Microtubule inhibitors have been shown to inhibit Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal transduction pathway in various ca... More
Microtubule inhibitors have been shown to inhibit Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal transduction pathway in various cancer cells. However, little is known of the mechanism by which the microtubule inhibitors inhibit STAT3 activity. In the present study, we examined the effect of a novel small-molecule microtubule inhibitor, MPT0B098, on STAT3 signaling in oral squamous cell carcinoma (OSCC). Treatment of various OSCC cells with MPT0B098 induced growth inhibition, cell cycle arrest and apoptosis, as well as increased the protein level of SOCS3. The accumulation of SOCS3 protein enhanced its binding to JAK2 and TYK2 which facilitated the ubiquitination and degradation of JAK2 and TYK2, resulting in a loss of STAT3 activity. The inhibition of STAT3 activity led to sensitization of OSCC cells to MPT0B098 cytotoxicity, indicating that STAT3 is a key mediator of drug resistance in oral carcinogenesis. Moreover, the combination of MPT0B098 with the clinical drug cisplatin or 5-FU significantly augmented growth inhibition and apoptosis in OSCC cells. Taken together, our results provide a novel mechanism for the action of MPT0B098 in which the JAK2/STAT3 signaling pathway is suppressed through the modulation of SOCS3 protein level. The findings also provide a promising combinational therapy of MPT0B098 for OSCC. Less
MicroRNAs (miRNAs) are implicated in the pathogenesis of oral squamous-cell carcinoma (OSCC). miR-101 is involved in the development and progression of OSCC, but the biol... More
MicroRNAs (miRNAs) are implicated in the pathogenesis of oral squamous-cell carcinoma (OSCC). miR-101 is involved in the development and progression of OSCC, but the biological functions and underlying molecular mechanisms of this miRNA remain largely unknown. In this study, we showed that miR-101 was underexpressed in OSCC tissues and cell lines. miR-101 downregulation was inversely correlated with zinc finger E-box binding homeobox 1 (ZEB1) expression, lymph-node metastasis, and poor prognosis in OSCC patients. Enhanced expression of miR-101 significantly inhibited OSCC cell proliferation, apoptosis resistance, migration and invasion in vitro, and suppressed tumor growth and lung metastasis in vivo. Bioinformatics analyses showed that miR-101 directly targeted ZEB1, as confirmed by a dual-luciferase reporter assay. The inhibitory effects of miR-101 on OSCC growth and metastasis were attenuated and phenocopied by ZEB1 overexpression and knockdown, respectively. Overall, our findings indicated that miRNA-101 reduced OSCC growth and metastasis by targeting ZEB1 and provided new evidence of miR-101 as a potential therapeutic target for OSCC patients. Less
Purpose Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week stora... More
Purpose Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed. Materials and Methods Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR. Results Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C. Conclusion HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture. Less
Porphyromonas gingivalis is a keystone pathogen of periodontitis. One of its bacterial characteristics is the ability to invade various host cells, including nonphagocyti... More
Porphyromonas gingivalis is a keystone pathogen of periodontitis. One of its bacterial characteristics is the ability to invade various host cells, including nonphagocytic epithelial cells and fibroblasts, which is known to facilitate P. gingivalis adaptation and survival in the gingival environment. In this study, we investigated two small compounds, Alop1 and dynasore, for their role in inhibition of P. gingivalis invasion. Using confocal microscopy, we showed that these two compounds significantly reduced invasion of P. gingivalis and its outer membrane vesicles into human oral keratinocytes in a dose-dependent manner. The inhibitory effects of dynasore, a dynamin inhibitor, on the bacterial entry is consistent with the notion that P. gingivalis invasion is mediated by a clathrin-mediated endocytic machinery. We also observed that microtubule arrangement, but not actin, was altered in the host cells treated with Alop1 or dynasore, suggesting an involvement of microtubule in this inhibitory activity. This work provides an opportunity to develop compounds against P. gingivalis infection. Less
Oral squamous cell carcinoma (OSCC) ranks as the fifth most common cancer worldwide with poor prognosis. Recently, tumor necrosis factor receptor-associated factor 4 (TRA... More
Oral squamous cell carcinoma (OSCC) ranks as the fifth most common cancer worldwide with poor prognosis. Recently, tumor necrosis factor receptor-associated factor 4 (TRAF4) has attracted increasing attenuation due to its overexpression in certain cancers. However, its function and underlying mechanism in OSCC remains elusive. In this study, the high expression of TRAF4 mRNA and protein levels was noted in OSCC cell lines. Its overexpression with pcDNA3.1-TRAF4 vector transfection dramatically promoted cell proliferation and inhibited cell apoptosis, indicating a pivotal role of TRAF4 in OSCC cell growth. Simultaneously, TRAF4 elevation also increased cell invasion and migration. Mechanism analysis confirmed that TRAF4 up-regulation induced the expression of β-catenin and the downstream target molecules of cyclinD1, c-myc, Bcl-2, MMP-9 and MMP-2, indicating that TRAF4 could induce the activation of Wnt/β-catenin pathway. After pretreatment with β-catenin siRNA, the pathway was remarkably silenced. Simultaneously, cell growth, invasion and migration induced by TRAF4 were strikingly abrogated, suggesting that TRAF4 may promote OSCC cell growth, invasion and migration by Wnt/β-catenin pathway. Together, this study confirmed that TRAF4 acts as an oncogene for the development and progression of OSCC. Therefore, our study may support a promising therapeutic target for the treatment of OSCC. Less
