Limits...
Overexpression of Heat Shock Transcription Factor 1 enhances the resistance of melanoma cells to doxorubicin and paclitaxel.

Vydra N, Toma A, Glowala-Kosinska M, Gogler-Piglowska A, Widlak W - BMC Cancer (2013)

Bottom Line: The expression of constitutively active mutant HSF1, also resulting in HSPs overproduction, did not reduce the sensitivity of melanoma cells to drugs, unlike in the case of dominant negative form expression.Cells overexpressing a full or dominant negative form of HSF1, but not a constitutively active one, had higher transcription levels of ABC genes when compared to control cells.Direct transcriptional activity of HSF1 is not necessary for increased expression of ABC genes, which is probably mediated by HSF1 regulatory domain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeże Armii Krajowej 15, Gliwice, Poland. nvydra@yahoo.co.uk.

ABSTRACT

Background: Heat Shock Transcription Factor 1 (HSF1) is activated under stress conditions. In turn, it induces expression of Heat Shock Proteins (HSPs), which are well-known regulators of protein homeostasis. Elevated levels of HSF1 and HSPs were observed in many types of tumors. The aim of the present study was to determine whether HSF1 could have an effect on the survival of cancer cells treated with chemotherapeutic cytotoxic agents.

Methods: We constructed mouse (B16F10) and human (1205Lu, WM793B) melanoma cells overexpressing full or mutant form of human HSF1: a constitutively active one with a deletion in regulatory domain or a dominant negative one with a deletion in the activation domain. The impact of different forms of HSF1 on the expression of HSP and ABC genes was studied by RT-PCR and Western blotting. Cell cultures were treated with increasing amounts of doxorubicin, paclitaxel, cisplatin, vinblastine or bortezomib. Cell viability was determined by MTT, and IC50 was calculated. Cellular accumulation of fluorescent dyes and side population cells were studied using flow cytometry.

Results: Cells overexpressing HSF1 and characterized by increased HSPs accumulation were more resistant to doxorubicin or paclitaxel, but not to cisplatin, vinblastine or bortezomib. This resistance correlated with the enhanced efflux of fluorescent dyes and the increased number of side population cells. The expression of constitutively active mutant HSF1, also resulting in HSPs overproduction, did not reduce the sensitivity of melanoma cells to drugs, unlike in the case of dominant negative form expression. Cells overexpressing a full or dominant negative form of HSF1, but not a constitutively active one, had higher transcription levels of ABC genes when compared to control cells.

Conclusions: HSF1 overexpression facilitates the survival of melanoma cells treated with doxorubicin or paclitaxel. However, HSF1-mediated chemoresistance is not dependent on HSPs accumulation but on an increased potential for drug efflux by ABC transporters. Direct transcriptional activity of HSF1 is not necessary for increased expression of ABC genes, which is probably mediated by HSF1 regulatory domain.

Show MeSH

Related in: MedlinePlus

Fluorescent dyes efflux is enhanced in melanoma cells overexpressing HSF1. Intracellular fluorescence of doxorubicin (A) and eFluxx-ID™ Green Detection Reagent (B) in hHSF1-overexpressing cells is shown in relation to control (Neo) cells (C). The percentage of dye-negative cells (side population, SP) following incubation with Hoechst 33342 in the absence or presence of verapamil is presented. Mean values ± SD from at least three experiments are shown (asterisks indicate p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231344&req=5

Figure 2: Fluorescent dyes efflux is enhanced in melanoma cells overexpressing HSF1. Intracellular fluorescence of doxorubicin (A) and eFluxx-ID™ Green Detection Reagent (B) in hHSF1-overexpressing cells is shown in relation to control (Neo) cells (C). The percentage of dye-negative cells (side population, SP) following incubation with Hoechst 33342 in the absence or presence of verapamil is presented. Mean values ± SD from at least three experiments are shown (asterisks indicate p < 0.05).

Mentions: To elucidate mechanisms of acquired doxorubicin resistance of cells overexpressing hHSF1 we estimated accumulation of the drug by flow cytometry. Cells were treated with doxorubicin (1 μg/ml) for 30 minutes and then the doxorubicin fluorescence was checked. Under those conditions doxorubicin accumulation was lower in hHSF1-transduced cells than in control cells, yet observed differences did not reach the level of statistical significance (Figure 2A, see also Additional file 3: Figure S2). The intracellular accumulation of doxorubicin is dependent on the activity of ABCB1 or other proteins belonging to the ABC transporters family. Therefore, we assessed the accumulation of a tracer dye eFluxx-ID™ Green Detection Reagent (Enzo Life Sciences). The reagent is a substrate for three main ABC transporter proteins, ABCB1, ABCC1/ABCC2 and ABCG2 and can serve as an indicator of these proteins’ activity in cells. We found that the dye-specific fluorescence was significantly lower in cells overexpressing hHSF1, and the most effective drug efflux occured in hHSF1-WM793B cells (Figure 2B). This indicates higher activity of ABC transporters in hHSF1-overexpressing melanoma cells leading to more effective drug efflux.


