ScRNA-seq revealed targeting regulator of G protein signaling 1 to mediate regulatory T cells in Hepatocellular carcinoma.

BACKGROUND: Regulatory T cells (Tregs) are central to determine immune response, thus targeting Tregs for immunotherapy is a promising strategy against tumor development and metastasis. OBJECTIVES: The objective of this study was to identify genes for targeting Tregs to improve the outcome of HCC. METHODS: We integrated expression data from different samples to remove batch effects and further applied embedding function in Scanpy to conduct sub-clustering of CD4+ T cells in HCC for each of two independent scRNA-seq data. The activity of transcription factors (TFs) was inferred by DoRothEA. Gene expression network analysis was performed in WGCNA R package. We finally used R packages (survminer and survival) to conduct survival analysis. Multiplex immunofluorescence analysis was performed to validate the result from bioinformatic analyses. RESULTS: We found that regulator of G protein signaling 1 (RGS1) expression was significantly elevated in Tregs compared to other CD4+ T cells in two independent public scRNA-seq datasets, and increased RGS1 predicted inferior clinical outcome of HCC patients. Multiplex immunofluorescence analysis supported that the higher expression of RGS1 in HCC Tregs in tumor tissue compared to it in adjacent tissue. Moreover, RGS1 expression in Tregs was positively correlated with the expression of marker genes of Tregs, C-X-C chemokine receptor 4 (CXCR4), and three CXCR4-dependent genes in both scRNA-seq and bulk RNA-seq data. We further identified that these three genes were selectively expressed in Tregs as compared to other CD4+ T cells. The activities of two transcription factors, recombination signal binding protein for immunoglobulin kappa J region (RBPJ) and yin yang 1 (YY1), were significantly different in HCC Tregs with RGS1 high and RGS1 low. CONCLUSIONS: Our findings suggested that RGS1 may regulate Treg function possibly through CXCR4 signaling and RGS1 could be a potential target to improve responses for immunotherapy in HCC.

Molecular biology of the SARs-CoV-2 spike protein: A review of current knowledge.

The global coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an unprecedented worldwide public health emergency. Despite the concerted efforts of the scientific field, by April 25, 2021, SARS-CoV-2 had spread to over 192 countries/regions, causing more than 146 million confirmed cases including 31 million deaths. For now, an established treatment for patients with COVID-19 remains unavailable. The key to tackling this pandemic is to understand the mechanisms underlying its infectivity and pathogenicity. As a predominant focus, the coronavirus spike (S) protein is the key determinant of host range, infectivity, and pathogenesis. Thereby comprehensive understanding of the sophisticated structure of SARS-CoV-2 S protein may provide insights into possible intervention strategies to fight this ongoing global pandemic. Herein, we summarize the current knowledge of the molecular structural and functional features of SARS-CoV-2 S protein as well as recent updates on the cell entry mechanism of the SARS-CoV-2, paving the way for exploring more structure-guided strategies against SARS-CoV-2.

Association of the hOGG1 Ser326Cys polymorphism with gynecologic cancer susceptibility: a meta-analysis.

The association between the hOGG1 Ser326Cys polymorphism and gynecologic cancer susceptibility is inconclusive. We performed a comprehensive meta-analysis to precisely estimate of the impact of the hOGG1 Ser326Cys polymorphism on gynecologic cancer susceptibility. Electronic databases including PubMed, Embase, WanFang, and the China National Knowledge Infrastructure were searched for relevant studies. Odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were determined to assess the strength of the association. Fourteen studies with 2712 cases and 3638 controls were included in the final meta-analysis. The pooled analysis yielded a significant association between the hOGG1 Ser326Cys polymorphism and overall gynecologic cancer susceptibility (dominant model: OR = 1.16, 95% CI = 1.03-1.30, P=0.017). A significantly higher gynecologic cancer risk was found for the European population (homozygous model: OR = 2.17, 95% CI = 1.80-2.61, P<0.001; recessive model: OR = 2.11, 95% CI = 1.41-3.17, P<0.001; dominant model: OR = 1.29, 95% CI = 1.12-1.48, P<0.001; and allele model: OR = 1.40, 95% CI = 1.13-1.74, P=0.002), but not in the Asian population. The stratified analysis by cancer type revealed endometrial cancer was significantly associated with the hOGG1 Ser326Cys polymorphism (dominant model: OR = 1.29, 95% CI = 1.09-1.54, P=0.003; and allele model: OR = 1.28, 95% CI = 1.02-1.60, P=0.031). In conclusion, the hOGG1 Ser326Cys polymorphism was associated with higher overall gynecologic cancer susceptibility, especially for endometrial cancer in the European population.

