Heat shock protein expression in canine osteosarcoma

Abnormal levels of heat shock proteins have been observed in a number of human neoplasms and demonstrate prognostic, predictive and therapeutic implications. Since osteosarcoma (OSA) in dogs provides an important model for the same disease in humans, the aim of this study was to evaluate the immunohistochemical expression of Hsp27, Hsp72, Hsp73 and Hsp90 in 18 samples of canine appendicular OSA, in relation to histological grade and overall survival (OS), in order to investigate their potential prognostic, predictive and/or therapeutic value. A semiquantitative method was used for the analysis of the results. Hsp27, Hsp73 and Hsp90 showed a variably intense, cytoplasmic and nuclear immunoreactivity that was not associated with histological type or grade. On the other hand, a high percentage of Hsp72 immunostaining was significantly associated with grade III (P < 0.01) and a lack of immunolabelling was significantly correlated to a longer OS (P = 0.006). Neoplastic emboli were occasionally positive for Hsp27, faintly immunoreactive for Hsp72 and intensely immunolabelled by Hsp73 and Hsp90. In conclusion, absence of Hsp72 immunosignal appears to be associated with a favourable prognosis whilst the widespread Hsp90 immunoreactivity detected in all tumour cases as well as in neoplastic emboli, suggests this protein could be targeted in the therapy of canine OSA, and likewise in its human counterpart.     

人口腔螺旋体热休克蛋白的初步研究

为分析牙周病的发病与人口爱螺旋体的热休克蛋白的关系。通过ＳＤＳ－ＰＡＧＥ电泳将菌体蛋白分离,转移电泳（Ｗｅｓｔｅｒｎ ｂｌｏｔ）检查螺旋体的热休克蛋白抗原,牙周病患者血清与螺旋体的热休克蛋白进行免疫印迹试验检查灯克蛋白抗体。结果为实验所用的螺旋体有４种可诱导产生质变休克蛋白,患者血清中有多种对于口腔螺旋体蛋白能起作用的本,其中有两名患者的血清对Ｔ．ｓｏｃｒａｎｓｋｉｉ３５５３５菌株的６０ｋＤ或６５     

Effects of prolonged stanozolol treatment on antioxidant enzyme activities, oxidative stress markers, and heat shock protein HSP72 levels in rat liver

The abuse of anabolic-androgenic steroids (AAS) to enhance physical performance is widespread in sport communities despite their reported side effects. Since the biochemical bases for the hepatotoxic effects of these compounds are largely unknown, this investigation was aimed at testing whether prolonged (8 weeks) treatment with high doses (2 mg kg⁻¹ body weight; 5 d wk⁻¹) of stanozolol (ST), either alone or in conjunction with treadmill-exercise training, induced changes in oxidative stress biomarker levels and antioxidant defence systems in rat liver. After ST oral administration, the mean values of serum parameters related to hepatic function were within normal ranges. No changes in protein carbonyl content and in the reduced to oxidized glutathione (GSH/GSSG) ratio were detected in liver homogenates of ST-treated rats, whereas thiobarbituric acid-reactive substances (TBARS) levels resulted increased (P<0.05). Total superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities were higher (P<0.05) in the liver of treated rats but mitochondrial SOD and glutathione reductase (GR) activities, and the 72 kDa heat shock protein (HSP72) level were not modified. Chronic exercise alone did not change any of the above parameters except for a remarkable enhancement of HSP72 expression; in no case training modified the effects of ST treatment. The present data show that 8 wk ingestion of ST, either with or without concurrent exercise training, can induce oxidative stress in rat liver despite the up-regulation of enzymatic antioxidant activities.     

Heat shock response in Actinobacillus actinomycetemcomitans

Abstract The heat shock response in Actinobacillus actinomycetemcomitans , a capnophilic Gram-negative bacterial species that is implicated in the development of certain forms of periodontitis, was characterized. Different strains of A. actinomycetemcomitans were grown at 37, 42 and 48°C in the presence of ³⁵S-methionine. The bacterial cells were lysed, run on SDS-PAGE and subsequently blotted on nitrocellulose paper. After autoradiography of the blots, several protein bands from the cultures at 42°C showed an increased intensity; major bands were observed at 90, 70, and 60 kDa, but increased protein synthesis was also detected at 54, 28 and 17 kDa. Nitrocellulose blots were also incubated with a panel of monoclonal and polyclonal antibodies directed to epitopes on different heat shock proteins. Strong reactivity was found with several antibodies at the position corresponding to a molecular mass of 60 kDa. The protein is probably the GroEL homologue in A. actinomycetemcomitans , a member of the ‘common bacterial antigen’ family.     

