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Small ubiquitin-like modifier (SUMO) 2/3 is known to conjugate to substrates in response to a variety of cellular stresses. However, whether and how SUMO2/3-specific proteases are involved in de-conjugation under cell stress is unclear. Here, we show that low doses of hydrogen peroxide (H(2)O(2)) induce an increase of the SENP3 protein, which removes SUMO2/3 from promyelocytic leukemia (PML). Low dose H(2)O(2) causes SENP3 to co-localize with PML bodies and reduces the number of PML bodies in a SENP3-dependent manner. Furthermore, de-conjugation of SUMO2/3 from PML is responsible for the accelerated cell proliferation caused by low dose H(2)O(2). Knocking down PML promotes basal cell proliferation as expected. This can be reversed by reconstitution with wild-type PML but not its mutant lacking SUMOylation, indicating that only the SUMOylated PML can play an inhibitory role for cell proliferation. Thus, SENP3 appears to be a key mediator in mild oxidative stress-induced cell proliferation via regulation of the SUMOylation status of PML. Furthermore, SENP3 is over-accumulated in a variety of primary human cancers including colon adenocarcinoma in which PML is hypo-SUMOylated. These results reveal an important role of SENP3 and the SUMOylation status of PML in the regulation of cell proliferation under oxidative stress.

Glutathione conjugation and transportation of glutathione conjugates of anticancer drugs out of cells are important for detoxification of many anticancer drugs. Inhibition of this detoxification system has recently been proposed as a strategy to treat drug-resistant solid tumors. Gallbladder carcinoma is resistant to many anticancer drugs, therefore, it is needed to develop a novel strategy for cancer therapy. In the present study, we tested the effect of emodin (1,3,8-trihydroxy-6-methylanthraquinone), a reactive oxygen species (ROS) generator reported by our group previously, in combination with cisplatin (CDDP), carboplatin (CBP) or oxaliplatin in treating the gallbladder carcinoma cell line SGC996. Our results showed that co-treatment with emodin could remarkably enhance chemosensitivity of SGC996 cells in comparison with cisplatin, carboplatin or oxaliplatin treatment alone. We found that the mechanisms may be attributed to reduction of glutathione level, and downregulation of multidrug resistance-related protein 1 (MRP1) expression in SGC996 cells. The experiments on tumor-bearing mice showed that emodin/cisplatin co-treatment inhibited the tumor growth in vivo via increasing tumor cell apoptosis and downregulating MRP1 expression. In conclusion, emodin can work as an adjunct to enhance the anticancer effect of platinum drugs in gallbladder cancer cells via ROS-related mechanisms.

Different groups of audiences in health care: health professionals and health consumers, each have different information needs. Health monographs targeting different audiences are created by leveraging readers' background knowledge. The NCI's Physician Data Query (PDQ®) Cancer Information Summaries provide parallel cancer information and education resources with different target audiences. In this paper, we used targeted audience-specific cancer information PDQs to measure characteristic differences on the element level between audiences. In addition, we compared vocabulary coverage. Results show a significant difference between the professional and patient version of cancer monographs in both content organization and vocabulary. This study provides a new view to assess targeted audience-specific health information, and helps editors to improve the quality and readability of health information.

Lambda-carrageenan is a sulfated galactan isolated from some red algae and have been reported to have many kinds of biological activities. Chondrus ocellatus is an important economic alga in China and many other parts of the world. The sample was obtained after lambda-carrageenan from the alga was degraded by microwave and its molecular weight is 9.3 kDa. In this study, tumor inhibiting activity of low molecular weight lambda-carrageenan and its mixture with 5-Fu on mice transplanted with S180 tumor was investigated. Weight of immune organ, proliferation ratio of lymphocyte concentration of TNF-alpha and histopathology of spleen and tumor from the transplanted S180 tumor mice were also determined. Results indicated that the degraded lambda-carrageenan could enhance antitumor activity of 5-Fu and improve immunocompetence damaged by 5-Fu.