Streptococcus mutans is the etiological agent of dental caries and one of the many bacterial species implicated in infective endocarditis. The expression of the collagen-... More
Streptococcus mutans is the etiological agent of dental caries and one of the many bacterial species implicated in infective endocarditis. The expression of the collagen-binding protein Cnm by S. mutans has been associated with extraoral infections, but its relevance for dental caries has only been theorized to date. Due to the collagenous composition of dentinal and root tissues, we hypothesized that Cnm may facilitate the colonization of these surfaces, thereby enhancing the pathogenic potential of S. mutans in advancing carious lesions. As shown for extraoral endothelial cell lines, Cnm mediates the invasion of oral keratinocytes and fibroblasts by S. mutans. In this study, we show that in the Cnm+ native strain, OMZ175, Cnm mediates stringent adhesion to dentinal and root tissues as well as collagen-coated surfaces and promotes both cariogenicity and carriage in vivo. In vitro, ex vivo, and in vivo experiments revealed that while Cnm is not universally required for S. mutans cariogenicity, it contributes to (i) the invasion of the oral epithelium, (ii) enhanced binding on collagenous surfaces, (iii) implantation of oral biofilms, and (IV) the severity of caries due to a native Cnm+ isolate. Taken together, our findings reveal that Cnm is a colonization factor that contributes to the pathogenicity of certain S. mutans strains in their native habitat, the oral cavity. Less
Radioresistance is still an emerging problem for radiotherapy of oral cancer. Aberrant epigenetic alterations play an important role in cancer development, yet the role o... More
Radioresistance is still an emerging problem for radiotherapy of oral cancer. Aberrant epigenetic alterations play an important role in cancer development, yet the role of such alterations in radioresistance of oral cancer is not fully explored. Using a methylation microarray, we identified promoter hypermethylation of FHIT (fragile histidine triad) in radioresistant OML1-R cells, established from hypo-fractionated irradiation of parental OML1 radiosensitive oral cancer cells. Further analysis confirmed that transcriptional repression of FHIT was due to promoter hypermethylation, H3K27me3 and overexpression of methyltransferase EZH2 in OML1-R cells. Epigenetic interventions or depletion of EZH2 restored FHIT expression. Ectopic expression of FHIT inhibited tumor growth in both in vitro and in vivo models, while also resensitizing radioresistant cancer cells to irradiation, by restoring Chk2 phosphorylation and G2/M arrest. Clinically, promoter hypermethylation of FHIT inversely correlated with its expression and independently predicted both locoregional control and overall survival in 40 match-paired oral cancer patient samples. Further in vivo therapeutic experiments confirmed that inhibition of DNA methylation significantly resensitized radioresistant oral cancer cell xenograft tumors. These results show that epigenetic silencing of FHIT contributes partially to radioresistance and predicts clinical outcomes in irradiated oral cancer. The radiosensitizing effect of epigenetic interventions warrants further clinical investigation. Less
Previous reports revealed that a significant decrease of miR-34a in oral cancer. But the role of miR-34a in oral cancer needs further research. In the present study, we w... More
Previous reports revealed that a significant decrease of miR-34a in oral cancer. But the role of miR-34a in oral cancer needs further research. In the present study, we will investigate the effect of miR-34a on oral cancer cell phenotypes. First, it was verified that miR-34a expression was lower in oral cancer tissues compared with their normal controls, so did the oral cancer cells. Next, it was showed that miR-34a overexpression in oral cancer cells could inhibit cell proliferation, G1 phase arrest, metastasis and epithelial mesenchymal transition. It was predicted that interleukin-6-receptor (IL6R) was a potential target gene of miR-34a by bioinformatics analysis and identified by luciferase assay. It was further showed that miR-34a inhibited oral cancer progression via IL6R. Collectively, our findings suggested that miR-34a may function as a tumor suppressor in oral cancer by targeting IL6R. Less
Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have g... More
Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM-MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion-induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM-MSCs. Seventy mice were pre-treated with BM-MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro-vascular endothelial cells (HPMVECs) were pre-conditioned with BM-MSCs by oxygen-glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI-treated mice, administration of BM-MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD-treated HPMVECs, co-culture with BM-MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM-MSCs decreased the level of PI3K class I and p-Akt while the expression of PI3K class III was increased. Finally, BM-MSCs-induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM-MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM-MSCs and will help to develop new cell-based therapeutic strategies in lung injury. Less