Gambogic acid and gambogenic acid induce a thiol-dependent heat shock response and disrupt the interaction between HSP90 and HSF1 or HSF2

Cancer cells rely on heat shock proteins (HSPs) for growth and survival. Especially HSP90 has multiple client proteins and plays a critical role in malignant transformation, and therefore different types of HSP90 inhibitors are being developed. The bioactive natural compound gambogic acid (GB) is a prenylated xanthone with antitumor activity, and it has been proposed to function as an HSP90 inhibitor. However, there are contradicting reports whether GB induces a heat shock response (HSR), which is cytoprotective for cancer cells and therefore a potentially problematic feature for an anticancer drug. In this study, we show that GB and a structurally related compound, called gambogenic acid (GBA), induce a robust HSR, in a thiol-dependent manner. Using heat shock factor 1 (HSF1) or HSF2 knockout cells, we show that the GB or GBA-induced HSR is HSF1-dependent. Intriguingly, using closed form ATP-bound HSP90 mutants that can be co-precipitated with HSF1, a known facilitator of cancer, we show that also endogenous HSF2 co-precipitates with HSP90. GB and GBA treatment disrupt the interaction between HSP90 and HSF1 and HSP90 and HSF2. Our study implies that these compounds should be used cautiously if developed for cancer therapies, since GB and its derivative GBA are strong inducers of the HSR, in multiple cell types, by involving the dissociation of a HSP90-HSF1/HSF2 complex.     

Heat shock protein expression analysis in canine osteosarcoma reveals HSP60 as a potentially relevant therapeutic target

Heat shock proteins (HSP) are highly conserved across eukaryotic and prokaryotic species. These proteins play a role in response to cellular stressors, protecting cells from damage and facilitating recovery. In tumor cells, HSPs can have cytoprotective effects and interfere with apoptotic cascades. This study was performed to assess the prognostic and predictive values of the gene expression of HSP family members in canine osteosarcoma (OS) and their potential for targeted therapy. Gene expressions for HSP were assessed using quantitative PCR (qPCR) on 58 snap-frozen primary canine OS tumors and related to clinic-pathological parameters. A significant increased expression of HSP60 was found in relation to shorter overall survival and an osteoblastic phenotype. Therefore, the function of HSP60 was investigated in more detail. Immunohistochemical analysis revealed heterogeneous staining for HSP60 in tumors. The highest immunoreactivity was found in tumors of short surviving dogs. Next HSP expression was shown in a variety of canine and human OS cell lines by qPCR and Western blot. In two highly metastatic cell lines HSP60 expression was silenced using siRNA resulting in decreased cell proliferation and induction of apoptosis in both cell lines. It is concluded that overexpression of HSP60 is associated with a poor prognosis of OS and should be evaluated as a new target for therapy.     

Nontoxic heat shock protein coinducer BRX-220 protects against acute pancreatitis in rats

Background: Nontoxic heat shock protein (HSP) inducer compounds open up promising therapeutic possibilities by activating one of the natural and highly conserved defense mechanisms of the organism. Aims: In the present experiments, we examined the effects of a HSP coinducer drug-candidate, BRX-220, on the cholecystokinin-octapeptide (CCK)-induced acute pancreatitis in rats. Methods: Male Wistar rats weighing 240 to 270 g were divided into two groups. In group B, 20 mg/kg BRX-220 was administered orally, followed by 75 μg/kg CCK subcutaneously three times, after 1, 3, and 5 h. This whole procedure was repeated for 5 d. The aminals in group B received physiological saline orally instead of BRX-220, but otherwise the protocol was the same as in group B. The rats were exsanguinated through the abdominal aorta 12 h after the last administration of CCK. We determined the serum amylase activity, the plasma trypsinogen activation peptide concentration, the pancreatic weight/body weight ratio, the DNA and total protein contents of the pancreas, the levels of pancreatic HSP60 and HSP72, the activities of pancreatic amylase, lipase, trypsinogen, and free radical scavenger enzymes (superoxide dismutase, catalase, and glutathione peroxidase), the degree of lipid peroxidation, protein oxidation, and the reduced glutathione level. Histopathological investigation of the pancreas was also performed in all cases. Results: Repeated CCK treatment resulted in the typical laboratory and morphological changes of experimentally induced pancreatitis. The pancreatic levels of HSP60 and HSP72 were significantly increased in the animals treated with BRX-220. In group B, the pancreatic total protein content and the amylase and trypsinogen activities were significantly higher vs. group B. The plasma trypsinogen activation peptide concentration, and the pancreatic lipid peroxidation, protein oxidation, and the activity of Cu/Zn-superoxide dismutase were significantly decreased in group B vs. group B, whereas the glutathione peroxidase activity was increased. The morphological damage in group B was significantly lower than that in group B. Conclusion: The HSP coinducer BRX-220, administered for 5 d, has a protective effect against CCK-induced acute pancreatitis.     

