OTUD4: A Potential Prognosis Biomarker for Multiple Human Cancers.

BACKGROUND: Deubiquitinase OTU domain containing 4 (OTUD4) is initially identified as a K48-specific deubiquitinase and plays an important role in DNA damage repair signaling transduction. However, the expression level, prognostic role, biological function and mechanism of OTUD4 in multiple human cancers are unclear. METHODS: GEPIA online (http://gepia.cancer-pku.cn/; The Cancer Genome Atlas (TCGA) database) was used to analyze the mRNA expression of OTUD4 in multiple human cancers. Kaplan-Meier plotter (KM plotter) database and TCGA database were used to evaluate the prognostic value of OTUD4 expression in multiple human cancers. MTT, Transwell and 3D culture assays were used to detect the role of OTUD4 in breast, liver and lung cancer cells. The correlation between OTUD4 and apoptosis signaling pathway and AKT signaling pathway was analyzed by Gene set enrichment analysis (GSEA). RESULTS: OTUD4 mRNA expression is significantly downregulated in multiple human cancer tissues. Survival analysis establishes that the downregulation of OTUD4 predicts poor prognosis in many solid tumors, including breast invasive carcinoma (BRCA), esophageal carcinoma (ESCA), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), and ovarian serous cystadenocarcinoma (OV). Furthermore, overexpression of OTUD4 could inhibit tumor cell proliferation, migration and invasion of breast, liver and lung cancer cells through inhibiting the AKT signaling pathway. CONCLUSION: This study found that OTUD4 may be a potential predictive factor for several human cancers and a tumor suppressor for breast, liver and lung cancer. The overexpression of OTUD4 restrained proliferation, migration and invasion of human breast, liver and lung cancer cells through promoting cancer cells apoptosis and inhibiting AKT signaling pathway. Notably, our results indicated that OTUD4 could be a useful biomarker for the prognosis of human cancers and a potential molecular target for diagnosis and treatment of breast, liver and lung cancer.

Knockdown of XB130 restrains cancer stem cell-like phenotype through inhibition of Wnt/ß-Catenin signaling in breast cancer.

The cancer stem cells (CSCs) is a subset of cancer cells that possess stem cell properties, which plays a crucial role in the occurrence, metastasis, and recurrence of the tumor. XB130 is a novel adapter protein potentially serves as a functional factor in CSCs. To determine the role of CSCs in breast cancer, we focused on the study of XB130. In our study, we found that XB130 expression was significantly upregulated in breast cancer and was closely related to the clinicopathologic characteristics, overall survival and poor prognosis of breast cancer patients. Functionally, we found that knockdown of XB130 was not only played an important role in proliferation, epithelial-mesenchymal transition (EMT), and metastasis in breast cancer cells but also exhibited potent antitumor activity in animal tumor models. Moreover, we demonstrated that silencing endogenous XB130 regulated the cancer stem cell-like properties of breast cancer, including the formation of self-renewing spheres and the proportion of breast cancer SP+ cells. Mechanistically, our studies indicated that downregulation of XB130 restrained the EMT and Wnt/ß-catenin signaling, so as to weaken the tumor-initiating cell-like phenotype of breast cancer cells. This study indicates that XB130 plays an important role in maintaining the EMT and stem cell-like characteristics of breast cancer cells, supporting the significance of XB130 as a new potential therapeutic target for early diagnosis and prognosis of breast cancer.

Downregulation of MMSET impairs breast cancer proliferation and metastasis through inhibiting Wnt/ß-catenin signaling.

