Differential expression of mitogen activating protein kinases in periodontitis

Aim Following toll‐like receptor ( TLR ) engagement, lipopolysaccharide ( LPS ) can stimulate the expression of pro‐inflammatory cytokines thus activating the innate immune response. The production of inflammatory cytokines results, in part, from the activation of kinase‐induced signalling cascades and transcriptional factors. Of the four distinct classes of mitogen‐activated protein kinases ( MAPK ) described in mammals, p38, c‐Jun N‐terminal activated kinases ( JNK 1‐3) and extracellular activated kinases ( ERK 1,2) are the best studied. Previous data have established that p38 MAPK signalling is required for inflammation and bone loss in periodontal disease pre‐clinical animal models. Materials & Methods In this study, we obtained healthy and diseased periodontal tissues along with clinical parameters and microbiological parameters. Excised fixed tissues were immunostained with total and phospho‐specific antibodies against p38, JNK and ERK kinases. Results Intensity scoring from immunostained tissues was correlated with clinical periodontal parameters. Rank correlations with clinical indices were statistically significantly positive ( p ‐value < 0.05) for total p38 (correlations ranging 0.49–0.68), phospho‐p38 (range 0.44–0.56), and total ERK (range 0.52–0.59) levels, and correlations with JNK levels also supported association (range 0.42–0.59). Phospho‐ JNK and phospho‐ ERK showed no significant positive correlation with clinical parameters of disease. Conclusion These data strongly implicate p38 MAPK as a major MAPK involved in human periodontal inflammation and severity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98997/1/jcpe12123.pd

Tristetraprolin Regulates Interleukin-6 Expression Through p38 MAPK-Dependent Affinity Changes with mRNA 3' Untranslated Region

Tristetraprolin (TTP) is a well-characterized, zinc finger-containing, RNA-binding protein. TTP targets tumor necrosis factor alpha for degradation via the 3- untranslated region (3-UTR). Although AU-rich elements (AREs) in the 3-UTR of interleukin-6 (IL-6) mRNA dictate mRNA degradation, the role of TTP in the post-transcriptional regulation of IL-6 gene expression is unclear. Here we used TTP-deficient mice to test the hypothesis that IL-6 expression is influenced by TTP. Genetic and siRNA-mediated knockdown of TTP resulted in increased IL-6 production and overexpression of TTP had the reverse effect. IL-6 and tumor necrosis factor alpha production were elevated after injection of IL-1- in TTP-deficient mice. Further, embryonic fibroblasts from these mice (mouse embryonic fibroblasts) exhibited greater IL-6 mRNA expression and longer half-life than wild-type mouse embryonic fibroblasts. Overexpression of TTP reduced IL-6 3-UTR luciferase reporter activity in an ARE-dependent manner. Proximal and distal regions of the 3-UTR acted synergistically to produce the full repression of TTP. Mutation-based luciferase assays show that ARE2, ARE3, and ARE4 are required for TTP-mediated repression. The constitutively activated p38-MK2 pathway abrogated TTP-mediated repression of IL-6 3-UTR reporter activity. RNA immunoprecipitation assay indicated that the deficiency of p38alpha resulted in the increased affinity of TTP to IL-6 mRNA. Taken together, we propose that TTP downregulates IL-6 gene expression at the post-transcriptional level by targeting ARE elements in the 3-UTR region.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90500/1/jir-2E2010-2E0154.pd

Adenovirus Encoding Human Platelet-Derived Growth Factor-B Delivered to Alveolar Bone Defects Exhibits Safety and Biodistribution Profiles Favorable for Clinical Use

Abstract Platelet-derived growth factor (PDGF) gene therapy offers promise for tissue engineering of tooth-supporting alveolar bone defects. To date, limited information exists regarding the safety profile and systemic biodistribution of PDGF gene therapy vectors when delivered locally to periodontal osseous defects. The aim of this preclinical study was to determine the safety profile of adenovirus encoding the PDGF-B gene (AdPDGF-B) delivered in a collagen matrix to periodontal lesions. Standardized alveolar bone defects were created in rats, followed by delivery of matrix alone or containing AdPDGF-B at 5.5-108 or 5.5-109 plaque-forming units/ml. The regenerative response was confirmed histologically. Gross clinical observations, hematology, and blood chemistries were monitored to evaluate systemic involvement. Bioluminescence and quantitative polymerase chain reaction were used to assess vector biodistribution. No significant histopathological changes were noted during the investigation. Minor alterations in specific hematological and blood chemistries were seen; however, most parameters were within the normal range for all groups. Bioluminescence analysis revealed vector distribution at the axillary lymph nodes during the first 2 weeks with subsequent return to baseline levels. AdPDGF-B was well contained within the localized osseous defect area without viremia or distant organ involvement. These results indicate that AdPDGF-B delivered in a collagen matrix exhibits acceptable safety profiles for possible use in human clinical studies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78106/1/hum.2008.114.pd