Heat shock protein responses in thermally stressed bay scallops, Argopecten irradians, and sea scallops, Placopecten magellanicus

The effects of thermal stress on the induction of heat shock proteins (HSPs) were examined in northern bay scallops, Argopecten irradians irradians, a relatively heat tolerant estuarine species, and sea scallops, Placopecten magellanicus, a species residing in cooler, deeper water. Polyclonal antibodies used in this work for analysis of inducible HSP70 and HSP40 only recognized proteins of 72 and 40 kDa respectively from the mantles of both scallop species. Additionally, HSP quantification using the antibody to HSP70 was equally effective by either immunoprobing of western blots or ELISA, demonstrating that either approach could be successfully employed for analysis of thermal response in scallops. Sea scallop HSP70 and HSP40 did not change when animals were heat-shocked for 3 h by raising the temperature from 10 °C to 20 °C; however, a 24 h treatment of the same magnitude elicited a significant response. Conversely, bay scallops displayed rapid and prolonged HSP70 and HSP40 responses during the recovery period following a 3 h heat shock from 20 °C to 30 °C. Temperature reduction from 20 °C to 3 °C for 3 h also caused significant HSP70 and HSP40 increases in bay scallops; this represents the first time cold shock was shown to induce HSP synthesis in bivalve mollusks. The onset of the HSP40 response was more rapid than for HSP70, occurring at the end of the cold shock itself prior to transfer to a recovery temperature. Both proteins responded maximally during recovery at control temperature. HSP responses of sea and bay scallops to thermal stress may be related to their habitat in the natural environment and they suggest a differential capacity for adaptation to temperature change. This is an important consideration in assessing the response of these scallops to different culture conditions.     

Heat shock protein 72 overexpression protects against hyperthermia, circulatory shock, and cerebral ischemia during heatstroke.

This study extends our earlier studies in rats by applying our heatstroke model to a new species. Additionally, transgenic mice are used to examine the role of heat shock protein (HSP) 72 in experimental heatstroke. Transgenic mice that were heterozygous for a porcine HSP70i gene ([+]HSP72), transgene-negative littermate controls ([-]HSP72), and normal Institute of Cancer Research strain mice (ICR) under pentobarbital sodium anesthesia were subjected to heat stress (40 degrees C) to induce heatstroke. In [-]HSP72 or ICR, the values for mean arterial pressure, the striatal blood flow, and the striatal PO2 after the onset of heatstroke were significantly lower than those in preheat controls. The core and brain temperatures, the extracellular concentrations of ischemic and injury markers in the striatum, and the striatal neuronal damage scores were significantly greater than those in the preheat controls. In [-]HSP72 or ICR, the body temperatures, cell ischemia content, and injury marker in the striatum were significantly higher, and the mean arterial pressure, striatal blood flow, and striatal PO2 concentration were significantly lower during heatstroke than in [+]HSP72. Accordingly, the latency and the survival times for [+]HSP72 significantly exceeded those of [-]HSP72 or ICR. These results demonstrate that the overexpression of HSP72 in multiple organs improves survival during heatstroke by reducing hyperthermia, circulatory shock, and cerebral ischemia and damage in mice.     

The heat shock proteins: Their roles as multi-component machines for protein folding

The roles played by heat shock proteins in fungi have been subjected to intense scrutiny. Presuming they only played roles in tolerance to stress gave way to the realization that many of them were essential for maintenance of cell physiology under all conditions. Recent progress has revealed their action as multi-component machines, playing roles in signalling and expansion of phenotypic plasticity, as well as their well-established function as molecular chaperones.     

