-
Journal of Experimental & Clinical... Apr 2019Abnormal expression or distribution of connexin 32 (Cx32) is associated with hepatocarcinogenesis, but the role of Cx32 and the underlying mechanisms are still unclear.
BACKGROUND
Abnormal expression or distribution of connexin 32 (Cx32) is associated with hepatocarcinogenesis, but the role of Cx32 and the underlying mechanisms are still unclear.
METHODS
The expression level of Cx32 in 96 hepatocellular carcinoma (HCC) specimens was determined using western blotting and immunohistochemistry. The correlation between Cx32 expression and clinicopathological parameters was analyzed. The cell apoptosis rate was examined using flow cytometry and western blotting. The role of Cx32 in the Src kinase and epidermal growth factor receptor (EGFR) signaling pathways was measured by quantitative real-time PCR, western blotting and coimmunoprecipitation (CO-IP). The effect of Cx32 overexpression on the streptonigrin (SN)-induced tumor growth suppression and apoptosis was assessed in nude mice.
RESULTS
Our study showed that overexpressed Cx32 accumulated in the cytoplasm and that Cx32-containing gap junctions (GJs) were nearly absent in HCC specimens. Upregulated Cx32 expression was highly correlated with advanced tumor-node-metastasis (TNM) stage and poor tumor differentiation and was an independent predictive marker for poor prognosis in HCC. Overexpression of Cx32 significantly inhibited SN-induced apoptosis by activating the EGFR signaling pathway in vitro and in vivo. Moreover, the expression levels of Cx32 and EGFR were positively correlated in HCC specimens. The CO-IP experiments demonstrated that Cx32 could bind to Src kinase, and the western blotting results revealed that Cx32 increased the levels of EGFR and p-EGFR by upregulating Src expression.
CONCLUSION
The present study demonstrated that overexpressed and internalized Cx32 was associated with advanced TNM stage and poor tumor differentiation and predicted poor prognosis in HCC. Cx32 facilitated HCC progression by blocking chemotherapy-induced apoptosis in vitro and in vivo via interacting with Src and thus promoting the phosphorylation of EGFR, subsequently activating the EGFR signaling pathway. Cx32 may be a potential biomarker and a new therapeutic target for HCC.
Topics: Adult; Aged; Animals; Apoptosis; Carcinoma, Hepatocellular; Connexins; ErbB Receptors; Humans; Liver Neoplasms; Male; Mice; Middle Aged; Signal Transduction; Transfection; Young Adult; Gap Junction beta-1 Protein
PubMed: 30947731
DOI: 10.1186/s13046-019-1142-y -
Marine Drugs Mar 2019Two new piperazine-triones lansai E and F (, ), together with four known secondary metabolites lansai D (), 1--methyl-()-albonoursin (), imidazo[4,5-]-1,2,4-triazine (),...
Two new piperazine-triones lansai E and F (, ), together with four known secondary metabolites lansai D (), 1--methyl-()-albonoursin (), imidazo[4,5-]-1,2,4-triazine (), and streptonigrin () were isolated from a deep-sea-derived sp. strain SMS636. The structures of the isolated compounds were confirmed by comprehensive spectroscopic analysis, including HRESIMS, 1D and 2D NMR. Compound exhibited moderate antibacterial activities against and methicillin resistant (MRSA) with Minimum Inhibitory Concentration (MIC) values of 12.5 and 25 μg/mL, respectively. Compound displayed significant antibacterial activities against , MRSA and Bacillus Calmette-Guérin (BCG) with MIC values of 0.78, 0.78 and 1.25 μg/mL, respectively.
Topics: Anti-Bacterial Agents; Candida albicans; Escherichia coli; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Piperazine; Pseudomonas aeruginosa; Staphylococcus aureus; Streptomycetaceae
PubMed: 30901830
DOI: 10.3390/md17030186 -
Accounts of Chemical Research Mar 2019Proteins are well-known to undergo a variety of post-translational modifications (PTMs). One such PTM is citrullination, an arginine modification that is catalyzed by a...
