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Alterations in ovarian cancer cell adhesion drive taxol resistance by increasing microtubule dynamics in a FAK-dependent manner.

McGrail DJ, Khambhati NN, Qi MX, Patel KS, Ravikumar N, Brandenburg CP, Dawson MR - Sci Rep (2015)

Bottom Line: Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics.Adhesion strength correlated best with Taxol-sensitivity, and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells.FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical &Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA.

ABSTRACT
Chemorefractory ovarian cancer patients show extremely poor prognosis. Microtubule-stabilizing Taxol (paclitaxel) is a first-line treatment against ovarian cancer. Despite the close interplay between microtubules and cell adhesion, it remains unknown if chemoresistance alters the way cells adhere to their extracellular environment, a process critical for cancer metastasis. To investigate this, we isolated Taxol-resistant populations of OVCAR3 and SKOV3 ovarian cancer cell lines. Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics. These changes in microtubule dynamics coincided with faster attachment rates and decreased adhesion strength, which correlated with increased surface β1-integrin expression and decreased focal adhesion formation, respectively. Adhesion strength correlated best with Taxol-sensitivity, and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells. FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics. Taken together, this work demonstrates that Taxol-resistance dramatically alters how ovarian cancer cells adhere to their extracellular environment causing down-stream increases in microtubule dynamics, providing a therapeutic target that may improve prognosis by not only recovering drug sensitivity, but also decreasing metastasis.

No MeSH data available.


Related in: MedlinePlus

Generation of Taxol-resistant populations and analysis of potential resistance mechanisms.(A) Viability of parental (-P) and Taxol-resistant (-T) populations of SKOV3 and OVCAR3 ovarian cancer cells in after incubation in varying concentrations of Taxol (* is significantly greater than solvent treated control, p < 0.05, N = 3). (B) Time-dependent rhodamine efflux over initial two hours was used to calculate efflux rates (k), which showed no significant difference among cell populations (N = 3). (C) Long-term 24 hour efflux showed no significant difference between cell populations (N = 3). (D) Viability after incubation with 25 μM Carboplatin relative to solvent-treated control. Values given as mean ± SEM.
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f1: Generation of Taxol-resistant populations and analysis of potential resistance mechanisms.(A) Viability of parental (-P) and Taxol-resistant (-T) populations of SKOV3 and OVCAR3 ovarian cancer cells in after incubation in varying concentrations of Taxol (* is significantly greater than solvent treated control, p < 0.05, N = 3). (B) Time-dependent rhodamine efflux over initial two hours was used to calculate efflux rates (k), which showed no significant difference among cell populations (N = 3). (C) Long-term 24 hour efflux showed no significant difference between cell populations (N = 3). (D) Viability after incubation with 25 μM Carboplatin relative to solvent-treated control. Values given as mean ± SEM.

Mentions: After establishing populations of SKOV3 and OVCAR3 ovarian cancer cell lines capable of growing in Taxol, we first verified this correlated with an increase in IC50 by treating cells with Taxol at varying concentrations for 72 hours. Consistent with previous reports25, SKOV3 and OVCAR3 parental populations (abbreviated as SKOV3-P and OVCAR3-P) showed IC50 values of 2.3 ± 0.3 nM and 4.1 ± 1.8 nM, respectively, which were increased an order of magnitude in the Taxol-resistant subpopulations (abbreviated as SKOV3-T and OVCAR3-T) to 22.1 ± 3.0 nM and 45.5 ± 4.9 nM (Fig. 1A). While parent populations show dose-dependent decreases in viability, Taxol-resistant cells showed significantly increased viability at concentrations of 5–10 nM Taxol beyond which viability decreased. To begin to investigate the mechanism of this resistance, we next evaluated the ability of the cells to efflux Rhodamine 123 as a model drug (Fig. 1B–C), as both Taxol and Rhodamine 123 are substrates for P-glycoprotein mediated efflux26. Evaluation of both efflux kinetics (Fig. 1B) and total efflux after twenty four hours (Fig. 1C) demonstrated no significant changes with Taxol resistance. Finally, to see if changes were the product of other pro-survival adaptions we assayed the ability of cells to survive in 25 μM Carboplatin and found no significant change in Carboplatin resistance in Taxol resistant subpopulations (Fig. 1D). These results suggest that alternative mechanisms must be underlying this Taxol resistance.


