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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.

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Expression of several ABC transporters is increased in cells overexpressing full HSF1 or its dominant negative form. Changes in ABC transporter genes expression were estimated based on semi-quantitative RT-PCR (after gel densitometry) in B16F10 cells (A) or using quantitative RT-PCR in WM793B (B) and 1205Lu (C) human cells. Fold changes were calculated in relation to expression levels in control (Neo) cells (1.0 value represented by a horizontal red line) after normalization against GAPDH gene expression. Results represent mean values ± SD from three experiments.
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Figure 5: Expression of several ABC transporters is increased in cells overexpressing full HSF1 or its dominant negative form. Changes in ABC transporter genes expression were estimated based on semi-quantitative RT-PCR (after gel densitometry) in B16F10 cells (A) or using quantitative RT-PCR in WM793B (B) and 1205Lu (C) human cells. Fold changes were calculated in relation to expression levels in control (Neo) cells (1.0 value represented by a horizontal red line) after normalization against GAPDH gene expression. Results represent mean values ± SD from three experiments.

Mentions: Increased efflux of drugs mediated by the ABC transporters is the most commonly encountered mechanism of drug resistance. We analyzed the expression of several ABC transporters in melanoma cells having different HSF1 status. We selected ABCB1, ABCC1, ABCC2, ABCC5, ABCB8, ABCD1 transporters, which were previously reported to be involved in doxorubicin resistance [25,26]. In cells overexpressing hHSF1 the most prominent was up-regulation of Abcb1b/ABCB1 gene transcription observed in both mouse and human cells (Figure 5). Transcription of other analyzed ABC genes (namely ABCB8, ABCC1, ABCC2, ABCC5 and ABCD1) was significantly elevated in human cells overexpressing hHSF1 (Figure 5B,C), but not in mouse cells (data not shown). We could not confirm differences in ABC protein levels between control and hHSF1-overexpressing cells due to unsatisfactory specificities of available antibodies, which showed substantial cross-reactivity to other proteins.


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)

Expression of several ABC transporters is increased in cells overexpressing full HSF1 or its dominant negative form. Changes in ABC transporter genes expression were estimated based on semi-quantitative RT-PCR (after gel densitometry) in B16F10 cells (A) or using quantitative RT-PCR in WM793B (B) and 1205Lu (C) human cells. Fold changes were calculated in relation to expression levels in control (Neo) cells (1.0 value represented by a horizontal red line) after normalization against GAPDH gene expression. Results represent mean values ± SD from three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Expression of several ABC transporters is increased in cells overexpressing full HSF1 or its dominant negative form. Changes in ABC transporter genes expression were estimated based on semi-quantitative RT-PCR (after gel densitometry) in B16F10 cells (A) or using quantitative RT-PCR in WM793B (B) and 1205Lu (C) human cells. Fold changes were calculated in relation to expression levels in control (Neo) cells (1.0 value represented by a horizontal red line) after normalization against GAPDH gene expression. Results represent mean values ± SD from three experiments.
Mentions: Increased efflux of drugs mediated by the ABC transporters is the most commonly encountered mechanism of drug resistance. We analyzed the expression of several ABC transporters in melanoma cells having different HSF1 status. We selected ABCB1, ABCC1, ABCC2, ABCC5, ABCB8, ABCD1 transporters, which were previously reported to be involved in doxorubicin resistance [25,26]. In cells overexpressing hHSF1 the most prominent was up-regulation of Abcb1b/ABCB1 gene transcription observed in both mouse and human cells (Figure 5). Transcription of other analyzed ABC genes (namely ABCB8, ABCC1, ABCC2, ABCC5 and ABCD1) was significantly elevated in human cells overexpressing hHSF1 (Figure 5B,C), but not in mouse cells (data not shown). We could not confirm differences in ABC protein levels between control and hHSF1-overexpressing cells due to unsatisfactory specificities of available antibodies, which showed substantial cross-reactivity to other proteins.

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