Forced expression of heat shock protein 27 (Hsp27) reverses P-glycoprotein (ABCB1)-mediated drug efflux and MDR1 gene expression in Adriamycin-resistant human breast cancer cells

Abstract

Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G(2)/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer.

FIGURE 1.

Inhibition of HSF-1 activity and Hsp27 expression and selective expression of P-glycoprotein in MCF-7 and MCF-7/adr cells. A, representative Western blots of HSF-1, Hsp27, Hsp70, Hsp90, p53, NF-κB, and P-gp. B, EMSA analysis of HSF-1 binding in HSE oligonucleotide. C, mRNA of HSF-1 and P-gp, determined by RT-PCR. D, luciferase reporter gene assay. A schematic illustration (top) shows the MDR1 promoter, both wild type and mutant, cloned upstream of the luciferase gene in pGL3B expression vector. The mutant MDR1 promoter (deleted between −198 and −156) was lacking the putative HSF-1 binding sequence (underlined), as shown in the figure. Luciferase activity (bottom) was determined in MDR1 (WT and mutant) promoter cloned PGL3 transfected cells. **, p < 0.01 (n = 3).

FIGURE 2.

Doxorubicin-induced activation of HSF-1 and expression of Hsp27 and p53 in MCF-7 and MCF-7/adr cells. A, Western blots of p53 and Hsp27 in Dox-treated (1 μg/ml) cells at different post-drug treatment time points as indicated in the figure. B, quantitative plots of p53 for MCF-7 and MCF-7/adr cells, obtained from three independent experiments (n = 3). Error bars, S.E. C, representative gel image of EMSA analysis of HSF-1 binding to HSE at different Dox concentrations in MCF-7 and MCF-7/adr cells as indicated in the figure. D, EMSSA in control and Dox-treated MCF-7 cells. Nuclear extracts were incubated with HSE (columns 1, 3, and 6) or with cold HSE (with no biotin tag; columns 2, 4, and 5). HSF-1 antibody was >100 than HSE in competitive binding assays (column 5).

FIGURE 3.

Overexpression of Hsp27 sensitizes MCF-7/adr cells to Dox. A, Western blots of Hsp27 overexpressed in MCF-7/adr cells using pCMV-SPORT6/Hsp27 vector. B, overexpression of Hsp27 in MCF-7 cells at the same conditions as in A. C, viability measurements, determined using MTT, in MCF-7/adr and Hsp27-overexpressing MCF-7/adr-Hsp27 cells. Error bars, S.E. (n = 3).

FIGURE 4.

Depletion of mutant p53, phosphorylation of Hsp27 and MAP kinase activation in Dox-treated MCF-7/adr and MCF-7/adr-Hsp27 cells. A, Western blots of p53, Hsp27 and its Ser-15 (S15)-phosphorylated isoform, p38 MAPK, phospho-p38 MAPK (p-p38MAP), and phospho-MAPKAP-2 (p-MAPKAP-2) in Dox-treated MCF-7/adr and MCF-7/adr-Hsp27 cells. B, quantitative plots of the change in phospho-Hsp27 (p-Hsp27) and p53 blot intensities in cells treated with increasing Dox concentration. Error bars, S.E. of three (n = 3) independent experiments. a.u., arbitrary unit.

FIGURE 5.

NF-κB inhibition in Dox-treated MCF-7/adr and MCF-7/adr-Hsp27 cells. A, Western blots of NF-κB precursor (p105), p50, p65, phosphorylated p65, IkBα, and IKKα in cytoplasmic and nuclear fractions of Dox-treated cells. B, quantitative plots of p65/p50 ratio in these cells in cytosolic and nuclear fractions. Error bars, S.E. (n = 3). The p65/p50 ratio progressively decreased in the nuclear fractions of MCF-7/adr/Hsp27 cells. ***, p < 0.0001 (n = 5). C, confocal fluorescence imaging of p65 in these cells. Left panels, phase-contrast microscopic images. Middle and right panels (different magnifications), p65 stained with Alexafluor488 (green fluorescence) and co-stained with DAPI (blue) for positive identification of nuclei.

FIGURE 6.

Attenuation of MDR1 gene expression, depletion of P-gp protein, and increased uptake of Dox in Hsp27-overexpressing MCF-7/adr cells. A, Western blots of P-gp in Dox-treated (5 and 10 μm) MCF-7/adr/Hsp27 and MCF-7/adr cells, determined 24 h post-treatment. B, P-gp mRNA level, determined by RT-PCR, at the conditions defined in A. C, confocal microscopic fluorescence images of Dox fluorescence, determined from the autofluorescence of Dox in these cells, at the conditions defined in A. Dox is mostly observed to concentrate in the nuclei and membrane. D, histograms of flow cytometry sorted using autofluorescence of Dox at the conditions in A. E, histograms of flow cytometric analysis of Dox and Dox + CMAC dye-stained MCF-7/adr and MCF-7/adr/Hsp27 cells. Cells were sorted with excitation and emission wavelengths specific to CAMC dye (353 and 466 nm). The left panel was obtained in cells treated with Dox alone, whereas the right panel was for Dox + CMAC-treated cells.

FIGURE 7.

Higher Dox-induced cytotoxicity in MCF-7/adr/Hsp27 cells. The cells were treated with different concentrations of Dox as indicated in the figure, and after 48 h, the cells were fixed in ethanol and subjected to flow cytometry. A, Western blots of p21, BCl-2, and Bax in these cells, treated with different concentrations of Dox as indicated. B, flow cytometry analysis of cell cycle arrest induced by overexpressed Hsp27 in MCF-7/adr/Hsp27 cells. C, quantitative plots of G₂/G₁ ratio, determined from three independent measurements. Error bars, S.E.

FIGURE 8.

Enhanced apoptotic signaling in MCF-7/adr/Hsp27 cells upon treatment with Dox. A, Western blots of PARP and cleaved PARP in MCF-7/adr and MCF-7/adr/Hsp27 cells. Western blots of caspase-9, caspase-7, and caspase-3 are also shown. B, schematic illustration of proposed mechanism of regulation of MDR-1 by HSF-1. In MCF-7 cells, HSF-1 is trimerized and translocated into nucleus and bound to HSE to transcribe various Hsps, including Hsp27. Also, HSF-1 homotrimer can bind to HSE in the MDR1 promoter region in competition with NF-κB, to repress MDR1 gene expression (29). Hsp27, upon phosphorylation, interacts with p53 to enhance its degradation (40). In MCF-7/adr cells, HSF-1 and Hsp27 are inhibited, and p53 mutation is enforced. Hence, the NF-κB pathway is more facile to induce MDR1/P-gp expression. Upon Hsp27 overexpression, this pathway is inhibited, so that MCF-7/adr/Hsp27 cells are sensitized to Dox.