miR-373 inhibits autophagy and further promotes apoptosis of cholangiocarcinoma cells by targeting ULK1.

Intrahepatic cholangiocarcinoma is a malignant tumor originating from intrahepatic bile ducts. Surgical therapy, radiotherapy, and chemotherapy are taken to treat this disease, but it is prone to recurrence and metastasis, with poor prognosis. Therefore, it is of great significance to explore new targets and molecular mechanisms for the development of cholangiocarcinoma cells. Clinical cholangiocarcinoma tissues from patients and four human cholangiocarcinoma cell lines were analyzed for microRNA-373 (miR-373) expression. For investigating whether miR-373 directly modulated unc-51 like autophagy activating kinase 1 (ULK1), dual-luciferase reporter assay was performed. In addition, CCK-8 assay, flow cytometry, western blot, and immunofluorescence were applied to evaluate the proliferation, apoptosis, and autophagy of cholangiocytic hepatocellular carcinoma cells. miR-373 downregulation was observed in clinical tissues and cell lines of cholangiocarcinoma. Overexpression of miR-373 reduced proliferation, enhanced apoptosis, and raised expression levels of pro-apoptosis proteins including BCL2 associated X (Bax), Caspase-3, and Caspase-9. Moreover, overexpression of miR-373 downregulated expression levels of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, Beclin-1, and promoted P62 expression on mRNA and protein levels. After miR-373 knockdown, all indexes of apoptosis and autophagy mentioned above were reversed. Luciferase activity was decreased after cotransfection of miR-373 mimic and wild-type ULK1 vector. Also, miR-373 overexpression inhibited ULK1 expression. Importantly, overexpression of miR-373 weakened expressions of ULK1, LC3, Beclin-1, and Bcl-2, and enhanced expressions of P62, Bax, Caspase-3, and Caspase-9. miR-373 mimic treatment and subsequent ULK1 overexpression, induced reverse regulation in expressions of these proteins, compared with overexpression of miR-373 only. miR-373 targeted ULK1 to initiate inhibition of autophagy and subsequent promotion of apoptosis in cholangiocarcinoma cells.

Mutation analysis of 21 autosomal short tandem repeats in Han population from Hunan, China.

Background: Short tandem repeats (STRs) are powerful genetic markers widely used in human genetics. Population data and locus-specific mutation rates of STRs are crucial for the evaluation and interpretation of genetic evidence in forensic and population genetics.Aim: To investigate the mutation rates of 21 autosomal STRs in a population from central south China.Subjects and methods: This study analysed 3420 paternity cases with a Combined Paternity Index >10,000 from Han population in Hunan. A total of 68,743 meiotic transfers were analysed and 62 mutations were identified.Results: The overall mutation rate of STR loci was 0.9 × 10-3 (95% CI, 0.0007-0.0011) and the locus-specific mutation rates were estimated ranging from 0.0000-0.0023. Locus D1S1656 exhibited the highest mutation rate of 2.3 × 10-3 (95% CI, 0.0005-0.0006), followed by D12S391 with a mutation rate of 2.0 × 10-3 (95% CI, 0.0007-0.0044). No mutation was observed at TPOX, D2S1338 or Penta D. One-step mutation cases accounted for 96.77% of total mutations and the ratio of paternal vs maternal mutations was ∼4.85:1. Inter-population comparisons of locus-specific mutation rates of several STRs revealed significant differences between Han in Hunan and Han in other regions of China. Conclusion: The data justified the use of geographical data in further genetic applications.

Highly informative single-copy nuclear microsatellite DNA markers developed using an AFLP-SSR approach in black spruce (Picea mariana) and red spruce (P. rubens).

