Glutaminase potentiates the glycolysis in esophageal squamous cell carcinoma by interacting with PDK1.

Increasing evidence has demonstrated that glutaminase (GLS) as a key mitochondrial enzyme plays a pivotal role in glutaminolysis, which widely participates in glutamine metabolism serving as main energy sources and building blocks for tumor growth. However, the roles and molecular mechanisms of GLS in esophageal squamous cell carcinoma (ESCC) remains unknown. Here, we found that GLS was highly expressed in ESCC tissues and cells. GLS inhibitor CB-839 significantly suppressed cell proliferation, colony formation, migration and invasion of ESCC cells, whereas GLS overexpression displayed the opposite effects. In addition, CB-839 markedly suppressed glucose consumption and lactate production, coupled with the downregulation of glycolysis-related proteins HK2, PFKM, PKM2 and LDHA, whereas GLS overexpression exhibited the adverse results. In vivo animal experiment revealed that CB-839 dramatically suppressed tumor growth, whereas GLS overexpression promoted tumor growth in ESCC cells xenografted nude mice. Mechanistically, GLS was localized in mitochondria of ESCC cells, which interacted with PDK1 protein. CB-839 attenuated the interaction of GLS and PDK1 in ESCC cells by suppressing PDK1 expression, which further evoked the downregulation of p-PDHA1 (s293), however, GLS overexpression markedly enhanced the level of p-PDHA1 (s293). These findings suggest that interaction of GLS with PDK1 accelerates the glycolysis of ESCC cells by inactivating PDH enzyme, and thus targeting GLS may be a novel therapeutic approach for ESCC patients.

Inhibition of nonsense-mediated decay rescues p53ß/γ isoform expression and activates the p53 pathway in MDM2-overexpressing and select p53-mutant cancers.

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non-small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53ß and p53γ, comprising exons 1 to 9ß or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53ß and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.

Matrilin-2 prevents the TNFα-induced apoptosis of WB-F344 cells via suppressing JNK pathway.

Oval cells, a kind of hepatic progenitor cell quiescent at normal condition, activates to proliferate and differentiate into hepatocytes under severe and long-term liver injury, which usually raises severe inflammation. However, how oval cell survives in the inflammatory milieu interne is still unclear. Tumor necrosis factor α (TNFα), mimicking inflammatory hepatic milieu interne, was used to treat oval cell line, WB-F344, to test the protective function of matrilin-2. In this study, our data suggested that matrilin-2 prevented TNFα-induced apoptosis in WB-F344 cells via inhibiting ASK1/MKK7/JNK pathway. In conclusion, we determined that matrilin-2 plays the key role in maintaining the survival of oval cell and guarantees its proliferation under various injury factors.

Drug-induced RAF dimerization is independent of RAS mutation status and does not lead to universal MEK dependence for cell survival in head and neck cancers.

Treatments for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) have limited efficacy. One potential therapeutic target for HNSCC is the RAS/RAF/MEK/ERK cascade, which is one of the major signaling pathways for HNSCC cell survival. In HNSCC, RAS can be activated either by HRAS mutation or by upstream signaling. The ABL inhibitor nilotinib acts as a weak RAF inhibitor that induces RAF dimerization and subsequent activation of MEK/ERK in other cancer cell lines with activated RAS, leading to an unexpected dependence on MEK/ERK for cell survival. We hypothesized that nilotinib and the MEK inhibitor MEK162 would be synergistic in HNSCC cell lines owing to the frequent activation of RAS. We treated HNSCC cell lines with nilotinib and performed immunoblotting and cell-viability experiments. We used an orthotopic mouse model to assess synergistic effects in vivo. Nilotinib induced significant BRAF-CRAF heterodimerization and ERK activation irrespective of RAS mutation status. In cell-viability assays, nilotinib synergized with MEK162. MEK162 alone induced G1 arrest that was minimally enhanced by nilotinib. In the mouse model, treatment with MEK162 alone or combined with nilotinib led to tumor growth inhibition. In HNSCC, nilotinib-induced RAF dimerization is independent of RAS mutation status, but this dimerization does not lead to MEK dependence for cell survival in all HNSCC cell lines. MEK inhibition alone leads to decreased proliferation both in vitro and in vivo. Although nilotinib has some synergistic effects with MEK162, other agents may be more effective against HNSCC when combined with MEK162.

