MeOE2, colchicine, STX140, ENMD1198 and STX243 was unaffected by the changes in class III beta-tubulin expression.

Conclusion: These data indicate that the effect of class III beta-tubulin overexpression may depend on where the drug's binding site is located on the tubulin. Therefore, this study highlights for the first time the potential key role of targeting the colchicine-binding site, to develop new treatment modalities for taxane-refractory breast cancer.

Figure 1

Structures. I: 2-MeOE2, II: STX140, III: STX243, IV: paclitaxel, V: vinorelbine, VI: ENMD1198 and VII: colchicine.

Figure 2

Class III β-tubulin mRNA and protein expression. (A) MCF-7 and MDA-MB-231 cells were transfected with the mock vector or the non-targeting siRNA (control), pALTER-TUBB3 or siRNA-tubb3 and the expression of class III β-tubulin mRNA was quantified by RT–PCR. Three or four independent experiments (n=3–4) were carried out in duplicate and the results presented are the average of the Ct values relative to the control Ct values, bars: mean±s.d., ^*P<0.05 and ^**P<0.01. The expression of class III β-tubulin protein, total β-tubulin and total α-tubulin was analysed in (B) MCF-7 and (C) in MDA-MB-231 24 h and 5 days after transfection by SDS–PAGE and immunoblotting. (1) Control 24 h, (2) pALTER-TUBB3 24 h, (3) siRNA-tubb3 24 h, (4) control 96 h, (5) pALTER-TUBB3 96 h and (6) siRNA-tubb3 96 h.

Figure 3

STX140, STX243, 2-MeOE2, paclitaxel, ENMD1198, vinorelbine and colchicine chemosensitivity in MCF-7 cells. MCF-7 cells were transfected with the mock vector or the non-targeting siRNA (control), (A) pALTER-TUBB3 or siRNA-tubb3 and exposed to STX140, (B) STX243, (C) paclitaxel, (D) 2-MeOE2, (E) ENMD1198, (F) vinorelbine or (G) colchicine for 4 days. The percent proliferation was determined by AlamarBlue assay and results presented are the average of three or four independent experiments (n=3–4) done in triplicate, ^*P<0.05 and ^**P<0.01.

Figure 4

STX140, STX243, 2-MeOE2, paclitaxel, ENMD1198, vinorelbine and colchicine chemosensitivity in MDA-MB-231 cells. MDA-MB-231 cells were transfected with the mock vector or the non-targeting siRNA (control), (A) pALTER-TUBB3 or siRNA-tubb3 and treated with STX140, (B) STX243, (C) paclitaxel, (D) 2-MeOE2, (E) ENMD1198, (F) vinorelbine or (G) colchicine for 4 days. The percent proliferation was determined by AlamarBlue assay and results presented are the average of three or four independent experiments (n=3–4) done in triplicate, ^*P<0.05, ^**P<0.01 and ^***P<0.001.

Figure 5

Fluorescent images of cells overexpressing class III β-tubulin after drug exposure. MCF-7-control, MCF-7-TUBB3, MDA-MB-231-control and MDA-MB-231-TUBB3 cells were treated for 48 h with STX140, paclitaxel or colchicine at concentrations close to IC₅₀ values obtained in control cells and stained with FITC-anti-tubulin (tubulin) and Hoechst 33342 (nucleus). Images were taken using a Zeiss inverted microscope at × 200 magnification.