Assay for transposase-accessible chromatin with high-throughput sequencing reveals radioresistance-related genes in oral squamous cell carcinoma cells.

Radiotherapy is commonly used to treat oral squamous cell carcinoma (OSCC), and radioresistance is a critical factor resulting in poor outcomes. Several genes have been reported to be therapeutic targets for radioresistance; however, the involvement of chromatin accessibility in radioresistance has not been clarified in OSCC cells. Accordingly, in this study, we evaluated chromatin accessibility in radioresistant (HSC-3) and radiosensitive (KOSC-2) cells, identified from nine OSCC cell lines using clonogenic survival assays after irradiation. Chromatin accessibility in radioresistant OSCC cells was assessed using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Gene expression was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunoblot analysis. Viability was assessed by MTS assay. We found 1273 peaks (open chromatin regions by ATAC-seq) related to 8 Gy irradiation in HSC-3 but not KOSC-2 cells, among which 235 genes located around the chromatin open peaks were identified by ChIPpeakAnno analysis. Subsequently, 12 genes were selected as signal transduction-related genes by Gene Ontology analysis, and gene expression was confirmed by RT-qPCR. Among these genes, adenylate cyclase 2 (ADCY2) was significantly upregulated after treatment with irradiation in HSC-3 but not KOSC-2 cells. To further evaluate ADCY2 function in radioresistant cells, we performed ADCY2 knockdown by transfection of HSC-3 cells with small interfering RNA (siADCY2). Cell viability after irradiation was significantly decreased in siADCY2-transfected cells compared with that in control cells. These results suggested that ADCY2 expression was related to the open chromatin region in radioresistant OSCC cells and that ADCY2 may have therapeutic efficacy when used in combination with radiotherapy in patients with OSCC.

Aberrant GIMAP2 expression affects oral squamous cell carcinoma progression by promoting cell cycle and inhibiting apoptosis.

GTPases of immunity-associated protein 2 (GIMAP2) is a GTPase family member associated with T cell survival. However, its mechanisms of action in oral squamous cell carcinoma (OSCC) remain largely unknown. Therefore, the present study aimed to elucidate the possible role of GIMAP2 in OSCC development by investigating its expression levels and molecular mechanisms in OSCC. Reverse transcription quantitative PCR, immunoblotting and immunohistochemistry indicated that GIMAP2 expression was significantly upregulated (P<0.05) in OSCC-derived cell lines and primary OSCC specimens compared with that in their normal counterparts. GIMAP2-knockdown OSCC cells exhibited decreased cell growth, which was associated with cyclin-dependent kinase (CDK)4, CDK6 and phosphorylated Rb downregulation and p53 and p21 upregulation. In addition to cell cycle arrest, GIMAP2 affected anti-apoptotic functions in GIMAP2-knockdown cells by upregulating Bcl-2 and downregulating Bax and Bak. These findings indicated that GIMAP2 may significantly influence OSCC development and apoptosis inhibition and thus is a potential biomarker of OSCC.

Ginkgolide B Regulates CDDP Chemoresistance in Oral Cancer via the Platelet-Activating Factor Receptor Pathway.

The platelet-activating factor receptor (PAFR) is a key molecule that participates in intracellular signaling pathways, including regulating the activation of kinases. It is involved in cancer progression, but the detailed mechanism of its chemosensitivity is unknown. The purpose of the current study was to elucidate the mechanism regulating cisplatin (CDDP) sensitivity through PAFR functions in oral squamous cell carcinoma (OSCC). We first analyzed the correlation between PAFR expression and CDDP sensitivity in seven OSCC-derived cell lines based upon cell viability assays. Among them, we isolated 2 CDDP-resistant cell lines (Ca9-22 and Ho-1-N-1). In addition to conducting PAFR-knockdown (siPAFR) experiments, we found that ginkgolide B (GB), a specific inhibitor of PAFR, enhanced both CDDP chemosusceptibility and apoptosis. We next evaluated the downstream signaling pathway of PAFR in siPAFR-treated cells and GB-treated cells after CDDP treatment. In both cases, we observed decreased phosphorylation of ERK and Akt and increased expression of cleaved caspase-3. These results suggest that PAFR is a therapeutic target for modulating CDDP sensitivity in OSCC cells. Thus, GB may be a novel drug that could enhance combination chemotherapy with CDDP for OSCC patients.