Tumor grafts derived from patients with head and neck squamous carcinoma authentically maintain the molecular and histologic characteristics of human cancers.

BACKGROUND: The patient-derived xenograft (PDX) model is likely to reflect human tumor biology more accurately than cultured cell lines because human tumors are implanted directly into animals; maintained in an in vivo, three-dimensional environment; and never cultured on plastic. PDX models of head and neck squamous cell carcinoma (HNSCC) have been developed previously but were not well characterized at the molecular level. HNSCC is a deadly and disfiguring disease for which better systemic therapy is desperately needed. The development of new therapies and the understanding of HNSCC biology both depend upon clinically relevant animal models. We developed and characterized the patient-derived xenograft (PDX) model because it is likely to recapitulate human tumor biology. METHODS: We transplanted 30 primary tumors directly into mice. The histology and stromal components were analyzed by immunohistochemistry. Gene expression analysis was conducted on patient tumors and on PDXs and cell lines derived from one PDX and from independent, human tumors. RESULTS: Five of 30 (17%) transplanted tumors could be serially passaged. Engraftment was more frequent among HNSCC with poor differentiation and nodal disease. The tumors maintained the histologic characteristics of the parent tumor, although human stromal components were lost upon engraftment. The degree of difference in gene expression between the PDX and its parent tumor varied widely but was stable up to the tenth generation in one PDX. For genes whose expression differed between parent tumors and cell lines in culture, the PDX expression pattern was very similar to that of the parent tumor. There were also significant expression differences between the human tumors that subsequently grew in mice and those that did not, suggesting that this model enriches for cancers with distinct biological features. The PDX model was used successfully to test targeted drugs in vivo. CONCLUSION: The PDX model for HNSCC is feasible, recapitulates the histology of the original tumor, and generates stable gene expression patterns. Gene expression patterns and histology suggested that the PDX more closely recapitulated the parental tumor than did cells in culture. Thus, the PDX is a robust model in which to evaluate tumor biology and novel therapeutics.

Revelation of p53-independent function of MTA1 in DNA damage response via modulation of the p21 WAF1-proliferating cell nuclear antigen pathway.

Although metastasis-associated protein 1 (MTA1), a component of the nucleosome remodeling and deacetylase (NuRD) complex, is a DNA-damage response protein and regulates p53-dependent DNA repair, it remains unknown whether MTA1 also participates in p53-independent DNA damage response. Here, we provide evidence that MTA1 is a p53-independent transcriptional corepressor of p21(WAF1), and the underlying mechanism involves recruitment of MTA1-histone deacetylase 2 (HDAC2) complexes onto two selective regions of the p21(WAF1) promoter. Accordingly, MTA1 depletion, despite its effect on p53 down-regulation, superinduces p21(WAF1), increases p21(WAF1) binding to proliferating cell nuclear antigen (PCNA), and decreases the nuclear accumulation of PCNA in response to ionizing radiation. In support of a p53-independent role of MTA1 in DNA damage response, we further demonstrate that induced expression of MTA1 in p53-null cells inhibits p21(WAF1) promoter activity and p21(WAF1) binding to PCNA. Consequently, MTA1 expression in p53-null cells results in increased induction of gamma H2AX foci and DNA double strand break repair, and decreased DNA damage sensitivity following ionizing radiation treatment. These findings uncover a new target of MTA1 and the existence of an additional p53-independent role of MTA1 in DNA damage response, at least in part, by modulating the p21(WAF1)-PCNA pathway, and thus, linking two previously unconnected NuRD complex and DNA-damage response pathways.

Regulation of NF-kappaB circuitry by a component of the nucleosome remodeling and deacetylase complex controls inflammatory response homeostasis.