Proteins are well-known to undergo a variety of post-translational modifications (PTMs). One such PTM is citrullination, an arginine modification that is catalyzed by a group of hydrolases called protein arginine deiminases (PADs). Hundreds of proteins are known to be citrullinated and hypercitrullination is associated with autoimmune diseases including rheumatoid arthritis (RA), lupus, ulcerative colitis (UC), Alzheimer's disease, multiple sclerosis (MS), and certain cancers. In this Account, we summarize our efforts to understand the structure and mechanism of the PADs and to develop small molecule chemical probes of protein citrullination. PAD activity is highly regulated by calcium. Structural studies with PAD2 revealed that calcium-binding occurs in a stepwise fashion and induces a series of dramatic conformational changes to form a catalytically competent active site. These studies also identified the presence of a calcium-switch that controls the overall calcium-dependence and a gatekeeper residue that shields the active site in the absence of calcium. Using biochemical and site-directed mutagenesis studies, we identified the key residues (two aspartates, a cysteine, and a histidine) responsible for catalysis and proposed a general mechanism of citrullination. Although all PADs follow this mechanism, substrate binding to the thiolate or thiol form of the enzyme varies for different isozymes. Substrate-specificity studies revealed that PADs 1-4 prefer peptidyl-arginine over free arginine and certain citrullination sites on a peptide substrate. Using high-throughput screening and activity-based protein profiling (ABPP), we identified several reversible (streptomycin, minocycline, and chlorotetracycline) and irreversible (streptonigrin, NSC 95397) PAD-inhibitors. Screening of a DNA-encoded library and lead-optimization led to the development of GSK199 and GSK484 as highly potent PAD4-selective inhibitors. Furthermore, use of an electrophilic, cysteine-targeted haloacetamidine warhead to mimic the guanidinium group in arginine afforded several mechanism-based pan-PAD-inhibitors including Cl-amidine and BB-Cl-amidine. These compounds are highly efficacious in various animal models, including those mimicking RA, UC, and lupus. Structure-activity relationships identified numerous covalent PAD-inhibitors with different bioavailability, in vivo stability, and isozyme-selectivity (PAD1-selective: D-Cl-amidine; PAD2-selective: compounds 16-20; PAD3-selective: Cl4-amidine; and PAD4-selective: TDFA). Finally, this Account describes the development of PAD-targeted and citrulline-specific chemical probes. While PAD-targeted probes were utilized for identifying off-targets and developing high-throughput inhibitor screening platforms, citrulline-specific probes enabled the proteomic identification of novel diagnostic biomarkers of hypercitrullination-related autoimmune diseases.
Topics: Animals; Aspartic Acid; Catalysis; Catalytic Domain; Citrullination; Cysteine; Enzyme Inhibitors; HEK293 Cells; Histidine; Humans; Mice; Models, Chemical; Mutation; Protein Processing, Post-Translational; Protein-Arginine Deiminases; Proteins
PubMed: 30844238
DOI: 10.1021/acs.accounts.9b00024 -
Organic & Biomolecular Chemistry Dec 2018Streptonigrin (STN, 1) is a highly functionalized aminoquinone alkaloid antibiotic with broad and potent antitumor activity. Previous isotope-labelling and genetic...
Streptonigrin (STN, 1) is a highly functionalized aminoquinone alkaloid antibiotic with broad and potent antitumor activity. Previous isotope-labelling and genetic studies suggested that a β-carboline alkaloid should be a key intermediate of STN biosynthesis and formed via a Pictet-Spengler (PS) reaction. Herein, StnK2 was biochemically characterized to be a Pictet-Spenglerase (PSase) catalysing the formation of a tetrahydro-β-carboline (TH-βC) scaffold from (2S,3S)-β-methyl tryptophan and d-erythrose-4-phosphate. StnK2 can tolerate the alteration of tryptophan but only accept d-erythrose-4-phosphate as the aldehyde substrate, and StnK2 was identified to be R-specific for the newly formed chiral center. This work increases the diversities of Pictet-Spenglerase in nature and set a stage for the generation of streptonigrin derivatives by precursor-directed pathway engineering based on the flexible substrate selectivity of StnK2.
