PURPOSE: Heat shock proteins (HSPs) are highly expressed during stress responses and cellular adaptation to environmental changes. One such protein is HSP27, a 27kDa protein that prevents cell death induced by many pro-apoptotic agents. Therefore, the aim of this study was to investigate the correlation between HSP27 expression and apoptosis induced by doxazosin treatment in prostate cancer cell line PC-3. MATERIALS AND METHODS: RT-PCR, Western blotting, and immunocytochemical staining were performed to determine whether HSP27 mRNA and protein are expressed in PC-3 cells. Next, to investigate the effects of doxazosin on apoptosis and HSP27 protein expression in PC-3 cells, the cells were stained using a TUNEL kit (to detect apoptotic cells) and with HSP27 antibody (to assess HSP27 protein expression) 6, 12, 24, and 48h after treatment with 25microM doxazosin. In addition, to determine whether HSP27 mRNA interference accelerates doxazosin-induced apoptosis of PC-3, we knocked down HSP27 with siRNA and then evaluated the rate of apoptosis after doxazosin treatment. RESULTS: HSP27 mRNA and protein were expressed in PC-3 cells. Furthermore, HSP27 mRNA and protein levels increased until 12 hours after 25microM doxazosin treatment, whereas the rate of apoptosis did not increased dramatically. After 12 hours, HSP27 expression decreased and then apoptosis was accelerated. In addition, siRNA-mediated knockdown of HSP27 induce higher apoptosis rate of PC-3 cells even before 12hrs after doxazosin treatment. CONCLUSIONS: By inhibiting apoptosis, HSP27 expression might play an important role in inhibiting progression to castration-refractory prostate cancer and resistance to anti-cancer treatment.
Apoptosis* ; Blotting, Western ; Cell Death ; Cell Line* ; Doxazosin ; Heat-Shock Proteins* ; Hot Temperature* ; HSP27 Heat-Shock Proteins* ; In Situ Nick-End Labeling ; Prostate* ; Prostatic Neoplasms* ; RNA, Messenger ; RNA, Small Interfering

Previous studies have demonstrated that oxidative stress involving generation of reactive oxygen species (ROS) is responsible for the cytotoxic action of TNF alpha. Protective effect of small heat shock proteins (small HSP) against diverse oxidative stress conditions has been suggested. Although overexpression of small hsp was shown to provide an enhanced survival of TNF alpha-sensitive cells when challenged with TNF alpha, neither the nature of TNF alpha-induced cytotoxicity nor the protective mechanism of small HSP has not been completely understood. In this study, we have attempted to determine whether TNF alpha induces oxidative DNA damage in TNF alpha-sensitive L929 cells. We chose to measure the level of 8-hydroxy-2'-deoxyguanosine (8 ohdG), which has been increasingly recognized as one of the most sensitive markers of oxidative DNA damage. Our results clearly demonstrated that the level of 8 ohdG increased in L929 cells in a TNF alpha dose-dependent manner. Subsequently, we asked whether small HSP has a protective effect on TNF alpha-induced oxidative DNA damage. To accomplish this goal, we have stably transfected L929 cells with mouse small hsp cDNA (hsp25) since these cells are devoid of endogenous small hsps. We found that TNF alpha-induced 8 ohdG was decreased in cells overexpressing exogenous small hsp. We also found that the cell killing activity of TNF alpha was decreased in these cells as measured by clonogenic survival. Taken together, results from the current study show that cytotoxic mechanism of TNF alpha involves oxidative damage of DNA and that overexpression of the small hsp reduces this oxidative damage. We suggest that the reduction of oxidative DNA damage is one of the most important protective mechanisms of small HSP against TNF alpha.
Animals ; DNA Damage* ; DNA* ; DNA, Complementary ; Heat-Shock Proteins* ; Heat-Shock Proteins, Small ; Homicide ; Hot Temperature* ; Mice ; Oxidative Stress ; Reactive Oxygen Species

