Functional polymorphism of the MMP-1 promoter (-1607 1G/2G) in potentially malignant and malignant head and neck lesions in an Indian population

Matrix metalloproteinases (MMP) are a family of zinc-dependent proteases that degrade the entire component of the extracellular matrix. Our study explores the association of the MMP1 gene promoter (-1607 1G/2G) polymorphisms in oral submucous fibrosis (OSMF) and head and neck squamous cell carcinoma (HNSCC) in an Indian population. The MMP1single-nucleotide polymorphism (SNP) was genotyped by polymerase chain reaction–restriction fragment length polymorphism analysis in 412 patients with OSMF, 422 with HNSCC and 426 controls. Our results showed that the frequency of 1G/2G or 2G/2G promoter genotypes having the 2G allele is associated with higher enzymatic activity and significantly increases in OSMF (p<0.001) and HNSCC cases (p<0.00). In this study, results concluded that SNPs in the MMP1 promoter region may be associated with susceptibility to OSMF as well as HNSCC in an Indian population and addiction habits such as areca nut chewing and alcohol abuse may enhance the expression of the 2G allele of MMP1 genes in OSMF and HNSCC cases.     

Platycodin D,a triterpenoid saponin from Platycodon grandiflorum,suppresses the growth and invasion of human oral squamous cell carcinoma cells via the NF‐κB pathway

This work was undertaken to explore the effects of platycodin D, a triterpenoid saponin from Platycodon grandiflorum, on the growth and invasiveness of human oral squamous cell carcinoma (OSCC). Platycodin D caused a significant, concentration‐dependent inhibition of cell viability and induced significant apoptosis in OSCC cells. Moreover, platycodin D significantly inhibited OSCC cell invasion. At the molecular level, platycodin D increased the amounts of IκBα protein and reduced the expression of phosphorylated NF‐κB p65, MMP‐2, and MMP‐9. Ectopic expression of constitutively active NF‐κB p65 prevented platycodin D‐mediated induction of apoptosis and suppression of invasion in OSCC cells. In vivo studies confirmed that platycodin D retarded the growth of subcutaneous SCC‐4 xenograft tumors and reduced phosphorylation of NF‐κB p65. Altogether, platycodin D shows inhibitory activity on OSCC growth and invasion through inactivation of the NF‐κB pathway and might provide therapeutic benefits in the treatment of OSCC.     

Effect of the interaction between MiR‐200b‐3p and DNMT3A on cartilage cells of osteoarthritis patients

The aim of this research is to explore the effect of miR‐200b‐3p targeting DNMT3A on the proliferation and apoptosis of osteoarthritis (OA) cartilage cells. Quantitative RT‐PCR was performed to analyse the expression of miR‐200b‐3p, DNMT3A, MMP1, MMP3, MMP9, MMP13 and COL II in normal and OA cartilage tissues. The dual‐luciferase reporter assay and Western blot assay were conducted to confirm the targeting relationship between miR‐200b‐3p and DNMT3A. We also constructed eukaryotic expression vector to overexpress miR‐200b‐3p and DNMT3A. We detected the expression level of MMPs and COL II in stable transfected cartilage cells using RT‐PCR and Western blot. Cell proliferation and apoptosis were evaluated using the MTS, pellet culture and Hoechst 33342 staining method. Finally, we explored the effect of miR‐200b‐3p targeting DNMT3A on the proliferation and apoptosis of OA cartilage cells. The results of RT‐PCR indicated that both miR‐200b‐3p and COL II were down‐regulated in OA cartilage tissues, while the expression of DNMT3A and MMPs was up‐regulated in OA cartilage tissues. The expressions of DNMT3A, MMPs and COL II detected by Western blot showed the same trend of the results of RT‐PCR. The dual‐luciferase reporter assay and Western blot assay confirmed the targeting relationship between miR‐200b‐3p and DNMT3A. In overexpressed miR‐200b‐3p cartilage cells, DNMT3A and MMPs were significantly down‐regulated, COL II was significantly up‐regulated, cell viability was enhanced and apoptosis rate was decreased (P < 0.05). In overexpressed DNM3T cartilage cells, MMPs were significantly up‐regulated, COL II was significantly down‐regulated, cell viability was weakened and apoptosis rate was increased (P < 0.05). MiR‐200b‐3p inhibited the secretion of MMPs, promoted the synthesis of COL II and enhanced the growth and proliferation of OA cartilage cells through inhibiting the expression of DNMT3A.     