Previously, we identified global epigenetic aberrations in smoking-associated oral squamous cell carcinoma (OSCC). We hypothesized that cigarette exposure triggers OSCC t... More
Previously, we identified global epigenetic aberrations in smoking-associated oral squamous cell carcinoma (OSCC). We hypothesized that cigarette exposure triggers OSCC through alteration of the methylome of oral cells. Here we report that cigarette smoke condensate (CSC) significantly changes the genomic 5-methyldeoxycytidine content and nuclear accumulation of DNA methyltransferase 1 (DNMT1) and DNMT3A in human untransformed oral cells. By using integrated analysis of cDNA and methylation arrays of the smoking-associated dysplastic oral cell line and OSCC tumors, respectively, we identified four epigenetic targets--UCHL1, GPX3, LXN, and LDOC1--which may be silenced by cigarette. Results of quantitative methylation-specific PCR showed that among these four genes, LDOC1 promoter was the most sensitive to CSC. LDOC1 promoter hypermethylation and gene silencing followed 3 weeks of CSC treatment. LDOC1 knockdown led to a proliferative response and acquired clonogenicity of untransformed oral cells. Immunohistochemistry showed that LDOC1 was downregulated in 53.3% (8/15) and 57.1% (20/35) of premalignant oral tissues and early stage OSCCs, respectively, whereas 76.5% (13/17) of normal oral tissues showed high LDOC1 expression. Furthermore, the microarray data showed that LDOC1 expression had decreased in the lung tissues of current smokers compared with that in those of never smokers and had significantly decreased in the lung tumors of smokers compared with that in normal lung tissues. Our data suggest that CSC-induced promoter methylation may contribute to LDOC1 downregulation, thereby conferring oncogenic features to oral cells. These findings also imply a tumor suppressor role of LDOC1 in smoking-related malignancies such as OSCC and lung cancer. Less
Background: MicroRNAs (miRNAs), small noncoding RNA molecules can function as oncogenes or tumor suppressors in tumorigenesis. Oral squamous cell carcinoma (OSCC) is one ... More
Background: MicroRNAs (miRNAs), small noncoding RNA molecules can function as oncogenes or tumor suppressors in tumorigenesis. Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide with a 5-year survival rate of approximately 50%. Methods: The expression of microRNA-99a (miR-99a) in OSCC tissues and cell lines was investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The functions of miR-99a in migration/invasion and lung colonization were determined by transwell and tail vein injection assays, respectively. Specific targets of miR-99a were determined by software prediction, correlation with target protein expression, and luciferase reporter assay. The signaling pathways involved in regulation of miR-99a were investigated using the kinase inhibitors. Results: We observed reduced levels of miR-99a, identified as one of the most downregulated miRNA in OSCC and all tested OSCC cell lines compared to normal oral keratinocytes. Ectopic miR-99a expression in OSCC cells markedly reduced migration and invasion in vitro as well as lung colonization in vivo. When evaluating the specific targets of miR-99a, we found that ectopic miR-99a expression downregulates insulin-like growth factor 1 receptor (IGF1R) protein and that the expression of miR-99a correlates negatively with IGF1R protein in OSCC cells. Insertion of the 3′UTR of IGF1R mRNA into the 3′UTR of a reporter gene markedly reduced luciferase activity in OSCC cells expressing miR-99a, suggesting that miR-99a reduces luciferase activity by targeting the 3′UTR of IGF1R mRNA. When evaluating the mechanisms of miR-99a downregulation, we observed the upregulation of miR-99a expression in serum-starved conditions and its suppression in response to insulin-like growth factor (IGF1) stimulation. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) kinase inhibited IGF1-induced suppression of miR-99a, suggesting the negative regulation of miR-99a expression by IGF1R signaling. Conclusion: Overall, results indicate that miR-99a functions as a tumor metastasis suppressor in OSCC cells and mutually regulates IGF1R expression in a reciprocal regulation. Less
Background: MicroRNAs (miRNAs), small noncoding RNA molecules can function as oncogenes or tumor suppressors in tumorigenesis. Oral squamous cell carcinoma (OSCC) is one ... More
Background: MicroRNAs (miRNAs), small noncoding RNA molecules can function as oncogenes or tumor suppressors in tumorigenesis. Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide with a 5-year survival rate of approximately 50%. Methods: The expression of microRNA-99a (miR-99a) in OSCC tissues and cell lines was investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The functions of miR-99a in migration/invasion and lung colonization were determined by transwell and tail vein injection assays, respectively. Specific targets of miR-99a were determined by software prediction, correlation with target protein expression, and luciferase reporter assay. The signaling pathways involved in regulation of miR-99a were investigated using the kinase inhibitors. Results: We observed reduced levels of miR-99a, identified as one of the most downregulated miRNA in OSCC and all tested OSCC cell lines compared to normal oral keratinocytes. Ectopic miR-99a expression in OSCC cells markedly reduced migration and invasion in vitro as well as lung colonization in vivo. When evaluating the specific targets of miR-99a, we found that ectopic miR-99a expression downregulates insulin-like growth factor 1 receptor (IGF1R) protein and that the expression of miR-99a correlates negatively with IGF1R protein in OSCC cells. Insertion of the 3′UTR of IGF1R mRNA into the 3′UTR of a reporter gene markedly reduced luciferase activity in OSCC cells expressing miR-99a, suggesting that miR-99a reduces luciferase activity by targeting the 3′UTR of IGF1R mRNA. When evaluating the mechanisms of miR-99a downregulation, we observed the upregulation of miR-99a expression in serum-starved conditions and its suppression in response to insulin-like growth factor (IGF1) stimulation. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) kinase inhibited IGF1-induced suppression of miR-99a, suggesting the negative regulation of miR-99a expression by IGF1R signaling. Conclusion: Overall, results indicate that miR-99a functions as a tumor metastasis suppressor in OSCC cells and mutually regulates IGF1R expression in a reciprocal regulation. Less