Reactive oxygen species (ROS) can drive the de‑differentiation of tumor cells leading to the process of epithelial-to-mesenchymal transition (EMT) to enhance invasion and metastasis. The invasive and metastatic phenotype of malignant cells is often linked to loss of E-cadherin expression, a hallmark of EMT. Recent studies have demonstrated that hypoxic exposure causes HIF-1-dependent repression of E-cadherin. However, the mechanism by which ROS and/or HIF suppresses E-cadherin expression remains less clear. In the present study, we found that ROS accumulation in ovarian carcinoma cells upregulated HIF-1α expression and subsequent transcriptional induction of lysyl oxidase (LOX) which repressed E-cadherin. Loss of E-cadherin facilitated ovarian cancer (OC) cell migration in vitro and promoted tumor growth in vivo. E-cadherin immunoreactivity correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, tumor differentiation and metastasis. Negative E-cadherin expression along with FIGO stage, tumor differentiation and metastasis significantly predicted for a lower 5-year survival rate. These findings suggest that ROS play an important role in the initiation of metastatic growth of OC cells and support a molecular pathway from ROS to aggressive transformation which involves upregulation of HIF-1α and its downstream target LOX to suppress E-cadherin expression leading to an increase in cell motility and invasiveness.

Background: Arsenic trioxide (As 2 O 3 ) can induce clinical remission in patients with acute promyelocytic leukemia via induction of differentiation and programmed cell death (apoptosis). We investigated the effects of As 2 O 3 on a panel of malignant lymphocytes to determine whether growth-inhibitory and apoptotic effects of As 2 O 3 can be observed in these cells at clinically achievable concentrations. Methods: Eight malignant lymphocytic cell lines and primary cultures of lymphocytic leukemia and lymphoma cells were treated with As 2 O 3 , with or without dithiothreitol (DTT) or buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis). Apoptosis was assessed by cell morphology, flow cytometry, annexin V protein level, and terminal deoxynucleotidyl transferase labeling of DNA fragments. Cellular proliferation was determined by 5-bromo-2'-deoxyuridine incorporation into DNA and flow cytometry and by use of a mitotic arrest assay. Mitochondrial transmembrane potential (ΔΨ m ) was measured by means of rhodamine 123 staining and flow cytometry. Protein expression was assessed by western blot analysis or immunofluorescence. Results: Therapeutic concentrations of As 2 O 3 (1-2 μM) had dual effects on malignant lymphocytes: 1) inhibition of growth through adenosine triphosphate (ATP) depletion and prolongation of cell cycle time and 2) induction of apoptosis. As 2 O 3 -induced apoptosis was preceded by ΔΨ m collapse. DTT antagonized and BSO enhanced As 2 O 3 -induced ATP depletion, ΔΨ m collapse, and apoptosis. Caspase-3 activation, usually resulting from ΔΨ m collapse, was not always associated with As 2 O 3 -induced apoptosis. As 2 O 3 induced PML (promyelocytic leukemia) protein degradation but did not modulate expression of cell cycle-related proteins, including c-myc, retinoblastoma protein, cyclin-dependent kinase 4, cyclin D1, and p53, or expression of differentiation-related antigens. Conclusions: Substantial growth inhibition and apoptosis without evidence of differentiation were induced in most malignant lymphocytic cells treated with 1-2 μM As 2 O 3 . As 2 O 3 may prove useful in the treatment of malignant lymphoproliferative disorders.

This paper introduced a LDA-based pseudo relevance feedback (PRF) model for cross language information retrieval. To validate the performance of PRF techniques in CLIR task, we conducted cross language query expansion experiments based on a self-constructed CLIR system, the LDA-based PRF model was applied before or after the query translating process, namely the pre-translation-PRF, the post-translation-PRF, and the combined-PRF strategy. We also compared this model with the classical VSM-based PRF algorithm. Experiment results showed that the proposed LDA-based PRF method was effective for improving the performance of CLIR.

Studies of Community-based question and answer services (cQA) have grown to be one of the emerging trends in Web information services. And one of the main tasks is to retrieve similar questions. To identify the fact that some questions with different expressions though may indeed have the same, or very similar, meaning, the similar questions are defined in syntactic, semantic and pragmatic aspects according to user retrieval intention. Five models including Language Model, Translation-based Language Model, Parser-based model, LDA and WordNet source-based model are selected as baselines. Integrated models which linearly combine WordNet, Stanford Parser and Language Model and further weighted by syntactic feature are proposed to integrate features of the three aspects. Experiment results show that our integrated models perform better than the basic models, especially when the linear combination of the WordNet model and the Stanford Parser model is weighted by syntactic information with proper noun phrases as representative. The integrated models are further verified by logistic analysis.