Effects of deer bone extract on the expression of pro-inflammatory cytokine and cartilage-related genes in monosodium iodoacetate-induced osteoarthritic rats

Deer bone extract has the potential to relieve the discomfort or the articular cartilaginous damage associated with osteoarthritic (OA) and may be useful as a natural supplement for OA treatment without serious side effects. We analyzed the expression of pro-inflammatory cytokine and cartilage-related genes in monosodium iodoacetate-induced OA rats. Increases in the levels of serum pro-inflammatory cytokines, such as interleukin-1β, interleukin-6, and tumor necrosis factor-α were significantly inhibited by the administration of deer bone extract (p?<?0.05). Decreases in the expression of collagen type II (COL2) and tissue inhibitors of metalloproteinases (TIMPs) mRNAs in the cartilage were significantly inhibited by deer bone extract treatment (p?<?0.05). The deer bone extract significantly suppressed the expression of matrix metalloproteinases (MMPs) mRNAs in the cartilage. The deer bone extract induced the up-regulation of COL2 and TIMP mRNAs and the down-regulation of MMP mRNAs by suppressing the expression of pro-inflammatory cytokine mRNAs.     

EphA8 acts as an oncogene and contributes to poor prognosis in gastric cancer via regulation of ADAM10

EphA8 is a member of the erythropoietin-producing hepatocellular receptor (Eph) family of receptor tyrosine kinases. Ephs and their ephrins ligands play crucial roles in many cellular processed by mediating intracellular signaling resulting from cell–cell interactions. But the underlying mechanisms of EphA8 in gastric cancer (GC) remains unclearly. 298 clinical specimens in tissues microarray, and was found to be significantly higher in GC tissues compared with nontumor tissues (p < 0.001). EphA8 expression was also strongly associated with differentiation level (p = 0.025), tumor-node-metastasis stage (p = 0.019), and poor 5 years survival (p < 0.001). A panel of GC cell lines showed reduced proliferation, invasion, and migration capacities after RNA-mediated knockdown of EphA8, concomitant with downregulation of the proliferation-related proteins (cyclin A, cyclin D1, and cyclin-dependent kinase 4) and the metastasis-related (matrix metalloproteinases MMP2, and MMP9). EphA8 knockdown also decreased expression of the protease ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) and ADAM10-related protein AKT, suggesting an interaction between EphA8 and ADAM10. In conclusion, we found that EphA8, which is highly expressed in GC tissues, stimulates proliferation, invasion, and migration of cancer cells, and is an independent risk factor for poor prognosis of GC. These dates suggest that EphA8 could be new diagnostic and/or therapeutic targets for GC.     

Exogenous pulmonary surfactant prevents the development of intra‐abdominal adhesions in rats

Intra‐abdominal adhesions are major post‐operative complications for which no effective means of prevention is available. We aimed to evaluate the efficacy of exogenous pulmonary surfactant administration in the prevention of post‐operative abdominal adhesions. Rats were randomly assigned to undergo laparotomy (L) or gastroenterostomy (GE) and then treated with surfactant (groups L‐S and GE‐S, respectively). Intra‐abdominal adhesions, collagen fibre content, metalloproteinase (MMP)‐9, expression of growth factors (TGF‐β, KGF and VEGF), type III procollagen (PCIII) and pro‐caspase 3, as well as isolectin B4 and ED1‐positive cells expressing MMP‐9, were evaluated. Groups treated with surfactant (GE‐S and L‐S) exhibited fewer adhesions. A significant reduction in collagen fibre content was observed in GE‐S compared to GE animals (P < 0.001). In situ and gelatin zymography analysis showed higher MMP‐9 expression and activity in the GE‐S group compared to the GE group (P < 0.05). ED1‐positive cell counts were significantly higher in the GE‐S group (P < 0.001) than in the GE group. Virtually all cells positive for ED1 were MMP‐9+. Double‐labelling of MMP‐9 with IB4 showed no significant differences between GE‐S and GE groups. TGF‐β, KGF, PCIII and pro‐caspase‐3 mRNA expression decreased significantly in GE‐S compared to GE animals (P < 0.05). Surfactant administration also reduced apoptosis in the GE‐S group. These findings suggest that surfactant reduces the intra‐abdominal adhesions triggered by laparotomy and gastrointestinal anastomosis, thus preventing fibrosis formation at the peritoneal surfaces. This preclinical study suggests an innovative treatment strategy for intra‐abdominal adhesions with surfactant and to endorse its putative mechanism of action.     