MicroRNA (miRNA) is a form of small noncoding RNA that regulates the expression of genes either by inhibiting mRNA translation or by inducing its degradation. Small micro... More
MicroRNA (miRNA) is a form of small noncoding RNA that regulates the expression of genes either by inhibiting mRNA translation or by inducing its degradation. Small microRNA play important roles in regulating a large number of cellular processes, including development, proliferation and apoptosis. This study examined the biological functions of miR-205 as a tumor suppressor in KB oral cancer cells. The results showed that miR-205 expression was significantly lower in KB oral cancer cells than in human normal oral keratinocytes. Furthermore, the miR-205 over-expressed in KB oral cancer cells increased the cell cytotoxicity and induced apoptosis through the activation of caspase-3/-7. The transfection of miR-205 into KB oral cancer cells strongly induced IL-24, a well known cytokine that acts as a tumor suppressor in a range of tumor tissues. In addition, miR-205 targeted the IL-24 promoter directly to induce gene expression. Overall, miR-205 has significant therapeutic potential to turn on silenced tumor suppressor genes by targeting them with miRNA. Keywords: apoptosis, human oral cancer, interleukin-24, microRNA-205 Less
MicroRNA (miRNA) is a form of small noncoding RNA that regulates the expression of genes either by inhibiting mRNA translation or by inducing its degradation. Small micro... More
MicroRNA (miRNA) is a form of small noncoding RNA that regulates the expression of genes either by inhibiting mRNA translation or by inducing its degradation. Small microRNA play important roles in regulating a large number of cellular processes, including development, proliferation and apoptosis. This study examined the biological functions of miR-205 as a tumor suppressor in KB oral cancer cells. The results showed that miR-205 expression was significantly lower in KB oral cancer cells than in human normal oral keratinocytes. Furthermore, the miR-205 over-expressed in KB oral cancer cells increased the cell cytotoxicity and induced apoptosis through the activation of caspase-3/-7. The transfection of miR-205 into KB oral cancer cells strongly induced IL-24, a well known cytokine that acts as a tumor suppressor in a range of tumor tissues. In addition, miR-205 targeted the IL-24 promoter directly to induce gene expression. Overall, miR-205 has significant therapeutic potential to turn on silenced tumor suppressor genes by targeting them with miRNA. Keywords: apoptosis, human oral cancer, interleukin-24, microRNA-205 Less
Areca nut has been proven to be correlated with various pathologic alterations in oral cavity. However, the mechanisms for such cytopathic effects are still elusive due m... More
Areca nut has been proven to be correlated with various pathologic alterations in oral cavity. However, the mechanisms for such cytopathic effects are still elusive due mostly to the limitations of cell culture systems. Here we discovered that areca nut extract (ANE) induced production of autophagosome vacuoles in cells cultured with rich medium but induced pyknosis and ballooning, two morphological alterations frequently observed in betel quid chewers, in cells under a serum-free culture condition. Permeability of the serum-starved cells to propidium iodide (PI) confirmed ANE induced novel necrosis with pyknosis (pyknotic necrosis), providing a possible explanation for inflammatory infiltration in chewers’ mucosa. In these serum-starved cells, ANE strongly induced reactive oxygen species (ROS), which acted as a key switch for the initiation of pyknotic necrosis. Calcium flux was also involved in the morphological alterations. Besides, inhibition of GSK3β by SB216763 significantly exacerbated the pyknotic necrosis either induced by ANE or H2O2 in serum-starved cells, suggesting that GSK3β is a critical regulator for ANE/ROS-mediated pyknotic necrosis. Interestingly, LC3-II transition and PARP cleavage were still detected in the serum-starved cells after ANE treatment, suggesting concurrent activation of apoptotic and autophagic pathways. Finally, insulin could counteract the effect of ANE-induced pyknotic necrosis. Taken together, these data provide a platform for studying ANE-induced cytopathogenesis and the first clinical implication for several pathological alterations, such as ballooning and inflammatory infiltration, in betel quid chewers. Less
Background: Snail is an important transcription factor implicated in several tumor progression and can be induced by reactive oxygen species (ROS). Areca quid chewing is ... More
Background: Snail is an important transcription factor implicated in several tumor progression and can be induced by reactive oxygen species (ROS). Areca quid chewing is a major risk factor of oral squamous cell carcinoma (OSCC). Therefore, we hypothesize that the major areca nut alkaloid arecoline may induce Snail via ROS and involve in the pathogenesis of areca quid chewing-associated OSCC. Less