Topics: Antibiotics, Antineoplastic; Biosynthetic Pathways; Carbolines; Stereoisomerism; Streptomyces; Streptonigrin; Substrate Specificity; Tryptophan
PubMed: 30483694
DOI: 10.1039/c8ob02710b -
Cancers Nov 2018In general, expression of transglutaminase 2 (TGase 2) is upregulated in renal cell carcinoma (RCC), resulting in p53 instability. Previous studies show that TGase 2...
In general, expression of transglutaminase 2 (TGase 2) is upregulated in renal cell carcinoma (RCC), resulting in p53 instability. Previous studies show that TGase 2 binds to p53 and transports it to the autophagosome. Knockdown or inhibition of TGase 2 in RCC induces p53-mediated apoptosis. Here, we screened a chemical library for TGase 2 inhibitors and identified streptonigrin as a potential therapeutic compound for RCC. Surface plasmon resonance and mass spectroscopy were used to measure streptonigrin binding to TGase 2. Mass spectrometry analysis revealed that streptonigrin binds to the N-terminus of TGase 2 (amino acids 95⁻116), which is associated with inhibition of TGase 2 activity in vitro and with p53 stabilization in RCC. The anti-cancer effects of streptonigrin on RCC cell lines were demonstrated in cell proliferation and cell death assays. In addition, a single dose of streptonigrin (0.2 mg/kg) showed marked anti-tumor effects in a preclinical RCC model by stabilizing p53. Inhibition of TGase 2 using streptonigrin increased p53 stability, which resulted in p53-mediated apoptosis of RCC. Thus, targeting TGase 2 may be a new therapeutic approach to RCC.
PubMed: 30463244
DOI: 10.3390/cancers10110455 -
Journal of Toxicology and Environmental... 2018The functional characterization of marine macroalgae toward their potential to strength genome protection is still scarce. Hence, the aim of this study was to assess the...
The functional characterization of marine macroalgae toward their potential to strength genome protection is still scarce. Hence, the aim of this study was to assess the antigenotoxic potential of Ulva rigida, Fucus vesiculosus, and Gracilaria species in Drosophila melanogaster following dietary exposure and adopting the somatic mutation and recombination test (SMART). All macroalgae displayed a genoprotection activity, namely against an exogenous challenge (streptonigrin). The action against subtler endogenous pressures was also noted indicating that supplementation level is a critical factor. Gracilaria species provided ambivalent indications, since 10% of G. vermiculophylla inhibited the egg laying and/or larvae development, while 10% of G. gracilis promoted spontaneous genotoxicity. The effects of U. rigida were modulated (in intensity) by the growing conditions, demonstrating higher genoprotection against streptonigrin-induced damage when grown in an aquaculture-controlled system, while the effectiveness against spontaneous genotoxicity was more apparent in specimens grown under wild conditions. In contrast, F. vesiculosus did not produce significant differences in its potential under varying growing conditions. Overall, these findings shed some light on the macroalgae ability toward genome protection, contributing to the development of algaculture industry, and reinforcing the concept of functional food and its benefits.
Topics: Animal Feed; Animals; Diet; Dietary Supplements; Drosophila melanogaster; Fucus; Gracilaria; Larva; Mutagenicity Tests; Mutagens; Protective Agents; Seaweed; Streptonigrin; Ulva
PubMed: 30156999
DOI: 10.1080/15287394.2018.1507856 -
Infection and Immunity Jul 2018Pneumococcal conjugate vaccines (PCV) elicit opsonophagocytic (opsonic) antibodies to pneumococcal capsular polysaccharides (PPS) and reduce nasopharyngeal (NP)...
