Inhibition of Elongation Factor-2 Kinase Augments the Antitumor Activity of Temozolomide against Glioma

Background

Glioblastoma multiforme (GBM), the most common form of brain cancer with an average survival of less than 12 months, is a highly aggressive and fatal disease characterized by survival of glioma cells following initial treatment, invasion through the brain parenchyma and destruction of normal brain tissues, and ultimately resistance to current treatments. Temozolomide (TMZ) is commonly used chemotherapy for treatment of primary and recurrent high-grade gliomas. Nevertheless, the therapeutic outcome of TMZ is often unsatisfactory. In this study, we sought to determine whether eEF-2 kinase affected the sensitivity of glioma cells to treatment with TMZ.

Methodology/Principal Findings

Using RNA interference approach, a small molecule inhibitor of eEF-2 kinase, and in vitro and in vivo glioma models, we observed that inhibition of eEF-2 kinase could enhance sensitivity of glioma cells to TMZ, and that this sensitizing effect was associated with blockade of autophagy and augmentation of apoptosis caused by TMZ.

Conclusions/Significance

These findings demonstrated that targeting eEF-2 kinase can enhance the anti-glioma activity of TMZ, and inhibitors of this kinase may be exploited as chemo-sensitizers for TMZ in treatment of malignant glioma.     

3-Deazaneplanocin A (DZNep), an Inhibitor of the Histone Methyltransferase EZH2, Induces Apoptosis and Reduces Cell Migration in Chondrosarcoma Cells

Objective

Growing evidences indicate that the histone methyltransferase EZH2 (enhancer of zeste homolog 2) may be an appropriate therapeutic target in some tumors. Indeed, a high expression of EZH2 is correlated with poor prognosis and metastasis in many cancers. In addition, 3-Deazaneplanocin A (DZNep), an S-adenosyl-L homocysteine hydrolase inhibitor which induces EZH2 protein depletion, leads to cell death in several cancers and tumors. The aim of this study was to determine whether an epigenetic therapy targeting EZH2 with DZNep may be also efficient to treat chondrosarcomas.

Methods

EZH2 expression was determined by immunohistochemistry and western-blot. Chondrosarcoma cell line CH2879 was cultured in the presence of DZNep, and its growth and survival were evaluated by counting adherent cells periodically. Apoptosis was assayed by cell cycle analysis, Apo2.7 expression using flow cytometry, and by PARP cleavage using western-blot. Cell migration was assessed by wound healing assay.

Results

Chondrosarcomas (at least with high grade) highly express EZH2, at contrary to enchondromas or chondrocytes. In vitro, DZNep inhibits EZH2 protein expression, and subsequently reduces the trimethylation of lysine 27 on histone H3 (H3K27me3). Interestingly, DZNep induces cell death of chondrosarcoma cell lines by apoptosis, while it slightly reduces growth of normal chondrocytes. In addition, DZNep reduces cell migration.

Conclusion

These results indicate that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach to treat chondrosarcomas.     

SAMSN1 Is Highly Expressed and Associated with a Poor Survival in Glioblastoma Multiforme

Objectives

To study the expression pattern and prognostic significance of SAMSN1 in glioma.

Methods

Affymetrix and Arrystar gene microarray data in the setting of glioma was analyzed to preliminarily study the expression pattern of SAMSN1 in glioma tissues, and Hieratical clustering of gene microarray data was performed to filter out genes that have prognostic value in malignant glioma. Survival analysis by Kaplan-Meier estimates stratified by SAMSN1 expression was then made based on the data of more than 500 GBM cases provided by The Cancer Genome Atlas (TCGA) project. At last, we detected the expression of SAMSN1 in large numbers of glioma and normal brain tissue samples using Tissue Microarray (TMA). Survival analysis by Kaplan-Meier estimates in each grade of glioma was stratified by SAMSN1 expression. Multivariate survival analysis was made by Cox proportional hazards regression models in corresponding groups of glioma.