CELF1 RNA-binding protein, otherwise called CUGBP1, associates and coordinates the degradation of GU-rich element (GRE) containing mRNA's encoding factors important for c... More
CELF1 RNA-binding protein, otherwise called CUGBP1, associates and coordinates the degradation of GU-rich element (GRE) containing mRNA's encoding factors important for cell growth, migration and apoptosis. Although many substrates of CELF1 have been identified, the biological significance of CELF1-mediated mRNA decay remains unclear. As the processes modulated by CELF1 are frequently disrupted in cancer, we investigated the expression and role of CELF1 in oral squamous cancer cells (OSCCs). We determined that CELF1 is reproducibly overexpressed in OSCC tissues and cell lines. Moreover, depletion of CELF1 reduced proliferation and increased apoptosis in OSCCs, but had negligible effect in non-transformed cells. We found that CELF1 associates directly with the 3'UTR of mRNAs encoding the pro-apoptotic factors BAD, BAX and JunD and mediates their rapid decay. Specifically, 3'UTR fragment analysis of JunD revealed that the GRE region is critical for binding with CELF1 and expression of JunD in oral cancer cells. In addition, silencing of CELF1 rendered BAD, BAX and JunD mRNAs stable and increased their protein expression in oral cancer cells. Taken together, these results support a critical role for CELF1 in modulating apoptosis and implicate this RNA-binding protein as a cancer marker and potential therapeutic target. Keywords: CELF1; caspase; mRNA stability; oral cancer; pro-apoptosis; translation. Less
Periodontal diseases are a class of pathologies wherein oral microbes induce harmful immune responses in a susceptible host. Therefore, an agent that can both reduce micr... More
Periodontal diseases are a class of pathologies wherein oral microbes induce harmful immune responses in a susceptible host. Therefore, an agent that can both reduce microbial burden and lessen pathogenesis of localized inflammation would have beneficial effects in periodontal disease; 2,4,4-trichloro-2-hydroxydiphenyl-ether [triclosan] is currently used in oral care products owing to broad spectrum antimicrobial and anti-inflammatory properties.Objective: To determine effects of triclosan on the response of oral epithelial cells to stimulation with the inflammatory microbial product lipopolysaccharide (LPS), a ligand for toll-like receptor 4 [TLR4]. Less
Despite the dismal prognosis for patients with squamous cell carcinoma of the head and neck (SCCHN), there have been no novel treatments in over 40 years. Identification ... More
Despite the dismal prognosis for patients with squamous cell carcinoma of the head and neck (SCCHN), there have been no novel treatments in over 40 years. Identification of novel tumor antigens in SCCHN will facilitate the identification of potential novel treatment targets. Tumor antigens are proteins selectively expressed by tumor cells and recognized by the host immune system. Phage-displayed tumor antigens were enriched by biopanning with normal and then SCCHN-specific serum. Ninety-six phage clones were sequenced for identification, and 21 clones were validated using Luminex. One of these proteins, L23, a novel tumor antigen in SCCHN, was validated as an oncogene. L23 is upregulated in SCCHN compared with normal keratinocytes. Knockdown of L23 inhibited proliferation, invasion and cell survival. Overexpression of L23 had the reverse effect. Overexpression of L23 in non malignant cells led to transformation. Injection of SCCHN cells with knockdown of L23 in mice, induced tumors that were significantly smaller than control tumors. In conclusion, the immunomic screen yielded a panel of antigens specific to SCCHN; one of these proteins, L23, is a novel oncogene in SCCHN. Less
Cisplatin is one of the commonly used chemotherapeutic drugs for the treatment of head and neck squamous cell carcinoma (HNSCC). However, acquisition of cisplatin resista... More
Cisplatin is one of the commonly used chemotherapeutic drugs for the treatment of head and neck squamous cell carcinoma (HNSCC). However, acquisition of cisplatin resistance is common in patients with HNSCC, and it often leads to local and distant failure. In this study, we showed that survivin expression is significantly upregulated in HNSCC primary tumors and cell lines. In addition, survivin levels were significantly higher in human papilloma virus-negative patients that normally respond poorly to cisplatin treatment. Survivin expression was further increased in cisplatin-resistant cells (CAL27-CisR) as compared with its parent cells (CAL27). Therefore, we hypothesized that targeting of survivin in HNSCC could reverse the resistant phenotype in tumor cells, thereby enhancing the therapeutic efficacy of cisplatin. We used both in vitro and in vivo models to test the efficacy of YM155, a small molecule survivin inhibitor, either as a single agent or in combination with cisplatin. YM155 significantly decreased survivin levels and cell proliferation in a dose-dependent manner. In addition, YM155 pretreatment significantly reversed cisplatin resistance in cancer cells. Interestingly, YM155 treatment altered the dynamic localization of survivin in cells by inducing a rapid reduction in cytoplasmic survivin, which plays a critical role in its antiapoptotic function. In a severe combined immunodeficient mouse xenograft model, YM155 significantly enhanced the antitumor and antiangiogenic effects of cisplatin, with no added systemic toxicity. Taken together, our results suggest a potentially novel strategy to use YM155 to overcome the resistance in tumor cells, thereby enhancing the effectiveness of the chemotherapy in HNSCC. Less