Pneumococcal conjugate vaccines (PCV) elicit opsonophagocytic (opsonic) antibodies to pneumococcal capsular polysaccharides (PPS) and reduce nasopharyngeal (NP) colonization by vaccine-included serotypes. However, nonopsonic antibodies may also be important for protection against pneumococcal disease. For example, 1E2, a mouse IgG1 monoclonal antibody (MAb) to the serotype 3 (ST3) PPS (PPS3), reduced ST3 NP colonization in mice and altered ST3 gene expression Here, we determined whether 1E2 affects ST3 gene expression during colonization of mice by performing RNA sequencing on NP lavage fluid from ST3-infected mice treated with 1E2, a control MAb, or phosphate-buffered saline. Compared to the results for the controls, 1E2 significantly altered the expression of over 50 genes. It increased the expression of the operon, which encodes an iron uptake system, and decreased the expression of , which encodes a protein critical for resistance to oxidative stress. 1E2-mediated effects on ST3 required divalent binding, as Fab fragments did not reduce NP colonization or alter ST3 gene expression. , 1E2 induced dose-dependent ST3 growth arrest and altered and expression, whereas an opsonic PPS3 MAb, 5F6, did not. 1E2-treated bacteria were more sensitive to hydrogen peroxide and the iron-requiring antibiotic streptonigrin, suggesting that 1E2 may increase iron import and enhance sensitivity to oxidative stress. Finally, 1E2 also induced rapid capsule shedding , suggesting that this may initiate 1E2-induced changes in sensitivity to oxidative stress and gene expression. Our data reveal a novel mechanism of direct, antibody-mediated antibacterial activity that could inform new directions in antipneumococcal therapy and vaccine development.
Topics: Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Bacterial Capsules; Female; Gene Expression; Mice; Mice, Inbred C57BL; Nasopharynx; Oxidative Stress; Pneumococcal Vaccines; Streptococcus pneumoniae
PubMed: 29735523
DOI: 10.1128/IAI.00300-18 -
Biochemistry Mar 2018Streptonigrin (CAS no. 3930-19-6) is a natural product shown to have antitumor activities in clinical trials conducted in the 1960s-1970s. However, its use in clinical...
Streptonigrin (CAS no. 3930-19-6) is a natural product shown to have antitumor activities in clinical trials conducted in the 1960s-1970s. However, its use in clinical studies eventually faded, and the molecular mechanisms of streptonigrin antitumor effects remain poorly defined. Despite its lack of current clinical use, efforts on its total synthesis have continued. Here, we show that streptonigrin binds and inhibits the SUMO-specific protease SENP1. NMR studies identified that streptonigrin binds to SENP1 on the surface where SUMO binds and disrupts SENP1-SUMO1 interaction. Site-directed mutations in combination with NMR chemical shift perturbation suggest key roles of aromatic π stacking interactions in binding streptonigrin. Treatment of cells with streptonigrin resulted in increased global SUMOylation levels and reduced level of hypoxia inducible factor alpha (HIF1α). These findings inform both the design of SENP1 targeting strategy and the modification of streptonigrin to improve its efficacy for possible future clinical use.
Topics: Cell Line, Tumor; Cysteine Endopeptidases; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Magnetic Resonance Spectroscopy; SUMO-1 Protein; Streptonigrin; Sumoylation
PubMed: 29481054
DOI: 10.1021/acs.biochem.7b00947 -
Cx32 suppresses extrinsic apoptosis in human cervical cancer cells via the NF‑κB signalling pathway.International Journal of Oncology Oct 2017Tumour necrosis factor α (TNFα) and TNF‑related apoptosis inducing ligand (TRAIL) usually trigger either survival or apoptosis signals in various cell types, and...