BACKGROUND: Recently, the biggest challenge in the treatment of breast cancer is the metastasis of breast cancer cells. Multiple myeloma SET protein (MMSET), a histone lysine methyltransferase, overexpressed in various human cancers, was reported to be associated with carcinogenesis of human cancers. METHODS: Expression of MMSET in breast cancer cell lines and tissues was quantified by real-time PCR and Western blotting. Immunohistochemistry was employed to analyze MMSET expression in 163 clinicopathologically characterized breast cancer cases. Cell functional assays such as MTT assay, colony formation, BrdU assay, flow cytometry, wound healing, Transwell assay, and 3D culture were used to investigate the effect of MMSET in the development and metastasis of human breast cancer. Effects of MMSET on Wnt/ß-catenin signaling pathway were further studied by using Western blotting analysis. RESULTS: Our results showed that MMSET expression was markedly overexpressed in breast cancer cells and clinical specimens and was significantly correlated with patients' clinicopatho-logic characteristics and prognosis. Moreover, silencing endogenous MMSET significantly inhibited the proliferation, migration, and metastasis of breast cancer cells through inhibiting the Wnt/ß-catenin pathway. CONCLUSION: This study found that the downregulated expression of MMSET impaired proliferation and metastasis of human breast cancer through inhibiting Wnt/ß-catenin signaling pathway. Notably, our results indicated that MMSET could be a useful biomarker for the prognosis of breast cancer.

CHP2 Promotes Cell Proliferation in Breast Cancer via Suppression of FOXO3a.

Calcineurin B homologous protein isoform 2 (CHP2), an essential cofactor for Na+/H+ exchanger isoform 1 (NHE1), is identified to be expressed in various malignant cell lines. However, the clinical significance and biological role of CHP2 in breast cancer remain to be established. Here, CHP2 was markedly overexpressed in breast cancer cells and clinical tumor specimens. Immunohistochemical analysis revealed that the expression of CHP2 was significantly correlated with patients' clinicopathologic characteristics like clinical stage, and breast cancer patients with high CHP2 expression had shorter overall survival compared with patients with low CHP2 expression. Moreover, it was demonstrated that overexpressing CHP2 significantly enhanced, whereas silencing endogenous CHP2 inhibited, the proliferation and tumorigenicity of breast cancer cells in vitro and in vivo In addition, overexpression of CHP2 accelerated, whereas inhibition of CHP2 retarded, G1-S phase cell-cycle transition in breast cancer cells. Mechanistically, overexpression of CHP2 activated AKT signaling and suppressed the transactivation of the forkhead box O3 (FOXO3/FOXO3a) transcription factor.Implications: This study discovers a previously unrecognized role of CHP2 in the progression of breast cancer and supports the significance of this gene as a novel prognostic biomarker and a potential therapeutic target for breast cancer. Mol Cancer Res; 16(10); 1512-22. ©2018 AACR.

TRIP6 promotes cell proliferation in hepatocellular carcinoma via suppression of FOXO3a.

Thyroid hormone receptor-interacting protein 6 (TRIP6), a member of LIM family, acts as an adaptor protein and is overexpressed in several tumor types. However, the clinical significance and biological role of TRIP6 in HCC remains unknown. In our study, we found that TRIP6 was markedly overexpressed in HCC cells and clinical specimens compared with normal hepatocytes and adjacent non-tumor tissues. Immunohistochemical and statistical analysis showed that the expression of TRIP6 significantly correlated with HCC patients' clinical stage and poor survival. Moreover, we demonstrated that overexpressing TRIP6 significantly enhanced, whereas silencing endogenous TRIP6 inhibited, the proliferation and the anchorage-independent growth ability of HCC cells. In addition, overexpression of TRIP6 accelerated, while inhibition of TRIP6 retarded, G1-S phase transition in HCC cells. We further found that overexpression of TRIP6 increased the activation of AKT and suppressed the transactivity of FOXO3a. Meanwhile, overexpression of TRIP6 leaded to the decreased expression of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 and increased expression of the cell cycle regulator cyclin D1. While silencing TRIP6 triggered the opposite effect. Taken together, these findings showed that TRIP6 plays an important role in promoting HCC cells proliferation and may serve as a novel prognostic biomarker and therapeutic target in HCC.

Human Adenovirus Serotype 3 Vector Packaged by a Rare Serotype 14 Hexon.