Cisplatin is one of the commonly used chemotherapeutic drugs for the treatment of head and neck squamous cell carcinoma (HNSCC). However, acquisition of cisplatin resista... More
Cisplatin is one of the commonly used chemotherapeutic drugs for the treatment of head and neck squamous cell carcinoma (HNSCC). However, acquisition of cisplatin resistance is common in patients with HNSCC, and it often leads to local and distant failure. In this study, we showed that survivin expression is significantly upregulated in HNSCC primary tumors and cell lines. In addition, survivin levels were significantly higher in human papilloma virus-negative patients that normally respond poorly to cisplatin treatment. Survivin expression was further increased in cisplatin-resistant cells (CAL27-CisR) as compared with its parent cells (CAL27). Therefore, we hypothesized that targeting of survivin in HNSCC could reverse the resistant phenotype in tumor cells, thereby enhancing the therapeutic efficacy of cisplatin. We used both in vitro and in vivo models to test the efficacy of YM155, a small molecule survivin inhibitor, either as a single agent or in combination with cisplatin. YM155 significantly decreased survivin levels and cell proliferation in a dose-dependent manner. In addition, YM155 pretreatment significantly reversed cisplatin resistance in cancer cells. Interestingly, YM155 treatment altered the dynamic localization of survivin in cells by inducing a rapid reduction in cytoplasmic survivin, which plays a critical role in its antiapoptotic function. In a severe combined immunodeficient mouse xenograft model, YM155 significantly enhanced the antitumor and antiangiogenic effects of cisplatin, with no added systemic toxicity. Taken together, our results suggest a potentially novel strategy to use YM155 to overcome the resistance in tumor cells, thereby enhancing the effectiveness of the chemotherapy in HNSCC. Less
Background: Tumor-derived cytokines play a significant role in the progression of head and neck squamous cell carcinoma (HNSCC). Targeting proteins, such as tristetraprol... More
Background: Tumor-derived cytokines play a significant role in the progression of head and neck squamous cell carcinoma (HNSCC). Targeting proteins, such as tristetraprolin (TTP), that regulate multiple inflammatory cytokines may inhibit the progression of HNSCC. However, TTP's role in cancer is poorly understood. The goal of the current study was to determine whether TTP regulates inflammatory cytokines in patients with HNSCC. Methods: TTP messenger RNA (mRNA) and protein expression were determined by quantitative real-time-polymerase chain reaction (Q-RT-PCR) and Western blot analysis, respectively. mRNA stability and cytokine secretion were evaluated by quantitative RT-PCR and enzyme-linked immunoadsorbent assay, respectively, after overexpression or knockdown of TTP in HNSCC. HNSCC tissue microarrays were immunostained for interleukin-6 (IL-6) and TTP. Results: TTP expression in HNSCC cell lines was found to be inversely correlated with the secretion of IL-6, vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE(2) )(.) Knockdown of TTP increased mRNA stability and the secretion of cytokines. Conversely, overexpression of TTP in HNSCC cells led to decreased secretion of IL-6, VEGF, and PGE(2) . Immunohistochemical staining of tissue microarrays for IL-6 demonstrated that staining intensity is prognostic for poor disease-specific survival (P = .023), tumor recurrence and development of second primary tumors (P = .014), and poor overall survival (P = .019). Conclusions: The results of the current study demonstrated that down-regulation of TTP in HNSCC enhances mRNA stability and promotes secretion of IL-6, VEGF, and PGE(2) . Furthermore, high IL-6 secretion in HNSCC tissue is a biomarker for poor prognosis. In as much as enhanced cytokine secretion is associated with poor prognosis, TTP may be a therapeutic target to reduce multiple cytokines concurrently in patients with HNSCC. Copyright © 2011 American Cancer Society. Less
Oral squamous cell carcinoma (OSCC) is a major health problem worldwide, and patients have a particularly poor 5-year survival rate. Thus, identification of the molecular... More
Oral squamous cell carcinoma (OSCC) is a major health problem worldwide, and patients have a particularly poor 5-year survival rate. Thus, identification of the molecular targets in OSCC and subsequent innovative therapies are greatly needed. Prolonged exposure to alcohol, tobacco, and pathogenic agents are known risk factors and have suggested that chronic inflammation may represent a potential common denominator in the development of OSCC. Microarray analysis of gene expression in OSCC cell lines with high basal NF-κB activity and OSCC patient samples identified dysregulation of many genes involved in inflammation, wound healing, angiogenesis, and growth regulation. In particular IL-8, CCL5, STAT1, and VEGF gene expression was up-regulated in OSCC. Moreover, IL-8 protein levels were significantly higher in OSCC cell lines as compared with normal human oral keratinocytes. Targeting IL-8 expression by siRNA significantly reduced the survival of OSCC cells, indicating that it plays an important role in OSCC development and/or progression. Inhibiting the inflammatory pathway by aspirin and the proteasome/NF-κB pathway by bortezomib resulted in marked reduction in cell viability in OSCC lines. Taken together our studies indicate a strong link between inflammation and OSCC development and reveal IL-8 as a potential mediator. Treatment based on prevention of general inflammation and/or the NF-κB pathway shows promise in OSCCs. Keywords: Gene Expression, Inflammation, Interleukin, Microarray, NF-κB, Anti-inflammatory, Biomarker, Oral Cancer, Meta-analysis Less
