Blockage of glioma cell survival by truncated TEAD-binding domain of YAP.

BACKGROUND: Gliomas are highly aggressive and lack of efficient targeted therapy. YAP, as a Hippo pathway downstream effector, plays a key role in promoting tumor development through the interaction with transcription factor TEAD on the NH3-terminal proline-rich domain. Therefore, targeting TEAD-interacting domain of YAP may provide a novel approach for the treatment of gliomas. MATERIALS AND METHODS: We generated a truncated YAP protein which includes the TEAD-binding domain (YAPBD), and supposed YAPBD can interact with endogenous TEAD but lost the function to activate YAP target gene expressions. The association of YAP expression with the malignant characters of glioma tissues were determined by immunohistochemistry. TEAD-binding capacity of YAPBD was determined by co-immunoprecipitation. The cell proliferation and migration were determined by MTT assay, xenograft assay, wound healing assay and transwell assay, respectively. YAP target genes were detected by Western blot. RESULTS: YAP was highly expressed in glioma tissues and associated with tumor malignancy. YAPBD could block the TEAD-YAP complex formation by competing with YAP binding to TEAD. YAPBD could inhibit glioma cell growth both in vitro and in vivo, through the induction of cell cycle arrest and apoptosis. The cell cycle-related gene cyclin D1 and c-myc, and anti-apoptotic gene Bcl-2, Bcl-xL and survivin were inhibited after YAPBD overexpression. Furthermore, YAPBD also decreased cell migration and invasion, and repressed epithelial-mesenchymal transition. CONCLUSION: YAPBD can block glioma cell survival and repress YAP-dependent gene expressions, indicating gene therapy which targets TEAD-YAP complex would be a potential and significant novel approach for human malignant gliomas.

Truncated TEAD-binding protein of TAZ inhibits glioma survival through the induction of apoptosis and repression of epithelial-mesenchymal transition.

Transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo pathway downstream effector, promotes tumor progression by serving as a transcriptional coactivator with TEAD. Here, we introduced a new construct which can express the TEAD-binding domain of TAZ protein (TAZBD), and determined its antitumor effect in malignant glioma both in vitro and in vivo. We first observed that TAZ was upregulated in glioma tissues and related to malignant clinicopathologic characteristic, indicating the crucial role of TAZ during glioma progression. In U87 and U251 cells, TAZBD expression increased the proportion of apoptotic cells, and suppressed the colony formation and tumorigenicity. Further, TAZBD also decreased cell metastasis through the repression of epithelial-mesenchymal transition. The mechanistic study showed that TAZBD suppression of glioma cells was predominantly through blocking the TAZ-TEAD complex formation by competing with endogenous TAZ. Thus, the gene therapy of malignant glioma through blocking TAZ-TEAD complex by TAZBD may provide a new way for the targeted therapy of glioma.

Investigation of the Shear Thickening Fluid Encapsulation in an Orifice Coagulation Bath.

The orifice coagulation bath method is proposed to encapsulate shear thickening fluid (STF) to form STF capsules, in an attempt to improve the combination of STF and the matrix as well as strengthen the flexibility and stability of the STF composites. By varying the calcium chloride concentration (10, 20 mg/mL), sodium alginate concentration (5, 7, 10 mg/mL) and the surfactant dosage (10%, 20%, 30%), optimal preparation conditions were studied, considering the capsule strength and encapsulation rate. The capsules were also characterized using a scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR) and a thermogravimetric analyzer (TGA). The results show that the optimal solution for the preparation of the capsules is composed of 30% surfactant, 10 mg/mL mass concentration of CaCl2, and 10 mg/mL mass concentration of sodium alginate. The rough surface and porous interior was observed by SEM. The average diameter of the capsules was 1.93 mm. The TGA curves indicate an improvement on the capsule thermal stability. This study thus provides a promising STF capsule preparation method.

Inhibition of eIF4F complex loading inhibits the survival of malignant glioma.

The eukaryotic initiation factor (eIF)4E­binding proteins (4E­BPs) regulate cap­dependent protein translation and control the assembly of the eIF4F complex. In the present study, a phosphorylation­deficient truncated 4E­BP2 (eIF4FD) was constructed into the eukaryotic expression vector pSecTag2, and the in vitro and in vivo effects on malignant glioma survival were determined through inhibiting eIF4F complex assembly. Cell cycle distribution analysis and TUNEL staining show that overexpression of eIF4FD suppressed cell proliferation and induced apoptosis in U251 cells. Western blotting showed that the cell cycle­related genes cyclin D1 and C­myc, and anti­apoptotic genes B­cell lymphoma 2 (Bcl­2), Bcl­extra large and survivin were reduced following the overexpression of eIF4FD. Furthermore, eIF4FD suppressed glioma vascularization via reductions in the expression of ß­catenin and vascular endothelial growth factor. In the orthotopic xenograft model, the stable expression of eIF4FD in U251 cells attenuated cell growth and increased the rate of apoptosis. Accordingly, pSecTag2­PTD­eIF4FD injection via the tail vein of mice also lead to cell growth inhibition and the induction of apoptosis. Therefore, the study showed that phosphorylation­deficient truncated 4E­BP2 efficiently inhibited eIF4E and prevented the formation of the eIF4F complex, which further contributed to the inhibition of cell proliferation and vascularization, and the induction of apoptosis. Therefore, the 4E­BP2­based phosphorylation­deficient truncation designed in the present study may represent a novel approach for the targeted therapy of human malignant glioma though inhibition of the translation initiation complex.