BACKGROUND: Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies. PRINCIPAL FINDINGS: A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean = 0.67) in black spruce and from 0.161 to 0.851 (mean = 0.62) in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies. SIGNIFICANCE: The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce and other Picea species for various genetics, genomics, breeding, forensics, conservation studies and applications.

FITC delivery into plant cells using magnetic single-walled carbon nanotubes.

In this paper, fluorescein isothiocyanate (FITC) was covalently bonded with magnetic single-walled carbon nanotubes (mSWCNTs) that were purified using our previous method. To demonstrate our design, mSWCNT-FITC was delivered into plant cells (canola and carrot cells) driven by external magnetic forces. From FACS results, the FITC delivery efficiency was about 100% for both two canola and carrot protoplasts, which were further confirmed by the confocal and sectional TEM images. Some mSWCNTs were found trapped both inside the endosomes of canola protoplast and outside endosome near the nuclear membrane of carrot protoplast according to the sectional TEM images. All results showed that mSWCNT is a good delivery carrier for biomolecules.

Primary human hepatocyte transplantation in the therapy of hepatic failure: 2 cases report.

Liver failure is the end stage of hepatopathy with unfavorable prognosis. In two patients with liver failure, viable primary human hepatocytes, obtained from resected liver tissue of patients with hepatolithiasis, were transplanted into the spleen by interventional therapy through femoral arterial cannula. After transplantation, the patients' clinical symptoms and liver function were significantly improved. However, their bilirubin increased within six days following transplantation. One suffered from hepatic coma and give up treatment and the other patient died fourteen days after transplantation. It is technically safe to treat liver failure by intrasplenic transplantation of adult hepatocytes and the clinical efficacy has been confirmed. How to make transplanted hepatic cells proliferate and functionally survive is the key point to maintain continuous improvement of the recipient's hepatic function.

[Effect of Mcl-1 antisense oligonucleotide on Hela cell biology and sensitivity of chemotherapy].

OBJECTIVE: To explore the effect of myeloid leukemia-1 (Mcl-1) gene on the proliferation and apoptosis of Hela cells and the sensitivity of cervical cancer chemotherapy by antisense technology. METHODS: Mcl-1 antisense oligonucleotide(AS-ODN)was transfected into Hela cells with lipofectamine 2000. The expression of Mcl-1 was analyzed by Western blot, the cell viability was detected by MTT assay, and apoptosis was evaluated by flow cytometry. RESULTS: Mcl-1 AS-ODN arrested the cell cycle at G1/S,greatly inhibited the cell growth and induced apoptosis. The sensitivity of Hela cells on chemotherapy was low. There was obvious increase in the apoptosis rate by chemotherapy drugs and growth inhibition rate after inhibiting the expression of Mc1-1. CONCLUSION: Mcl-1 AS-ODN can not only inhibit Hela cell proliferation and induce apoptosis, but also increase the sensitivity of chemotherapy. Mcl-1 may be a potential target gene for cervical cancer chemotherapy.

Down-regulation of c-Myc expression inhibits the invasion of bile duct carcinoma cells.

Cholangiocarcinoma is the second most common primary hepatic tumour originating from biliary tract epithelial cells with poor prognosis. Enhanced c-Myc protein expression contributes to many aspects of tumour cell biology. Although the ability of c-Myc to drive unrestricted cell proliferation and to inhibit cell differentiation had been well recognized, whether down-regulated c-Myc expression can inhibit tumour cell invasion still remains to be explored. The c-Myc ASODN (antisense oligodeoxyribonucleotide) and NSODN (nonsense oligodeoxyribonucleotide) were designed, synthesized and transfected into human QBC939 bile duct carcinoma cells using the Lipofectamine 2000 reagent. The protein expression of c-Myc was detected by Western blot. A transwell experiment was applied to evaluate the invasive capacity of the QBC939 cells. c-Myc ASODN could significantly suppress the c-Myc protein expression (P<0.05) and the invasion (P<0.01) of QBC939 cells transfected with c-Myc ASODN compared with that in the control and c-Myc NSODN-transfected group. Thus in the present study we show that down-regulation of c-Myc expression can inhibit the invasion of QBC939 cells in vitro.