Recombinant adenovirus serotype 3 (rAd3), which infects cells through the receptor desmoglein 2 (DSG2), has been investigated as a vector for gene therapy or vaccination. However, pre-existing anti-vector immunity may limit the practical application of rAd3. In this study, we investigated the seroprevalence and neutralizing antibody (NAb) titers to Ad3 and alternate serotypes in normal healthy adults in southern China. Sera samples had a high seroprevalence (80.00%) against Ad3 and Ad7 (85.83%), compared with Ad14 (22.50%). Furthermore, 19.17% and 25.83% of samples had high-titer neutralizing antibodies to Ad3 and Ad7, respectively, compared with 3.33% against Ad14. We constructed a chimeric adenovirus, rAd3H14, designed to evade anti-vector immunity by replacing the enhanced green fluorescent protein (EGFP)-expressing hexon of the rAd3EGFP vector with a hexon from Ad14. The chimeric vector rAd3H14 was not neutralized in vitro efficiently by Ad3 NAbs using sera from mice and normal healthy human volunteers. Furthermore, in contrast to the unmodified vector rAd3EGFP, rAd3H14 induced robust antibody responses against EGFP in mice with high levels of pre-existing anti-Ad3 immunity. In conclusion, the chimeric vector rAd3H14 may be a useful alternative vector in adult populations with a high prevalence of Ad3 NAbs.

Prevalence of neutralizing antibodies to common respiratory viruses in intravenous immunoglobulin and in healthy donors in southern China.

BACKGROUND: Acute respiratory infections (ARIs) are a leading cause of death among children under the age of 5. However, there are no effective drugs for most of these severe viral infections. Passive immunotherapy with convalescent plasma or hyperimmune intravenous immunoglobulin (H-IVIG) is a potential therapeutic option for serious viral infections. It is important to find a suitable source of convalescent plasma and of H-IVIG containing high titer neutralizing antibodies (NAbs). METHODS: Sera from 96 healthy adult donors in southern China and commercially available IVIG were analyzed for the titers of NAb to several most common respiratory viruses including respiratory syncytial virus (RSV), seasonal influenza A (InfA), enterovirus 71 (EV71), coxsackievirus A16 (CA16), adenovirus type 3 (Ad3) and a recent epidemic adenovirus type 55 (Ad55) by microneutralization test. RESULTS: A high proportion of samples from healthy adult donors were positive for NAbs (>16) to all the viruses except Ad55. A different proportion of these samples had high NAb titers (>512) for InfA (25%), Ad3 (17.71%), RSV (9.38%), EV71 (1.04%), CA16 (3.13%), and Ad55 (4.17%). Commercially available IVIG had high NAb titers to InfA and Ad3 (>1,000) and lower NAb titers to RSV [320], EV71 [160], and CA16 [160]. Strikingly, IVIG also had a high NAb titer to Ad55 (>1,000). CONCLUSIONS: Convalescent plasma could be screened from healthy blood volunteers to establish blood banks and to prepare specific H-IVIG for treating severe ARIs caused by common respiratory viruses.

Mapping the epitope of neutralizing monoclonal antibodies against human adenovirus type 3.

Human adenovirus type 3 (HAdV-3) has produced a global epidemic in recent years causing serious diseases such as pneumonia in both pediatric and adult patients. Development of an effective neutralizing monoclonal antibody (MAb) and identification of its neutralizing epitope is important for the control of HAdV-3 infection. In this study, three neutralizing MAbs were generated, of which MAb 3D7 had a high neutralization titer of 4096 (approximately 0.5 µg/ml) against HAdV-3 infection. In indirect enzyme-linked immunosorbent assays, all three MAbs specifically recognized HAdV-3 virus particles and hexon protein, but did not react with the virus particles or the hexon protein of HAdV-7. Analyses using a series of peptides and chimeric adenovirus particles of epitope mutants revealed that all three MAbs bound to the same exposed region (amino acid positions 244-254 of hexon) in hypervariable region 4 (HVR4), which is highly conserved among global HAdV-3 strains. The amino acids T246 and G250 may be the critical amino acids recognized by these MAbs. MAb 3D7 reduced the recombinant enhanced green fluorescent protein-expressing HAdV-3 (rAd3EGFP) load recovered in the lungs of mice at 3 days post-infection. The generation of MAb 3D7 and the identification of its neutralizing epitope may be useful for therapeutic treatment development, subunit vaccine construction, and virion structural analysis for HAdV-3.