Role of sialic acid‐containing glycans of matrix metalloproteinase‐9 (MMP‐9) in the interaction between MMP‐9 and staphylococcal superantigen‐like protein 5

Staphylococcal superantigen‐like proteins (SSL) show no superantigenic activity but have recently been considered to act as immune suppressors. It was previously reported that SSL5 bound to P‐selectin glycoprotein ligand‐1 (PSGL‐1) and matrix metalloproteinase (MMP)‐9, leading to inhibition of leukocyte adhesion and invasion. These interactions were suggested to depend on sialic acid‐containing glycans of MMP‐9, but the roles of sialic acids in the interaction between SSL5 and MMP‐9 are still controversial. In the present study, we prepared recombinant glutathione S‐transferase‐tagged SSL5 (GST‐SSL5) and analyzed its binding capacity to MMP‐9 by pull‐down assay after various modifications of its carbohydrate moieties. We observed that GST‐SSL5 specifically bound to MMP‐9 from a human monocytic leukemia cell line (THP‐1 cells) and inhibited its enzymatic activity in a concentration‐dependent manner. After MMP‐9 was treated with neuraminidase, its binding activity towards GST‐SSL5 was markedly decreased. Furthermore, recombinant MMP‐9 produced by sialic acid‐deficient Lec2 mutant cells showed much lower affinity for SSL5 than that produced by wild‐type CHO‐K1 cells. Treatment of MMP‐9 with PNGase F to remove N‐glycan resulted in no significant change in the GST‐SSL5/MMP‐9 interaction. In contrast, the binding of GST‐SSL5 to MMP‐9 secreted from THP‐1 cells cultured in the presence of an inhibitor for the biosynthesis of O‐glycan (benzyl‐GalNAc) was weaker than the binding of GST‐SSL5 to MMP‐9 secreted from untreated cells. These results strongly suggest the importance of the sialic acid‐containing O‐glycans of MMP‐9 for the interaction of MMP‐9 with GST‐SSL5.

     

Gene promoter methylation signature predicts survival of head and neck squamous cell carcinoma patients

Infection with high-risk types of human papilloma virus (HPV) is currently the best-established prognostic marker for head and neck squamous cell carcinoma (HNSCC), one of the most common and lethal human malignancies worldwide. Clinical trials have been launched to address the concept of treatment de-escalation for HPV-positive HNSCC with the final aim to reduce treatment related toxicity and debilitating long-term impacts on the quality of life. However, HPV-related tumors are mainly restricted to oropharyngeal SCC (OPSCC) and there is an urgent need to establish reliable biomarkers for all patients at high risk for treatment failure. A patient cohort (n = 295) with mainly non-OPSCC (72.9%) and a low prevalence of HPV16-related tumors (8.8%) was analyzed by MassARRAY to determine a previously established prognostic methylation score (MS). Kaplan-Meier revealed a highly significant correlation between a high MS and a favorable survival for OPSCC (P = 0.0004) and for non-OPSCC (P<0.0001), which was confirmed for all HNSCC by multivariate Cox regression models (HR: 9.67, 95% CI [4.61–20.30], P<0.0001). Next, we established a minimal methylation signature score (MMSS), which consists of ten most informative of the originally 62 CpG units used for the MS. The prognostic value of the MMSS was confirmed by Kaplan-Meier analysis for all HNSCC (P<0.0001) and non-OPSCC (P = 0.0002), and was supported by multivariate Cox regression models for all HNSCC (HR: 2.15, 95% CI [1.36–3.41], P = 0.001). In summary, the MS and the MMSS exhibit an excellent performance as prognosticators for survival, which is not limited by the anatomical site, and both could be implemented in future clinical trials.     

Gene expression,protein profiling,and chemotactic activity of infrapatellar fat pad mesenchymal stem cells in pathologies of the knee joint