[Expression of Toll like receptor 4 in peripheral-blood mononuclear cells of patients with acute cerebral infarction and its correlation with clinical subtypes and the severity of the disease].

AIM: To investigate the expression of Toll like receptor 4 (TLR4) in peripheral-blood mononuclear cells (PBMC) of patients with acute cerebral infarction and its correlation with clinical subtypes and the severity of the disease. METHODS: The levels of mRNA and protein of TLR4 in PBMC of 66 patients with acute cerebral infraction were examined by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry (FCM), respectively. Clinical neurological deficit score and OCSP (Oxford Community Stroke Project) clinical subtype were performed in the patients within 3 days after admission. RESULTS: The expression of TLR4 in PBMC of patients with acute cerebral infraction (positive rate: 66.87+/-16.27%, fluorescence intensity: 11.62+/-4.39) was significantly higher than normal controls (P<0.01, respectively). 66 patients were divided into 4 groups based on OCSP. There were 9 patients with TACI, 39 patients with PACI, 7 patients with POCI, and 11 patients with LACI. The differences in the expression levels of TLR4 protein and mRNA in these groups have no statistic significance. Meanwhile, there were 27 mild cases, 30 median cases, and 9 severe cases according to the neurological deficit score. The differences in the expression levels of TLR4 protein and mRNA in these groups have statistic significance (P<0.05). The expression of TLR4 protein and mRNA were correlated positively with the severity of the disease (r=0.85, 0.87; P=0.01, 0.01). CONCLUSION: The increased expression of TLR4 may be associated with the progression of acute cerebral infarction. It may be a risk factor of acute cerebral infarction.

The POM monoclonals: a comprehensive set of antibodies to non-overlapping prion protein epitopes.

PrP(Sc), a misfolded and aggregated form of the cellular prion protein PrP(C), is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrP(C) in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrP(C) and PrP(Sc). Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrP(C). Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrP(C). Some of these antibodies recognize epitopes that are resilient to protease digestion in PrP(Sc). Other antibodies immunoprecipitate PrP(C), but not PrP(Sc). A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrP(C) and PrP(Sc). Amino-proximal antibodies were found to react with repetitive PrP(C) epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.

Extracellular heat shock protein 70 inhibits tumour necrosis factor-alpha induced proinflammatory mediator production in fibroblast-like synoviocytes.

INTRODUCTION: It was recently suggested that heat shock protein (HSP)70, an intracellular protein, is a potential mediator of inflammatory disease when it is released into the extracellular compartment. Although elevated HSP70 levels have been identified in rheumatoid arthritis (RA) synovial tissues and RA synovial fluid compared with patients with osteoarthritis and healthy individuals, it remains unclear what role extracellular HSP70 plays in the pathogenesis of RA. This study was conducted to investigate the effects of extracellular HSP70 on the production of RA-associated cytokines in fibroblast-like synoviocytes from patients with RA and to elucidate the mechanisms involved. METHODS: IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 levels in culture supernatants were measured using enzyme-linked immunosorbent assays. Activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated protein kinases (ERKs), c-Jun amino-terminal kinase (JNK) and p38 MAPK, was detected using Western blotting. Nuclear translocation of nuclear factor-kappaB (NF-kappaB) and degradation of the inhibitory protein IkappaBalpha were examined using immunohistochemistry and Western blotting. RESULTS: Human HSP70 downregulated IL-6, IL-8 and MCP-1 production in RA fibroblast-like synoviocytes induced by tumour necrosis factor (TNF)-alpha in a concentration dependent manner. HSP70 inhibited the activation of ERK, JNK and p38 MAPK in fibroblast-like synoviocytes stimulated by TNF-alpha. Furthermore, HSP70 also significantly inhibited nuclear translocation of nuclear factor-kappaB and degradation of IkappaBalpha induced by TNF-alpha. CONCLUSION: Extracellular HSP70 has an anti-inflammatory effect on RA by downregulating production of IL-6, IL-8 and MCP-1 in fibroblast-like synoviocytes, which is mediated through inhibited activation of the MAPKs and NF-kappaB signal pathways.