Community-run, formal evaluations and manually annotated text corpora are critically important for advancing biomedical text-mining research. Recently in BioCreative V, a new challenge was organized for the tasks of disease named entity recognition (DNER) and chemical-induced disease (CID) relation extraction. Given the nature of both tasks, a test collection is required to contain both disease/chemical annotations and relation annotations in the same set of articles. Despite previous efforts in biomedical corpus construction, none was found to be sufficient for the task. Thus, we developed our own corpus called BC5CDR during the challenge by inviting a team of Medical Subject Headings (MeSH) indexers for disease/chemical entity annotation and Comparative Toxicogenomics Database (CTD) curators for CID relation annotation. To ensure high annotation quality and productivity, detailed annotation guidelines and automatic annotation tools were provided. The resulting BC5CDR corpus consists of 1500 PubMed articles with 4409 annotated chemicals, 5818 diseases and 3116 chemical-disease interactions. Each entity annotation includes both the mention text spans and normalized concept identifiers, using MeSH as the controlled vocabulary. To ensure accuracy, the entities were first captured independently by two annotators followed by a consensus annotation: The average inter-annotator agreement (IAA) scores were 87.49% and 96.05% for the disease and chemicals, respectively, in the test set according to the Jaccard similarity coefficient. Our corpus was successfully used for the BioCreative V challenge tasks and should serve as a valuable resource for the text-mining research community. Database URL: http://www.biocreative.org/tasks/biocreative-v/track-3-cdr/

Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R(724)KEFAKFEEER(734). In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R(724)KEFAKFEEER(734), each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E(724)AERKFERKFE(734), but not in cells expressing wild type αIIbβ3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.

Gallbladder carcinoma is known to be an aggressive malignancy and non-sensitive to routine chemotherapy; its prognosis is quite poor. In this study, we show that emodin (1,3,8-trihydroxy-6-methylanthraquinone), an active component from Chinese medicinal herbs, can enhance apoptosis of gallbladder cancer cells induced by cisplatin (CDDP) in a reactive oxygen species (ROS)-dependent manner. The expression of survivin, which is involved in the inhibition of apoptosis, was measured after drug treatment and it was found that this could be suppressed by CDDP. Co-treatment with emodin additively inhibited survivin expression in a ROS-dependent manner. Further experiments proved that emodin potentiated the antitumor effects of CDDP in vivo by downregulating the expression of survivin without causing detectable toxic effects on normal tissues.

Menadione and diquat cause toxicity in isolated hepatocytes. The toxicities of both menadione and diquat are primarily due to redox cycling and consequent oxidative stress. Menadione toxicity, however, has another component as the compound also possesses alkylating and oxidating properties allowing it to interact directly with cellular nucleophiles. Sulfite afforded considerable protection of isolated rat hepatocytes against the toxicity of menadione. This protective effect of sulfite may have several components. Sulfite effectively competed with glutathione (GSH) for conjugation with menadione, sparing intracellular GSH which may continue to detoxify reactive oxygen species formed through menadione redox cycling. The menadione sulfite conjugate undergoes much slower redox cycling than both menadione and the menadione glutathione conjugate. Sulfite also showed some degree of protection of hepatocytes from the toxicity of diquat. Diquat is a "pure" redox cycling agent and the protective effect of sulfite may involve the liberation of GSH from GSSG by sulfitolysis. This would again bolster intracellular GSH levels allowing further GSH-dependent detoxification of reactive oxygen species through cellular GSH peroxidases. In conclusion, our data illustrate the potential of inorganic sulfite to support the intracellular detoxification function of GSH, both against reactive electrophilic metabolites and against agents undergoing redox cycling.

Lambda-carrageenan is a sulfated galactan isolated from some red algae and have been reported to have many kinds of biological activities. Chondrus ocellatus is an important economic alga in China and many other parts of the world. The sample was obtained after lambda-carrageenan from the alga was degraded by microwave and its molecular weight is 9.3 kDa. In this study, tumor inhibiting activity of low molecular weight lambda-carrageenan and its mixture with 5-Fu on mice transplanted with S180 tumor was investigated. Weight of immune organ, proliferation ratio of lymphocyte concentration of TNF-alpha and histopathology of spleen and tumor from the transplanted S180 tumor mice were also determined. Results indicated that the degraded lambda-carrageenan could enhance antitumor activity of 5-Fu and improve immunocompetence damaged by 5-Fu.