To screen molecular chaperones similar to small heat shock proteins (sHsps), but without alpha-crystalline domain, heat-stable proteins from Schizosaccharomyces pombe were analyzed by 2-dimensional electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Sixteen proteins were identified, and four recombinant proteins, including cofilin, NTF2, pyridoxin biosynthesis protein (Snz1) and Wos2 that has an alpha-crystalline domain, were purified. Among these proteins, only Snz1 showed the anti-aggregation activity against thermal denaturation of citrate synthase. However, pre-heating of NTF2 and Wos2 at 70degrees C for 30 min, efficiently prevented thermal aggregation of citrate synthase. These results indicate that Snz1 and NTF2 possess molecular chaperone activity similar to sHsps, even though there is no alpha-crystalline domain in their sequences.
alpha-Crystallins ; Citrate (si)-Synthase ; Electrophoresis ; Heat-Shock Proteins, Small* ; Mass Screening* ; Mass Spectrometry ; Molecular Chaperones* ; Pyridoxine ; Recombinant Proteins ; Schizosaccharomyces*

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.
Carcinoma, Non-Small-Cell Lung* ; Cell Death ; Down-Regulation ; Glioblastoma ; Heat-Shock Proteins ; Homeostasis ; Hot Temperature ; Oxidative Stress ; RNA Stability ; RNA, Messenger ; RNA, Small Interfering ; Shock ; Transcriptional Activation*

The influence of spinal cord injury (SCI) on protein expression in the rat urinary bladder was assessed by proteomic analysis at different time intervals post-injury. After contusion SCI between T9 and T10, bladder tissues were processed by 2-DE and MALDI-TOF/MS at 6 hr to 28 days after SCI to identify proteins involved in the healing process of SCI-induced neurogenic bladder. Approximately 1,000 spots from the bladder of SCI and sham groups were visualized and identified. At one day after SCI, the expression levels of three protein were increased, and seven spots were down-regulated, including heat shock protein 27 (Hsp27) and heat shock protein 20 (Hsp20). Fifteen spots such as S100-A11 were differentially expressed seven days post-injury, and seven proteins including transgelin had altered expression patterns 28 days after injury. Of the proteins with altered expression levels, transgelin, S100-A11, Hsp27 and Hsp20 were continuously and variably expressed throughout the entire post-SCI recovery of the bladder. The identified proteins at each time point belong to eight functional categories. The altered expression patterns identified by 2-DE of transgelin and S100-A11 were verified by Western blot. Transgelin and protein S100-A11 may be candidates for protein biomarkers in the bladder healing process after SCI.
Animals ; Biological Markers/metabolism ; Electrophoresis, Gel, Two-Dimensional ; Female ; HSP20 Heat-Shock Proteins/metabolism ; HSP27 Heat-Shock Proteins/metabolism ; Microfilament Proteins/metabolism ; Muscle Proteins/metabolism ; Proteome/*biosynthesis ; Proteomics ; Rats ; Rats, Sprague-Dawley ; S100 Proteins/metabolism ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Spinal Cord Injuries/*metabolism/pathology ; Urinary Bladder/*metabolism ; *Wound Healing