Nuclear heat shock protein 72 as a negative regulator of oxidative stress (hydrogen peroxide)-induced HMGB1 cytoplasmic translocation and release.

In response to inflammatory stimuli (e.g., endotoxin, proinflammatory cytokines) or oxidative stress, macrophages actively release a ubiquitous nuclear protein, high-mobility group box 1 (HMGB1), to sustain an inflammatory response to infection or injury. In this study, we demonstrated mild heat shock (e.g., 42.5 degrees C, 1 h), or enhanced expression of heat shock protein (Hsp) 72 (by gene transfection) similarly rendered macrophages resistant to oxidative stress-induced HMGB1 cytoplasmic translocation and release. In response to oxidative stress, cytoplasmic Hsp72 translocated to the nucleus, where it interacted with nuclear proteins including HMGB1. Genetic deletion of the nuclear localization sequence (NLS) or the peptide binding domain (PBD) from Hsp72 abolished oxidative stress-induced nuclear translocation of Hsp72-DeltaNLS (but not Hsp72-DeltaPBD), and prevented oxidative stress-induced Hsp72-DeltaPBD-HMGB1 interaction in the nucleus. Furthermore, impairment of Hsp72-DeltaNLS nuclear translocation, or Hsp72-DeltaPBD-HMGB1 interaction in the nucleus, abrogated Hsp72-mediated suppression of HMGB1 cytoplasmic translocation and release. Taken together, these experimental data support a novel role for nuclear Hsp72 as a negative regulator of oxidative stress-induced HMGB1 cytoplasmic translocation and release.

Hydrogen peroxide stimulates macrophages and monocytes to actively release HMGB1.

High mobility group box 1 (HMGB1) can be actively secreted by macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli (such as bacterial endotoxin, TNF-alpha, IL-1, and IFN-gamma) or passively released by necrotic cells and mediates innate and adaptive inflammatory responses to infection and injury. Here, we demonstrated that a reactive oxygen species, hydrogen peroxide (H(2)O(2)), induces active and passive HMGB1 release from macrophage and monocyte cultures in a time- and dose-dependent manner. At nontoxic doses (e.g., 0.0125-0.125 mM), H(2)O(2) induced HMGB1 cytoplasmic translocation and active release within 3-24 h. At higher concentrations (e.g., 0.25 mM), however, H(2)O(2) exhibited cytotoxicity to macrophage and monocyte cell cultures and consequently, triggered active and passive HMGB1 release. In addition, H(2)O(2) stimulated potential interaction of HMGB1 with a nuclear export factor, chromosome region maintenance (CRM1), in macrophage/monocyte cultures. Inhibitors specific for the JNK (SP600125) and MEK (PD98059), but not p38 MAPK (SB203580), abrogated H(2)O(2)-induced, active HMGB1 release. Together, these data establish an important role for oxidative stress in inducing active HMGB1 release, potentially through a MAPK- and CRM1-dependent mechanism.

The inhibition of LPS-induced production of inflammatory cytokines by HSP70 involves inactivation of the NF-kappaB pathway but not the MAPK pathways.

The objective of this study was to evaluate the negative regulatory role of heat shock protein 70 (HSP70) on endotoxin-induced activation of inflammatory cytokine signaling pathways in a macrophage cell line. Our studies show that elevation of HSP70 either by activation of the heat shock response (HSR) or through forced expression of the hsp70.1 gene downregulates cytokine expression. Our experiments showed that activation of the HSR and HSP70 overexpression could inhibit LPS-mediated expression of the proinflammatory cytokines TNF-alpha and IL-1 at the mRNA and protein levels. We also investigated the effects of HSP70 elevation on signaling pathways downstream of LPS and its receptors, including the NF-kappaB and mitogen-activated protein kinase (MAPK) pathways. The effects of HSP70 on cytokine expression were correlated with its effects on activation of NF-kappaB, a known activator of the tnfalpha and Il-1 genes. Overexpression of HSP70 inhibited the nuclear translocation of p65, the transcriptionally active component of the NF-kappaB complex, and prevented the degradation of IkappaBalpha, the regulator of NF-kappaB activity. However, HSP70 elevation did not markedly inhibit signaling through the MAPK arm of the LPS-induced pathway, suggesting that the effects of HSP70 are mediated primarily through the NF-kappaB cascade. Our experiments therefore suggested that elevated levels of HSP70 inhibit LPS-induced production of inflammatory cytokines by a mechanisms involving inactivation of NF-kappaB but cast doubt on significant role for the MAPK pathway in these effects.

