The effects of dysregulation of SPHK1 expression on epirubicin induced-apoptosis <i>in vivo</i>.

<p>(A) Tumor volumes were measured on the indicated days. Left panel, indicated cells (2×10⁶) were injected in the flank of nude mice. When the mean tumor volume reached approximately 50 mm³, the mice were injected i.p. with epirubicin (5 mg/kg), every 3 days, up to 15 days. Right panel, U87MG-vector cells (2×10⁶) were injected in the flank of nude mice. When the mean tumor volume reached approximately 50 mm³, the mice were randomized into two groups (n = 5) and injected intraperitoneally either with epirubicin (Epi, 5 mg/kg) or epirubicin (Epi, 5 mg/kg) plus SK1-I (50 mg/kg) every 3 days, up to 15days. (B) Mean tumor weights were measured. Immunofluorescent images (C) and quantification (D) of TUNEL positive cells. For (B) and (D), error bars represent mean ± SD from three independent experiments with similar results *, <i>p</i><0.05.</p

The effect of SPHK1 on Akt/FOXO3a/Bim pathway can be inhibited by PI3K inhibitor or SPHK1 inhibitor.

<p>U87MG-SPHK1 and LN-382-SPHK1 cells were incubated with 50 µM LY294002 (A) or 5 µM SK-I (B) for 24 h and cell lysates were harvested and subjected to WB. phospho-Akt (p-Akt), total Akt, p-FOXO3a, total FOXO3a and Bim were detected with specific antibodies, respectively.</p

FOXO3a phosphorylation, transcriptional activity and Akt phosphorylation are regulated by SPHK1 in glioma cells.

<p>(A) SPHK1 phosphorylates FOXO3a in glioma cells. (B) FOXO3a-dependent transcription activity is regulated by overexpression (left panel) or knockdown (right panel) of SPHK1. Error bars represent mean ± SD from three independent experiments with similar results. *, <i>p</i><0.05. The GFP expression was used to indicate the transfection efficiency. (C) WB analysis of nuclear FOXO3a protein in indicated cells. (D) SPHK1 knockdown-induced upregulation of Bim could be reversed by silencing of FOXO3a in indicated glioma cells. (E) The phosphorylation status of Akt at Thr³⁰⁸ and Ser⁴⁷³ were assessed by WB in SPHK1 overexpressed and knocked-down glioma cell lines.</p

Bmi-1 promotes an angiogenesis phenotype in glioma cells via activation of the NF-κB.

<p>Bmi-1-overexpressing glioma cells were treated with a NF-κB inhibitor, JSH-23. (A) Representative images (left) and quantification (right) of HUVEC formed tube-like structures on Matrigel-coated plates with CM derived from indicated cultured cells. (B) Quantification of migrated HUVEC cells treated with indicated conditioned medium analyzed in a Transwell migration assay. (C) Real-time PCR quantification of <i>VEGF-C</i> mRNA expression levels in DMSO (control) and JSH-23 treated cells. Levels of mRNA expression are presented as fold increase relative to vector-control cells and normalized to GAPDH. (D) ELISA for VEGF-C protein expression in cell supernatants. Error bars represent the mean ± SD of three independent experiments; ** <i>P</i><0.01.</p

Glioma cells overexpressing SPHK1 protein are less sensitive to UV- or adriamycin-induced apoptosis.

<p>(A) Overexpression of SPHK1 in glioma cell lines analyzed by WB. α-tubulin was used as a loading control. (B) SPHK1 enzymatic activity in SPHK1-overexpressed glioma cells was markedly increased. (C) Overexpression of SPHK1 inhibited cell death induced by adriamycin (left panel) or UV irradiation (right panel). Seventy-two hours after treatment with adriamycin or UV irradiation, cell viability was assessed by the trypan blue exclusion method. (D) SPHK1 prevented adriamycin-induced apoptosis of glioma cells. Annexin-V binding and TUNEL assays of indicated cells were performed after incubation with adriamycin for 6 h and 24 h, respectively. Quantification of TUNEL positive cells (left panel) and Annexin V⁺/PI⁻ cells (right panel). Results are expressed as percentages of total cells. (E) PARP cleavage and cleaved caspase 3 levels were assessed in indicated cells treated with UV irradiation (40 J/m²) or adriamycin (1.0 µM) for 24 h via WB. α-tubulin was used as a loading control. For (B), (C) and (D), error bars represent mean ± SD from three independent experiments with similar results. *, <i>p</i><0.05.</p

Downregulation of SPHK1 expression decreases the resistance of glioma cells to apoptosis.

<p>(A) SPHK1 knockdown was achieved by introducing specific shRNAs in glioma cells. α-tubulin was used as a loading control. (B) Silencing of SPHK1 led to significant decrease of enzymatic activity in glioma cells. (C) Knockdown of SPHK1 enhances cell death. (D) SPHK1 knockdown enhanced the sensitivity of glioma cells to adriamycin-induced apoptosis. Quantification of TUNEL positive cells (left panel) and Annexin V⁺/PI⁻ cells (right panel). Results are expressed as percentages of total cells. (E) PARP cleavage and cleaved caspase 3 levels were assessed in indicated cell lines treated with UV irradiation (40 J/m²) or adriamycin (1.0 µM) for 24 h via WB. α-tubulin was used as a loading control. For (B), (C) and (D), error bars represent mean ± SD from three independent experiments with similar results. *, <i>p</i><0.05.</p

The expression of Bim is correlated with SPHK1 <i>in vitro</i> and <i>in vivo</i>.