Tumor Suppressive Circular RNA-102450: Development of a Novel Diagnostic Procedure for Lymph Node Metastasis from Oral Cancer.

Circular RNAs (circRNAs), which form as covalently closed loop structures, have several biological functions such as regulation of cellular behavior by adsorbing microRNAs. However, there is limited information of circRNAs in oral squamous cell carcinoma (OSCC). Here, we aimed to elucidate the roles of aberrantly expressed circRNAs in OSCC. CircRNA microarray showed that circRNA-102450 was down-regulated in OSCC cells. Clinical validation of circRNA-102450 was performed using highly sensitive droplet digital PCR in preoperative liquid biopsy samples from 30 OSCC patients. Interestingly, none of 16 studied patients with high circRNA-102450 had regional lymph node metastasis (RLNM), whereas 4 of 14 studied patients (28.5%) with low expression had pathologically proven RLNM. Overexpressed circRNA-102450 significantly inhibited the tumor metastatic properties of cell proliferation, migration, and invasion. Furthermore, circRNA-102450 directly bound to, and consequently down-regulated, miR-1178 in OSCC cells. Taken together, circRNA-102450 has a tumor suppressive effect via the circRNA-102450/miR-1178 axis and may be a novel potential marker of RLNM in OSCC patients.

Engineered exosomes delivering specific tumor-suppressive RNAi attenuate oral cancer progression.

Exosomes are involved in a wide range of biological processes in human cells. Considerable evidence suggests that engineered exosomes (eExosomes) containing therapeutic agents can attenuate the oncogenic activity of human cancer cells. Despite its biomedical relevance, no information has been available for oral squamous cell carcinoma (OSCC), and therefore the development of specific OSCC-targeting eExosomes (octExosomes) is urgently needed. We demonstrated that exosomes from normal fibroblasts transfected with Epstein-Barr Virus Induced-3 (EBI3) cDNA were electroporated with siRNA of lymphocyte cytoplasmic protein 1 (LCP1), as octExosomes, and a series of experiments were performed to evaluate the loading specificity/effectiveness and their anti-oral cancer cell activities after administration of octExosomes. These experiments revealed that octExosomes were stable, effective for transferring siLCP1 into OSCC cells and LCP1 was downregulated in OSCC cells with octExosomes as compared with their counterparts, leading to a significant tumor-suppressive effect in vitro and in vivo. Here we report the development of a new valuable tool for inhibiting tumor cells. By engineering exosomes, siLCP1 was transferred to specifically suppress oncogenic activity of OSCC cells. Inhibition of other types of human malignant cells merits further study.

Tspan15 plays a crucial role in metastasis in oral squamous cell carcinoma.

Tetraspanin 15 (Tspan15) is a member of the tetraspanin family, which is associated with various biological events and several diseases, however, its role in human oral squamous cell carcinoma (OSCC) remains unknown. The current study aimed to clarify the role of Tspan15 in OSCC. The mRNA and protein expression levels of Tspan15 were up-regulated in OSCC cases and OSCC-derived cell lines. Significant up-regulated Tspan15 expression was found in the advanced OSCC cases; primary tumoral size (P = 0.042), regional lymph node metastasis (P = 0.036) and TNM classification (P = 0.024). The decreased expression of Tspan15 did not significantly affect cellular proliferation, whereas tumoral invasion and migration activities were suppressed in Tspan15-down-regulated cells, suggesting that Tspan15 might activate metastasis-related signaling. Moreover, in the Tspan15-down-regulated cells, the expression of a disintegrin and metalloproteinase (ADAM) 10 was also down-regulated and the cells secreted less soluble N-cadherin compared with control cells. And weak immunoreactivity of ß-catenin in the nucleus was detected in Tspan15-down-regulated cells compared with the control cells. These findings suggested that overexpression of Tspan15 positively regulates development of OSCC, and that ADAM10, N-cadherin, ß-catenin might be involved in the Tspan15-mediated pathway. These unusual conditions of cell adhesion molecules may lead to high metastasis rate found in Tspan15-overexpressing cases.