Results

With the expression data of SAMSN1 and 68 other genes, high-grade glioma could be classified into two groups with clearly different prognoses. Gene and large sample tissue microarrays showed high expression of SAMSN1 in glioma particularly in GBM. Survival analysis based on the TCGA GBM data matrix and TMA multi-grade glioma dataset found that SAMSN1 expression was closely related to the prognosis of GBM, either PFS or OS (P<0.05). Multivariate survival analysis with Cox proportional hazards regression models confirmed that high expression of SAMSN1 was a strong risk factor for PFS and OS of GBM patients.

Conclusion

SAMSN1 is over-expressed in glioma as compared with that found in normal brains, especially in GBM. High expression of SAMSN1 is a significant risk factor for the progression free and overall survival of GBM.     

H-rev107 regulates prostaglandin D2 synthase-mediated suppression of cellular invasion in testicular cancer cells

Background

H-rev107 is a member of the HREV107 type II tumor suppressor gene family which includes H-REV107, RIG1, and HRASLS. H-REV107 has been shown to express at high levels in differentiated tissues of post-meiotic testicular germ cells. Prostaglandin D2 (PGD2) is conjectured to induce SRY-related high-mobility group box 9 (SOX9) expression and subsequent Sertoli cell differentiation. To date, the function of H-rev107 in differentiated testicular cells has not been well defined.

Results

In the study, we found that H-rev107 was co-localized with prostaglandin D2 synthase (PTGDS) and enhanced the activity of PTGDS, resulting in increase of PGD2 production in testis cells. Furthermore, when H-rev107 was expressed in human NT2/D1 testicular cancer cells, cell migration and invasion were inhibited. Also, silencing of PTGDS would reduce H-rev107-mediated increase in PGD2, cAMP, and SOX9. Silencing of PTGDS or SOX9 also alleviated H-rev107-mediated suppression of cell migration and invasion.

Conclusions

These results revealed that H-rev107, through PTGDS, suppressed cell migration and invasion. Our data suggest that the PGD2-cAMP-SOX9 signal pathway might play an important role in H-rev107-mediated cancer cell invasion in testes.     

Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines

Introduction

Glioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43) and estrogen receptors (ERs). Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2) on Cx43 expression in two glioma cell lines with variable native expression of Cx43.

Materials and Methods

F98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERβ and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique.

Results

E2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERβ was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERβ in C6 cultures, while it decreased ERα expression in F98 glioma cells.

Discussion

These findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma.     

Decreased miR-204 in H. pylori-Associated Gastric Cancer Promotes Cancer Cell Proliferation and Invasion by Targeting SOX4

Background

The molecular mechanism between Helicobacter pylori (H. pylori) infection and gastric cancer remained largely unknown. In this study, we determined the role of miRNA in H. pylori induced gastric cancer.

Methods and Results

We found that miR-204 was decreased in H. pylori positive tissues by qRT-PCR. Knockdown of miR-204 enhanced the invasion and proliferation ability of gastric cancer cells in vitro. Luciferase assay revealed that SOX4 was target gene of miR-204, which was found up-regulated in H. pylori positive tissues. Down-regulation of miR-204 and over-expression of SOX4 promoted epithelial-mesenchymal transition process.

Conclusion

Taken together, our findings demonstrated that miR-204 may act as a tumor suppressor in H. pylori induced gastric cancer by targeting SOX4.     

B7-H1 Expression Is Associated with Poor Prognosis in Colorectal Carcinoma and Regulates the Proliferation and Invasion of HCT116 Colorectal Cancer Cells

Background And Objective

The investigation concerning the B7-H1 expression in colorectal cancer cells is at an early stage. It is unclear whether B7-H1 expression may have diagnostic or prognostic value in colorectal carcinoma. Additionally, how B7-H1 is associated with the clinical features of colorectal carcinoma is not known. In order to investigate the relationship between B7-H1 and colorectal cancer, we analyzed B7-H1 expression and its effect in clinical specimens and HCT116 cells.

Methods

Paraffin-embedded specimens from 143 eligible patients were used to investigate the expression of CD274 by immunohistochemistry. We also examined whether B7-H1 itself may be related to cell proliferation, apoptosis, migration and invasion in colon cancer HCT116 cells.

