Punit Kaur

Heat shock protein-containing exosomes in mid-trimester amniotic fluids

Exosomes are multivesicular bodies formed by inverse membrane budding into the lumen of an endocytic compartment. Fusion with the plasma membrane leads to their release into the external milieu. The incorporation of heat shock proteins into exosomes has been associated with immune regulatory activity. We have examined whether heat shock protein-containing exosomes are present in mid-trimester amniotic fluid. Exosomes were isolated from mid-trimester amniotic fluids by sequential low-speed and high-speed centrifugation followed by sucrose density gradient centrifugation. Biochemical characterization included floatation pattern in sucrose gradients, acetylcholinesterase (AChE) activity and Western blot analysis for exosome-containing proteins. Exosomes were present in each of 23 amniotic fluids tested. They banded at a density of 1.17g/ml in sucrose gradients, were positive for AChE activity and contained tubulin, the inducible 72kDa heat shock protein, Hsp72 and the constitutively expressed heat shock protein, Hsc73; they were negative for calnexin. Exosome concentrations correlated positively with the number of pregnancies. Heat shock protein-containing exosomes are constituents of mid-trimester amniotic fluids and may contribute to immune regulation within the amniotic cavity.

Adaptogens exert a stress-protective effect by modulation of expression of molecular chaperones

Adaptogens are medicinal plants that augment resistance to stress, and increase concentration, performance and endurance during fatigue. Experiments were carried out with BALB/c mice taking ADAPT-232 forte, a fixed combination of three genuine (native) extracts of Eleutherococcus senticocus, Schisandra chinensis and Rhodiola rosea, characterised for the content of active markers eleutherosides, schisandrins, salidroside, tyrosol and rosavin and in doses of about 30, 90 and 180 mg/kg for seven consecutive days followed by forced swimming test to exhaustion. ADAPT-232 forte strongly augments endurance of mice, increasing the time taken to exhaustion (TTE) in a dose-dependent manner from 3.0+/-0.5 to 21.1+/-1.7 min, approximately seven fold. Serum Hsp72 was measured by EIA both in normal and stressful conditions before and after swimming test. Repeated administration of adaptogen dose dependently increases basal level of Hsp72 in serum of mice from 0.8-1.5 to 5.5-6.3 pg/ml. This effect is even stronger than the effect of stress, including both physical (swimming) and emotional impacts: 3.2+/-1.2 pg/ml. Cumulative effect of stress and adaptogen was clearly observed in groups of animals treated with adaptogen after swimming to exhaustion, when serum Hsp72 increased to 15.1+/-1 pg/ml and remained at almost the same level during the 7 days. It can be concluded that adaptogens induce increase of serum Hsp72, regarded as a defense response to stress, and increase tolerance to stress (in our model combination of physical and emotional stresses). It can be suggested that increased tolerance to stress induced by adaptogen is associated with its stimulation of expression of Hsp70 and particularly with Hsp72 production and release into systemic circulation, which is known as a mediator of stress response involved in reparation of proteins during physical load. Our studies suggest that this could be one of the mechanisms of action of plant adaptogens.

A mouse model for triple-negative breast cancer tumor-initiating cells (TNBC-TICs) exhibits similar aggressive phenotype to the human disease

BackgroundTriple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease--recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems.MethodsTo understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis.ResultsWe isolated tumor-initiating cells (TICs) by sorting for CD24+/CD44high/ALDH1+ cells from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24+/CD44high/ALDH1+ cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24-/CD44-/ALDH1- cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A).ConclusionsTaken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC.

