Downregulation of LRIG2 expression inhibits angiogenesis of glioma via EGFR/VEGF-A pathway.

Active angiogenesis is the basic pathological feature of glioma. Tumor angiogenesis is involved in vascular endothelial cell migration to the tumor tissue and in the formation of tube-like structures. The present study aimed to investigate the role of leucine-rich repeats and immunoglobulin-like domains 2 (LRIG2) in glioma angiogenesis. Glioma (n=50) and normal brain (n=20) tissue samples were collected from patients to detect the expression of LRIG2, epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGF-A), and cluster of differentiation 31 (CD31) using immunohistochemistry. In addition, the association between the expression of LRIG2 in glioma tissue and the microvessel density (MVD) was analyzed. In vitro, the expression of LRIG2 in human glioma U87 and U251 cell lines was knocked down. Subsequently, cell migration and tube formation assays of human umbilical vein endothelial cells (HUVECs) were performed using a coculture system. The protein expression levels of LRIG2, EGFR, phosphorylated-EGFR and VEGF-A were determined using western blotting. The results demonstrated that the expression levels of LRIG2, EGFR, VEGF-A and CD31 were highly upregulated in glioma tissue samples. Furthermore, LRIG2 expression in glioma tissue samples was significantly correlated with the MVD. In vitro, the downregulation of LRIG2 inhibited HUVEC migration and tube formation induced by coculture with glioma cells. The downregulation of LRIG2 resulted in decreased expression of EGFR and VEGF-A. The effects of the LRIG2 knockdown were reversed following EGF treatment. These findings suggest that LRIG2 is a potential target for the inhibition of glioma angiogenesis, which is possibly mediated via the EGFR/VEGF-A signaling pathway.

Hedyotis diffusa Willd extract inhibits the growth of human glioblastoma cells by inducing mitochondrial apoptosis via AKT/ERK pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Hedyotis diffusa Willd (Rubiaceae) (HDW) has been widely applied for the treatment of tumors, inflammation and toxication in traditional Chinese medicine. The antitumor effect of HDW on glioblastoma has been rarely reported. We aim to evaluate the activity of this extract and explore the underlying mechanism in U87 human glioblastoma cell line. MATERIALS AND METHODS: Cytotoxicity of HDW extract on U87 cells was measured by MTT assay. Apoptosis, cell cycle arrest and mitochondrial membrane potential (MMP) collapse induced by HDW extract were determined by flow cytometry. Caspase activity was analyzed based on colorimetric assay with a microplate spectrophotometer. Protein expression was examined by Western blot. RESULTS: HDW extract suppressed U87 cells growth in a dose- and time-dependent manner. Flow cytometry showed that HDW extract induced significant apoptosis, S/G2-M phase arrest and MMP collapse in U87 cells. Furthermore, dose-dependent activation of caspase-3, Bcl-2, Bax and ERK was observed with HDW extract treatment. Decreased Bcl-2/Bax ratio and Akt suppression were readily found as well. CONCLUSIONS: Induction of mitochondria-mediated apoptosis played an essential role in antitumor activity of HDW extract in U87 cells, in which ERKs and Akt signaling proteins were also involved. These findings contributed to the feasibility of using HDW extract in glioblastoma treatment and the understanding of the molecular mechanism.

[Differentiation treatment by all-trans retinoic acid reduces phenotype of glioma stem cells].

OBJECTIVE: To explore the effects of all-trans retinoic acid (ATRA) on glioma stem cell phenotype. METHODS: The glioma stem cell (GSC) from surgically resected human glioma specimens were isolated and enriched by neurosphere assay and then its differentiation was induced with all-trans retinoic acid (ATRA, 1 µmol/L) for 1 week. Markers were determined by flow cytometry, Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Side population cells were analyzed by flow cytometry. Growth characteristics were detected by neurosphere formation assay and cell cycle analysis. GSC and the differentiated cells (1×10(5)) were implanted stereotactically and intracranially into the Balb/c nude mice to compare the survival time. All data were analyzed with the SPSS software version 17.0. RESULTS: ATRA potently induced the differentiation of GSC and reduced glioma stem cell phenotype. And there were an elevated expression of glial fibrillary acidic protein (GFAP) and a reduced expression of such stem cell makers as CD133 and Nestin. The side population rate decreased. ATRA inhibited the neurosphere formation of GSC and induced the arrest of cell growth. ATRA could prolong the survival time. CONCLUSION: GSC may be differentiated efficiently by ATRA. The phenotype of GSC decreases obviously after the differentiation of ATRA and the survival time is prolonged. Thus ATRA may be applied for targeted therapies of glioma stem cell.