[Screen of inflammatory genes regulated by heat shock factor 1 and corroboration with SOCS3 gene].

OBJECTIVE: To screen the inflammatory mediators genes regulated by HSF1, and explore the mechanism of downstream genes regulated by HSF1. METHODS: HSF-/- and HSF1+/+ mice were injected with 15 mg/kg LPS intraperitoneally (ip), respectively, and were treated as previous after HSR. The total RNA of lung tissues were extracted and filtrated by SuperArray gene Microarry. The promoter of candidate genes were analyzed by transcription element search software to search for heat shock element (HSE). Select the suppressor of cytokine signaling 3 (SOCS3) with HSE. Macrophage cells were stimulated with 400 ng/mL LPS, and were treated as previous after HSR, then the total RNA was extracted respectively. RT-PCR and northern blot assay were performed to detect the expression levels of SOCS3 mRNA. RESULTS: Fifteen genes were repressed by HSF1, including 9 genes with complete HSE. Eleven genes were accelerated by HSF1 possibly, including 8 genes with complete HSE. The promoter of SOCS3 gene contained one complete HSE. LPS stimulation obviously increased the levels of SOCS3 mRNA in macrophages of RAW264.7 mice, which was inhibited by HSR and over-expression of HSF1. CONCLUSION: HSR or HSF1 inhibits LPS induced expression of SOCS3 mRNA; HSF1 might inhibit LPS-induced expression of SOCS3 mRNA by binding to HSE in the promoter of SOCS3 gene.

Heat shock pretreatment inhibited the release of Smac/DIABLO from mitochondria and apoptosis induced by hydrogen peroxide in cardiomyocytes and C2C12 myogenic cells.

Oxidative stress may cause apoptosis of cardiomyocytes in ischemia-reperfused myocardium, and heat shock pretreatment is thought to be protective against ischemic injury when cardiac myocytes are subjected to ischemia or simulated ischemia. However, the detailed mechanisms responsible for the protective effect of heat shock pretreatment are currently unclear. The aim of this study was to determine whether heat shock pretreatment exerts a protective effect against hydrogen peroxide(H2O2)-induced apoptotic cell death in neonatal rat cardiomyocytes and C2C12 myogenic cells and whether such protection is associated with decreased release of second mitochondria-derived activator of caspase-direct IAP binding protein with low pl (where IAP is inhibitor of apoptosis protein) (Smac/DIABLO) from mitochondria and the activation of caspase-9 and caspase-3. After heat shock pretreatment (42 +/- 0.3 degrees C for 1 hour, recovery for 12 hours), cardiomyocytes and C2C12 myogenic cells were exposed to H2O2 (0.5 mmol/L) for 6, 12, 24, and 36 hours. Apoptosis was evaluated by Hoechst 33258 staining and DNA laddering. Caspase-9 and caspase-3 activities were assayed by caspase colorimetric assay kit and Western analysis. Inducible heat shock proteins (Hsp) were detected using Western analysis. The release of Smac/DIABLO from mitochondria to cytoplasm was observed by Western blot and indirect immunofluorescence analysis. (1) H2O2 (0.5 mmol/L) exposure induced apoptosis in neonatal rat cardiomyocytes and C2C12 myogenic cells, with a marked release of Smac/DIABLO from mitochondria into cytoplasm and activation of caspase-9 and caspase-3, (2) heat shock pretreatment induced expression of Hsp70, Hsp90, and alphaB-crystallin and inhibited H2O2-mediated Smac/DIABLO release from mitochondria, the activation of caspase-9, caspase-3, and subsequent apoptosis. H2O2 can induce the release of Smac/DIABLO from mitochondria and apoptosis in cardiomyocytes and C2C12 myogenic cells. Heat shock pretreatment protects the cells against H2O2-induced apoptosis, and its mechanism appears to involve the inhibition of Smac release from mitochondria.