The MTA1 coregulator (metastatic tumor antigen 1), a component of the nucleosome remodeling and deacetylase (NuRD) complex, has been intimately linked with human cancer, but its role in inflammatory responses remains unknown. Here, we discovered that MTA1 is a target of inflammation, and stimulation of macrophages with Escherichia coli lipopolysaccharide (LPS) stimulates MTA1 transcription via the NF-kappaB pathway. Unexpectedly, we found that MTA1 depletion in LPS-stimulated macrophages impairs NF-kappaB signaling and expression of inflammatory molecules. MTA1 itself acts as a transcriptional coactivator of inflammatory cytokines in LPS-stimulated macrophages, and in contrast, it acts as a corepressor in resting primary macrophages as its depletion induced cytokine expression. LPS stimulates S-nitrosylation of histone deacetylase 2 (HDAC2) and interferes with its binding to MTA1, which, in turn, resulted in the loss of corepressor behavior of MTA1.HDAC complex in activated macrophages. Consequently, the net levels of inflammatory cytokines in LPS-stimulated macrophages from MTA1(-/-) mice were high compared with wild-type mice. Accordingly, MTA1(-/-) mice were much more susceptible than control mice to septic shock induced by LPS, revealing that MTA1 protects mice from deregulated host inflammatory response. These findings reveal a previously unrecognized, critical homeostatic role of MTA1, both as a target and as a component of the NF-kappaB circuitry, in the regulation of inflammatory responses.

Association of tumor downstaging after neoadjuvant chemotherapy with survival in patients with locally advanced nasopharyngeal carcinoma: a retrospective cohort study.

PURPOSE: Assessing the downstaging effects of neoadjuvant chemotherapy (NACT) in patients with locally advanced nasopharyngeal carcinoma (LANPC) and predicting response to treatment remain challenging. The present study aimed to evaluate the long-term prognosis of downstaging after NACT in patients with LANPC and to investigate the prognostic value of post-NACT tumor downstaging on treatment outcomes in the era of concurrent chemoradiotherapy (CCRT). METHODS: This retrospective study included 226 patients with stage III (n = 188) and IVA (n = 38) NPC admitted to Haikou People's Hospital between 1 October 2009 and 1 October 2012. The patients were grouped as downstaging or no after NACT. Overall survival (OS), locoregional failure-free survival (LFFS), and distant failure-free survival (DFFS) were analyzed. RESULTS: Among 226 patients, 196 (86.7%) were in the downstaging group and 30 (13.3%) were in the non-downstaging group. The longest follow-up was 76 months, and the median was 45 months. The 3-year OS rates of the downstaging group and non-downstaging group were 91.0% (95% CI 0.89-0.93) and 69.5% (95% CI 0.66-0.72) (P = 0.005). The 5-year OS rates were 81.6% (95% CI 0.78-0.86) and 53.3% (95% CI 0.49-0.61) (P = 0.001). N downstaging (3-year OS, HR 0.491, 95% CI 0.221-0.881, P = 0.022; 5-year OS, HR = 0.597, 95% CI 0.378-0.878, P = 0.021) was independently associated with OS. CONCLUSION: In the treatment of LANPC, the patients with downstaging after NACT have a better prognosis than those without downstaging. This study suggests that NACT can improve the prognosis for patients with LANPC if there is downstaging.

Novel Anaplastic Thyroid Cancer PDXs and Cell Lines: Expanding Preclinical Models of Genetic Diversity.

CONTEXT: Anaplastic thyroid cancer (ATC) is a rare, aggressive, and deadly disease. Robust preclinical thyroid cancer models are needed to adequately develop and study novel therapeutic agents. Patient-derived xenograft (PDX) models may resemble patient tumors by recapitulating key genetic alterations and gene expression patterns, making them excellent preclinical models for drug response evaluation. OBJECTIVE: We developed distinct ATC PDX models concurrently with cell lines and characterized them in vitro and in vivo. METHODS: Fresh thyroid tumor from patients with a preoperative diagnosis of ATC was surgically collected and divided for concurrent cell line and PDX model development. Cell lines were created by generating single cells through enzymatic digestion. PDX models were developed following direct subcutaneous implantation of fresh tumor on the flank of immune compromised/athymic mice. RESULTS: Six ATC PDX models and 4 cell lines were developed with distinct genetic profiles. Mutational characterization showed one BRAF/TP53/CDKN2A, one BRAF/CDKN2A, one BRAF/TP53, one TP53 only, one TERT-promoter/HRAS, and one TERT-promoter/KRAS/TP53/NF2/NFE2L2 mutated phenotype. Hematoxylin-eosin staining comparing the PDX models to the original patient surgical specimens show remarkable resemblance, while immunohistochemistry stains for important biomarkers were in full concordance (cytokeratin, TTF-1, PAX8, BRAF). Short tandem repeats DNA fingerprinting analysis of all PDX models and cell lines showed strong concordance with the original tumor. PDX successful establishment rate was 32%. CONCLUSION: We have developed and characterized 6 novel ATC PDX models with 4 matching cell lines. Each PDX model harbors a distinct genetic profile, making them excellent tools for preclinical therapeutic trials.