<p>(A) The expression of BimEL protein level was significantly decreased in SPHK1 overexpression cells (left panel) and increased in SPHK1 knocked-down cells (right panel). (B) Overexpression of SPHK1 significantly decreased mRNA level of Bim (left panel), while knockdown of SPHK1 increased transcriptional level of Bim (right panel). (C) Examples of Bim expression in correlation with SPHK1 in human primary glioma specimens. Left panel, IHC staining of SPHK1 and Bim in glioma. Right panel, correlation of Bim and SPHK1 (n = 82; <i>p</i> = 0.021). Each data point represents one glioma specimen, and 24 of 33 (72.7%) glioma specimens with high SPHK1 expression displayed low expression of Bim, whereas 34 of 49 (69.4%) glioma specimens that showed low SPHK1 expression exhibited high expression of Bim. 0, no staining; 1, low staining; 2, moderate staining; 3, high staining.</p

Bmi-1 Promotes Glioma Angiogenesis by Activating NF-κB Signaling

<div><p>Angiogenesis in glioma is associated with the poor prognosis of the disease and closely correlates with the highly invasive phenotype of glioma cells, which represents the most challenging impediment against the currently glioma treatments. Bmi-1, an onco-protein, has been implicated in the progression of various human cancers, including gliomas, whereas its role in glioma angiogenesis remains unclear. Our current study examined the effects of Bmi-1 on glioma angiogenesis in vitro as well as in vivo. We found that overexpression of Bmi-1 enhanced, whereas knockdown of Bmi-1 diminished, the capability of glioma cells to induce tubule formation and migration of endothelial cells and neovascularization in chicken chorioallantoic membrane. <em>In vivo</em>, Bmi-1 overexpression and knockdown, respectively, promoted and inhibited angiogenesis in orthotopically transplanted human gliomas. Furthermore, NF-κB activity and VEGF-C expression was induced by Bmi-1 overexpression, whereas Bmi-1 knockdown attenuated NF-κB signaling and decreased VEGF-C expression. Additionally suppression of NF-κB activity using a specific chemical inhibitor abrogated the NF-κB activation and the pro-angiogenic activities of glioma cells. Together, our data suggest that Bmi-1 plays an important role in glioma angiogenesis and therefore could represent a potential target for anti-angiogenic therapy against the disease.</p> </div

MACC1 as a Prognostic Biomarker for Early-Stage and AFP-Normal Hepatocellular Carcinoma

<div><p>Background</p><p>The metastasis-associated in colon cancer 1 gene (MACC1) has been found to be associated with cancer development and progression. The aim of this study was to investigate the prognostic value of MACC1 in early-stage and AFP-normal hepatocellular carcinoma (HCC).</p><p>Methods</p><p>mRNA and protein levels of MACC1 expression in one normal liver epithelial cells THLE3 and 15 HCC cell lines were examined using reverse transcription-PCR and Western blot. MACC1 expression was also comparatively studied in 6 paired HCC lesions and the adjacent non-cancerous tissue samples. Immunohistochemistry was employed to analyze MACC1 expression in 308 clinicopathologically characterized HCC cases. Statistical analyses were applied to derive association between MACC1 expression scores and clinical staging as well as patient survival.</p><p>Results</p><p>Levels of MACC1 mRNA and protein were higher in HCC cell lines and HCC lesions than in normal liver epithelial cells and the paired adjacent noncancerous tissues. Significant difference in MACC1 expression was found in patients of different TNM stages (<i>P</i><0.001). Overall survival analysis showed that high MACC1 expression level correlated with lower survival rate (<i>P</i> = 0.001). Importantly, an inverse correlation between MACC1 level and patient survival remained significant in subjects with early-stage HCC or with normal serum AFP level.</p><p>Conclusions</p><p>MACC1 protein may represent a promising biomarker for predicting the prognosis of HCC, including in early-stage and AFP-normal patients.</p></div

Bmi-1 induces NF-κB transcriptional activity.

<p>(A) Luciferase reporter assay of NF-κB transcriptional activity in Bmi-1 overexpressing (Bmi-1) and Bmi-1 silenced glioma cells (Bmi-1-RNAi), compared to the vector control cells respectively. (B) Real-time PCR analysis of NF-κB-regulated gene expression in vector-control, Bmi-1 overexpressing and Bmi-1-silenced glioma cells; GAPDH was used as the control gene. (C) Left, schematic illustration of luciferase reporter gene construction using cloned fragments of a human <i>VEGF-C</i> promoter. Right, transactivation activity of luciferase reporter genes driven by <i>VEGF-C</i> promoter fragments (as indicated on the left) in vector-control, Bmi-1 overexpressing and Bmi-1 silenced glioma cells. Luciferase activity was normalized to <i>Renilla</i> luciferase activity. Vector: pMSCV-vector, Vector-RNAi: pSuper-retro-puro-vector. Error bars represent the mean ± SD of three independent experiments; ** <i>P</i><0.01.</p