Role of Smac/DIABLO in hydrogen peroxide-induced apoptosis in C2C12 myogenic cells.

Smac/DIABLO was recently identified as a protein released from mitochondria in response to apoptotic stimuli which promotes apoptosis by antagonizing inhibitors of apoptosis proteins. Furthermore, Smac/DIABLO plays an important regulatory role in the sensitization of cancer cells to both immune-and drug-induced apoptosis. However, little is known about the role of Smac/DIABLO in hydrogen peroxide (H(2)O(2))-induced apoptosis of C2C12 myogenic cells. In this study, Hoechst 33258 staining was used to examine cell morphological changes and to quantitate apoptotic nuclei. DNA fragmentation was observed by agarose gel electrophoresis. Intracellular translocation of Smac/DIABLO from mitochondria to the cytoplasm was observed by Western blotting. Activities of caspase-3 and caspase-9 were assayed by colorimetry and Western blotting. Full-length Smac/DIABLO cDNA and antisense phosphorothioate oligonucleotides against Smac/DIABLO were transiently transfected into C2C12 myogenic cells and Smac/DIABLO protein levels were analyzed by Western blotting. The results showed that: (1) H(2)O(2) (0.5 mmol/L) resulted in a marked release of Smac/DIABLO from mitochondria to cytoplasm 1 h after treatment, activation of caspase-3 and caspase-9 4 h after treatment, and specific morphological changes of apoptosis 24 h after treatment; (2) overexpression of Smac/DIABLO in C2C12 cells significantly enhanced H(2)O(2)-induced apoptosis and the activation of caspase-3 and caspase-9 (P<0.05). (3) Antisense phosphorothioate oligonucleotides against Smac/DIABLO markedly inhibited de novo synthesis of Smac/DIABLO and this effect was accompanied by decreased apoptosis and activation of caspase-3 and caspase-9 induced by H(2)O(2) (P<0.05). These data demonstrate that H(2)O(2) could result in apoptosis of C2C12 myogenic cells, and that release of Smac/DIABLO from mitochondria to cytoplasm and the subsequent activation of caspase-9 and caspase-3 played important roles in H(2)O(2)-induced apoptosis in C2C12 myogenic cells.

[HSP70 inhibits smac release from the mitochondria and protects against H2O2-induced apoptosis in C2C12 myogenic cells].

OBJECTIVE: To observe whether HSP70 could protect against H2O2-induced apoptosis in C2C12 myogenic cells by inhibiting Smac release from the mitochondria. METHODS: HSP70 gene and full length Smac gene was transiently transfected in C2C12 myogenic cells by lipofectamine and the protein levels of HSP70 and Smac were analysed by Western blotting. Hoechst 33 258 staining was used to examine cell morphological changes and to calculate percentage of apoptotic nuclei. DNA ladder pattern on agarose gel electrophoresis was used to observe the DNA fragmentation. Activities of caspase-3 and caspase-9 were assayed with Western blotting. The release of Smac from the mitochondria to the cytoplasm was observed by immunofluorescence. RESULTS: H2O2 ( 0.5 mmol/L ) activated caspase-3, caspase-9 8 h after the treatment and specific morphological changes of apoptosis 12 h after the treatment, and overexpression of Smac significantly promoted H2O2-induced activation of caspase-3, caspase-9 and apoptosis in C2C12 myogenic cells. HSP70 overexpression significantly inhibited H2O2-induced and Smac-promoted apoptosis, as shown by no specific DNA ladder pattern in agarose gel electrophoresis, decrease of percentage of apoptotic nuclei, and marked inactivation of caspase-3 and caspase-9. HSP70 could inhibit the release of Smac from the mitochondria to the cytoplasm 2 h after the treatment by H2O2. CONCLUSION: HSP70 inhibits Smac release from the mitochondria and protects against H2O2-induced apoptosis in C2C12 myogenic cells.