A High-throughput Approach to Identify Effective Systemic Agents for the Treatment of Anaplastic Thyroid Carcinoma.

BACKGROUND: Despite the use of aggressive multimodality treatment, most anaplastic thyroid carcinoma (ATC) patients die within a year of diagnosis. Although the combination of BRAF and MEK inhibitors has recently been approved for use in BRAF-mutated ATC, they remain effective in a minority of patients who are likely to develop drug resistance. There remains a critical clinical need for effective systemic agents for ATC with a reasonable toxicity profile to allow for rapid translational development. MATERIAL AND METHODS: Twelve human thyroid cancer cell lines with comprehensive genomic characterization were used in a high-throughput screening (HTS) of 257 compounds to select agents with maximal growth inhibition. Cell proliferation, colony formation, orthotopic thyroid models, and patient-derived xenograft (PDX) models were used to validate the selected agents. RESULTS: Seventeen compounds were effective, and docetaxel, LBH-589, and pralatrexate were selected for additional in vitro and in vivo analysis as they have been previously approved by the US Food and Drug Administration for other cancers. Significant tumor growth inhibition (TGI) was detected in all tested models treated with LBH-589; pralatrexate demonstrated significant TGI in the orthotopic papillary thyroid carcinoma model and 2 PDX models; and docetaxel demonstrated significant TGI only in the context of mutant TP53. CONCLUSIONS: HTS identified classes of systemic agents that demonstrate preferential effectiveness against aggressive thyroid cancers, particularly those with mutant TP53. Preclinical validation in both orthotopic and PDX models, which are accurate in vivo models mimicking tumor microenvironment, may support initiation of early-phase clinical trials in non-BRAF mutated or refractory to BRAF/MEK inhibition ATC.

mTOR inhibition overcomes primary and acquired resistance to Wee1 inhibition by augmenting replication stress in epithelial ovarian cancers.

Epithelial ovarian cancer is characterized by universal TP53 mutations, which result in G1/S checkpoint deficiencies. Therefore, it is hypothesized that the abrogation of the G2/M checkpoint with Wee1 inhibitor might preferentially sensitize TP53-defective ovarian cancer cells. Given the extremely high molecular diversity in ovarian cancer, one approach to improving the clinical efficacy is to identify drug combinations that either broaden the applicable spectrum or circumvent resistance. Here, through a high-throughput unbiased proteomic profiling (RPPA), we found the complementary activated mTOR pathway contributes greatly to Wee1 inhibitor resistance. A combination of Wee1 and mTOR inhibits synergistically inhibiting tumor growth in ovarian cancer cell lines and patient-derived xenograft that closely mimic the heterogeneity of patient tumors. Mechanistically, dual Wee1/mTOR inhibition induced massive DNA replication stress, leading to fork stalling and DNA damage. Moreover, we found that the addition of nucleotide metabolic substrate dNTPs alleviated replication stress, restored the cell cycle and reduced apoptosis to some extent, supporting dNTPs depletion is necessary for the synergy between Wee1 and mTOR inhibits. These results suggest that our study opening up a wider therapeutic window of Wee1 inhibitor for the treatment in epithelial ovarian cancers.

Non-canonical cMet regulation by vimentin mediates Plk1 inhibitor-induced apoptosis.

To address the need for improved systemic therapy for non-small-cell lung cancer (NSCLC), we previously demonstrated that mesenchymal NSCLC was sensitive to polo-like kinase (Plk1) inhibitors, but the mechanisms of resistance in epithelial NSCLC remain unknown. Here, we show that cMet was differentially regulated in isogenic pairs of epithelial and mesenchymal cell lines. Plk1 inhibition inhibits cMet phosphorylation only in mesenchymal cells. Constitutively active cMet abrogates Plk1 inhibitor-induced apoptosis. Likewise, cMet silencing or inhibition enhances Plk1 inhibitor-induced apoptosis. Cells with acquired resistance to Plk1 inhibitors are more epithelial than their parental cells and maintain cMet activation after Plk1 inhibition. In four animal NSCLC models, mesenchymal tumors were more sensitive to Plk1 inhibition alone than were epithelial tumors. The combination of cMet and Plk1 inhibition led to regression of tumors that did not regrow when drug treatment was stopped. Plk1 inhibition did not affect HGF levels but did decrease vimentin phosphorylation, which regulates cMet phosphorylation via ß1-integrin. This research defines a heretofore unknown mechanism of ligand-independent activation of cMet downstream of Plk1 and an effective combination therapy.

