Sensitivity of docetaxel-resistant MCF-7 breast cancer cells to microtubule-destabilizing agents including vinca alkaloids and colchicine-site binding agents

Abstract

Introduction: One of the main reasons for disease recurrence in the curative breast cancer treatment setting is the development of drug resistance. Microtubule targeted agents (MTAs) are among the most commonly used drugs for the treatment of breaset cancer and therefore overcoming taxane resistance is of primary clinical importance. Our group has previously demonstrated that the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitivity to docetaxel due to the distinct expression profiles of β-tubulin isotypes in addition to the high expression of p-glycoprotein (ABCB1). In the present investigation we examined whether taxane-resistant breast cancer cells are more sensitive to microtubule destabilizing agents including vinca alkaloids and colchicine-site binding agents (CSBAs) than the non-resistant cells.

Methods: Two isogenic MCF-7 breast cancer cell lines were selected for resistance to docetaxel (MCF-7TXT) and the wild type parental cell line (MCF-7CC) to examine if taxane-resistant breast cancer cells are sensitive to microtubule-destabilizing agents including vinca alkaloids and CSBAs. Cytotoxicity assays, immunoblotting, indirect immunofluorescence and live imaging were used to study drug resistance, apoptosis, mitotic arrest, microtubule formation, and microtubule dynamics.

Results: MCF-7TXT cells were demonstrated to be cross resistant to vinca alkaloids, but were more sensitive to treatment with colchicine compared to parental non-resistant MCF-7CC cells. Cytotoxicity assays indicated that the IC50 of MCF-7TXT cell to vinorelbine and vinblastine was more than 6 and 3 times higher, respectively, than that of MCF-7CC cells. By contrast, the IC50 of MCF-7TXT cell for colchincine was 4 times lower than that of MCF-7CC cells. Indirect immunofluorescence showed that all MTAs induced the disorganization of microtubules and the chromatin morphology and interestingly each with a unique pattern. In terms of microtubule and chromain morphology, MCF-7TXT cells were more resistant to vinorelbine and vinblastine, but more sensitive to colchicine compared to MCF-7CC cells. PARP cleavage assay further demonstrated that all of the MTAs induced apoptosis of the MCF-7 cells. However, again, MCF-7TXT cells were more resistant to vinorelbine and vinblastine, and more sensitive to colchicine compared to MCF-7CC cells. Live imaging demonstrated that the microtubule dynamics of MCF-7TXT cells were less sensitive to vinca alkaloids, and more sensitive to colchicine. MCF-7TXT cells were also noted to be more sensitive to other CSBAs including 2MeOE2, ABT-751 and phosphorylated combretastatin A-4 (CA-4P).

Conclusion: Docetaxel-resistant MCF-7TXT cells have demonstrated cross-resistance to vinca alkaloids, but appear to be more sensitive to CSBAs (colchicine, 2MeOE2, ABT-751 and CA-4P) compared to non-resistant MCF-7CC cells. Taken together these results suggest that CSBAs should be evaluated further in the treatment of taxane resistant breast cancer.

Fig 1. Dose-response curve to drug treatment.

Cytotoxicity of docetaxel, vinorelbine, vinblastine and colchicine at various concentrations for MCF-7_CC and MCF-7_TXT cells was determined following a 24 h treatment with (A) docetaxel; (B) vinorelbine. (C) Treatment with vinblastine. (D) Treatment with colchicine. (E) IC₅₀s calculated from A-D. The means of at least three independent experiments are plotted.Error bars are the standard errors.

Fig 2. The effects of docetaxel on the microtubule formation, M-phase arrest and cell apoptosis in the selected MCF-7 cell lines.

MCF-7_CC and MCF-7_TXT cells were seeded on coverslip and treated with docetaxel of indicated concentrations for 24 hours. The microtubule formation, M-phase arrest and cell apoptosis were determined by fluorescence microscopy. The red indicates the α-tubulin and the blue is the DAPI stain for DNA. Size bar: 10 μm.

