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

An increased resistance of melanoma cells to doxorubicin is not coupled with HSF1 transcriptional activity. A. Detection of transcripts of HSF1 and HSP genes in B16F10, WM793B and 1205Lu cells containing either the empty vector (Neo) or HSF1 mutants. Where indicated, cells were subjected to heat shock (HS) for 1 h at 42°C with subsequent recovery at 37°C for 30 minutes. B. Western blot detection of HSF1 and HSPs in cells modified and treated as above. HSF1 was detected directly after HS while HSPs were detected after a 6-hour recovery. C. Viability of cells treated with various concentrations of doxorubicin for 48 h (B16F10) or 72 h (WM793B and 1205Lu). Results of the MTT assay are shown in relation to the untreated cells; mean values ± SD from three experiments are presented (asterisks indicate p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: An increased resistance of melanoma cells to doxorubicin is not coupled with HSF1 transcriptional activity. A. Detection of transcripts of HSF1 and HSP genes in B16F10, WM793B and 1205Lu cells containing either the empty vector (Neo) or HSF1 mutants. Where indicated, cells were subjected to heat shock (HS) for 1 h at 42°C with subsequent recovery at 37°C for 30 minutes. B. Western blot detection of HSF1 and HSPs in cells modified and treated as above. HSF1 was detected directly after HS while HSPs were detected after a 6-hour recovery. C. Viability of cells treated with various concentrations of doxorubicin for 48 h (B16F10) or 72 h (WM793B and 1205Lu). Results of the MTT assay are shown in relation to the untreated cells; mean values ± SD from three experiments are presented (asterisks indicate p < 0.05).

Mentions: To further investigate the mechanism of HSF1-dependent resistance of melanoma cells to doxorubicin we tested two mutant forms of HSF1: constitutively active one and dominant-negative one. The constitutively active form (aHSF1) corresponds to the human HSF1 with a deletion in a heat-responsive regulatory domain (RD; residues 221–315). Dominant-negative form (hHSF1-DN) corresponds to the human HSF1 with a deletion in the C-terminal transcriptional activation domain (residues 453–523) (see Additional file 1: Figure S1). It has been previously shown that deletion of amino acids 221–315 conferred on HSF1 the ability to bind DNA and to induce HSPs expression in the absence of heat shock [6,7], while deletion of amino acids within C-terminal domain led to DNA-binding activity of HSF1 without the ability to activate HSPs expression during heat shock [19]. We established mouse (B16F10) and human (WM793B and 1205Lu) cells overexpressing these mutant forms of HSF1. The shorter mutant forms of HSF1 were present in the modified cells in addition to the longer endogenous HSF1 form (Figure 3). Stably transfected cells were tested for HSPs expression in the absence or after heat shock. Increased expression of several HSP genes (HSPH1, HSPB1, HSPA1) was detected in cells overexpressing aHSF1 already at physiological temperature. On the other hand, induction of the same HSP genes was partially blocked following hyperthermia in mouse B16F10 cells overexpressing hHSF1-DN (Figure 3A). In human cells, introduction of hHSF1-DN was associated with a slightly higher expression of some HSPs (HSPA1, HSPH1) at physiological temperature, which suggested that introduced dominant negative HSF1 could form heterotrimers with endogenous HSF1 leading to basal transcriptional activity [19,24]. However, in the presence of hHSF1-DN hyperthermia-induced accumulation of HSPs was suppressed in both mouse and human cells (Figure 3B). We have concluded that overexpression of aHSF1 mimicked transcriptional activity of HSF1 during stress conditions, while hHSF1-DN was able to suppress strong induction of HSF1-dependent HSP genes normally observed after heat shock, plausibly by blocking the endogenous HSF1 binding.


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)

An increased resistance of melanoma cells to doxorubicin is not coupled with HSF1 transcriptional activity. A. Detection of transcripts of HSF1 and HSP genes in B16F10, WM793B and 1205Lu cells containing either the empty vector (Neo) or HSF1 mutants. Where indicated, cells were subjected to heat shock (HS) for 1 h at 42°C with subsequent recovery at 37°C for 30 minutes. B. Western blot detection of HSF1 and HSPs in cells modified and treated as above. HSF1 was detected directly after HS while HSPs were detected after a 6-hour recovery. C. Viability of cells treated with various concentrations of doxorubicin for 48 h (B16F10) or 72 h (WM793B and 1205Lu). Results of the MTT assay are shown in relation to the untreated cells; mean values ± SD from three experiments are presented (asterisks indicate p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: An increased resistance of melanoma cells to doxorubicin is not coupled with HSF1 transcriptional activity. A. Detection of transcripts of HSF1 and HSP genes in B16F10, WM793B and 1205Lu cells containing either the empty vector (Neo) or HSF1 mutants. Where indicated, cells were subjected to heat shock (HS) for 1 h at 42°C with subsequent recovery at 37°C for 30 minutes. B. Western blot detection of HSF1 and HSPs in cells modified and treated as above. HSF1 was detected directly after HS while HSPs were detected after a 6-hour recovery. C. Viability of cells treated with various concentrations of doxorubicin for 48 h (B16F10) or 72 h (WM793B and 1205Lu). Results of the MTT assay are shown in relation to the untreated cells; mean values ± SD from three experiments are presented (asterisks indicate p < 0.05).
Mentions: To further investigate the mechanism of HSF1-dependent resistance of melanoma cells to doxorubicin we tested two mutant forms of HSF1: constitutively active one and dominant-negative one. The constitutively active form (aHSF1) corresponds to the human HSF1 with a deletion in a heat-responsive regulatory domain (RD; residues 221–315). Dominant-negative form (hHSF1-DN) corresponds to the human HSF1 with a deletion in the C-terminal transcriptional activation domain (residues 453–523) (see Additional file 1: Figure S1). It has been previously shown that deletion of amino acids 221–315 conferred on HSF1 the ability to bind DNA and to induce HSPs expression in the absence of heat shock [6,7], while deletion of amino acids within C-terminal domain led to DNA-binding activity of HSF1 without the ability to activate HSPs expression during heat shock [19]. We established mouse (B16F10) and human (WM793B and 1205Lu) cells overexpressing these mutant forms of HSF1. The shorter mutant forms of HSF1 were present in the modified cells in addition to the longer endogenous HSF1 form (Figure 3). Stably transfected cells were tested for HSPs expression in the absence or after heat shock. Increased expression of several HSP genes (HSPH1, HSPB1, HSPA1) was detected in cells overexpressing aHSF1 already at physiological temperature. On the other hand, induction of the same HSP genes was partially blocked following hyperthermia in mouse B16F10 cells overexpressing hHSF1-DN (Figure 3A). In human cells, introduction of hHSF1-DN was associated with a slightly higher expression of some HSPs (HSPA1, HSPH1) at physiological temperature, which suggested that introduced dominant negative HSF1 could form heterotrimers with endogenous HSF1 leading to basal transcriptional activity [19,24]. However, in the presence of hHSF1-DN hyperthermia-induced accumulation of HSPs was suppressed in both mouse and human cells (Figure 3B). We have concluded that overexpression of aHSF1 mimicked transcriptional activity of HSF1 during stress conditions, while hHSF1-DN was able to suppress strong induction of HSF1-dependent HSP genes normally observed after heat shock, plausibly by blocking the endogenous HSF1 binding.

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