Resveratrol Targets Urokinase-Type Plasminogen Activator Receptor Expression to Overcome Cetuximab-Resistance in Oral Squamous Cell Carcinoma.

Drug resistance to anti-cancer agents is a major concern regarding the successful treatment of malignant tumors. Recent studies have suggested that acquired resistance to anti-epidermal growth factor receptor (EGFR) therapies such as cetuximab are in part caused by genetic alterations in patients with oral squamous cell carcinoma (OSCC). However, the molecular mechanisms employed by other complementary pathways that govern resistance remain unclear. In the current study, we performed gene expression profiling combined with extensive molecular validation to explore alternative mechanisms driving cetuximab-resistance in OSCC cells. Among the genes identified, we discovered that a urokinase-type plasminogen activator receptor (uPAR)/integrin ß1/Src/FAK signal circuit converges to regulate ERK1/2 phosphorylation and this pathway drives cetuximab-resistance in the absence of EGFR overexpression or acquired EGFR activating mutations. Notably, the polyphenolic phytoalexin resveratrol, inhibited uPAR expression and consequently the signaling molecules ERK1/2 downstream of EGFR thus revealing additive effects on promoting OSCC cetuximab-sensitivity in vitro and in vivo. The current findings indicate that uPAR expression plays a critical role in acquired cetuximab resistance of OSCC and that combination therapy with resveratrol may provide an attractive means for treating these patients.

Centromere Protein N Participates in Cellular Proliferation of Human Oral Cancer by Cell-Cycle Enhancement.

Centromere protein N (CENP-N), an important member of the centromere protein family, is essential for kinetochore assembly and chromosome segregation; however, the relevance of CENP-N in cancers remains unknown. The aim of this study was to investigate CENP-N expression and its functional mechanisms in oral squamous cell carcinoma (OSCC). CENP-N expression was up-regulated significantly in vitro and in vivo in OSCCs. Overexpressed CENP-N was closely (p < 0.05) correlated with tumor growth using quantitative reverse transcriptase-polymerase chain reaction, immunoblot analysis, and immunohistochemistry. CENP-N knockdown (shCENP-N) cells showed depressed cellular proliferation by cell-cycle arrest at the G1 phase with up-regulation of p21Cip1 and p27Kip1 and down-regulation of cyclin D1, CDK2, and CDK4. Interestingly, we newly discovered that calcitriol (1, 25-dihydroxyvitamin D3) controlled the CENP-N expression level, leading to inhibition of tumor growth similar to shCENP-N cells. These results suggested that CENP-N plays a critical role in determining proliferation of OSCCs and that calcitriol might be a novel therapeutic drug for OSCCs by regulating CENP-N.

UNC93B1 promotes tumoral growth by controlling the secretion level of granulocyte macrophage colony-stimulating factor in human oral cancer.

Unc-93 homolog B1 (UNC93B1), a transmembrane protein, is correlated with immune diseases, such as influenza, herpes simplex encephalitis, and the pathogenesis of systemic lupus erythematosus; however, the role of UNC93B1 in cancers including human oral squamous cell carcinomas (OSCCs) remains unknown. In the current study, we investigated the UNC93B1expression level in OSCCs using quantitative reverse transcription-polymerase chain reaction, immunoblot analysis, and immunohistochemistry. Our data showed that UNC93B1 mRNA and protein expressions increased markedly (p < 0.05) in OSCCs compared with normal cells and tissues and that high expression of UNC93B1 in OSCCs was related closely to tumoral size. UNC93B1 knockdown (shUNC93B1) OSCC cells showed decreased cellular proliferation by cell-cycle arrest in the G1 phase with up-regulation of p21Cip1 and down-regulation of CDK4, CDK6, cyclin D1, and cyclin E. We also found that granulocyte macrophage colony-stimulating factor (GM-CSF) was down-regulated significantly (p < 0.05) in shUNC93B1 OSCC cells. Moreover, inactivation of GM-CSF using neutralization antibody led to cell-cycle arrest at the G1 phase similar to the phenotype of the shUNC93B1 cells. The current findings indicated that UNC93B1 might play a crucial role in OSCC by controlling the secretion level of GM-CSF involved in tumoral growth and could be a potential therapeutic target for OSCCs.