Antigenic variability among two subtypes of human adenovirus serotype 7.

Human adenovirus type 7 (HAdV-7) is one of the major serotypes responsible for acute respiratory infection. It is important to investigate the antigenic variabilities of different HAdV-7 genomic subtypes for vaccine development. Phylogenetic analysis of global HAdV-7 strains and major antigen proteins showed that HAdV-7 could be classified into two subtypes. There were three highly variable regions (HVR1, HVR4, and HVR7) in the hexon protein that varied between subtypes. Within each of the subtypes, these regions were conserved. Two subtype HAdV-7 strains isolated in China were used to immunize mice for antigenic characterization. Mice immunized with one subtype strain showed 4-8-fold lower neutralizing antibody titers against another subtype strain. ELISA results showed that the variation in HVR1, 4, and 7 regions contributed to antigenic change, and it may be concluded that the three regions contain subtype-specific epitopes. In summary, strains of HAdV-7 could be divided into two subtypes using genome sequence and antigenic analysis; our results could be important for HAdV-7 vaccine development.

Construction and characterization of a recombinant human adenovirus type 3 vector containing two foreign neutralizing epitopes in hexon.

The "antigen capsid-incorporation" strategy has been developed for adenovirus-based vaccines in the context of several diseases. Exogenous antigenic peptides incorporated into the adenovirus capsid structure can induce a robust and boosted antigen-specific immune response. Recently, we sought to generate a multivalent adenovirus type 3 (Ad3) vaccine vector by incorporating multiple epitopes into the major adenovirus capsid protein, hexon. In the present study, a multivalent recombinant Ad3 vaccine (R1R2A3) was constructed by homologous recombination, displaying two neutralizing epitopes from enterovirus type 71 (EV71) in hexon. The recombinant virus was confirmed by PCR, immunoblotting, and enzyme-linked immunosorbent assay, and injected into mice to analyze the epitope-specific humoral response. No differences were found between the viruses with two epitopes incorporated into the hypervariable regions (HVR1 and HVR2) of hexon and Ad3EGFP, based on thermostability and growth kinetic tests. Both the epitopes are thought to be exposed on the hexon-modified intact virion surface. The repeated administration of the modified adenovirus R1R2A3 to BALB/c mice boosted the humoral immune response against both epitopes. Immunization with recombinant virus R1R2A3 elicited higher IgG titers and higher neutralization titers against EV71 in vitro than immunization with the modified adenovirus with only one epitope incorporated into HVR1. In this study, the recombinant R1R2A3 virus expressing two exogenous neutralizing epitopes in hexon HVR1 and HVR2 induced specific immune responses to both foreign epitopes. Our study contributes to a better understanding of hexon-modified Ad vector as a multiple-epitope delivery vehicle.

Protection against enterovirus 71 with neutralizing epitope incorporation within adenovirus type 3 hexon.

Enterovirus 71 (EV71) is responsible for hand, foot and mouth disease with high mortality among children. Various neutralizing B cell epitopes of EV71 have been identified as potential vaccine candidates. Capsid-incorporation of antigens into adenovirus (Ad) has been developed for a novel vaccine approach. We constructed Ad3-based EV71 vaccine vectors by incorporating a neutralizing epitope SP70 containing 15 amino acids derived from capsid protein VP1 of EV71 within the different surface-exposed domains of the capsid protein hexon of Ad3EGFP, a recombinant adenovirus type 3 (Ad3) expressing enhanced green fluorescence protein. Thermostability and growth kinetic assays suggested that the SP70 epitope incorporation into hypervariable region (HVR1, HVR2, or HVR7) of the hexon did not affect Ad fitness. The SP70 epitopes were thought to be exposed on all hexon-modified intact virion surfaces. Repeated administration of BALB/c mice with the modified Ads resulted in boosting of the anti-SP70 humoral immune response. Importantly, the modified Ads immunization of mother mice conferred protection in vivo to neonatal mice against the lethal EV71 challenge, and the modified Ads-immunized mice serum also conferred passive protection against the lethal challenge in newborn mice. Compared with the recombinant GST-fused SP70 protein immunization, immunization with the Ads containing SP70 in HVR1 or HVR2 elicited higher SP70-specific IgG titers, higher neutralization titers, and conferred more effective protection to neonatal mice. Thus, this study provides valuable information for hexon-modified Ad3 vector development as a promising EV71 vaccine candidate and as an epitope-delivering vehicle for other pathogens.