lambda-Carrageenan is a sulfated galactan isolated from some red algae and have been reported to have many kinds of biological activities. lambda-Carrageenan from Chondrus ocellatus, an important economic alga in China and many other parts of the world, was degraded by microwave, and obtained five products that have different molecular weight: 650, 240, 140, 15, 9.3 kDa. Analytical results confirmed that microwave degradation might not change the chemical components and structure of polysaccharides under certain condition. In this study, tumor-inhibiting activities, weight of immune organ, nature killer cells activity, lymphocyte proliferation ratio and pathological slice of spleen and tumor cells from the control group and lambda-carrageenan-treated mice of transplanted S 180 and H22 tumor were investigated. The results indicated that the five lambda-carrageenan samples all showed antitumor and immunomodulation activities in different degree. Molecular weight of polysaccharides had notable effect on the activities. In addition, their antitumor and immunomodulation have some relevance and the five lambda-carrageenans probably inhibited tumor by means of activating the immunocompetence of the body. Among all the experiment results, samples with the highest activities are PC4 and PC5 whose molecular weight are 15 and 9.3 kDa. (C) 2003 Elsevier Ltd. All rights reserved.

To test whether splicing overlapping extension(SOE) method can be a tool for obtaining rare fusion gene's transcripts and to study the tumorigenic capacity of a novel fusion gene AML1-MTG16.SOE method was used to obtain AML1- MTG16 fusion gene's transcripts. MTG16, AML1-MTG16 and AML1-MTG16 without III,VI conserved domains of MTG16 segment were inserted into pEGFP- C1,pDsRed-N1 vector respectively,then transfected NIH3T3 cell line by lipofection. Forty-eight hours later, the transfected cells were examined by laser-scanning confocal microscopy. Stable transfected cells were obtained by G418 500ug/ul selection for one month. Growth curve, soft agar colonies formation tumorigenesis in nude mice were done to compare the difference between stable transfected cells.Recombined AML1-MTG16 by SOE contained its CDS. NIH3T3 expressing AML1-MTG16 had a faster proliferation in medium, colony growth in soft agar. AML1-MTG16 expression cells also induced tumors formation following injection into nude mouse. MTG16,AML1-MTG16 and AML1-MTG16 without III,VI conserved domains of MTG16 were colocalized in the nucleus of cotransfected NIH3T3 cells under the examination of laser-scanning confocal microscope.SOE is an effective method to get rare fusion gene's transcripts. AML1-MTG16 plays an important role in leukemogenesis. MTG16 may also have a carcinogenic property within the AML1-MTG16 fusion gene. Carcinogenic property of AML1-MTG16 is restricted to its localization in the nuclear matrix. N terminal of MTG16 may play an important part in the carcinogenic activity of AML1-MTG16.

The involvement of platelets in tumor progression is well recognized. The depletion of circulating platelets or pharmacologic inhibitors of platelet activation decreases the metastatic potential of circulating tumor cells in metastasis mouse models. The platelet ADP receptor P2Y12 amplifies the initial hemostatic responses activated by a variety of platelet agonists and stabilizes platelet aggregation, playing a crucial role in granule secretion, integrin activation and thrombus formation. However, the relationship between P2Y12 and tumor progression is not clear. In our study, the Lewis Lung Carcinoma (LLC) spontaneous metastatic mouse model was used to evaluate the role of P2Y12 in metastasis. The results demonstrated that P2Y12 deficiency significantly reduced pulmonary metastasis. Further studies indicated that P2Y12 deficiency diminished the ability of LLC cells to induce platelet shape change and release of active TGFβ1 by a non-contact dependent mechanism resulting in a diminished, platelet-induced EMT-like transformation of the LLC cells, and that transformation probably is a prerequisite of LLC cell metastasis. Immunohistochemical analyses indicated an obvious P2Y12 deficiency related attenuation of recruitment of VEGFR1+ bone marrow derived cell clusters, and extracellular matrix fibronectin deposition in lungs, which presumably are required for pre-metastatic niche formation. In contrast to the LLC cells, non-epithelial melanoma B16 cells induced platelet aggregation in a cell number and P2Y12-dependent manner. Also, a platelet induced EMT-like transformation of B16 cells is dependent on P2Y12. In agreement with the LLC cell model, platelet P2Y12 deficiency also results in significantly less lung metastasis in the B16 melanoma experimental metastasis model. These results demonstrate that P2Y12 is a safe drug target for anti-thrombotic therapy, and that P2Y12 may serve as a new target for inhibition of tumor metastasis.