Critical role of deoxynucleotidyl transferase terminal interacting protein 1 in oral cancer.

Deoxynucleotidyl transferase terminal interacting protein 1 (DNTTIP1) forms a complex with histone deacetylase (HDAC); however, the relevance of DNTTIP1 in cancer remains unknown. The aim of this study was to examine DNTTIP1 expression and its functional mechanisms in oral squamous cell carcinomas (OSCCs). DNTTIP1 expression was analyzed by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting analysis, and immunohistochemistry. The expression of DNTTIP1 was upregulated significantly in vitro and in vivo, and in patients with OSCC in whom DNTTIP1 was overexpressed and the expression level was correlated significantly (P < 0.05) with tumoral growth. DNTTIP1 knockdown (siDNTTIP1) cells showed depressed cellular proliferation by cell-cycle arrest at the G1 phase with high acetylation of p53 and upregulation of p21Cip1. Moreover, resveratrol, a HDAC inhibitor, controlled not only acetylated p53 status but also DNTTIP1 expression, leading to a similar phenotype of siDNTTIP1 cells. A marked (P < 0.05) reduction of tumoral growth in mouse xenograft models was observed with lower DNTTIP1 expression under the presence of this chemical reagent. Taken together, our results suggested that DNTTIP1-HDAC interaction promotes tumoral growth through deacetylation of p53 and that DNTTIP1 might be a critical therapeutic target in OSCCs.

Multiple coagulation factor deficiency protein 2 as a crucial component in metastasis of human oral cancer.

Multiple coagulation factor deficiency protein 2 (MCFD2), a binding partner of lectin mannose binding 1 (LMAN1), causes combined deficiencies of coagulation factors V and VIII. MCFD2 function in inherited hematologic disorders is well elucidated; however, little is known about its role in human tumorigenesis. The aim of the current study was to investigate the states of MCFD2 in oral squamous cell carcinoma (OSCC). The expression of MCFD2 was up-regulated significantly in all cell lines examined. Evaluation of the cellular functions associated with tumoral metastasis showed that MCFD2 knockdown (shMCFD2) cells exhibited significantly lower cellular invasiveness and migration and higher cellular adhesion compared with shControl cells. Of note, shMCFD2 cells also showed weak immunoreactivity of LMAN1 and a lower secretion level of galactoside-binding soluble 3 binding protein (LGALS3BP). In addition to in vitro validation, clinical data on 70 patients with OSCC indicated that state of MCFD2 expression level is associated with regional lymph node metastasis. Altogether, we have demonstrated that MCFD2 promotes cancer metastasis by regulating LMAN1 and LGALS3BP expression levels. Hence, MCFD2 may represent a promising candidate for a novel therapeutic target for patients with metastatic OSCCs.

UBE2S associated with OSCC proliferation by promotion of P21 degradation via the ubiquitin-proteasome system.

Ubiquitin-conjugating enzyme E2S (UBE2S), a family of E2 protein in the ubiquitin-proteasome system, is highly expressed in several types of cancers; however, its roles in oral squamous cell carcinoma (OSCC) have not yet been well elucidated. The purpose of this study was to clarify the functional activities of UBE2S in OSCCs. We analyzed the expression levels of UBE2S in nine OSCC cell lines and primary OSCC tissues by quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry (IHC). The correlations between UBE2S expression and clinical classifications of OSCCs were analyzed using the IHC scoring system. We also used UBE2S knockdown OSCC cells for functional assays (proliferation assay, flow cytometry, and Western blotting). UBE2S was overexpressed in OSCCs in vitro and in vivo and was correlated significantly (P < 0.05) with the primary tumoral size. The cellular growth was decreased and the cell-cycle was arrested in the G2/M phase in the UBE2S knockdown (shUBE2S) cells. The expression level of P21, a target of the ubiquitin-proteasome system, was increased in the shUBE2S cells because of lower anaphase activity that promotes complex subunit 3 (APC3), an E3 ubiquitin ligase, compared with shMock cells. These findings might promote the understanding of the relationship between UBE2S overexpression and oral cancer proliferation, indicating that UBE2S would be a potential biomarker of and therapeutic target in OSCCs.

TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase.

TEA domain transcription factor 4 (TEAD4), which has critical functions in the process of embryonic development, is expressed in various cancers. However, the important role of TEAD4 in human oral squamous cell carcinomas (OSCCs) remain unclear. Here we investigated the TEAD4 expression level and the functional mechanism in OSCC using quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. Furthermore, TEAD4 knockdown model was used to evaluate cellular proliferation, cell-cycle analysis, and the interaction between TEAD4 and Yes-associated protein (YAP) which was reported to be a transcription coactivator of cellular proliferation. In the current study, we found that TEAD4 expression increased significantly in vitro and in vivo and correlated with tumoral size in OSCC patients. TEAD4 knockdown OSCC cells showed decreased cellular proliferation resulting from cell-cycle arrest in the G1 phase by down-regulation of cyclins, cyclin-dependent kinases (CDKs), and up-regulation of CDK inhibitors. We also found that the TEAD4-YAP complex in the nuclei may be related closely to transcriptions of G1 arrest-related genes. Taken together, we concluded that TEAD4 might play an important role in tumoral growth and have potential to be a therapeutic target in OSCCs.

SIPA1 promotes invasion and migration in human oral squamous cell carcinoma by ITGB1 and MMP7.

Signal-induced proliferation-associated protein 1 (SIPA1) is known to be a GTPase activating protein. Overexpressed SIPA1 is related to metastatic progression in breast and prostate cancers; however, the relevance of SIPA1 in oral squamous cell carcinoma (OSCC) is still unknown. The aim of this study was to examine SIPA1 expression and its functional mechanisms in OSCC. SIPA1 mRNA and protein expressions were analyzed by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. The expressions of SIPA1 were up-regulated significantly in vitro and in vivo. Moreover, SIPA1 expression was correlated with regional lymph node metastasis. We next assessed the cellular functions associated with tumoral metastasis using SIPA1 knockdown (shSIPA1) cells and analyzed the downstream molecules of SIPA1, i.e., bromodomain containing protein 4(BRD4), integrin beta1 (ITGB1), and matrix metalloproteinase 7 (MMP7). The shSIPA1 cells showed decreased invasiveness and migratory activities, however cellular adhesion ability was maintained at a high level. In addition, ITGB1 expression was greater in shSIPA1 cells, whereas MMP7 expression was lower than in control cells. This research is the first to establish that SIPA1 promotes cancer metastasis by regulating the ITGB1 and MMP7. Therefore, SIPA1 might be a novel therapeutic target for patients with lymph node metastasis of OSCC.

Novel mechanism of aberrant ZIP4 expression with zinc supplementation in oral tumorigenesis.

Zrt-Irt-like protein 4 (ZIP4) is critical molecule for proper mammalian development and releasing zinc from vesicular compartments. Recent studies suggested that ZIP4 plays an important role of tumor progression in pancreatic, prostate, and hepatocellular cancers, however, little is known about the detail mechanism of ZIP4 in their cancers. In the present study, we examined the possibility of ZIP4 as a new molecular target for oral squamous cell carcinoma (OSCC). We evaluated ZIP4 expression in OSCC-derived cell lines and primary OSCC samples by quantitative RT-PCR, immunoblotting, and immunohistochemistry (IHC). We also analyzed the clinical correlation between ZIP4 status and clinical behaviors in patients with OSCC. In addition, ZIP4 knockdown cells (shZIP4 cells) and ZnCl2 treatment were used for functional experiments, including cellular proliferation assay, zinc uptake assay, and cell-cycle analysis. ZIP4 mRNA and protein were up-regulated significantly in OSCCs compared with normal counterparts in vitro and in vivo. IHC showed that ZIP4 expression in the primary OSCC was positively correlated with primary tumoral size. The shZIP4 cells showed decrease accumulation of intercellular zinc and decreased cellular growth by cell-cycle arrest at the G1 phase, resulting from up-regulation of cyclin-dependent kinase inhibitors and down-regulation of cyclins and cyclin-dependent kinases. Since cellular growth of OSCC cells after treatment with zinc was significantly greater than control cells, we speculated that intercellular ZnCl2 accumulation is an important factor for cellular growth. Consistent with our hypothesis, not only decreased zinc uptake by ZIP4 knockdown but also chelating agent, N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), showed inhibitory effects of cellular proliferation. Therefore, our data provide evidence for an essential role of ZIP4 and intracellular zinc for tumoral growth in OSCC, suggesting that zinc uptake might be a potential therapeutic targeting event for OSCCs.