Construction and characterization of human adenovirus serotype 3 packaged by serotype 7 hexon.

Human adenovirus serotype 3 (Ad3) and serotype 7 (Ad7) are important pathogens causing respiratory tract diseases such as acute respiratory disease in pediatric and adult patients, but the immunodominant targets of Ad3- and Ad7-specific neutralizing antibodies (NAbs) remain unclear. A chimeric Ad vector, Ad3/H7, was constructed by replacing the Ad3 hexon gene (H3) with the hexon gene (H7) of Ad7. The chimeric viruses were successfully rescued in HEp-2 cells, and the Ad7 hexon was able to encapsidate the Ad3 genome, and functioned as efficiently as the Ad3 hexon. Furthermore, we tested the host neutralization responses against the viruses using BALB/C mice. Up to 97% of the NAbs produced by mice that were infected with these viruses were specific for the hexon protein in vitro. Preimmunization of mice with one of Ad7 and Ad3/H7 significantly prevented subsequent intranasal infection of the other type in vivo. In contrast, preimmunization of mice with one of Ad3 and Ad3/H7 did not remarkably prevent subsequent infection of the other type. We next evaluated the functional significance of hexon and other structural proteins specific NAbs to suppress the immunogenicity of Ad3/H3 and Ad3/H7 vectors expressing EGFP in mice preimmunized with wild type Ad. Preimmunization of mice with Ad7 evidently suppressed EGFP-specific humoral immune responses elicited by Ad3/H7, and did not exert suppressive effects on Ad3/H3. But contrary to the in vitro neutralization results, EGFP-specific humoral immune responses elicited by Ad3/H7 was remarkably inhibited in Ad3-preimmunization mice. The whole genome of the Ad7 strain was sequenced and aligned with Ad3. The major differences between Ad3 and Ad7 were only observed in the fiber and hexon among all structural proteins, and the variation between the hexons only located in four hypervariable regions (HVRs), HVR-1, -2, -5, and -7. These results thus suggest that Ad3- and Ad7-specific NAbs are directed primarily against the hexon proteins both in vitro and in vivo. But high titer Ad3 fiber-specific NAbs may also play an important role in blunting Ad3 immunogenicity in vivo. These studies contribute to a more profound understanding of Ad immunogenicity and have relevance for the design of novel Ad vaccine.

Pyridine-derived γ-secretase modulators.

SAR of a novel series of pyridine-derived γ-secretase modulators is described. Compound 5 was found to be a potent modulator in vitro, which on further profiling, was found to decrease Aß42 and Aß40, and maintain (or increase) the levels of total Aß. Furthermore, representative compounds 1 and 5 demonstrated in vivo efficacy to lower Aß42 in the brain without altering Notch processing in the peripheral.

Complete genome analysis of a novel E3-partial-deleted human adenovirus type 7 strain isolated in Southern China.

Human adenovirus (HAdV) is a causative agent of acute respiratory disease, which is prevalent throughout the world. Recently there are some reports which found that the HAdV-3 and HAdV-5 genomes were very stable across 50 years of time and space. But more and more recombinant genomes have been identified in emergent HAdV pathogens and it is a pathway for the molecular evolution of types. In our paper, we found a HAdV-7 GZ07 strain isolated from a child with acute respiratory disease, whose genome was E3-partial deleted. The whole genome was 32442 bp with 2864 bp deleted in E3 region and was annotated in detail (GenBank: HQ659699). The growth character was the same as that of another HAdV-7 wild strain which had no gene deletion. By comparison with E3 regions of the other HAdV-B, we found that only left-end two proteins were remained: 12.1 kDa glycoprotein and 16.1 kDa protein. E3 MHC class I antigen-binding glycoprotein, hypothetical 20.6 kDa protein, 20.6 kDa protein, 7.7 kDa protein., 10.3 kDa protein, 14.9 kDa protein and E3 14.7 kDa protein were all missing. It is the first report about E3 deletion in human adenovirus, which suggests that E3 region is also a possible recombination region in adenovirus molecular evolution.