Abstract 3356 The platelet ADP receptor P2Y12 plays a prominent role in amplifying platelet activation, aggregation and thrombus formation, and the P2Y12 inhibitor Clopidogrel is widely used clinically to treat coronary artery, peripheral vascular and cerebrovascular diseases. Recently, several publications analyzed the TRITON-TIMI 38 clinical trial (test the efficacy and safety of Prasugrel, a newly FDA approved thienopyridine P2Y12 inhibitor) revealing an increase in multiple types of solid tumors with Prasugrel use, casting doubt on the safety of anti-platelet therapy targeting P2Y12. In this study, we used animal models to investigate the safety of targeting P2Y12, and to elucidate the probable mechanism for the effects of P2Y12 antagonists on metastasis. Tumor growth and metastasis are basic features of malignant cancer. Pulmonary metastasis in the experimental, and spontaneous mouse metastasis models were used to identify the role of P2Y12, and the effects of its inhibitors in tumor growth and metastasis. In the experimental pulmonary metastasis group, one week before tail vein injection of 2×105 melanoma B16 cells, wild type (WT) and P2y12–/– mice were treated every other day with PBS, Clopidogrel or Prasugrel by oral gavage and continue in whole experiment process. Tumor nodules were counted, and the average nodule sizes measured at 20 days after B16 cell injection. P2y12–/– mice had fewer lung metastatic loci than the lungs of the WT controls. The experimental and control lungs had similar size nodules. But Clopidogrel dose dependently (1.5mg/kg and 30mg/kg) caused an increase of metastatic loci and loci size in both WT and P2y12–/– mice. Prasugrel (0.2mg/kg and 4mg/kg) did not affect the number or size of metastatic loci produced in either strain. These results demonstrated that p2y12 deficiency negatively affected cancer metastasis.In contrast, Clopidogrel, but not Prasugrel, promotes metastasis and cancer growth. Importantly, Clopidogrel induced metastasis promotion and growth was not P2Y12 dependent. A plausible explanation of this difference between the two drugs may reside in the fact that both are pro-drugs of thienopyridine family. Presumably, activation of the pro-drug clopidogrel, but not Prasugrel produces one or more metabolites able to promote cancer growth and metastasis in vivo. This supposition was tested, in part by asking if the effects of Clopidogrel are limited to the B16 model of metastasis. The Lewis lung carcinoma (LLC) spontaneous pulmonary metastasis model was used for this purpose. LLC cells (2.0×106) were implanted intradermally in each mouse. Surgery was done 14 days after cell implantation to completely remove the primary tumors (about 1.0g or 1cm3 in size). One month later, lungs were removed, rinsed with PBS, and pulmonary metastatic nodules counted and measured. P2Y12 deficiency decreased the spontaneous formation of pulmonary metastatic loci, but had no effect on the primary tumor. Also, Clopidogrel, dose dependently promoted lung metastasis. Next, the mechanism of the effect of P2Y12 absence on tumor metastasis was investigated. In vitro aggregation experiments revealed that B16 melanoma cells directly interact with p2y12+/+ platelets and cause platelet-cancer cell aggregation. In contrast, P2Y12 deficiency results in significantly less or no cancer cell-platelet aggregation. Interestingly, LLC cells do not cause platelet aggregation. Immunohistochemistry and immunofluorescence analyses of lung samples from the spontaneous metastasis model indicated an obvious attenuation of recruitment of VEGFR1+ bone marrow derived cell clusters, and extracellular matrix fibronectin deposition in lungs, which presumably are required for metastatic niche formation. Moreover, this effect is transplantable, as wild-type mice reconstituted with p2y12–/– bone marrow have a similar phenotype as P2y12–/– mice. These results imply that the mechanism of P2Y12 function on cancer metastasis is complicated, probably mediated through regulation of both cancer cell-platelet direct interaction and metastatic niche formation. Further work is needed to resolve this issue. In summary, it is clear that P2Y12 is a safe drug target for anti-thrombotic therapy. But, metabolic derivatives of the pro-drug Clopidogrel apparently promote cancer metastasis and growth; whereas the newly approved pro-drug Prasugrel lacks that effect in animal models, and hopefully in humans. Disclosures: No relevant conflicts of interest to declare.