Fig 3. The effects of vinca alkaloids on the microtubule formation, M-phase arrest and cell apoptosis in the selected MCF-7 cell lines.

MCF-7_CC and MCF-7_TXT cells were seeded on coverslip and treated with vinorelbine or vinblastine of indicated concentrations for 24 hours. The microtubule formation, M-phase arrest and cell apoptosis were determined by fluorescence microscopy. The red indicates the α-tubulin and the blue is the DAPI stain for DNA. Size bar: 10 μm.

Fig 4. The effects of colchicine on the microtubule formation, M-phase arrest and cell apoptosis on the selected MCF-7 cell lines.

MCF-7_CC and MCF-7_TXT cells were seeded on coverslip and treated with colchicine of indicated concentrations for 24 hours. The microtubule formation, M-phase arrest and cell apoptosis were determined by fluorescence microscopy. The red indicates the α-tubulin and the blue is the DAPI stain for DNA. Size bar: 10 μm.

Fig 5. The effects of docetaxel, vinorelbine, vinblastine and colchicine on the apoptosis of MCF-7_CC and MCF-7_TXT cells.

The apoptotic cells were determined by chromatin condensation and nuclear morphology as revealed by DAPI stain in Figs 2–4. Quantification of cell apoptosis was expressed as the percentage of the apoptotic cells of the total cells. For each data, 300 cells from at least three independent experiments were examined. Error bars are the standard errors.

Fig 6. The effects of docetaxel, vinorelbine, vinblastine and colchicine on the cleavage of PARP in selected MCF-7 cell lines.

MCF-7_CC and MCF-7_TXT cells were treated with various drugs at indicated concentration for 24 h. (A) The cleavage of PARP in MCF-7_CC and MCF-7_TXT cells was determined by immunoblotting with antibody to PARP. (B) Quantification of the results from three independent experiments as described in panel A. The intensity of the bands of various tubulin proteins was normalized against the intensity of the actin loading. Error bars are the standard errors. Statistical analysis with paired t-Test indicated that the difference between MCF-_7CC and MCF-7_TXT cells was statistically significant with p<0.01 for all four drugs including docetaxel, vinorelbine, vinblastine, and cochicine.

Fig 7. The effects of MTAs (docetaxel, vinorelbine and colchicine) on the microtubule dynamics of MCF-7_TXT an MCF-7_CC cells.

The Live imaging was performed following the transfection of the cells with GFP-tagged α-tubulin for 24 h. Cells were incubated with various MTAs at 2μM for 2 h. Live images of microtubule dynamics of MCF-7_CC and MCF-7_TXT cells were recorded every 3 min. Selected images from the live imaging (S1–S6 Videos) of MCF-7_CC and MCF-7_TXT cells were shown. Arrow indicates the crystallization of tubulins. Size bar, 10 μm.

Fig 8. Dose-response curve to CSBA treatment.

Cytotoxicity of CA-4P, ABT-751, and 2MeOE₂ for MCF-7_CC and MCF-7_TXT cells was determined at various concentrations following 24 h drug treatment. Treatments with CA-4P (A), ABT-751 (B), 2MeOE₂ (C) are shown IC₅₀s calculated from A-C are given in (D). The means of at least three independent experiments are plotted. The error bars are the standard errors.

Fig 9. The effects of CA-4P, ABT-751, and 2MeOE₂ on the microtubule formation, M-phase arrest and cell apoptosis in the selected MCF-7 cell lines.

MCF-7_CC and MCF-7_TXT cells were seeded on coverslip and treated with CA-4P, ABT-751, and 2MeOE₂ of indicated concentrations for 24 h. Microtubule formation, M-phase arrest and cell apoptosis were determined by fluorescence microscopy. The red staining indicates the α-tubulin and the blue is the DAPI stain for DNA. Size bar: 10 μm.