Tricyclic compounds containing nonenolizable cyano enones. A novel class of highly potent anti-inflammatory and cytoprotective agents.

Forty-four novel tricycles containing nonenolizable cyano enones (TCEs) were designed and synthesized on the basis of a semisynthetic pentacyclic triterpenoid, bardoxolone methyl, which is currently being developed in phase II clinical trials for the treatment of severe chronic kidney disease in diabetic patients. Most of the TCEs having two different kinds of nonenolizable cyano enones in rings A and C are highly potent suppressors of induction of inducible nitric oxide synthase stimulated with interferon-γ and are highly potent inducers of the cytoprotective enzymes heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Among these compounds, (±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile ((±)-31) is the most potent in these bioassays in our pool of drug candidates including semisynthetic triterpenoids and synthetic tricycles. These facts strongly suggest that an essential factor for potency is not a triterpenoid skeleton but the cyano enone functionality. Notably, TCE 31 reduces hepatic tumorigenesis induced with aflatoxin in rats. Further preclinical studies and detailed mechanism studies on 31 are in progress.

Construction and characterization of a replication-competent human adenovirus type 3-based vector as a live-vaccine candidate and a viral delivery vector.

In southern China, as well as in neighboring Asian regions, human adenovirus type 3 (HAdV-3) outbreaks have become very prevalent in recent years. To address this problem regionally and globally, a recombinant virus has been constructed, containing a full-length infectious genomic clone of HAdV-3, to act as a vaccine. This was constructed by using a bacterial homologous recombination mechanism and was based on the cloning, manipulation and maintenance of the full-length adenovirus genome as a stable plasmid in E. coli. The resultant recombinant viral DNA was screened, identified and characterized by duplex PCR, Western blot, indirect immunofluorescence assay and electron microscopy. This putative vaccine strain was shown to be fully infectious in permissive cells, and no genome mutations were found in the recombinant plasmid. To demonstrate the utility of such a vaccine, a recombinant HAdV-3 plasmid expressing the reporter molecule eGFP was also constructed. This confirmed the recombinant protein expression capability. Mice immunized with this recombinant eGFP adenovirus by either intramuscular injection, intragastric or intranasal inoculation routes raised a significant antibody response to eGFP. Our results have provided a solid foundation for development of a recombinant live vaccine and potential more effective adenovirus vector-based delivery system for immune and gene therapy.

[Epidemiological study of human type 5 adenovirus in Guangzhou using chemiluminescence for neutralizing antibody assay].

OBJECTIVE: To establish a high-throughput chemiluminescence assay of serotype 5 specific neutralizing antibody and understand the epidemiology of this antibody in the healthy adults and children in Guangzhou. METHODS: Using rAd5 carrying the reporter gene of secreted alkaline phosphatases (SEAP), serum samples from 116 healthy adults and 94 healthy children were examined with chemiluminescence assay to detect the presence of Ad5 neutralizing antibody. The reliability of this assay was tested against conventional cytopathic effect observation. RESULTS: The chemiluminescence assay using secreted alkaline phosphatases (SEAP) as the reporter allowed rapid, sensitive, specific and reproducible detection of serotype 5 specific neutralizing antibody for epidemiological study of Ad5, which was positive in 26.72% of the adults and 17.02% of the children in this study. CONCLUSION: Ad5 neutralizing antibody is prevalent in the population of Guangzhou, suggesting the necessity of developing other serotype adenovectors for better vaccination and therapeutic effects.

Promoter hypermethylation of multiple genes in gastric lymphoma.