PDK1 Mediates NOTCH1-Mutated Head and Neck Squamous Carcinoma Vulnerability to Therapeutic PI3K/mTOR Inhibition.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is driven largely by the loss of tumor suppressor genes, including NOTCH1, but lacks a biomarker-driven targeted therapy. Although the PI3K/mTOR pathway is frequently altered in HNSCC, the disease has modest clinical response rates to PI3K/mTOR inhibitors and lacks validated biomarkers of response. We tested the hypothesis that an unbiased pharmacogenomics approach to PI3K/mTOR pathway inhibitors would identify novel, clinically relevant molecular vulnerabilities in HNSCC with loss of tumor suppressor function.Experimental Design: We assessed the degree to which responses to PI3K/mTOR inhibitors are associated with gene mutations in 59 HNSCC cell lines. Apoptosis in drug-sensitive cell lines was confirmed in vitro and in vivo. NOTCH1 pathway components and PDK1 were manipulated with drugs, gene editing, knockdown, and overexpression. RESULTS: PI3K/mTOR inhibition caused apoptosis and decreased colony numbers in HNSCC cell lines harboring NOTCH1 loss-of-function mutations (NOTCH1 MUT) and reduced tumor size in subcutaneous and orthotopic xenograft models. In all cell lines, NOTCH1 MUT was strongly associated with sensitivity to six PI3K/mTOR inhibitors. NOTCH1 inhibition or knockout increased NOTCH1 WT HNSCC sensitivity to PI3K/mTOR inhibition. PDK1 levels dropped following PI3K/mTOR inhibition in NOTCH1 MUT but not NOTCH1 WT HNSCC, and PDK1 overexpression rescued apoptosis in NOTCH1 MUT cells. PDK1 and AKT inhibitors together caused apoptosis in NOTCH1 WT HNSCC but had little effect as single agents. CONCLUSIONS: Our findings suggest that NOTCH1 MUT predicts response to PI3K/mTOR inhibitors, which may lead to the first biomarker-driven targeted therapy for HNSCC, and that targeting PDK1 sensitizes NOTCH1 WT HNSCC to PI3K/mTOR pathway inhibitors.

Estrogen induces expression of BCAS3, a novel estrogen receptor-alpha coactivator, through proline-, glutamic acid-, and leucine-rich protein-1 (PELP1).

We recently reported that the breast carcinoma amplified sequence-3 (BCAS3) gene is regulated by estrogen receptor (ER) alpha. However, the role of ERalpha coactivators in the regulation of BCAS3 expression remains unknown, and information regarding the function of the BCAS3 protein is lacking. Here, we define the contribution of ERalpha coactivators to BCAS3 regulation and identify BCAS3 itself as an ERalpha coactivator in breast cancer cells. We found that PELP1 (proline-, glutamic acid-, and leucine-rich protein-1), a newly described ERalpha coregulator, is recruited to BCAS3 chromatin and activates its expression. Analysis of the BCAS3 sequence for functional motifs and evidence from biochemical fractionation suggested that BCAS3 acts as a transcriptional coactivator. Results from chromatin immunoprecipitation, reporter assays, and expression studies further validated the coactivator function of BCAS3 for ERalpha. BCAS3 physically associated with histone H3 and histone acetyltransferase complex protein P/CAF (p300/CBP-associated factor) and possessed histone acetyltransferase activity. Unexpectedly, BCAS3 required PELP1 to function as a coactivator in ERalpha transactivation activity. In brief, these results highlight a mechanism whereby ERalpha activation triggers a positive feedback loop leading to signal amplification in the cell.

A Novel Method for Quantifying Total Thoracic Tumor Burden in Mice.