Radiation therapy induces circulating serum Hsp72 in patients with prostate cancer

BACKGROUND AND PURPOSE Hsp72 found in the extracellular milieu has been shown to play an important role in immune regulation. The impact of common cancer therapies on extracellular release of Hsp72 however, has been to date undefined. MATERIALS AND METHODS Serum from 13 patients undergoing radiation therapy (XRT) for prostate cancer with or without hormonal therapy (ADT) was measured for levels of circulating serum Hsp72 and pro-inflammatory cytokines (IL-6 and TNF-alpha) using the classical sandwich ELISA technique and the relative expression of CD8(+) T lymphocytes and natural killer (NK) cells was measured using flow cytometry. Mouse orthotopic xenograft of human prostate cancer tumors (DU-145 and PC-3) were used to validate and further characterize the response noted in the clinical setting. The biological significance of tumor released Hsp72 was studied in human dendritic cells (DC) in vitro. RESULTS Circulating serum Hsp72 levels increased an average of 3.5-fold (median per patient 4.8-fold) with XRT but not with ADT (p=0.0002). Increases in IL-6 (3.3-fold), TNF-alpha (1.8-fold), CD8(+) CTL (2.1-fold) and NK cells (3.2-fold) also occurred. Using PC-3 and DU-145 human prostate cancer xenograft models in mice, we confirmed that XRT induces Hsp72 release primarily from implanted tumors. In vitro studies using supernatant recovered from irradiated human prostate cancer cells point to exosomes containing Hsp72 as a possible stimulator of pro-inflammatory cytokine production and costimulatory molecules expression in human DC. CONCLUSIONS The current study confirms for the first time in an actual clinical setting elevation of circulating serum Hsp72 with XRT. The accompanying studies in mice and in vitro identify the released exosomes containing Hsp72 as playing a pivotal role in stimulating pro-inflammatory immune responses. These findings, if validated, may lead to new treatment paradigms for common human malignancies.

Adaptogens Stimulate Neuropeptide Y and Hsp72 Expression and Release in Neuroglia Cells

The beneficial stress–protective effect of adaptogens is related to the regulation of homeostasis via mechanisms of action associated with the hypothalamic–pituitary–adrenal axis and the regulation of key mediators of the stress response, such as molecular chaperones, stress-activated c-Jun N-terminal protein kinase, forkhead box O transcription factor, cortisol, and nitric oxide (NO). However, it still remains unclear what the primary upstream targets are in response to stimulation by adaptogens. The present study addresses this gap in our knowledge and suggests that an important target for adaptogen mediated stress–protective effector functions is the stress hormone neuropeptide Y (NPY). We demonstrated that ADAPT-232, a fixed combination of adaptogens Eleutherococcus senticosus root extract, Schisandra chinensis berry extract, Rhodiola rosea root extract SHR-5, and its active constituent salidroside, stimulated the expression of NPY and 72 kDa heat shock protein (Hsp72) in isolated human neuroglia cells. The central role of NPY was validated in experiments in which pre-treatment of human neuroglia cells with NPY-siRNA and HSF1-siRNA resulted in the significant suppression of ADAPT-232-induced NPY and Hsp72 release. Taken together our studies suggest that the stimulation and release of the stress hormones, NPY and Hsp72, into systemic circulation is an innate defense response against mild stressors (ADAPT-232), which increase tolerance and adaptation to stress.

Silencing Hsp25/Hsp27 gene expression augments proteasome activity and increases CD8+ T-cell-mediated tumor killing and memory responses.

Relatively high expression of Hsp27 in breast and prostate cancer is a predictor of poor clinical outcome. This study elucidates a hitherto unknown mechanism by which Hsp27 regulates proteasome function and modulates tumor-specific T-cell responses. Here, we showed that short-term silencing of Hsp25 or Hsp27 using siRNA or permanent silencing of Hsp25 using lentivirus RNA interference technology enhanced PA28α mRNA expression, PA28α protein expression, and proteasome activity; abrogated metastatic potential; induced the regression of established breast tumors by tumor-specific CD8+ T cells; and stimulated long-lasting memory responses. The adoptive transfer of reactive CD8+ T cells from mice bearing Hsp25-silenced tumors efficiently induced the regression of established tumors in nontreated mice which normally succumb to tumor burden. The overexpression of Hsp25 and Hsp27 resulted in the repression of normal proteasome function, induced poor antigen presentation, and resulted in increased tumor burden. Taken together, this study establishes a paradigm shift in our understanding of the role of Hsp27 in the regulation of proteasome function and tumor-specific T-cell responses and paves the way for the development of molecular targets to enhance proteasome function and concomitantly inhibit Hsp27 expression in tumors for therapeutic gain. Cancer Prev Res; 5(1); 122–37. ©2011 AACR.

