Karthikeyan Krishnamurthy

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

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 G2/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.

Heat shock factor-1 knockout induces multidrug resistance gene, MDR1b, and enhances P-glycoprotein (ABCB1)-based drug extrusion in the heart

Heat-shock factor 1 (HSF-1), a transcription factor for heat-shock proteins (HSPs), is known to interfere with the transcriptional activity of many oncogenic factors. In the present work, we have discovered that HSF-1 ablation induced the multidrug resistance gene, MDR1b, in the heart and increased the expression of P-glycoprotein (P-gp, ABCB1), an ATP binding cassette that is usually associated with multidrug-resistant cancer cells. The increase in P-gp enhanced the extrusion of doxorubicin (Dox) to alleviate Dox-induced heart failure and reduce mortality in mice. Dox-induced left ventricular (LV) dysfunction was significantly reduced in HSF-1−/− mice. DNA-binding activity of NF-κB was higher in HSF-1−/− mice. IκB, the NF-κB inhibitor, was depleted due to enhanced IκB kinase (IKK)-α activity. In parallel, MDR1b gene expression and a large increase in P-gp and lowering Dox loading were observed in HSF-1−/− mouse hearts. Moreover, application of the P-gp antagonist, verapamil, increased Dox loading in HSF-1−/− cardiomyocytes, deteriorated cardiac function in HSF-1−/− mice, and decreased survival. MDR1 promoter activity was higher in HSF-1−/− cardiomyocytes, whereas a mutant MDR1 promoter with heat-shock element (HSE) mutation showed increased activity only in HSF-1+/+ cardiomyocytes. However, deletion of HSE and NF-κB binding sites diminished luminescence in both HSF-1+/+ and HSF-1−/− cardiomyocytes, suggesting that HSF-1 inhibits MDR1 activity in the heart. Thus, because high levels of HSF-1 are attributed to poor prognosis of cancer, systemic down-regulation of HSF-1 before chemotherapy is a potential therapeutic approach to ameliorate the chemotherapy-induced cardiotoxicity and enhance cancer prognosis.

Heat Shock Protein 25-Enriched Plasma Transfusion Preconditions the Heart against Doxorubicin-Induced Dilated Cardiomyopathy in Mice

Extracellular heat shock proteins (eHsps) in the circulation have recently been found to activate both apoptotic and protective signaling in the heart. However, the role of eHsps in doxorubicin (Dox)-induced heart failure has not yet been studied. The objective of the present study was to determine how Dox affects circulating eHsp25 in blood plasma and how eHsp25 affects Dox-induced dilated cardiomyopathy. Wild-type mice [HSF-1(+/+)] were pretreated with 100 μl of heterozygous heat shock factor-1 [HSF-1(+/−)] mouse plasma (which contained 4-fold higher eHsp25 compared with wild-type mice), HSF-1(+/+) plasma, or saline, before treatment with Dox (6 mg/kg). After 4 weeks of this treatment protocol, HSF-1(+/−) plasma-pretreated mice showed increased eHsp25 in plasma and improved cardiac function (percentage of fractional shortening 37.3 ± 2.1 versus 26.4 ± 4.0) and better life span (31 ± 2 versus 22 ± 3 days) compared with the HSF-1(+/+) plasma or saline-pretreated mice. Preincubation of isolated adult cardiomyocytes with HSF-1(+/−) plasma or recombinant human Hsp27 (rhHsp27) significantly reduced Dox-induced activation of nuclear factor-κB and cytokine release and delayed cardiomyocyte death. Moreover, when cardiomyocytes were incubated with fluorescence-tagged rhHsp27, a saturation in binding was observed, suggesting that eHsp25 can bind to surface receptors. Competitive assays with a Toll-like receptor 2 (TLR2) antibody reduced the rhHSP27 binding, indicating that Hsp25 interacts with TLR2. In conclusion, transfusion of Hsp25-enriched blood plasma protected the heart from Dox-induced cardiotoxicity. Hsp25 antagonized Dox binding to the TLR2 receptor on cardiomyocytes.