The infrapatellar fat pad (IPFP) is a periarticular adipose knee tissue. This tissue contains a large number of mesenchymal stem cells (MSCs). In the present work, we wanted to study the IPFP MSCs and their relationship and differences in two groups, anterior cruciate ligament (ACL) ruptures knees and ostheoarthrosis (OA). The IPFP of 42 patients with OA or ACL rupture were analyzed. Isolation, primary culture, and a genetic and proteomic study of MSCs from IPFP were performed. Gene expression of IL-6, tumor necrosis factor (TNF), IL-8, HSPA1A (Hsp70), CXCL10, RANTES, MMP1, MMP3, TIMP1, and BMP7 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). We analyzed MSCs from from 12 diferents patients in two cellular pools (6 from AO disease and 6 from ALC rupture to form two cell pool), for the iTRAQ Proteomic Assay. The conditional media were used in quantitative analysis of MSC soluble factors by Luminex and for de migration assay. A higher gene expression of IL-6, TNF, CXCL10, RANTES, and MMP1 and OPG in MSCs from OA versus ACL (p < 0.05) was observed. Conversely HSPA1A, TIMP1, and RANKL showed a significant lower expression in OA-MSCs (p < 0.05). In the secretome analysis, adipsin and visfantin levels in the supernatants from OA-MSCs were lower (p < 0.05) respect to ACL-MSCs. Also, the monocytic cells migrated two-folds in the presence of conditioned media from OA-MSCs patients versus patients with ACL-MSC. The infrapatellar pad should be considered as an adipose tissue capable of producing and excreting inflammatory mediators directly in the knee joint, influencing the development and progression of knee joint pathologies.     

TFPI‐2 silencing increases tumour progression and promotes metalloproteinase 1 and 3 induction through tumour‐stromal cell interactions

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI‐2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI‐2 is frequently down‐regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI‐2 down‐regulation in the National Cancer Institute (NCI)‐H460 non‐small cell lung cancer cell line using specific micro interfering micro‐interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI‐2 down‐regulation enhanced cell adhesion to collagen IV and laminin via an increase in α₁ integrin on cell surface, and increased MMP expression (mainly MMP‐1 and ‐3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI‐2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP‐1, ‐3 and ‐7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.     

Hesperidin suppressed metastasis,angiogenesis and tumour growth in Balb/c mice model of breast cancer

Considering the unfavourable response of breast cancer (BC) to treatment, we assessed the therapeutic potential hesperidin in mice bearing 4T1 BC tumours. Anti-tumour effects were assessed by measuring pathologic complete response (pCR), survival analysis, immunohistochemistry for E-cadherin, VEGF, MMP9, MMP2 and Ki-67, serum measurement of IFNγ and IL-4, and gene expression analysis of CD105, VEGFa, VEGFR2 and COX2. Survival of tumour-bearing mice was the highest in mice receiving a combination of hesperidin and doxorubicin (Dox) (80%) compared to the normal saline (43%), hesperidin 5 (54%), 10 (55.5%), 10 (60.5%) and 40 (66%) mg/kg, and 10 mg/kg Dox-treated (73%) groups (p < 0.0001 for all). Compared to the normal saline group, there was a significant elevation in IFNγ level in the animals receiving 20 (p = 0.0026) and 40 (p < 0.001) mg/kg hesperidin, 10 mg/kg Dox (p < 0.001), and combined hesperidin (20 mg/kg) and Dox (10 mg/kg) (p < 0.001). A significant reduction in the gene expression of CD 105 (p = 0.0106), VEGFa (p < 0.0001), VEGFR2 (p < 0.0001), and Cox2 (p = 0.034) and a significant higher pCR score (p = 0.006) were noticed in mice treated with 10 mg/kg Dox + 20 mg/kg hesperidin compared to those treated with 10 mg/kg Dox alone. Immunohistochemical staining showed significant reductions in Ki-67 (p < 0.001) and VEGF (p < 0.001) and a significant elevation in E-cadherin (p = 0.005) in the 10 mg/kg Dox + 20 mg/kg treatment group than in 10 mg/kg Dox alone group. Hesperidin can be considered as a potentially suitable anti-cancer agent for BC that can synergize with other chemotherapeutics.     

NHE1 mediates migration and invasion of HeLa cells via regulating the expression and localization of MT1‐MMP

Na⁺/H⁺ exchanger 1 (NHE1), acting as an important regulator of intracellular pH (pH_i) and extracellular pH (pH_e), has been known to play a key role in the metastasis of many solid tumours. However, the exact mechanism underlying these processes, especially in cervical cancer, is still poorly understood. In the current study, we first showed that the inhibition of NHE1 activity by the specific inhibitor cariporide could suppress migration and invasion of HeLa cells in vitro. Moreover, cariporide also reversed the enhanced migration and invasion in HeLa cells by overexpressed membrane‐type 1 matrix metalloproteinase (MT1‐MMP). Subsequently, our results showed that NHE1 regulated the expression of MT1‐MMP at both messenger RNA and protein levels as well as its localization. Meanwhile, we observed slight modification in the morphology of HeLa cell after treating with cariporide. The present work indicates that NHE1 mediates HeLa cell metastasis via regulating the expression and localization of MT1‐MMP and provides a theoretical basis for the development of novel therapeutic strategies targeting cervical cancer. Copyright © 2011 John Wiley & Sons, Ltd.     