The cap-translation inhibitor 4EGI-1 induces mitochondrial dysfunction via regulation of mitochondrial dynamic proteins in human glioma U251 cells.

Translation initiation factors (eIFs) are over-activated in many human cancers and may contribute to their progression. The small molecule 4EGI-1, a potent inhibitor of translation initiation through disrupting eIF4E/eIF4G interaction, has been shown to exert anti-cancer effects in human cancer cells. The goal of the present study was to evaluate the anti-cancer effects of 4EGI-1 in human glioma U251 cells. We found that 4EGI-1 impaired the assembly of the eIF4F complex, and inhibited proliferation of U251 cells via inducing apoptosis. 4EGI-1 treatment induced collapse of mitochondrial membrane potential (MMP) and production of intracellular reactive oxygen species (ROS), which were prevented by the ROS scavenger N-acetyl-cysteine (NAC). In addition, 4EGI-1 inhibited mitochondrial ATP synthesis via suppressing complex I activity, but had no effects on mitochondrial biogenesis. The results of fluorescence staining showed that 4EGI-1 indeed fragmented the mitochondrial network of U251 cells. We found a significant decrease in optic atrophy type 1 (Opa-1) and mitofusin 1 (Mfn-1) related to fusion proteins as well as an increase in fission protein dynamin-related protein 1 (Drp-1). Furthermore, the anti-cancer effects of 4GI-1 were partially nullified by knock down of Drp-1 using siRNA. These data indicate that the use of inhibitors that directly target the translation initiation complex eIF4F could represent a potential novel approach for human glioma therapy.

RASSF7 expression and its regulatory roles on apoptosis in human intervertebral disc degeneration.

Apoptosis plays an important role in intervertebral disc degeneration (IDD). Overwhelming evidence indicates that RASSF7 is essential for cell growth and apoptosis. Recently, it has been noted that the JNK signaling can be negatively regulated by suppressing phosphorylated-MKK7 activation during pro-apoptosis. We aimed to investigate the RASSF7 expression level in human degenerative nucleus pulposus (NP) cells and non-degenerative NP cells and the link between RASSF7-JNK with NP cells apoptosis. We harvested NP tissues from 20 IDD patients as disease group and 8 cadaveric donors as normal controls. We detected RASSF7 expression by Real-time-PCR and western blotting. Consequently, we found that the expression of RASSF7 was higher in non-degenerative group than in degenerative group (P<0.05). Overexpression of RASSF7 in degenerative NP cells led to decreased apoptosis rate than that in scramble group (P<0.05). Collectively, our findings suggest that RASSF7 plays an important role in human IDD and RASSF7 might be potentially developed as a curative agent.

[An epidemiological study on HIV sexual transmission in married spouse in Yunnan province].

OBJECTIVE: To understand the status of HIV sexual transmission among HIV-sero-discordant spouses and HIV-sero-accordant spouses in Yunnan province, to discuss the related factors and to provide evidence for HIV prevention and control strategy. METHODS: Five places with serious epidemic and 3 moderate ones were voluntarily, randomly selected. According to time sequence, 300 spouses (600 people) with stable marriage were interviewed with questionnaire. RESULTS: HIV-sero-accordant spouses occupied for 40.7% of the total spouses under survey, with the others were HIV-sero-discordant ones. Among the ones that had already been diagnosed in the families, sexual transmission was their main mode of transmission, which was accounted for 68.3%, followed by IDU as 19.7%. After disclosed the HIV test outcomes to their spouses, 63.4% HIV-sero-discordant spouses and 47.0% HIV-sero-accordant ones changed their sexual behaviors. The rates of consistent condom use among the HIV-sero-discordant spouses increased from 16.8% to 95.0%, and in HIV-sero-accordant spouses increased from 8.2% to 60.9%. Data were analyzed by multi-factor logistic regression. Factors on influencing the sexual transmission in spouses would include condom use, frequency of sexual contacts and sexual transmission disease (STD) status etc. CONCLUSION: The main transmission mode for the first HIV infected spouse was sexual transmission. Factors influencing sexual transmission in spouses would include condom use, frequency of sexual contacts, STD situation and husband was the first one being infected in the families, etc. Disclosure of the HIV results to the spouses could make a significant changes in the frequencies of sexual contact as well as the rate of condom use.

Overexpression of ZNF217 in glioblastoma contributes to the maintenance of glioma stem cells regulated by hypoxia-inducible factors.