Pretreatment dietary intake is associated with tumor suppressor DNA methylation in head and neck squamous cell carcinomas

Diet is associated with cancer prognosis, including head and neck cancer (HNC), and has been hypothesized to influence epigenetic state by determining the availability of functional groups involved in the modification of DNA and histone proteins. The goal of this study was to describe the association between pretreatment diet and HNC tumor DNA methylation. Information on usual pretreatment food and nutrient intake was estimated via food frequency questionnaire (FFQ) on 49 HNC cases. Tumor DNA methylation patterns were assessed using the Illumina Goldengate Methylation Cancer Panel. First, a methylation score, the sum of individual hypermethylated tumor suppressor associated CpG sites, was calculated and associated with dietary intake of micronutrients involved in one-carbon metabolism and antioxidant activity, and food groups abundant in these nutrients. Second, gene specific analyses using linear modeling with empirical Bayesian variance estimation were conducted to identify if methylation at individual CpG sites was associated with diet. All models were controlled for age, sex, smoking, alcohol and HPV status. Individuals reporting in the highest quartile of folate, vitamin B12 and vitamin A intake, compared with those in the lowest quartile, showed significantly less tumor suppressor gene methylation, as did patients reporting the highest cruciferous vegetable intake. Gene specific analyses identified differential associations between DNA methylation and vitamin B12 and vitamin A intake when stratifying by HPV status. These preliminary results suggest that intake of folate, vitamin A and vitamin B12 may be associated with the tumor DNA methylation profile in HNC and enhance tumor suppression

Pretreatment dietary intake is associated with tumor suppressor DNA methylation in head and neck squamous cell carcinomas

Diet is associated with cancer prognosis, including head and neck cancer (HNC), and has been hypothesized to influence epigenetic state by determining the availability of functional groups involved in the modification of DNA and histone proteins. The goal of this study was to describe the association between pretreatment diet and HNC tumor DNA methylation. Information on usual pretreatment food and nutrient intake was estimated via food frequency questionnaire (FFQ) on 49 HNC cases. Tumor DNA methylation patterns were assessed using the Illumina Goldengate Methylation Cancer Panel. First, a methylation score, the sum of individual hypermethylated tumor suppressor associated CpG sites, was calculated and associated with dietary intake of micronutrients involved in one-carbon metabolism and antioxidant activity, and food groups abundant in these nutrients. Second, gene specific analyses using linear modeling with empirical Bayesian variance estimation were conducted to identify if methylation at individual CpG sites was associated with diet. All models were controlled for age, sex, smoking, alcohol and HPV status. Individuals reporting in the highest quartile of folate, vitamin B12 and vitamin A intake, compared with those in the lowest quartile, showed significantly less tumor suppressor gene methylation, as did patients reporting the highest cruciferous vegetable intake. Gene specific analyses identified differential associations between DNA methylation and vitamin B12 and vitamin A intake when stratifying by HPV status. These preliminary results suggest that intake of folate, vitamin A and vitamin B12 may be associated with the tumor DNA methylation profile in HNC and enhance tumor suppression

Biomarkers in advanced larynx cancer

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102058/1/lary24245.pd

Stromal‐Derived Factor‐1α (CXCL12) Levels Increase in Periodontal Disease

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142300/1/jper0845.pd

The CS1 segment of fibronectin is involved in human OSCC pathogenesis by mediating OSCC cell spreading, migration, and invasion

<p>Abstract</p> <p>Background</p> <p>The alternatively spliced V region or type III connecting segment III (IIICS) of fibronectin is important in early development, wound healing, and tumorigenesis, however, its role in oral cancer has not been fully investigated. Thus, we investigated the role of CS-1, a key site within the CSIII region of fibronectin, in human oral squamous cell carcinoma (OSCC).</p> <p>Methods</p> <p>To determine the expression of CS-1 in human normal and oral SCC tissue specimens immunohistochemical analyses were performed. The expression of CS1 was then associated with clinicopathological factors. To investigate the role of CS-1 in regulating OSCC cell spreading, migration and invasion, OSCC cells were assayed for spreading and migration in the presence of a CS-1 peptide or a CS-1 blocking peptide, and for invasion using Matrigel supplemented with these peptides. In addition, integrin α4siRNA or a focal adhesion kinase (FAK) anti-sense oligonucleotide was transfected into OSCC cells to examine the mechanistic role of integrin α4 or FAK in CS1-mediated cell spreading and migration, respectively.</p> <p>Results</p> <p>CS-1 expression levels were significantly higher in OSCC tissues compared to normal tissues (p < 0.05). Also, although, high levels of CS-1 expression were present in all OSCC tissue samples, low-grade tumors stained more intensely than high grade tumors. OSCC cell lines also expressed higher levels of CS-1 protein compared to normal human primary oral keratinocytes. There was no significant difference in total fibronectin expression between normal and OSCC tissues and cells. Inclusion of CS-1 in the in vitro assays enhanced OSCC cell spreading, migration and invasion, whereas the CS1 blocking peptide inhibited these processes. Suppression of integrin α4 significantly inhibited the CS1-mediated cell spreading. Furthermore, this migration was mediated by focal adhesion kinase (FAK), since FAK suppression significantly blocked the CS1-induced cell migration.</p> <p>Conclusion</p> <p>These data indicate that the CS-1 site of fibronectin is involved in oral cancer pathogenesis and in regulating OSCC cell spreading, migration and invasion.</p