Background and objective: The mechanism behind the survival of periodontal ligament fibroblasts is critical for the maintenance of periodontal ligament tissue. However, t... More
Background and objective: The mechanism behind the survival of periodontal ligament fibroblasts is critical for the maintenance of periodontal ligament tissue. However, the number of known proteins that are involved in this action is limited. The aim of this study was to examine the role of a novel molecule, secreted mammalian Ly-6/urokinase-type plasminogen activator receptor-related protein 1 (SLURP-1), in periodontal ligament fibroblast survival. Material and methods: Human periodontal ligament fibroblasts were isolated from eight healthy human donors using established protocols. Gene expression for SLURP-1 was analysed using the reverse transcriptase-polymerase chain reaction, while protein expression was examined by immunoblotting with a SLURP-1 antibody. In addition, the apoptotic effect was examined using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Results: Messenger RNA for SLURP-1 was expressed in the periodontal ligament, gingival fibroblasts, oral keratinocytes and bone. Moreover, the protein was secreted by both periodontal ligament and gingival fibroblasts. Functional analysis revealed that SLURP-1 substantially enhanced cell survival in periodontal ligament fibroblasts by the anti-apoptotic signal phosphatidylinositol 3-kinase. Conclusion: These findings suggest that SLURP-1 may play an important role in the control and maintenance of the periodontal ligament by protecting the periodontal ligament fibroblasts from apoptosis. Less
The use of smokeless tobacco products is often associated with an oral injury at the site of repeated use. To further our understanding of this injury process, the effect... More
The use of smokeless tobacco products is often associated with an oral injury at the site of repeated use. To further our understanding of this injury process, the effect of reference moist smokeless tobacco extract (STE) on cell death, oxidative stress, and MAPK signaling in a human oral keratinocyte cell line, HOK-16B, was investigated. STE caused dose-dependent cell death and reactive oxygen species (ROS) production within 30 min to 3h of exposure. This same insult enhanced the activity of ERK1/2, JNK1/2, p38 MAPK and ASK1, an upstream activator of JNK1/2 and p38 MAPK. Inhibition of JNK1/2 and to a lesser extent p38 MAPK, but not ERK1/2, suppressed STE-induced cell death. Pretreatment with antioxidants and an iron chelator, deferoxamine suppressed ROS production, ASK1, JNK1/2 and p38 MAPK activation, and reduced cell death after STE exposure. Interestingly, extracellular free iron levels in STE (29.4+/-0.5 microM) were significantly elevated as compared with cell culture medium (4.9+/-0.6 microM) and the addition of extracellular free iron (14, 30 or 70 microM) to HOK-16B cultures (without STE) caused dose-dependent cell death after 3h. Thus, acute exposure to STE leads to HOK-16B cell death in part through oxidative stress via activation of ASK1 and the JNK1/2 and p38 MAPK pathways. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved. Less
Tumor metastasis is the dominant cause of death in cancer patients, including patients with oral tongue squamous cell carcinoma (TSCC). Previously, we reported that reduc... More
Tumor metastasis is the dominant cause of death in cancer patients, including patients with oral tongue squamous cell carcinoma (TSCC). Previously, we reported that reduced miR-138 level is correlated with enhanced metastatic potential in TSCC cells. Here, we demonstrate that miR-138 suppresses TSCC cell migration and invasion by regulating two key genes in the Rho GTPase signaling pathway: RhoC and ROCK2. Direct targeting of miR-138 to specific sequences located in the 3′-untranslated regions of both RhoC and ROCK2 mRNAs were confirmed using luciferase reporter gene assays. Ectopic transfection of miR-138 reduced the expression of both RhoC and ROCK2 in TSCC cells. These reduced expressions, in consequence, led to the reorganization of the stress fibers and the subsequent cell morphology change to a round bleb-like shape, as well as the suppression of cell migration and invasion. In contrast, knockdown of miR-138 in TSCC cells enhanced the expression of RhoC and ROCK2, which resulted in an altered, elongated cell morphology, enhanced cell stress fiber formation, and accelerated cell migration and invasion. Taken together, our results suggest that miR-138 plays an important role in TSCC cell migration and invasion by concurrently targeting RhoC and ROCK2, and miR-138 may serve as a novel therapeutic target for TSCC patients at risk of metastatic disease. Keywords: miR-138, RhoC, ROCK2, migration, invasion Less
The human homolog of the Drosophila headcase (HECA) belongs to a new class of cell differentiation regulators. In Drosophila, the HECA protein regulates the proliferation... More