Glioblastoma multiforme (GBM) is the most aggressive and common kind of primary brain tumor in adults, and is thought to be driven by a subpopulation of glioma stem cells (GSCs). GSCs reside in a specialized hypoxic niche, which can regulate the tumorigenic capacity of GSCs primarily through the hypoxia-inducible factors (HIFs), HIF1α and HIF2α. ZNF217 is an oncogene frequently amplified in many kinds of tumors. It is associated with aggressive tumor behavior and poor clinical prognosis, but its role in gliomas is poorly known. Gene expression and copy number analysis from TCGA data reveal that ZNF217 is amplified in 32% and overexpressed in 71.2% of GBMs. Quantitative RT-PCR and western blotting of a cohort of glioma samples showed that ZNF217 was highly expressed in gliomas and increased with tumor grade. Analysis of a molecular database demonstrated that ZNF217 expression correlated with poor survival of glioma patients. Investigation of ZNF217 expression in GSCs, non-GSCs and normal neural stem cells (NSCs) indicated that ZNF217 was more highly expressed in GSCs than in non-GSCs and NSCs. Knockdown of ZNF217 in GSCs by small-interfering RNA (siRNA) inhibited their growth and promoted their differentiation. Interestingly, ZNF217 was upregulated in GSCs and the GBM cell line U87 when exposed to the hypoxic environment of 1% oxygen. Knockdown of either HIF1α or HIF2α, which has a central role in the hypoxia-induced responses of these cells, inhibited ZNF217 expression. In addition, ZNF217 upregulation was compromised under hypoxia in U87 and GSCs when either HIF1α or HIF2α was targeted by siRNA. HIF2α knockdown inhibited ZNF217 expression more efficiently in both normoxia and hypoxia than HIF1α knockdown. Therefore, ZNF217 is overexpressed in GBMs and contributes to the maintenance of GSCs, which is regulated by HIFs released by the hypoxic environment of the tumor.

The validation of the standard Chinese version of the European Organization for Research and Treatment of Cancer Quality of Life Core Questionnaire 30 (EORTC QLQ-C30) in pre-operative patients with brain tumor in China.

BACKGROUND: Health related quality of life (HRQOL) has increasingly emphasized on cancer patients. The psychometric properties of the standard Chinese version of the European Organization for Research and Treatment of Cancer Quality of Life Core Questionnaire 30 (EORTC QLQ-C30, version 3.0) in brain tumor patients wasn't proven, and there was no baseline HRQOL in brain tumor patients prior to surgery. METHODS: The questionnaire EORTC QLQ-C30 (version 3.0) was administered at three time points: T1, the first or the second day that patients were hospitalized after the brain tumor suspected or diagnosed by MRI or CT; T2, 1 to 2 days after T1, (T1 and T2 were both before surgery); T3, the day before discharge. Clinical variables included disease histologic types, cognitive function, and Karnofsky Performance Status. RESULTS: Cronbach's alpha coefficients for multi-item scales were greater than .70 and multitrait scaling analysis showed that most of the item-scale correlation coefficients met the standards of convergent and discriminant validity, except for the cognitive functioning scale. All scales and items exhibited construct validity. Score changes over peri-operation were observed in physical and role functioning scales. Compared with mixed cancer patients assessed after surgery but before adjuvant treatment, brain tumor patients assessed pre-surgery presented better function and fewer symptoms. CONCLUSIONS: The standard Chinese version of the EORTC QLQ-C30 was overall a valid instrument to assess HRQOL in brain tumor patients in China. The baseline HRQOL in brain tumor patients pre-surgery was better than that in mixed cancer patients post-surgery. Future study should modify cognitive functioning scale and examine test-retest reliability and response validity.

Quantitative detection of multiple gene promoter hypermethylation in tumor tissue, serum, and cerebrospinal fluid predicts prognosis of malignant gliomas.

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the malignant transformation in gliomas. We hypothesized that quantitative analysis of methylated genes will provide prognostic values in malignant glioma patients. We used an immunocapturing approach followed by real-time polymerase chain reaction analysis to detect altered patterns of promoter methylation in O-6-methylguanine-DNA methyltransferase (MGMT), p16INK4a, tissue inhibitor of metalloproteinase-3 (TIMP-3), and thrombospondin 1 (THBS1). The tumor tissue and paired serum as well as cerebrospinal fluid (CSF) from 66 patients with malignant gliomas were studied. Serum and CSF from 20 age-matched noncancer individuals were used as control. Promoter hypermethylation in MGMT, p16INK4a, TIMP-3, and THBS1 was detected at high frequencies in tumor tissue, serum, and CSF. None of the control serum or CSF showed aberrant methylation. Hypermethylation in serum and CSF DNA was all accompanied with methylation in the corresponding tumor tissues with 100% specificity. Highly elevated MGMT, p16INK4a, and THBS1 methylation levels in gliomas serum were the sole independent factors predicting inferior overall survival in this cohort. For progression-free survival, hypermethylation of MGMT and THBS1 in CSF were the independent prognostic factors. Multiple gene promoter hypermethylation analysis appears to be promising as a prognostic factor in glioma and as a mini-invasive tumor marker in serum and/or CSF DNA. Evaluation of these changes may help in selecting glioma patients for optimal adjuvant treatments and modifying chemotherapy.