Tumour necrosis factor α (TNFα) and TNF‑related apoptosis inducing ligand (TRAIL) usually trigger either survival or apoptosis signals in various cell types, and nuclear factor κB (NF‑κB) is a key factor that regulates their biological effects. Connexin 32 (Cx32) is a gap junction (GJ) protein that plays vital roles in tumourigenesis and tumour progression. Our previous study explored abnormal Cx32 expression in para‑nuclear areas, exacerbated prognostic parameters and suppressed streptonigrin/cisplatin-induced apoptosis in human cervical cancer (CaCx) cells. In this study, we investigated the role of Cx32 in the extrinsic apoptosis pathway of CaCx cells. In transgenic HeLa cells and C-33A cells, Cx32 expression was manipulated using doxycycline or Cx32 siRNA. GJ inhibitors or low density culturing was used to change the status of gap junction intracellular communication (GJIC). We found that apoptosis induced by TNFα and TRAIL was suppressed by Cx32 expression despite the presence or absense of GJIC. We also found that Cx32 upregulated the expression of nuclear NF‑κB and its downstream targets c-IAP1, MMP‑2, and MMP‑9 in HeLa‑Cx32 and C-33A cells. Following our previous study design, our clinical data showed that NF‑κB and MMP‑2 levels increased in human CaCx specimens with high Cx32 expression compared to levels in para‑carcinoma of cervical specimens. SC75741 and JSH-23, NF‑кB signalling pathway inhibitors, inhibited the anti-apoptotic effects of Cx32. In conclusion, Cx32 suppressed TNFα /TRAIL-induced extrinsic apoptosis by upregulating the NF‑κB signalling pathway. This study demonstrates a novel mechanism for Cx32's anti-apoptotic effect and provides a reasonable explanation for the pro-tumour effect of Cx32 in human CaCx cells.
Topics: Apoptosis; Cell Line, Tumor; Connexins; Doxycycline; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; NF-kappa B; RNA, Small Interfering; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Uterine Cervical Neoplasms; Gap Junction beta-1 Protein
PubMed: 28902345
DOI: 10.3892/ijo.2017.4106 -
Oncology Reports Nov 2017Tumor necrosis factor α (TNFα) and TNF-related apoptosis-inducing ligand (TRAIL) can trigger the extrinsic apoptosis pathway. Our previous study indicated that...
Tumor necrosis factor α (TNFα) and TNF-related apoptosis-inducing ligand (TRAIL) can trigger the extrinsic apoptosis pathway. Our previous study indicated that connexin32 (Cx32) inhibited streptonigrin-induced intrinsic apoptosis via the epidermal growth factor receptor (EGFR) pathway. However, whether Cx32 can exert effects on the extrinsic apoptosis pathway through EGFR signaling remains unclear. In the present study, we investigated the role of Cx32 in extrinsic apoptosis induced by treatment with TNFα + cycloheximide (CHX) or afatinib in human cervical cancer (CaCx) cells. In stable inducible Cx32-transfected HeLa cells (HeLa-Cx32), Cx32 expression was induced by treatment with doxycycline (Dox). Furthermore, C-33A cells, which natively express high levels of Cx32, were used as a cell model for knockdown of Cx32 with siRNA. To determine the non-junctional function of Cx32 in apoptosis, 18α-glycyrrhetinic acid (18α-GA), a gap junction intracellular communication (GJIC) inhibitor, was used. Our results showed that Cx32 could inhibit apoptosis induced by TNFα + afatinib with or without the GJIC inhibitor. In clinical cervical tissue samples, we found that the expression of survivin was markedly higher in CaCx than in normal cervix tissue, which was in accordance with the expression of Cx32 in our previous study. In HeLa-Cx32 cells, we also found that Cx32 upregulated the expression of Cox-2. In addition, Cx32 upregulated EGFR expression in low-density culture (lacking GJ formation). Cx32 could also promote the expression of EGFR, phospho-STAT3 and phospho-ERK in HeLa-Cx32 cells following TNFα treatment. After knocking down Cx32 in C-33A cells, the expression levels of survivin and TNFα were downregulated. The present study verifies that Cx32 exerts an inhibitory effect on extrinsic apoptosis in CaCx cells, and suggests that Cx32 may regulate the progression and micro-environment of CaCx cells.
Topics: Afatinib; Apoptosis; Connexins; Cycloheximide; Doxycycline; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Inhibitor of Apoptosis Proteins; Phosphorylation; Quinazolines; Survivin; Tumor Necrosis Factor-alpha; Uterine Cervical Neoplasms; Gap Junction beta-1 Protein
PubMed: 28901517
DOI: 10.3892/or.2017.5950