ECT2 exprssion in OSCC-derived cell lines.

<p>(<b>A</b>) Quantification of <i>ECT2</i> mRNA levels in OSCC-derived cell lines by qRT-PCR analysis. To determine mRNA expression of <i>ECT2</i> in Oral cancer, we performed qRT-PCR analysis using six OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, H1, Ca9-22, and Sa3) and HNOKs. Significant up-regulation of <i>ECT2</i> mRNA is seen in six OSCC-derived cell lines compared with that in the HNOKs. Data are expressed as the means ± SEM of values from three assays (*<i>p</i><0.05; Mann-Whitney <i>U</i> test). (<b>B</b>) Western blot analysis of ECT2 protein in the OSCC-derived cell lines and HNOKs. To investigate protein expression of ECT2 in Oral cancer, we performed Western blot analysis using six OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, H1, Ca9-22, and Sa3) and HNOKs. ECT2 protein expression is up-regulated in OSCC-derived cell lines compared with HNOKs. Densitometric ECT2 protein data are normalized to α-tubulin protein levels. The values are expressed as a percentage of the HNOKs.</p

shECT2 promotes G1 arrest.

<p>To investigate cell cycle progression, we analyzed Flow cytometric determination of DNA content by a FACScalibur in the G0–G1, S, and, G2–M phases. We then determined the expression level of cyclin-dependent kinase inhibitors (p16^INK4A, p21^cip1, and p27^kip1), cyclin D1, cyclin E, and CDK4 to identify the mechanism by which ECT2 blocks G1 progression. (<b>A</b>) Flow cytometric analysis was performed to investigate cell cycle in shECT2- and Mock-transfected cells. The number of cells in the G1 has increased markedly in the ECT2 knockdown cells. (<b>B</b>) qRT-PCR was performed to investigate mRNA levels of cell cycle related genes. PCR shows up-regulation of <i>p21^cip1</i> and <i>p27^kip1</i> and down-regulation of <i>cyclin D1</i>, <i>cyclin E</i>, <i>and CDK4</i>. Data are expressed as the means ± SEM of values from three assays (*<i>p</i><0.05; Mann-Whitney <i>U</i> test).</p

Correlation between ECT2 expression and clinical classification in OSCCs.

<p>*<i>p</i><0.05. ECT2(+), ECT2-positive case; ECT2(−), ECT2-negative case.</p

Comparison of <i>ECT2</i> mRNA expression levels between primary OSCCs and matched normal oral tissues.

<p>To investigate the <i>ECT2</i> mRNA expression levels in primary OSCCs and paired normal oral tissues from 96 patients, we performed qRT-PCR analysis. The relative mRNA expression levels in primary OSCCs and the matched oral tissues (n = 96) range from 0.005 to 4.39 (median, 0.289) and 0.003 to 1.632 (median, 0.081), respectively. <i>ECT2</i> mRNA expression was up-regulated in 75 (78%) of 96 primary OSCCs compared with the matched normal oral tissues. Significantly higher <i>ECT2</i> mRNA expression was observed in primary OSCCs than matched normal oral tissues (<i>P</i><0.05; Mann-Whitney <i>U</i> test).</p

Evaluation of ECT2 protein expression in primary OSCCs.

<p>(<b>A</b>, <b>B</b>) Representative IHC results of ECT2 in normal oral tissue and primary OSCC. (<b>A</b>) Normal oral tissue has no ECT2 protein expression. Original magnification, ×100. Scale bars, 50 µm. (<b>B</b>) ECT2-positive cases of OSCC. Positive immunoreaction for ECT2 is detected in the nucleus and cytoplasm. Original magnification, ×400. Scale bars, 10 µm. (<b>C</b>) State of ECT2 protein expression in nomal oral tissue and primary OSCC. To investigate protein expression of ECT2 in primary OSCCs, we carried out IHC. The ECT2 IHC scores are calculated as follows: IHC score = 1×(number of weak stained cells in the field)+2×(number of moderately stained cells in the field)+3×(number of intensely stained cells in the field). The ECT2 IHC scores for OSCCs and normal oral tissues range from 55.67 to 211.33 (median, 163.33) and 8.33 to 85.33 (median, 44.00), respectively. The ECT2 protein expression level in OSCCs is significantly higher than that in normal oral tissues (<i>p</i><0.001; Mann-Whitney <i>U</i> test).</p