Sirtuin-3 (SIRT3), a novel potential therapeutic target for oral cancer

BACKGROUND: Several sirtuin family members (SIRT1-7), which are evolutionarily conserved NAD-dependent deacetylases, play an important role in carcinogenesis. However, their role in oral cancer has not yet been investigated. Therefore, the objective of this study was to investigate whether sirtuins play a role in oral cancer carcinogenesis. METHODS: The expression levels of all sirtuins in several oral squamous cell carcinoma (OSCC) cell lines were compared with normal human oral keratinocytes and observed that SIRT3 was highly expressed. Therefore, tissue microarrays were used to evaluate the clinical relevance of this overexpression. SIRT3 down-regulation in OSCC cell proliferation and survival was investigated and analyzed by using cell-proliferation and cell-viability assays. Ionizing radiation and cisplatin were used to investigate whether SIRT3 down-regulation could increase the sensitivity of OSCC to both treatments. To further assess the in vivo role of SIRT3 in OSCC carcinogenesis, a floor-of-mouth oral cancer murine model was used to study the effect of SIRT3 down-regulation on OSCC tumor growth in immunodeficient mice. RESULTS: The current results demonstrated for the first time that SIRT3 is overexpressed in OSCC in vitro and in vivo compared with other sirtuins. Down-regulation of SIRT3 inhibited OSCC cell growth and proliferation and increased OSCC cell sensitivity to radiation and cisplatin treatments in vitro. SIRT3 down-regulation also reduced tumor burden in vivo. CONCLUSIONS: The current investigation revealed a novel role for SIRT3 in oral cancer carcinogenesis as a promoter of cell proliferation and survival, thus implicating SIRT3 as a new potential therapeutic target to treat oral cancer. Cancer 2011. © 2010 American Cancer Society.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/84409/1/25676_ftp.pd

Effect of erlotinib on epidermal growth factor receptor and downstream signaling in oral cavity squamous cell carcinoma

Background The purpose of this study was to determine if there are differences in biomarker modulation and epidermal growth factor receptor (EGFR) degradation between the tumor and the normal mucosa after treatment with an EGFR inhibitor, erlotinib, in head and neck cancer. Methods Patients with primary oral cavity squamous cell cancers received a course of erlotinib, 150 mg every day for 7 days before surgical resection. Tumor and normal mucosa biopsies were obtained both pre‐erlotinib and post‐erlotinib. Changes in known markers of EGFR activity (phospho, AKT, STAT3) were measured by immunoblotting, whereas changes in tissue distribution were analyzed by immunohistochemical analysis. Results Twelve patients were enrolled; 7 had evaluable paired tumors and normal mucosa biopsies pretreatment and posttreatment. Expression of EGFR was higher in tumors compared to the normal mucosa ( p = .005). Erlotinib administration was associated with marked inhibition of phosphorylated epidermal growth factor receptor (pEGFR) and reduction in total EGFR protein ( p = .004, p = .007) in tumors, whereas there was heterogeneity in EGFR inhibition in the normal mucosa ( p = .10 [pEGFR], and p = .07 [EGFR]). Reduced levels of pSrc and pSTAT3 and enhanced p27 levels were noted in tumors after erlotinib. Cell culture studies confirmed that EGFR is degraded in tumor cells after prolonged treatment with erlotinib. Conclusion Our results show that EGFR inhibition by erlotinib led to a marked reduction in EGFR protein levels in patients. Differential effects of erlotinib on tumors compared to the normal mucosa suggest there may be individual patient heterogeneity. These preliminary data suggest EGFR degradation should be further analyzed as a potential biomarker in selecting patients likely to benefit from EGFR inhibitors. © 2012 Wiley Periodicals, Inc. Head Neck, 2013Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/99603/1/23128_ftp.pd