The promoter of mitochondria-localized small heat shock protein gene in Lycopersicon esculentum (Lehsp23.8) is characterized as strongly heat-inducible. In this study, to determine how the expression of Lehsp23.8 is regulated, we conducted five expression vectors carrying the gus gene driven by the 5' deletion products of the Lehsp23.8 promoter. The corresponding transgenic tobacco plants were generated via Agrobacterium tumefaciens-mediated transformation. Transgenic plants were identified by PCR and Southern blotting analysis. GUS activities under heat-shock conditions were characterized in transgenic tobacco plants. After heat shock, obvious GUS staining was detected in the leaves, shoots, roots, flowers and fruits of the transgenic tobacco plants. The result of fluorometric GUS assays in leaves showed that the heat-induced GUS activity of the 565 bp promoter was the strongest, while that of the 255 bp promoter was the lowest. Deletion analysis shows that the smallest promoter fragment (-255 bp to -23 bp) is sufficient for heat induction. It also indicates that the sequences between -255 bp and -565 bp serve as enhancers, while the sequences between -565 bp and -871 bp can repress the heat-induced activity of the Lehsp23.8 promoter.
Gene Expression Regulation, Plant ; genetics ; Genetic Vectors ; Heat-Shock Proteins ; genetics ; Heat-Shock Proteins, Small ; genetics ; metabolism ; Hot Temperature ; Lycopersicon esculentum ; genetics ; Mitochondria ; genetics ; metabolism ; Plants, Genetically Modified ; genetics ; Promoter Regions, Genetic ; genetics ; Tobacco ; genetics

BACKGROUND: Anti-apoptotic proteins may be involved in tumor development, progression and the response to treatment. Bcl-2 is by far the most studied anti-apoptotic protein. A novel infibitor of apoptosis, designat ed survivin, and the heat shock proteins (HSPs) have recently been found in many human cancers. Immunohistochemical methods were used to determine the expression level of survivin, HSP 70 and bcl-2 in non-small cell lung cancer (NSCLC) to evaluate their clinical significance. METHODS: Tissue array slides were obtained from 99 surgically resected NSCLCs. Immunohistochemical staining was performed by an immuno-peroxidase technique using an avidin-biotinylated horseradish peroxidase complex. Anti-survivin rabbit polyclonal antibodies, anti-HSP70 mouse monoclonal antibodies and anti-bcl-2 mouse monoclonal antibodies were used as the primary antibodies. RESULTS: Positive ataining of survivin was detected in 33.3% of the cases. Survivin positivity is associated with to females and recurrence. A nonstatistically significant trend toward increased survivin expression was observed in non-smokers, and its expression inversely correlated with the number of cigarettes smoked in smokers. HSP70 was detected in 84.8% but this did not correlate with the clinicopathologic characteristics. Bcl-2 was detected in 18.2% and its and its expression correlated to tumor recurrence. No significant difference in the median survival time was noted in a comparison of al cases with survivin expression and those without. There was no association betwwen HSP70 or bcl-2 expression and survival. CONCLUSIONS: Survivin expression was significantly associated with females and tumor recurrence. In addition its expression was inversely associated with the number of cigarettes smoked. However, HSP70 and bcl-2 expression were not associated with the clinical parameters or survival. This suggests that measuring the survivin levels may be useful in identifying patients at high risk for disease recurrence. Therefore, survivin might be a new diagnostic/therapeutic target in cancer.
Animals ; Antibodies ; Antibodies, Monoclonal ; Apoptosis ; Apoptosis Regulatory Proteins ; Carcinoma, Non-Small-Cell Lung ; Female ; Heat-Shock Proteins ; Horseradish Peroxidase ; Humans ; Immunohistochemistry ; Lung* ; Mice ; Recurrence ; Smoke ; Tobacco Products

OBJECTIVETo study the effect of RNA interference (RNAi) on small heat shock protein (sHSP) Sjp40 of Schistosoma japonicum and its synergistic effect on the expression of SjHSP60, SjHSP70, and SjHSP90 mRNA, and observe the mRNA expression levels of Sjp40, SjHSP60, SjHSP70, and SjHSP90 in different stages of S.japonicum.

METHODSDouble-stranded RNA (dsRNA) of Sjp40 (dsSjp40) and a control dsRNA of green fluorescent protein (dsGFP) were generated by in vitro transcription and transfected into adult worm by immersing the worm in dsRNA solution. The total RNA and proteins were isolated simultaneously from the adult worms using TRIzol reagent 7 days after transfection. The expression levels of Sjp40, SjHSP60, SjHSP70, and SjHSP90 mRNA and the expression level of Sjp40 protein were determined by quantitative real-time PCR (qPCR) and Western blotting, respectively. The mRNA expression of HSPs of S. japonicum in different stages was evaluated by qPCR.