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

<div><p>Background</p><p>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).</p><p>Methods</p><p>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.</p><p>Results</p><p>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 (p21^Cip1 and p27^Kip1) 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).</p><p>Conclusion</p><p>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.</p></div

Lysophosphatidylcholine Acyltransferase1 Overexpression Promotes Oral Squamous Cell Carcinoma Progression via Enhanced Biosynthesis of Platelet-Activating Factor

<div><p>Background</p><p>The relevance of lysophosphatidylcholine acyltransferase1 (LPCAT1), a cytosolic enzyme in the remodeling pathway of phosphatidylcholine metabolism, in oral squamous cell carcinoma (OSCC) is unknown. We investigated LPCAT1 expression and its functional mechanism in OSCCs.</p><p>Methods</p><p>We analyzed LPCAT1 mRNA and protein expression levels in OSCC-derived cell lines. Immunohistochemistry was performed to identify correlations between LPCAT1 expression levels and primary OSCCs clinicopathological status. We established LPCAT1 knockdown models of the OSCC-derived cell lines (SAS, Ca9-22) for functional analysis and examined the association between LPCAT1 expression and the platelet-activating factor (PAF) concentration and PAF-receptor (PAFR) expression.</p><p>Results</p><p>LPCAT1 mRNA and protein were up-regulated significantly (p<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemistry showed significantly (p<0.05) elevated LPCAT1 expression in primary OSCCs compared with normal counterparts and a strong correlation between LPCAT1-positive OSCCs and tumoral size and regional lymph node metastasis. In LPCAT1 knockdown cells, cellular proliferation and invasiveness decreased significantly (p<0.05); cellular migration was inhibited compared with control cells. Down-regulation of LPCAT1 resulted in a decreased intercellular PAF concentration and PAFR expression.</p><p>Conclusion</p><p>LPCAT1 was overexpressed in OSCCs and correlated with cellular invasiveness and migration. LPCAT1 may contribute to tumoral growth and metastasis in oral cancer.</p></div

Cell-cycle analysis of SEMA7A knockdown cells.

<p>(<b>A</b>) Flow cytometric analysis was performed to investigate cell-cycle progression in the shSEMA7A and shMock cells (SAS and KOSC-2-derived transfectants) after synchronization at the G2/M phase to using nocodazole. The percentage of cells at the G1 phase in the shSEMA7A cells is increased markedly compared with the shMock cells. (<b>B</b>) Immunobloting analysis shows up-regulation of p21^Cip1 and p27^Kip1 and down-regulation of cyclin D1, cyclin E, CDK2, CDK4, and CDK6 in the shSEMA7Acells (SAS and KOSC-2-derived transfectants) compared with the shMock cells.</p

Evaluation of SEMA7A expression in OSCC-derived cell lines.

<p>(<b>A</b>) Quantification of <i>SEMA7A</i> mRNA expression in OSCC-derived cell lines by qRT-PCR analysis. Significant (^✽P<0.05, Student’s t-test) up-regulation of <i>SEMA7A</i> mRNA is seen in seven OSCC-derived cell lines compared with the HNOKs. Data are expressed as the mean ± SEM of triplicate results. (<b>B</b>) Immunoblotting analysis of SEMA7A protein in OSCC-derived cell lines and HNOKs. SEMA7A protein expression is up-regulated in OSCC-derived cell lines compared with that in the HNOKs. Densitometric SEMA7A protein data are normalized to the GAPDH protein levels. The values are expressed as a percentage of the HNOKs.</p

Inactivation of the ERK1/2 and AKT pathways in SEMA7A knockdown cells.

<p>(<b>A, B</b>) Immunoblotting analysis shows that SEMA7A knockdown results in decreased levels of pERK1/2 and pAKT compared with the shMock cells (SAS and KOSC-2-derived transfectants). Densitometric pERK1/2, ERK1/2, pAKT, and AKT protein data are normalized to GAPDH protein levels.</p