Semaphorin7A Promotion of Tumoral Growth and Metastasis in Human Oral Cancer by Regulation of G1 Cell Cycle and Matrix Metalloproteases: Possible Contribution to Tumoral Angiogenesis.

BACKGROUND: Semaphorins (SEMAs) consist of a large family of secreted and membrane-anchored proteins that are important in neuronal pathfinding and axon guidance in selected areas of the developing nervous system. Of them, SEMA7A has been reported to have a chemotactic activity in neurogenesis and to be an immunomodulator; however, little is known about the relevance of SEMA7A in the behaviors of oral squamous cell carcinoma (OSCC). METHODS: We evaluated SEMA7A expression in OSCC-derived cell lines and primary OSCC samples using quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and semiquantitative immunohistochemistry (sq-IHC). In addition, SEMA7A knockdown cells (shSEMA7A cells) were used for functional experiments, including cellular proliferation, invasiveness, and migration assays. We also analyzed the clinical correlation between SEMA7A status and clinical behaviors in patients with OSCC. RESULTS: SEMA7A mRNA and protein were up-regulated significantly (P<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. The shSEMA7A cells showed decreased cellular growth by cell-cycle arrest at the G1 phase, resulting from up-regulation of cyclin-dependent kinase inhibitors (p21Cip1 and p27Kip1) and down-regulation of cyclins (cyclin D1, cyclin E) and cyclin-dependent kinases (CDK2, CDK4, and CDK6); and decreased invasiveness and migration activities by reduced secretion of matrix metalloproteases (MMPs) (MMP-2, proMMP-2, pro-MMP-9), and expression of membrane type 1- MMP (MT1-MMP). We also found inactivation of the extracellular regulated kinase 1/2 and AKT pathways, an upstream molecule of cell-cycle arrest at the G1 phase, and reduced secretion of MMPs in shSEMA7A cells. sq-IHC showed that SEMA7A expression in the primary OSCCs was significantly (P = 0.001) greater than that in normal counterparts and was correlated with primary tumoral size (P = 0.0254) and regional lymph node metastasis (P = 0.0002). CONCLUSION: Our data provide evidence for an essential role of SEMA7A in tumoral growth and metastasis in OSCC and indicated that SEMA7A may play a potential diagnostic/therapeutic target for use in patients with OSCC.

Targeting phosphodiesterase 3B enhances cisplatin sensitivity in human cancer cells.

We previously reported that human squamous cell carcinoma (SCC) cell lines refractory to cis-diaminedichloro-platinum II (cisplatin [CDDP]) had significant upregulation of the phosphodiesterase 3B gene (PDE3B), suggesting that inhibiting PDE3B suppresses CDDP resistance. shRNA-mediated PDE3B depletion in CDDP-resistant cells derived from SCC cells and Hela cells and induced CDDP sensitivity and inhibited tumor growth with elevated cyclic GMP induction resulting in upregulation of the multidrug-resistant molecule, but this did not occur in the 5-fluorouracil-resistant hepatocellular carcinoma cell lines. Furthermore, the antitumor growth effect of the combination of a PDE3B inhibitor (cilostazol) and CDDP in vivo was also greater than with either cilostazol or CDDP alone, with a significant increase in the number of apoptotic and cell growth-suppressive cancer cells in CDDP-resistance cell lines. Our results provided novel information on which to base further mechanistic studies of CDDP sensitization by inhibiting PDE3B in human cancer cells and for developing strategies to improve outcomes with concurrent chemotherapy.

High prevalence of epigenetic inactivation of the human four and a half LIM domains 1 gene in human oral cancer.