Comparative study on the stem cell phenotypes of C6 cells under different culture conditions.

BACKGROUND: Glioma stem cell (GSC) hypothesis posits that a subpopulation of cells within gliomas have true clonogenic and tumorigenic potential. Significantly, a more controversial correlate to GSC is that cells in different culture conditions might display distinct stem cell properties. Considering these possibilities, we applied an approach comparing stem cell characteristics of C6 glioma cells under different culture conditions. METHODS: C6 cells were cultured under three different growth conditions, i.e., adherent growth in conventional 10% serum medium, non-adherent spheres growth in serum-free medium, as well as adherent growth on laminin-coated flask in serum-free medium. Growth characteristics were detected contrastively through neurosphere formation assay and cell cycle analysis. Markers were determined by immunofluorescence, relative-quantitative reverse transcription (RT)-PCR, Western blotting and flow cytometry. Side population cells were analyzed via flow cytometry. Tumor models were detected by magnetic resonance imaging and hematoxylin & eosin staining. Data analyses were performed with SPSS software (17.0). RESULTS: C6 cells (C6-Adh, C6-SC-Sph and C6-SC-Adh) showed distinctive growth patterns and proliferation capacity. Compared to suspending C6-SC-Sph, adherent C6-Adh and C6-SC-Adh displayed higher growth ratio. C6-SC-Sph and C6-SC-Adh showed enhanced capability of neurosphere formation and self-renewal. High side population ratio was detected in C6-SC-Sph and C6-SC-Adh. CD133 was not detected in all three kinds of cells. Conversely, Nestin and ß-III-tubulin were demonstrated positive, nonetheless with no statistical significance (P > 0.05). Interestingly, lower expression of glial fibrillary acidic protein was demonstrated in C6-SC-Sph and C6-SC-Adh. C6-Adh, C6-SC-Sph and C6-SC-Adh were all displayed in situ oncogenicity, while statistical difference of survival time was not confirmed. CONCLUSIONS: C6 glioma cell line is endowed with some GSC phenotypes that can be moderately enriched in vitro when transferred into stem cell culture condition. The resultant tumor-spheres may be not a prerequisite or sound source of GSCs and adherent culture in stem cell medium is not a growth condition in favor of GSCs expanding in vivo.

Effect of RNAi-mediated LRIG3 gene silencing on proliferation of glioma GL15 cells and expression of PCNA and Ki-67.

BACKGROUND AND OBJECTIVE: Leucine-rich repeats and immunoglobin-like domains 3 (LRIG3), a member of LRIG gene family, is down-regulated in various human cancers, but its functions are still unclear. This study was to explore the effect of RNA interference (RNAi)-mediated LRIG3 gene silencing on the proliferation of glioma GL15 cells and the expression of proliferating cell nuclear antigen (PCNA) and Ki-67, and investigate possible mechanisms. METHODS: The plasmids pGenesil2-LRIG3-shRNA1 and pGenesil2-LRIG3-shRNA2 which containing U6 promoter and LRIG3-specific short hairpin RNA (shRNA) and the plasmid pGenesil2-negative-shRNA containing unspecific shRNA were transfected into GL15 cells. Stable cell clones were selected by G418. The mRNA and protein levels of LRIG3 were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation was detected by MTT assay. The expression of PCNA and Ki-67 in GL15 cells were examined by SABC immunohistochemistry. RESULTS: Compared with those in control cells, the mRNA levels of LRIG3 transcripts were reduced by 52.4% and 63.8% in shRNA1- and shRNA2-transfected cells, respectively; its protein levels were reduced by 50.9% and 67.4%, respectively. Cell proliferation was enhanced by LRIG3 shRNA transfection. The positive rate of PCNA was significantly higher in shRNA1- and shRNA2-transfected cells than in control cells [(72.13 +/- 5.64)% and (81.93 +/- 5.23)% vs. (35.40 +/- 5.69)%, p < 0.01]. The positive rate of Ki-67 was also significantly higher in shRNA1- and shRNA2-transfected cells than in control cells [(82.27 +/- 5.50)% and (88.67 +/- 3.52)% vs. (49.73 +/- 5.73)%, p < 0.01]. PCNA expression was positively correlated to Ki-67 expression (r =0.932, p < 0.001). CONCLUSION: Down-regulating LRIG3 gene expression can improve the proliferation of glioma GL15 cells.