Abstract 3252 PTEN (Phosphatase and tensin homologue deleted in chromosome 10) is a well-known tumor suppressor. PTEN dephosphorylates PtdIns(3,4,5)P3 into PtdIns(4,5)P2 and thereby negatively regulates PIP3-downstream signaling. According to present studies, PTEN is also a protein tyrosine phosphatase. In 2010 (Blood. 2010;116(14):2579–81), we reported that hematopoietic specific PTEN deficiency causes an increased platelet count, a shortened tail vein bleeding time, and enhanced platelet activation in response to stimulation by collagen and other agonists along with augmented collagen-induced phosphorylation of platelet Akt at Ser473. The PI3K specific inhibitor LY294002 almost completely blocked collagen-induced WT platelet aggregation, but had less effect on PTEN-deficient platelets. This result implies that PTEN may regulate collagen-induced platelet activation via both PI3K/Akt signaling and an yet to be identified pathway. In this study, we investigated the mechanism underlying PTEN regulation of collagen-induced platelet activation. It is well-known that the GPVI receptor signaling cascade is similar to that of T- and B-cell immune receptors. This signaling cascade relies on the formation of a signalosome composed of the transmembrane adapter LAT (linker for activation of T cells), the two cytosolic adapters SLP-76 and Gads, and the activation of PLCγ2. Accordingly, we investigated the phosphorylation of LAT, SLP-76 and PLCγ2 in PTEN-deficient and WT platelets in response to low level collagen (2μg/ml), with and without LY294002 pre-incubation. The platelet lysate was immunoprecipitated with 4G10, a specific anti-phosphotyrosine monoclonal antibody, to detect tyrosine phosphorylation. Interestingly, we found that in the presence of LY294002, the phosphorylation levels of LAT, SLP-76 and PLCγ2 were very high in PTEN-deficient platelets while the phosphorylation of these molecules was hardly detectable in WT platelets, all in response to low level collagen. These results suggested that PTEN may also regulate platelet activation through LAT, SLP-76 and PLCγ2. This hypothesis was supported by co-immunopricipitation experiments demonstrating that PTEN interacts with LAT in platelets upon collagen stimulation. Moreover, we also found that PTEN Ser380 phosphorylation was correlated with collagen-induced WT platelet activation without LY294002, and PTEN Ser380 phosphorylation could be inhibited by CK2 inhibitor DMAT and PDK1 inhibitor II, but not by Akt inhibitor SH6 or the GSK3β inhibitor LiCl. In contrast, the PDK1 inhibitor II blocked collagen-induced activation of CK2. Radioactivity assays indicated that collagen-induced enzyme activity activation of CK2 was blocked by the PDK1 inhibitor II. Ser380 phosphorylation is thought to reflect the loss of PTEN phosphatase function. So, because inhibition of PI3K, and PDK1 do not result in phosphorylation of PTEN S380 and the resulting loss of PTEN phosphatase function, it may be true that PDK1 inactivates PTEN. Therefore, we conclude that the mechanism of PTEN involvement in collagen-induced platelet activation is complicated, and PTEN plays a key role in GPVI mediated platelet activation probably through down-regulating both the PI3K/Akt and PI3K-PDK1-LAT pathways. We also investigated PTEN's function in αIIbβ3 mediated platelet activation. Platelet spreading and clot retraction experiments were performed. We found that PTEN deficiency significantly promoted clot retraction, but had no effect on platelet spreading on immobilized fibrinogen. Moreover, U46619 and thrombin induced PTEN ser380 phosphorylation was inhibited in β3 deficient platelet. These results indicate that PTEN is also involved in αIIbβ3 mediated outside-in signaling. Although the molecular mechanism of PTEN modulation of αIIbβ3 mediated outside-in signaling is unclear, our results may partially explain why PTEN deficiency also enhances platelet aggregation and secretion in response to agonists other than collagen. Disclosures: No relevant conflicts of interest to declare.

Sulfite is rapidly oxidized to sulfate in the liver. This was shown both in isolated rat hepatocytes and isolated perfused liver. In addition sulfite treatment resulted in release of GSH originating probably from low molecular disulfides such as GSSG and/or mixed disulfides between GSH and protein sulfhydryl groups. Sulfite was demonstrated to be an efficient precursor for sulfate conjugation. This was demonstrated using paracetamol as a substrate. Sulfite was even more efficient in supplying sulfate for sulfate conjugation than inorganic sulfate. Sulfite was furthermore shown to be protective against the toxicity of both N-acetyl-p-benzoquinone imine (NAPQI), the reactive paracetamol metabolite, and acrolein, a reactive aldehyde which is a metabolite of allyl alcohol. This protection is most likely due to direct reaction between sulfite and these reactive metabolites in a manner similar to that occurring with GSH and other thiols. When NAPQI and acrolein were generated intracellularly in isolated hepatocytes from paracetamol and allyl alcohol, respectively, toxicity was also expressed. In this case sulfite only protected against allyl alcohol induced toxicity and not against paracetamol induced toxicity. The reason for this discrepancy is not clear but may depend on factors such as site of generation of the reactive metabolite or the reactivity of the reactive metabolite.