Mouse models are powerful tools to study lung cancer initiation and progression in vivo and have contributed significantly to recent advances in therapy. Using micro-computed tomography to monitor and study parenchymal and extra-parenchymal metastases in existing murine models of lung cancer is challenging owing to a lack of radiographic contrast and difficulty in achieving respiratory gating. To facilitate the analysis of these in vivo imaging studies and study of tumor progression in murine models we developed a novel, rapid, semi-automated method of calculating thoracic tumor burden from computed tomography images. This method, in which commercially available software is used to calculate the mass of the thoracic cavity (MTC), takes into account the aggregate tumor burden in the thoracic cavity. The present study showed that in tumor-free mice, the MTC does not change over time and is not affected by breathing, whereas in tumor-bearing mice, the increase in the MTC is a measure of tumor mass that correlates well with tumor burden measured by lung weight. Tumor burden calculated with our MTC method correlated with that measured by lung weight as well as or better than that calculated using four established methods. To test this method, we assessed metastatic tumor development and response to a pharmacologic PLK1 inhibitor in an orthotopic xenograft mouse model. PLK1 inhibition significantly inhibited tumor growth. Our results demonstrate that the MTC method can be used to study dynamic changes in tumor growth and response to therapeutics in genetically engineered mouse models and orthotopic xenograft mouse models of lung cancer.

Comprehensive pharmacogenomic profiling of human papillomavirus-positive and -negative squamous cell carcinoma identifies sensitivity to aurora kinase inhibition in KMT2D mutants.

To address the unmet need for effective biomarker-driven targeted therapy for human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) and cervical cancer, we conducted a high-throughput drug screen using 1122 compounds in 13 HPV-positive and 11 matched HPV-negative cell lines. The most effective drug classes were inhibitors of polo-like kinase, proteasomes, histone deacetylase, and Aurora kinases. Treatment with a pan-Aurora inhibitor, danusertib, led to G2M arrest and apoptosis in vitro. Furthermore, danusertib decreased tumor size compared with controls in patient derived xenograft models of HNSCC. To identify biomarkers predicting response, we determined associations between mutations and drug sensitivity. Our data and the Genomics of Drug Sensitivity in Cancer database showed that cancer cells with KMT2D mutations were more sensitive to Aurora kinase inhibitors than were cells without mutations. Knockdown of KMT2D in wild-type cells led to increased Aurora kinase inhibitor-induced apoptosis. We identified Aurora kinase inhibitors as effective and understudied drugs in HNSCC and CESC. This is the first published study to demonstrate that mutations in KMT2D, which are common in many cancers, correlate with drug sensitivity in two independent datasets.

One-step method embedding superoxide dismutase and gold nanoparticles in silica sol-gel network in the presence of cysteine for construction of third-generation biosensor.

A new and one-step method has been developed for the fabrication of superoxide dismutase (SOD) based biosensor. This method was used to form a silica sol-gel (SG) thin film and to immobilize SOD and gold nanoparticles (GNPs) in silica SG network for the fabrication of biosensor. The immobilized superoxide dismutase realized direct electron transfer between the enzyme and electrode surface, and the rate constants of the electrochemical process (ks) of SOD was markedly enhanced by GNPs. The electrochemical performance and influencing factors of the resulting biosensor were studied in detail. The resulting biosensor exhibited fast amperometric response to superoxide anion. The calibration range of superoxide anion was from 0.05 to 0.4 micromol L(-1). The proposed method exhibited the benefits of the advantages of self-assembly, nanoparticles and SG techniques. The fabrication of the SOD-modified electrode was easy and simple. The biosensor exhibited high sensitivity and long-term stability.

Clinicopathological and molecular characteristics of synchronous gastric adenocarcinoma and gastrointestinal stromal tumors.

Synchronous gastric tumors that consist of both gastrointestinal stromal tumor (GIST) and adenocarcinoma are rare. We studied the clinicopathological and molecular characteristics of six cases containing both gastric adenocarcinoma and GIST. By means of immunohistochemical analysis, all GIST cells expressed CD117, CD34 and Dog1 in all six synchronous gastric adenocarcinomas with GIST, and in GIST alone. Sequencing analysis demonstrated that exon 11 c-kit mutations were present in two of six synchronous tumors and four of five GISTs. One of the two exon 11 c-kit mutations in synchronous adenocarcinomas with GISTs was an uncommon mutation of CTT > CCA at amino acid 576, and the other was a GTT deletion at amino acid 560. The mutation was a homozygous A > G mutation in exon 12 (amino acid 567) of PDGFR-α. We concluded that the exon 11 mutations were the most important in both cases of synchronous gastric adenocarcinoma with GIST and GIST alone. The mutation rate was higher in GIST alone than in synchronous adenocarcinoma with GIST.