The human homolog of the Drosophila headcase (HECA) belongs to a new class of cell differentiation regulators. In Drosophila, the HECA protein regulates the proliferation and differentiation of cells during adult morphogenesis. There is growing evidence that HECA plays an important role in human carcinogenesis. In different tumor entities, an altered HECA expression was found (colorectal, pancreatic and renal cancer). Colorectal cancer studies also suggested HECA as a marker for early disease stages. Therefore, we speculated whether human HECA affects cell cycle progression and proliferation in head and neck cancer cells. In vivo, we found a distinct HECA protein expression in basal and superficial cells of a healthy oral epithelium via immunohistochemistry, whereas in tissues of oral squamous cell carcinoma (OSCC), a weaker staining was observed, particularly in basal cells. In vitro, mRNA and protein expression analyses of OSCC cell lines exhibited that HECA expression correlates with the state of cellular differentiation. In further investigations, we overexpressed HECA in the OSCC cell line PCI 13 and performed functional assays. HECA-overexpressing OSCC cells revealed a significant extended doubling time (up to 45%, 17 h) and yielded a lower number of proliferating cells (up to 30%) than controls. Flow cytometry analyses have shown that HECA-overexpressing OSCC cells forced to hold in the G(2)/M-Phase. In summary, our results show that human HECA slows down cell division of OSCC cells and may therefore act as a tumor suppressor in head and neck cancer. Less
MAGE-A-antigens are an immunologic marker for many cancers. The goal of this study was to compare the expression profiles of MAGE-A2, -A3, -A4, -A6 and -A10 in foetal and... More
MAGE-A-antigens are an immunologic marker for many cancers. The goal of this study was to compare the expression profiles of MAGE-A2, -A3, -A4, -A6 and -A10 in foetal and adult keratinocytes with an oral squamous cell carcinoma (OSCC) cell line. Expression of MAGE-A2, -A3, -A4, -A6 and -A10-antigens were detected with PCR in foetal and adult keratinocyte cell lines and in an OSCC cell line (pT4N1M0). Quantitative expression of the single MAGE-A-antigens was measured with rtq-PCR. The results were compared to the reference value of the adult keratinocyte cell line. MAGE-A-antigens were detected in all cell lines. Expression profiles of adult and foetal keratinocyte cell lines differed significantly. Expression profiles of foetal and carcinoma cell lines differed significantly also. MAGE-A-antigens were detected in foetal keratinocyte cell line and oral squamous cell carcinoma cell line but differ in their expression profiles. Up to now MAGE-A-antigens were not detected in foetal keratinocytes. Their role is still unknown. Less
During infection and inflammation, bacterial and inflammatory proteases break down extracellular matrices into macromolecular fragments. Fibronectin fragments are associa... More
During infection and inflammation, bacterial and inflammatory proteases break down extracellular matrices into macromolecular fragments. Fibronectin fragments are associated with disease severity in arthritis and periodontitis. The mechanisms by which these fragments contribute to disease pathogenesis are unclear. One likely mechanism is that fibronectin fragments induce apoptosis of resident cells, which can be further modulated by nitric oxide. Nitric oxide levels are increased at inflammatory sites in periodontitis patients. The aim of this study was to examine whether a proapoptotic fibronectin matrix (AFn) exerts its action by inducing nitric oxide and whether priming by bacterial and inflammatory components exacerbates this mechanism. Our data demonstrate that AFn increased the levels of nitric oxide and inducible nitric oxide synthase (iNOS) dose and time dependently in periodontal ligament (PDL) cells. These effects and apoptosis were inhibited by iNOS suppression and enhanced by iNOS overexpression. Nitric oxide and iNOS induction were paralleled by increased c-Jun N-terminal kinase 1 (JNK-1) phosphorylation. JNK-1 overexpression enhanced the expression of nitric oxide and iNOS, whereas inhibiting JNK-1 by small interfering RNA or a kinase mutant reversed these findings. Priming PDL cells with Porphyromonas gingivalis, its lipopolysaccharide (LPS), or gamma interferon (IFN-gamma) further increased nitric oxide levels and apoptosis. Escherichia coli and Streptococcus mutans induced lesser effects. Gingival fibroblasts and neutrophils responded to a lesser degree to these stimuli, whereas keratinocytes were resistant to apoptosis. Thus, proapoptotic matrices trigger nitric oxide release via JNK-1, promoting further apoptosis in host cells. LPS and IFN-gamma accentuate this mechanism, suggesting that during inflammation, the affected matrices and bacterial and inflammatory components combined exert a greater pathogenic effect on host cells. Less
Th17 cells are a distinct lineage of effector CD4(+) T cells characterized by their production of interleukin (IL)-17. We demonstrate that Th17 cells also expressed IL-22... More
Th17 cells are a distinct lineage of effector CD4(+) T cells characterized by their production of interleukin (IL)-17. We demonstrate that Th17 cells also expressed IL-22, an IL-10 family member, at substantially higher amounts than T helper (Th)1 or Th2 cells. Similar to IL-17A, IL-22 expression was initiated by transforming growth factor beta signaling in the context of IL-6 and other proinflammatory cytokines. The subsequent expansion of IL-22-producing cells was dependent on IL-23. We further demonstrate that IL-22 was coexpressed in vitro and in vivo with both IL-17A and IL-17F. To study a functional relationship among these cytokines, we examined the expression of antimicrobial peptides by primary keratinocytes treated with combinations of IL-22, IL-17A, and IL-17F. IL-22 in conjunction with IL-17A or IL-17F synergistically induced the expression of beta-defensin 2 and S100A9 and additively enhanced the expression of S100A7 and S100A8. Collectively, we have identified IL-22 as a new cytokine expressed by Th17 cells that synergizes with IL-17A or IL-17F to regulate genes associated with skin innate immunity. Less
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