Molecular Chaperones as Mediators of Stress Protective Effect of Plant Adaptogens

The ability of plant adaptogens to enhance the “state of non-specific resistance” of an organism to stress by augmenting resistance to physical, biological, chemical and psychological stresses, and increasing concentration, performance and endurance during fatigue have placed it in a unique position among medicinal plants. However, the molecular mechanism by which plant adaptogens exerts its beneficial effects is thus far incompletely understood. This chapter focuses on recent advances in the understanding the molecular mechanism exerted by ADAPT-232 forte, a plant adaptogens consisting of a fixed combination of three extracts of Eleutherococcus senticocus, Schisandra chinensis and Rhodiola rosea. Our studies suggest that ADAPT-232 exerts its beneficial effect, in part, by a mechanism dependent on the upregulation of Hsp70 expression. A concise discussion of the effect of adaptogens on endurance and a comparison of hormetins and adaptogens will also be discussed

ROLE OF HEAT SHOCK PROTEINS IN OBESITY AND TYPE 2 DIABETES

Heat shock proteins (HSP) play an important role in human health and physiology and are known to function intracellularly as cytoprotection proteins by protecting cells against a wide variety of stressors, and extracellularly as chaperokines by stimulating the synthesis of pro-inflammatory cytokines, chemokines, and upregulates co-stimulatory molecule expression on antigen presenting cells, and enhancing natural killer (NK) cell-mediated migration and general anti-tumor responses. Obesity is known to be associated with raised serum inflammatory markers suggesting a state of heightened immune activation. The recent findings that antibody titers to several HSP are elevated in dyslipidaemic patients and individuals with established vascular disease, and that patients with Type 2 diabetes have reduced gene expression of Hsp72 which correlates with reduced insulin sensitivity point to an important role for HSP in obesity and Type 2 diabetes. This chapter briefly reviews recent advances in our understanding of the role of Hsp70 in obesity and Type 2 diabetes

Heat Shock Protein-Based Therapies

Heat shock proteins (HSP) are a family of evolutionary conserved proteins induced by cellular stressors. HSP are essential players in the development and progression of cancer inducing resistance to conventional treatment in a wide range of human tumors. Overexpression of HSP27, a member of HSP family, is associated with apoptosis inhibition, enhanced migration and metastasis and several clinical features of cancer including drug resistance promotion. At present, HSP27 could be a promising strategy to enhance sensitivity of tumors to cancer treatment. A plethora of novel compounds present in the diet, including flavonoids, can efficiently inhibit the growth of tumor cells by acting as natural “chemopreventers”. Many works reported the efficient targeting of HSP27 by using small inhibitors and dietary natural compounds in order to enhance the effectiveness of cancer therapies. In this chapter, we reviewed the current status of treatments based in dietary components targeting HSP27 as a novel strategy to circumvent chemotherapy cytotoxicity in cancer patients. Moreover we addressed the current status of HSP27 overexpression in many types of human cancers and highlighted the prominent role of targeting HSP27 as a novel therapeutic strategy in cancer.

Heat Shock Proteins and Plants

Small heat shock proteins are one of the fi ve classes of heat shock proteins, a family named after their expression in response to heat shock. Despite their name some members of this family have been shown to express during a gamut of non-stress conditions in a variety of plant species. Small HSPs have been known to accumulate during plant developmental stages like pollen development, seed maturation stages, early seed germination and also in storage organs. Interestingly, aging induced accumulation of small HSPs has also been observed in a few species. The spatial and temporal accumulation pattern of small HSPs also correlates well with other seed abundant proteins like late embryogenesis abundant (LEA) proteins. Regulation of these developmental stages responsive and non-stress induced small HSPs is also distinct from the heat stress regulated transcript induction in terms of involvement of some novel and exclusive transcription activators like ABI3 and HsfA9. Small HSPs are known to function as molecular chaperone and thus their role in plant development especially during seed development has been discussed in the light of their functional implication during these stages.