Overexpression of Heat Shock Transcription Factor 1 enhances the resistance of melanoma cells to doxorubicin and paclitaxel.

Vydra N, Toma A, Glowala-Kosinska M, Gogler-Piglowska A, Widlak W - BMC Cancer (2013)

Fluorescent dyes efflux is enhanced in melanoma cells overexpressing HSF1. Intracellular fluorescence of doxorubicin (A) and eFluxx-ID™ Green Detection Reagent (B) in hHSF1-overexpressing cells is shown in relation to control (Neo) cells (C). The percentage of dye-negative cells (side population, SP) following incubation with Hoechst 33342 in the absence or presence of verapamil is presented. Mean values ± SD from at least three experiments are shown (asterisks indicate p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231344&req=5

Figure 2: Fluorescent dyes efflux is enhanced in melanoma cells overexpressing HSF1. Intracellular fluorescence of doxorubicin (A) and eFluxx-ID™ Green Detection Reagent (B) in hHSF1-overexpressing cells is shown in relation to control (Neo) cells (C). The percentage of dye-negative cells (side population, SP) following incubation with Hoechst 33342 in the absence or presence of verapamil is presented. Mean values ± SD from at least three experiments are shown (asterisks indicate p < 0.05).
Mentions: To elucidate mechanisms of acquired doxorubicin resistance of cells overexpressing hHSF1 we estimated accumulation of the drug by flow cytometry. Cells were treated with doxorubicin (1 μg/ml) for 30 minutes and then the doxorubicin fluorescence was checked. Under those conditions doxorubicin accumulation was lower in hHSF1-transduced cells than in control cells, yet observed differences did not reach the level of statistical significance (Figure 2A, see also Additional file 3: Figure S2). The intracellular accumulation of doxorubicin is dependent on the activity of ABCB1 or other proteins belonging to the ABC transporters family. Therefore, we assessed the accumulation of a tracer dye eFluxx-ID™ Green Detection Reagent (Enzo Life Sciences). The reagent is a substrate for three main ABC transporter proteins, ABCB1, ABCC1/ABCC2 and ABCG2 and can serve as an indicator of these proteins’ activity in cells. We found that the dye-specific fluorescence was significantly lower in cells overexpressing hHSF1, and the most effective drug efflux occured in hHSF1-WM793B cells (Figure 2B). This indicates higher activity of ABC transporters in hHSF1-overexpressing melanoma cells leading to more effective drug efflux.

Bottom Line: The expression of constitutively active mutant HSF1, also resulting in HSPs overproduction, did not reduce the sensitivity of melanoma cells to drugs, unlike in the case of dominant negative form expression.Cells overexpressing a full or dominant negative form of HSF1, but not a constitutively active one, had higher transcription levels of ABC genes when compared to control cells.Direct transcriptional activity of HSF1 is not necessary for increased expression of ABC genes, which is probably mediated by HSF1 regulatory domain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeże Armii Krajowej 15, Gliwice, Poland. nvydra@yahoo.co.uk.

ABSTRACT

Background: Heat Shock Transcription Factor 1 (HSF1) is activated under stress conditions. In turn, it induces expression of Heat Shock Proteins (HSPs), which are well-known regulators of protein homeostasis. Elevated levels of HSF1 and HSPs were observed in many types of tumors. The aim of the present study was to determine whether HSF1 could have an effect on the survival of cancer cells treated with chemotherapeutic cytotoxic agents.

Methods: We constructed mouse (B16F10) and human (1205Lu, WM793B) melanoma cells overexpressing full or mutant form of human HSF1: a constitutively active one with a deletion in regulatory domain or a dominant negative one with a deletion in the activation domain. The impact of different forms of HSF1 on the expression of HSP and ABC genes was studied by RT-PCR and Western blotting. Cell cultures were treated with increasing amounts of doxorubicin, paclitaxel, cisplatin, vinblastine or bortezomib. Cell viability was determined by MTT, and IC50 was calculated. Cellular accumulation of fluorescent dyes and side population cells were studied using flow cytometry.

Results: Cells overexpressing HSF1 and characterized by increased HSPs accumulation were more resistant to doxorubicin or paclitaxel, but not to cisplatin, vinblastine or bortezomib. This resistance correlated with the enhanced efflux of fluorescent dyes and the increased number of side population cells. The expression of constitutively active mutant HSF1, also resulting in HSPs overproduction, did not reduce the sensitivity of melanoma cells to drugs, unlike in the case of dominant negative form expression. Cells overexpressing a full or dominant negative form of HSF1, but not a constitutively active one, had higher transcription levels of ABC genes when compared to control cells.

Conclusions: HSF1 overexpression facilitates the survival of melanoma cells treated with doxorubicin or paclitaxel. However, HSF1-mediated chemoresistance is not dependent on HSPs accumulation but on an increased potential for drug efflux by ABC transporters. Direct transcriptional activity of HSF1 is not necessary for increased expression of ABC genes, which is probably mediated by HSF1 regulatory domain.

Show MeSH
Related in: MedlinePlus