Matrix metalloproteinases and their endogenous regulators in squamous cervical carcinoma (A review of own results)

Own results of long-term studies of expression of matrix metalloproteinases (MMPs) and their endogenous regulators examined in fibroblasts transformed by oncogene E7 HPV16 (TF), immortalized fibroblasts (IF), cell lines associated with HPV16 and HPV18, and tumor tissue samples from patients with squamous cervical carcinoma (SCC) associated with HPV16 have been summarized. Transfection of fibroblasts with the E7 HPV16 oncogen was accompanied by induction of collagenase (MMP-1, MMP-14) and gelatinase (MMP-9) gene expression and the increase in catalytic activity of these MMP, while gelatinase MMP-2 expression remained unchanged. MMP expression correlated with the tumorigenic of transformed clones. Expression of MMP-9 was found only in TF. In TF expression mRNA TIMP-1 decreased, while expression of the genatinase inhibitor, TIMP-2, increased. Collagenase activity and expression of the MMP-14 (collagenase) mRNA increased, while gelatinase activity remained unchanged. The destructive potential of TF is associated with induction of collagenases, gelatinase MMP-9 and decreased levels of MMP inhibitors. MMP-9 may serve as a TF marker. Invasive potential of cell lines associated with HPV18 (HeLa and S4-1) was more pronounced than that of cell lines associated with HPV16 (SiHa and Caski). In most cell lines mRNA levels of collagenases MMP-1 and MMP-14 and the activator (uPA) increased, while gelatinase MMP-2 mRNA and tissue inhibitors mRNAs changed insignificantly. MMP-2 activity significantly increased in Caski and HeLa cell lines, while MMP-9 expression in these cell lines was not detected. The comparative study of expression MMP of and their endogenous regulators performed using SCC tumor samples associated with HPV16 has shown that the invasive and metastatic potentials of tumor tissue in SCC is obviously associated with increased expression of collagenases MMP-1, MMP-14 and gelatinase MMP-9, as well as decreased expression of inhibitors (TIMP-1 and TIMP-2), and to a lesser extent with increased expression of MMP-2. MMP-1 and MMP-9 can serve as markers of invasive and metastatic potential of the SCC tumor. The morphologically normal tissue adjacent to the tumor tissue is characterized by significant expression of MMP-1, MMP-2, and MMP-9. This also contributes to the increased destructive potential of the tumor.     

Cardiotoxin III suppresses hepatocyte growth factor–stimulated migration and invasion of MDA‐MB‐231 cells

The hepatocyte growth factor (HGF)/c‐Met signalling pathway is deregulated in most cancers and associated with a poor prognosis in breast cancer. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. In this study, we use HGF as an invasive inducer to investigate the effect of CTX III on MDA‐MB‐231 cells. When cells were treated with non‐toxic doses of CTX III, CTX III inhibited the HGF‐promoted cell migration and invasion. CTX III significantly suppressed the HGF‐induced c‐Met phosphorylation and downstream activation of phosphatidylinositol 3‐kinase (PI3k)/Akt and extracellular signal‐regulated kinase (ERK) 1/2. Additionally, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an upstream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by HGF. This effect was paralleled by a significant reduction in phosphorylation of IκBα kinase and IκBα and nuclear translocation of nuclear factor κB (NF‐κB) as well as a reduction of matrix metalloproteinase‐9 (MMP‐9) activity. Furthermore, the c‐Met inhibitor PHA665752 inhibited HGF‐induced MMP‐9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occurs downstream of c‐Met activation. Taken together, these findings suggest that CTX III inhibits the HGF‐induced invasion and migration of MDA‐MB‐231 cells via HGF/c‐Met‐dependent PI3K/Akt, ERK1/2 and NF‐κB signalling pathways, leading to the downregulation of MMP‐9 expression. Copyright © 2014 John Wiley & Sons, Ltd.     