Heat shock factor-1 knockout enhances cholesterol 7α-hydroxylase (CYP7A1) and multidrug transporter (MDR1) gene expressions to attenuate atherosclerosis

AIMS Stress response, in terms of activation of stress factors, is known to cause obesity and coronary heart disease such as atherosclerosis in human. However, the underlying mechanism(s) of these pathways are not known. Here, we investigated the effect of heat shock factor-1 (HSF-1) on atherosclerosis. METHODS AND RESULTS HSF-1 and low-density lipoprotein receptor (LDLr) double knockout (HSF-1(-/-)/LDLr(-/-)) and LDLr knockout (LDLr(-/-)) mice were fed with atherogenic western diet (WD) for 12 weeks. WD-induced weight gain and atherosclerotic lesion in aortic arch and carotid regions were reduced in HSF-1(-/-)/LDLr(-/-) mice, compared with LDLr(-/-) mice. Also, repression of PPAR-γ2 and AMPKα expression in adipose tissue, low hepatic steatosis, and lessened plasma adiponectins and lipoproteins were observed. In HSF-1(-/-)/LDLr(-/-) liver, higher cholesterol 7α-hydroxylase (CYP7A1) and multidrug transporter [MDR1/P-glycoprotein (P-gp)] gene expressions were observed, consistent with higher bile acid transport and larger hepatic bile ducts. Luciferase reporter gene assays with wild-type CYP7A1 and MDR1 promoters showed lesser luminescence than with mutant promoters (HSF-1 binding site deleted), indicating that HSF-1 binding is repressive of CYP7A1 and MDR1 gene expressions. CONCLUSION HSF-1 ablation not only eliminates heat shock response, but it also transcriptionally up-regulates CYP7A1 and MDR1/P-gp axis in WD-diet fed HSF-1(-/-)/LDLr(-/-) mice to reduce atherosclerosis.

Small Heat Shock Proteins and Doxorubicin-Induced Oxidative Stress in the Heart

Doxorubicin (Dox) and its derivatives are used as chemotherapeutics, either alone or in combination with other agents. Dilated cardiomyopathy and congestive heart failure due to cardiotoxicity continues to be the most serious side effect, imposing severe limitations in the use of these agents despite the arrival of new classes of Dox-derivatives and new formulations. In this chapter we summarize the recent understanding of the mechanism of Dox-induced cardiotoxicity and its relevance to the stress-inducible proteins, with special emphasis on the small heat shock proteins such as Hsp27, Hsp20, etc. The heat shock proteins are expressed as a response to the oxidative stress in the heart due to the redox reactions of these drugs and the generation of reactive oxygen species (ROS). On the other hand, ROS are also known to induce various MAP kinases and phosphorylate and activate the stress-responding transcription factors, including the heat shock factors (HSF). Activation of HSF-1 leads to the induction of a series of heat shock proteins, depending upon the type of exerted stress. Recent studies have confirmed that Dox-induced oxidative stress indeed leads to HSF-1 activation to induction of heat shock proteins, especially small Hsps in the heart. The Dox-induced small Hsps have been found to be involved in cell signaling and can be either cardioprotective or detrimental. Additionally, a few transgenic animal models have shown that selective overexpression of these proteins can be cardioprotective against Dox. These results establish the fact that proper regulation of the function of small Hsps could eliminate cardiotoxicity and serve as a potential therapeutic target to protect the heart from Dox-induced toxicity.

Abstract 1744: Hsp27 overexpression enhances the chemosensitivity to MCF7 doxorubicin resistance cells

Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy and its inhibition is an effective way to reverse cancer drug resistance. In the present study we report that over expression of HSP27 down-regulated MDR1 expression in MCF7/Doxorubicin (MCF7/DOX), a human breast MDR cancer cell line. In MCF7/Dox cells, mutant p53 was found to be accumulated, whereas Hsp27 level is completely depleted. Since Hsp27 was shown to enhance Akt phosphorylation and G2/M cell cycle arrest, a common pathway of Dox-induced cell death in MCF7 cells, we hypothesized that over expression of Hsp27 in resistant cell will sensitize for chemotherapy. HSP27 over expression significantly inhibited mutant p53 and MDR1 (P-gp) expression and MDR1 mRNA. The suppression of MDR1 was accompanied by partial recovery of intracellular drug accumulation and increased cytotoxicity of Doxorubicin, indicating that HSP27 reversed the MDR phenotype by inhibiting the drug efflux function of MDR1. Moreover, nuclear factor-KB activity and IKB degradation were inhibited by HSP27 over expression through down regulation of mutant p53. Interestingly, the over expression of HSP27 negatively regulated the cell growth with significant G 2 /M arrest of cell cycle, and it could up-regulate endogenous p21 in a p53 independent manner leading to enhanced caspase activation to promote intracellular apoptotic signaling for cell death. Taken together, our results suggested that HSP27 is down regulated in MCF7/Dox resistant cells, and when re-expressed, it down regulates MDR1 expression via p53 and NF-KB., which results in growth suppression and higher sensitivity to Doxorubicin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1744. doi:10.1158/1538-7445.AM2011-1744