Aberrant hypermethylation of CpG islands in the promoter region of tumor suppressor and other important genes in neoplastic cells of lymphoma has been demonstrated to be one of the mechanisms for epigenetic loss of gene function. In this study, we analyzed promoter hypermethylation of the following genes in 49 cases of primary gastric lymphoma (PGL): ATM, p16INK4a(CDKN2A), hMLH1, MGMT, DAPK, and CDH1(ECAD). The PGL cases studied included 26 (53%) cases of diffuse large B-cell lymphoma (DLBCL), 12 (25%) cases of extranodal marginal zone lymphoma (MZL), 7 (14%) cases of MZL with large cell transformation (MZL/DLBCL), 1 (2%) case of follicular lymphoma (FL), one (2%) case of Burkitt-like lymphoma (BL), one case (2%) of lymphoplasmacytic lymphoma (LPL) and one case (2%) of peripheral T-cell lymphoma. Available pathologic data regarding to extragastric involvement at the time of resection of the PGLs were reviewed and correlated. Promoter hypermethylation was detected in 6 of 49 (12.2%) cases for ATM; 13 of 49 (26.5%) for p16INK4a, 19 of 49 (38.8%) for hMLH1; 22 of 49 (44.9%) for MGMT; 27 of 49 (55.1%) for DAPK and 16 of 49 (32.7%) for CDH1. A total of 85% of the PGLs had promoter hypermethylation in at least one of these genes. With different histologic subtypes, promoter hypermethylation of DAPK, hMLH1, and CDH1 genes occurred in 70%, 42%, and 42% respectively for DLBCL, which appeared to be higher than combined MZL and MZL/DLBCL subgroup. Approximately 81% PGLs demonstrated H. pylori infection by immunohistochemistry. H. pylori status did not appear to be statistically correlated with promoter hypermethylation of the genes. Of 37 PGL cases, 19 cases had extragastric involvement at the time of resection, indicating relatively higher stage disease. The frequencies of promoter methylation in those cases were 58% for DAPK, 42% for hMLH1, 37% for CDH1, 26% for p16INK4a and 11% for ATM respectively. The promoter methylation at MGMT gene was significantly higher in the PGLs without extragastric involvement (61%) as compared to those with extragastric involvement (26%).

Promoter hypermethylation for molecular nodal staging in non-small cell lung cancer.

CONTEXT: Even among cases of non-small cell lung cancer (NSCLC) in the most favorable stage (IA), the disease-specific mortality is 25% or greater. One plausible explanation implicates the simplistic standard pathologic procedures used to designate lymph node involvement. A more sensitive assessment of the nodal status may improve staging. OBJECTIVE: To determine the prognostic impact of detecting an abnormal molecular event (promoter hypermethylation in a set of relevant genes) in histologically uninvolved lymph nodes in resected NSCLC. DESIGN: In this retrospective analysis of archived material, we examined DNA extracted from lymph nodes of stage I NSCLC (n = 180). Patients underwent surgery between 1991 and 1995 in a single institution. Methylation-specific polymerase chain reaction was used to detect promoter hypermethylation in a panel of 8 genes. Survival data were extracted from the computerized database at the Tumor Registry. RESULTS: Evidence of promoter hypermethylation in at least 1 gene was detected in 67% of these N0 nodes. The most commonly hypermethylated gene was E-cadherin (53%). The hypermethylation frequency for the remaining genes were as follows: APC, 5%; p16, 9%; MGMT, 11%; hMLH1, 15%; RASSF1A, 4%; DAP kinase, 9%; and ATM, 19%. The presence of promoter hypermethylation in 2 or more genes did not influence the overall, median, or 5-year survival rates. CONCLUSIONS: Identifying promoter hypermethylation (in our panel) in N0 lymph nodes in stage I NSCLC cannot be recommended for clinical decision making. Molecular abnormalities, including those found in cancer by qualitative methylation-specific polymerase chain reaction, are not synonymous with established, histologically detectable metastasis.

Novel tricyclic compounds having acetylene groups at C-8a and cyano enones in rings A and C: highly potent anti-inflammatory and cytoprotective agents.

Novel C-8a functionalized tricyclic compounds having cyano enones in rings A and C have been synthesized and biologically evaluated. Among them, compounds with acetylene groups at C-8a show extremely high potency in in vitro and in vivo bioassays for anti-inflammatory and cytoprotective activities. Both in vitro and in vivo potencies are markedly higher than those of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), which is being evaluated as an anticancer drug in phase I clinical trials.