Heat shock protein expression in fish

Heat shock proteins (HSP) are a family of proteins expressed in response to a wide range of biotic and abiotic stressors. They are thus also referred to as stress proteins. Their extraordinarily high degree of identity at the amino acid sequence level and the fact that this cellular stress response has been described in nearly all organisms studied, make this group of proteins unique. We provide a brief historical overview of HSP research, as a background to summarizing what is known about HSP expression in fish. The expression of HSPs in fish has been described in cell lines, primary cultures of various cells, and in the tissues of whole organisms. Collectively, the data show that the expression of HSPs are affected in a wide variety of fish cells and tissues, in response both to biological stressors such as infectious pathogens, as well as to abiotic stressors such as heat and cold shock, and environmental contaminants. HSP research in fish is in its early stages and many studies are describing the expression of proteins in response to various stressors. Several studies have contributed to our understanding of the molecular nature and the molecular biology of HSPs in fish. Recent studies have shown a relationship between HSP expression and the generalized stress response in fish, but further research is needed to clarify the complex relationships between stress hormones and the cellular HSP response. In general, the HSP response seems to be related to the sensing of the stressor and the subsequent cellular effects which may adapt the cells to cope with the stressors. Consequently, such data may be of central importance in understanding the significance of HSP expression to the whole organism. We conclude with sections on laboratory methods used in HSP research and on potential applications of this knowledge in biomonitoring.     

Potential roles of hepatic heat shock protein 25 and 70i in protection of mice against acetaminophen-induced liver injury

The aim of the present study was to assess the contribution of the level of expression of heat shock protein 25 (HSP25), 60 (HSP60), 70 (HSC70) and 70i (HSP70i) in mouse livers after a lethal dose of acetaminophen (APAP) to their survival. We examined changes in survival ratio, plasma APAP level and alanine aminotransferase (ALT) activity, and hepatic reduced glutathione (GSH), HSP25, HSP60, HSC70 and HSP70i levels following treatment of mice with APAP (500 mg/kg, p.o.). The plasma APAP level increased rapidly, and reached a maximum 0.5 h after APAP treatment. Hepatic GSH decreased rapidly, and was almost completely depleted 1 h after APAP treatment. Plasma ALT activity, an index of liver injury, significantly increased from 3 h onwards after APAP treatment. The survival ratios 9 h, 24 h and 48 h after APAP treatment were 96%, 38% and 36%, respectively. We found a remarkable difference in the patterns of hepatic HSP25 and HSP70i induction in mice that survived after APAP treatment. HSP70i levels increased from 1 h onwards after APAP treatment in a time-dependent manner, and reached a maximum at 9 h. In contrast, HSP25 could be detected just 24 h after APAP treatment, and maximal accumulation was observed at 48 h. Other HSPs examined were unchanged. Notably, the survival ratio dropped by only 2% after HSP25 expression. Recently, a novel role for HSP25 as an anti-inflammatory factor was suggested. We have already shown that 48-h treatment with APAP induces severe centrilobular necrosis with inflammatory cell infiltration in mouse livers. Taken together, the level of expression of hepatic HSP25 may be a crucial determinant of the fate of mice exposed to APAP insult.     

Heat shock protein 20 promotes sirtuin 1-dependent cell proliferation in induced pluripotent stem cells

BACKGROUNDHeat shock proteins (HSPs) are molecular chaperones that protect cells against cellular stresses or injury. However, it has been increasingly recognized that they also play crucial roles in regulating fundamental cellular processes. HSP20 has been implicated in cell proliferation, but conflicting studies have shown that it can either promote or suppress proliferation. The underlying mechanisms by which HSP20 regulates cell proliferation and pluripotency remain unexplored. While the effect of HSP20 on cell proliferation has been recognized, its role in inducing pluripotency in human-induced pluripotent stem cells (iPSCs) has not been addressed.AIMTo evaluate the efficacy of HSP20 overexpression in human iPSCs and evaluate the ability to promote cell proliferation. The purpose of this study was to investigate whether overexpression of HSP20 in iPSCs can increase pluripotency and regeneration.METHODSWe used iPSCs, which retain their potential for cell proliferation. HSP20 overexpression effectively enhanced cell proliferation and pluripotency. Overexpression of HSP20 in iPSCs was characterized by immunocytochemistry staining and real-time polymerase chain reaction. We also used cell culture, cell counting, western blotting, and flow cytometry analyses to validate HSP20 overexpression and its mechanism.RESULTSThis study demonstrated that overexpression of HSP20 can increase the pluripotency in iPSCs. Furthermore, by overexpressing HSP20 in iPSCs, we showed that HSP20 upregulated proliferation markers, induced pluripotent genes, and drove cell proliferation in a sirtuin 1 (SIRT1)-dependent manner. These data have practical applications in the field of stem cell-based therapies where the mass expansion of cells is needed to generate large quantities of stem cell-derived cells for transplantation purposes. CONCLUSIONWe found that the overexpression of HSP20 enhanced the proliferation of iPSCs in a SIRT1-dependent manner. Herein, we established the distinct crosstalk between HSP20 and SIRT1 in regulating cell proliferation and pluripotency. Our study provides novel insights into the mechanisms controlling cell proliferation that can potentially be exploited to improve the expansion and pluripotency of human iPSCs for cell transplantation therapies. These results suggest that iPSCs overexpressing HSP20 exert regenerative and proliferative effects and may have the potential to improve clinical outcomes.     