RESULTSCompared with those in the control worms transfected with dsGFP, Sjp40 mRNA level was decreased by 80% in the worms transfected with dsSjp40, and the level of Sjp40 protein showed also a significant decrease. The mRNA expression levels of SjHSP60, SjHSP70, and SjHSP90 did not show an obvious synergism after Sjp40 RNAi. The expression profiles of Sjp40, SjHSP60, SjHSP70, and SjHSP90 showed significant differences in different stages of S. japonicum, and the expression level of Sjp40 mRNA in the egg stage was much higher than that of other HSP genes.

CONCLUSIONdsSjp40-RNAi can induce effective suppression of Sjp40 gene expression at both mRNA and protein levels, but no obvious synergism occurs in the mRNA expressions of SjHSP60, SjHSP70, and SjHSP90.

Animals ; Gene Expression Profiling ; Heat-Shock Proteins, Small ; genetics ; Helminth Proteins ; genetics ; RNA Interference ; RNA, Double-Stranded ; RNA, Messenger ; genetics ; Schistosoma japonicum ; genetics

BACKGROUND: The mesenchymal-epithelial transition factor (MET) receptor can be overexpressed in solid tumors, including small cell lung cancer (SCLC). However, the molecular mechanism regulating MET stability and turnover in SCLC remains undefined. One potential mechanism of MET regulation involves the C-terminus of Hsp70-interacting protein (CHIP), which targets heat shock protein 90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, we investigated the functional effects of CHIP expression on MET regulation and the control of SCLC cell apoptosis and invasion. METHODS: To evaluate the expression of CHIP and c-Met, which is a protein that in humans is encoded by the MET gene (the MET proto-oncogene), we examined the expression pattern of c-Met and CHIP in SCLC cell lines by western blotting. To investigate whether CHIP overexpression reduced cell proliferation and invasive activity in SCLC cell lines, we transfected cells with CHIP and performed a cell viability assay and cellular apoptosis assays. RESULTS: We found an inverse relationship between the expression of CHIP and MET in SCLC cell lines (n=5). CHIP destabilized the endogenous MET receptor in SCLC cell lines, indicating an essential role for CHIP in the regulation of MET degradation. In addition, CHIP inhibited MET-dependent pathways, and invasion, cell growth, and apoptosis were reduced by CHIP overexpression in SCLC cell lines. CONCLUSION: CHIP is capable of regulating SCLC cell apoptosis and invasion by inhibiting MET-mediated cytoskeletal and cell survival pathways in NCI-H69 cells. CHIP suppresses MET-dependent signaling, and regulates MET-mediated SCLC motility.
Apoptosis ; Blotting, Western ; Cell Line ; Cell Proliferation ; Cell Survival ; Heat-Shock Proteins ; Humans ; Lung Neoplasms ; Small Cell Lung Carcinoma* ; Ubiquitin ; Ubiquitination

OBJECTIVETo study the characteristics of the mutation of small heat-shock protein 22 (HSP22) gene in Chinese patients with Charcot-Marie-Tooth (CMT) disease.

METHODSA CMT2L proband with 423(G--> T) mutation in HSP22 gene had been studied and reported by the present authors. In this study, mutation analysis of HSP22 gene was performed using polymerase chain reaction and DNA direct sequencing in 114 CMT probands.

RESULTSIn the 114 CMT probands, a 582(C--> T)(T194T)samesense mutation was found in two unrelated families.

CONCLUSIONThe rate of HSP22 gene mutation in Chinese patients with CMT is as low as 0.87%(1/115).

Asian Continental Ancestry Group ; genetics ; Charcot-Marie-Tooth Disease ; ethnology ; genetics ; China ; DNA Mutational Analysis ; Heat-Shock Proteins, Small ; genetics ; Humans ; Mutation ; Polymerase Chain Reaction