The expression of MMP‐1 and MMP‐9 is up‐regulated by smooth muscle cells after their cross‐talk with macrophages in high glucose conditions

Patients with diabetes mellitus have an increased risk of myocardial infarction and coronary artery disease‐related death, exhibiting highly vulnerable plaques. Many studies have highlighted the major role of macrophages (MAC) and smooth muscle cells (SMC) and the essential part of metalloproteases (MMPs) in atherosclerotic plaque vulnerability. We hypothesize that in diabetes, the interplay between MAC and SMC in high glucose conditions may modify the expression of MMPs involved in plaque vulnerability. The SMC‐MAC cross‐talk was achieved using trans‐well chambers, where human SMC were grown at the bottom and human MAC in the upper chamber in normal (NG) or high (HG) glucose concentration. After cross‐talk, the conditioned media and cells were isolated and investigated for the expression of MMPs, MCP‐1 and signalling molecules. We found that upon cross‐talk with MAC in HG, SMC exhibit: (i) augmented expression of MMP‐1 and MMP‐9; (ii) significant increase in the enzymatic activity of MMP‐9; (iii) higher levels of soluble MCP‐1 chemokine which is functionally active and involved in MMPs up‐regulation; (iv) activated PKCα signalling pathway which, together with NF‐kB are responsible for MMP‐1 and MMP‐9 up‐regulation, and (v) impaired function of collagen assembly. Taken together, our data indicate that MCP‐1 released by cell cross‐talk in diabetic conditions binds to CCR2 and triggers MMP‐1 and MMP‐9 over‐expression and activity, features that could explain the high vulnerability of atherosclerotic plaque found at diabetic patients.     

Porphyromonas gingivalis promotes invasion of oral squamous cell carcinoma through induction of proMMP9 and its activation

Recent epidemiological studies have revealed a significant association between periodontitis and oral squamous cell carcinoma (OSCC). Furthermore, matrix metalloproteinase 9 (MMP9) is implicated in the invasion and metastasis of tumour cells. We examined the involvement of Porphyromonas gingivalis, a periodontal pathogen, in OSCC invasion through induced expression of proMMP and its activation. proMMP9 was continuously secreted from carcinoma SAS cells, while P. gingivalis infection increased proenzyme expression and subsequently processed it to active MMP9 in culture supernatant, which enhanced cellular invasion. In contrast, Fusobacterium nucleatum, another periodontal organism, failed to demonstrate such activities. The effects of P. gingivalis were observed with highly invasive cells, but not with the low invasivetype. P. gingivalis also stimulated proteinase‐activated receptor 2 (PAR2) and enhanced proMMP9 expression, which promoted cellular invasion. P. gingivalis mutants deficient in gingipain proteases failed to activate MMP9. Infected SAS cells exhibited activation of ERK1/2, p38, and NF‐kB, and their inhibitors diminished both proMMP9‐overexpression and cellular invasion. Together, our results show that P. gingivalis activates the ERK1/2‐Ets1, p38/HSP27, and PAR2/NF‐kB pathways to induce proMMP9 expression, after which the proenzyme is activated by gingipains to promote cellular invasion of OSCC cell lines. These findings suggest a novel mechanism of progression and metastasis of OSCC associated with periodontitis.     

Genetic Effects on Postprandial Variations of Inflammatory Markers in Healthy Individuals

Circulating levels of inflammatory markers predict the risk of cardiovascular disease (CVD), mediated perhaps in part by dietary fat intake, through mechanisms only partially understood. To evaluate post‐fat load changes in inflammatory markers and genetic influences on these changes, we administered a standardized high‐fat meal to 838 related Amish subjects as part of the Heredity and Phenotype Intervention (HAPI) Heart Study and measured a panel of inflammatory markers, including C‐reactive protein (CRP), interleukin‐1β (IL‐1β), matrix metalloproteinase‐1 and ‐9 (MMP‐1 and MMP‐9), and white blood cell (WBC) count, before and 4 h after fat challenge (CRP prechallenge only). Heritabilities (h² ± s.d.) of basal inflammatory levels ranged from 16 ± 8% for MMP‐9 (P = 0.02) to 90 ± 7% for MMP‐1 (P < 0.0001). Post‐fat load, circulating levels of WBC, MMP‐1, and MMP‐9 increased by 16, 32, and 43% (all P < 0.0001), with no significant changes in IL‐1β. Postprandial changes over the 4‐h period were modestly heritable for WBC (age‐ and sex‐adjusted h² = 14 ± 9%, P = 0.04), but the larger MMP‐1 and MMP‐9 changes appeared to be independent of additive genetic effects. These results reveal that a high‐fat meal induces a considerable inflammatory response. Genetic factors appear to play a significant role influencing basal inflammatory levels but to have minimal influence on post‐fat intake inflammatory changes.