Alterations in ovarian cancer cell adhesion drive taxol resistance by increasing microtubule dynamics in a FAK-dependent manner.

McGrail DJ, Khambhati NN, Qi MX, Patel KS, Ravikumar N, Brandenburg CP, Dawson MR - Sci Rep (2015)

Generation of Taxol-resistant populations and analysis of potential resistance mechanisms.(A) Viability of parental (-P) and Taxol-resistant (-T) populations of SKOV3 and OVCAR3 ovarian cancer cells in after incubation in varying concentrations of Taxol (* is significantly greater than solvent treated control, p < 0.05, N = 3). (B) Time-dependent rhodamine efflux over initial two hours was used to calculate efflux rates (k), which showed no significant difference among cell populations (N = 3). (C) Long-term 24 hour efflux showed no significant difference between cell populations (N = 3). (D) Viability after incubation with 25 μM Carboplatin relative to solvent-treated control. Values given as mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400875&req=5

f1: Generation of Taxol-resistant populations and analysis of potential resistance mechanisms.(A) Viability of parental (-P) and Taxol-resistant (-T) populations of SKOV3 and OVCAR3 ovarian cancer cells in after incubation in varying concentrations of Taxol (* is significantly greater than solvent treated control, p < 0.05, N = 3). (B) Time-dependent rhodamine efflux over initial two hours was used to calculate efflux rates (k), which showed no significant difference among cell populations (N = 3). (C) Long-term 24 hour efflux showed no significant difference between cell populations (N = 3). (D) Viability after incubation with 25 μM Carboplatin relative to solvent-treated control. Values given as mean ± SEM.
Mentions: After establishing populations of SKOV3 and OVCAR3 ovarian cancer cell lines capable of growing in Taxol, we first verified this correlated with an increase in IC50 by treating cells with Taxol at varying concentrations for 72 hours. Consistent with previous reports25, SKOV3 and OVCAR3 parental populations (abbreviated as SKOV3-P and OVCAR3-P) showed IC50 values of 2.3 ± 0.3 nM and 4.1 ± 1.8 nM, respectively, which were increased an order of magnitude in the Taxol-resistant subpopulations (abbreviated as SKOV3-T and OVCAR3-T) to 22.1 ± 3.0 nM and 45.5 ± 4.9 nM (Fig. 1A). While parent populations show dose-dependent decreases in viability, Taxol-resistant cells showed significantly increased viability at concentrations of 5–10 nM Taxol beyond which viability decreased. To begin to investigate the mechanism of this resistance, we next evaluated the ability of the cells to efflux Rhodamine 123 as a model drug (Fig. 1B–C), as both Taxol and Rhodamine 123 are substrates for P-glycoprotein mediated efflux26. Evaluation of both efflux kinetics (Fig. 1B) and total efflux after twenty four hours (Fig. 1C) demonstrated no significant changes with Taxol resistance. Finally, to see if changes were the product of other pro-survival adaptions we assayed the ability of cells to survive in 25 μM Carboplatin and found no significant change in Carboplatin resistance in Taxol resistant subpopulations (Fig. 1D). These results suggest that alternative mechanisms must be underlying this Taxol resistance.

Bottom Line: Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics.Adhesion strength correlated best with Taxol-sensitivity, and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells.FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical &Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA.

ABSTRACT
Chemorefractory ovarian cancer patients show extremely poor prognosis. Microtubule-stabilizing Taxol (paclitaxel) is a first-line treatment against ovarian cancer. Despite the close interplay between microtubules and cell adhesion, it remains unknown if chemoresistance alters the way cells adhere to their extracellular environment, a process critical for cancer metastasis. To investigate this, we isolated Taxol-resistant populations of OVCAR3 and SKOV3 ovarian cancer cell lines. Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics. These changes in microtubule dynamics coincided with faster attachment rates and decreased adhesion strength, which correlated with increased surface β1-integrin expression and decreased focal adhesion formation, respectively. Adhesion strength correlated best with Taxol-sensitivity, and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells. FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics. Taken together, this work demonstrates that Taxol-resistance dramatically alters how ovarian cancer cells adhere to their extracellular environment causing down-stream increases in microtubule dynamics, providing a therapeutic target that may improve prognosis by not only recovering drug sensitivity, but also decreasing metastasis.

No MeSH data available.


Related in: MedlinePlus