Heat shock protein 72 associated with CD44v6 in human colonic adenocarcinoma

CD44v6 is a splice variant of CD44 (CD44v), probably promoting cancer cell adherence to vascular endothelium and base membranes and enhancing the invasion and metastasis of colonic carcinomas. Heat shock protein 72 (HSP72) as a molecular chaperone has been confirmed to be overexpressed in epithelial carcinoma cells. There may be a possible association between the expression of HSP72 and CD44v6 during the growth and progression of colonic carcinoma cells. The aim of the study was to investigate the interaction between heat shock protein 72 and CD44v6 in human colonic carcinomas. The localization of HSP72 and CD44v6 in human colonic carcinomas was determined by immunohistochemistry and confocal laser microscopy. The interaction between HSP72 and CD44v6 in colonic carcinoma cells was analyzed by immunoprecipitation and Western immunoblots. Our results revealed that colonic carcinoma synchronously co-expressed higher levels of HSP72 and CD44v6 than that in adjacent normal colonic tissues. HSP72 and CD44v6 were mainly immunolocalized in the cytoplasm, and also immunolabelled on the cell membrane. Based on immunoprecipitation and Western immunoblots, we found that HSP72 was associated with CD44v6 precursor fragments in human colonic carcinoma cells. The interaction between HSP72 and CD44v6 in human colonic carcinoma cells may contribute to study the pathogenesis and immunotherapy of colonic carcinoma.     

热休克蛋白27在胃癌的表达及意义

目的探讨热休克蛋白27(HSP27)在胃癌及癌旁组织中的表达及其意义。方法用免疫组织化学法检测68例胃癌及57例癌旁组织中HSP27的表达,比较其阳性表达率。结果HSP27在胃癌中的表达率为47.1%,明显高于癌旁组织26.32%(P<0.05),并且与胃癌的分化程度相关,但与肌层浸润深度及淋巴结转移无关。结论HSP27在胃癌组织中过度表达,提示其在胃癌的发生发展中起着重要作用。     

Adaptation to heat of cardiomyoblasts in culture protects them against heat shock: role of nitric oxide and heat shock proteins

Dosed adaptation to environmental factors is an efficient non-drug means for increasing the resistance of organs or the body as a whole. We demonstrated earlier that nitric oxide (NO) plays an important role in adaptive defense of the organism, in particular due to activation of heat shock protein (HSP) synthesis. A key question remained open—to what extent the formation of adaptive defense depends on central mechanisms and to what extent on the intracellular mechanisms immediately responding to the adapting factor, and whether the NO-dependent activation of HSP synthesis plays a role in adaptation of isolated cells. In the present study we looked into the possibility of producing a protective effect of adaptation to heat in cell culture. A 6-day adaptation to heat limited to 17% the decrease in metabolic activity induced by heat shock in H9c2 cardiomyoblasts. The development of adaptation was associated with increased NO production. Treatment of cells with the inhibitor of NO synthase L-NNA (100 M) prevented the development of adaptive protection. Adaptation of cell culture enhanced synthesis of HSP70 but not HSP27. Blockade of HSP70 synthesis with quercetin (50 M) left unchanged the protective effect of adaptation. Inhibition of NO synthesis restricted the adaptation-induced HSP70 synthesis. Therefore, the formation of adaptation at the cell level may result from a direct action of an environmental factor without participation of neurohumoral factors. Such adaptation involves NO-dependent mechanisms divorced from the activation of HSP70 synthesis.