The four and a half LIM domains 1 (FHL1) gene has been related to carcinogenesis. However, the expression status of FHL1 in human oral squamous cell carcinoma (OSCC) remains unclear and the detailed mechanism of gene silencing is poorly understood. The aim of this study was to examine the FHL1 expression level and its regulatory mechanism in OSCCs. Quantitative reverse-transcriptase-polymerase chain reaction (PCR) and western blotting showed significant downregulation of FHL1 in all OSCC-derived cell lines (Sa3, HSC-2, HSC-3, HSC-4, HO-1-u-1, HO-1-N-1, KON and Ca9-22) compared to human normal oral keratinocytes. We also found that FHL1 mRNA expression was frequently downregulated (P<0.01) in 51 (86.4%) of 59 primary OSCCs compared with the corresponding normal oral tissues, while there was no significant difference between the status of the FHL1 protein expression in OSCCs and the clinicopathological features. Using methylation-specific PCR, we detected methylated FHL1 in all cell lines and treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine restored the FHL1 expression. However, no significant restoration of FHL1 expression was observed using sodium butyrate, an inhibitor of histone deacetylase and chromatin immunoprecipitation showed that histone H3 lysine 9 in the FHL1 promoter region was significantly acetylated. In addition, no mutation in the entire coding region of the FHL1 gene was found. Therefore, our data suggested that inactivation of the FHL1 gene is a frequent event during oral carcinogenesis and that the mechanism of FHL1 downregulation in OSCCs is through DNA methylation of the promoter region rather than histone deacetylation or mutation.

Gibberellic acid induces α-amylase expression in adipose-derived stem cells.

Salivary α-amylase is the most important enzyme for oral digestion of dietary starch. Therefore, regeneration of the salivary glands via a tissue engineering approach is clearly required for patients with salivary gland dysfunction. Early during seed germination, the embryo synthesizes gibberellic acid (GA3), a plant hormone that activates the synthesis and secretion of α-amylase. The purpose of this study was to explore an approach for differentiation of stem cells into salivary glands using GA3. We isolated adipose-derived stem cells (ASCs), which are positive for mesenchymal stem cell markers (CD73, CD90 and CD105) and possess pluripotency to osteoblasts, adipocytes and neural cells, from human buccal fat pads, which are a readily available source for dentists and oral surgeons. In addition, we investigated the cytotoxicity of GA3 for human ASCs. GA3 neither affects cell morphology nor cell viability in a dose- or time-dependent manner. ASCs were incubated with GA3 to assess mRNA and protein expression of α-amylase by reverse transcriptase-polymerase chain reaction and western blot analyses. α-amylase mRNA expression on 21 days after treatment with GA3 (1 mM) was seven times greater than that in resting condition (Day 0). While we did not detect α-amylase bands on Day 0, α-amylase protein was detectable 7 days after treatment with GA3, reaching a maximal level on Day 21. Our results indicated that GA3 can increase cellular α-amylase expression and that our induction method would be useful for therapeutic application for salivary gland regeneration.

State of heat shock factor 1 expression as a putative diagnostic marker for oral squamous cell carcinoma.

Heat shock factor 1 (HSF1) is responsible for expres-- sion of a large class of heat shock proteins that have been implicated in the malignant phenotype of human cancers. Little is known about the effect of a high level of HSF1 on the behavior of oral squamous cell carcinoma (OSCC). In this study, we assessed the value of HSF1 for predicting clinical outcomes in OSCC. Quantitative reverse transcriptase-polymerase chain reaction and Western blotting showed that the expressions of HSF1 mRNA and protein in OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, Sa3, Ca9-22, KON and Ho-1-u-1) were elevated compared with those in human normal oral keratinocytes (P<0.05). Similar to in vitro data, HSF1 mRNA expression in primary OSCCs (n=50) was significantly greater than in normal counterparts (P<0.05). Since HSF1 was observed in the nucleus and cytoplasm by immu-- nohistochemistry, we investigated the correlation between the HSF1 expression status at each subcellular location and the clinical behavior of OSCCs. Among the clinical classifications, higher nuclear HSF1 expression was closely related to tumor size and histopathologic types (P<0.05). These results showed for the first time that nuclear HSF1 expression may contribute to cancer progression and that HSF1 might be a potential diagnostic biomarker and a therapeutic target for OSCCs.