Results

Our results show that B7-H1 was highly expressed in colorectal carcinoma and was significantly associated with cell differentiation status and TNM (Tumor Node Metastasis) stage. Patients with positive B7-H1 expression showed a trend of shorter survival time. Using multivariate analysis, we demonstrate that positive B7-H1 expression is an independent predictor of colorectal carcinoma prognosis. Our results indicate that B7-H1 silencing with siRNA inhibits cell proliferation, migration and invasion. Furthermore, cell apoptosis was also increased by B7-H1 inhibition.

Conclusions

Positive B7-H1 expression is an independent predictor for colorectal carcinoma prognosis. Moreover, knockdown of B7-H1 can inhibit cell proliferation, migration and invasion.     

Knockdown of FRAT1 Expression by RNA Interference Inhibits Human Glioblastoma Cell Growth,Migration and Invasion

Background

FRAT1 positively regulates the Wnt/β-catenin signaling pathway by inhibiting GSK-3-mediated phosphorylation of β-catenin. It was originally characterized as a protein frequently rearranged in advanced T cell lymphoma, but has recently also been identified as a proto-oncogene involved in tumorigenesis. Our previous studies showed that FRAT1 was dramatically overexpressed in gliomas and its expression level was significantly increased along with clinicopathological grades.

Methods

In the current study, we used RT-PCR and Western blotting to assess the mRNA and protein levels of FRAT1 in three glioma cell lines. In addition, to evaluate its functional role in gliomas, we examined the effects of FRAT1 knockdown on proliferation, migration and invasion in vitro and tumor growth in vivo using glioblastoma U251 cells and RNAi.

Results

FRAT1 was highly expressed in all three glioma cell lines. RNAi-mediated down-regulation of endogenous FRAT1 in human glioblastoma U251 cells resulted in suppression of cell proliferation, arrest of cell cycle, inhibition of cell migration and invasion in vitro. Moreover, FRAT1 depletion significantly impaired tumor xenograft growth in nude mice.

Conclusions

Our results highlight the potential role of FRAT1 in tumorigenesis and progression of glioblastoma. These findings provide a biological basis for FRAT1 as a potential molecular marker for improved pathological grading and as a novel candidate therapeutic target for glioblastoma management.     

Suppression of miR-221 inhibits glioma cells proliferation and invasion via targeting SEMA3B

Background

Gliomas are the most common primary tumors in the central nervous system. Due to complicated signaling pathways involved in glioma progression, effective targets for treatment and biomarkers for prognosis prediction are still scant.

Results

In this study we revealed that a new microRNA (miR), the miR-221, was highly expressed in the glioma cells, and suppression of miR-221 resulted in decreased cellular proliferation, migration, and invasion in glioma cells. Mechanistic experiments validated that miR-221 participates in regulating glioma cells proliferation and invasion via suppression of a direct target gene, the Semaphorin 3B (SEMA3B). The rescue experiment with miR-221 and SEMA3B both knockdown results in significant reversion of miR-221 induced phenotypes.

Conclusion

Taken together, our findings highlight an unappreciated role for miR-221 and SEMA3B in glioma.     

Human Omental-Derived Adipose Stem Cells Increase Ovarian Cancer Proliferation,Migration, and Chemoresistance

Objectives

Adipose tissue contains a population of multipotent adipose stem cells (ASCs) that form tumor stroma and can promote tumor progression. Given the high rate of ovarian cancer metastasis to the omental adipose, we hypothesized that omental-derived ASC may contribute to ovarian cancer growth and dissemination.

Materials and Methods

We isolated ASCs from the omentum of three patients with ovarian cancer, with (O-ASC4, O-ASC5) and without (O-ASC1) omental metastasis. BM-MSCs, SQ-ASCs, O-ASCs were characterized with gene expression arrays and metabolic analysis. Stromal cells effects on ovarian cancer cells proliferation, chemoresistance and radiation resistance was evaluated using co-culture assays with luciferase-labeled human ovarian cancer cell lines. Transwell migration assays were performed with conditioned media from O-ASCs and control cell lines. SKOV3 cells were intraperitionally injected with or without O-ASC1 to track in-vivo engraftment.

Results

O-ASCs significantly promoted in vitro proliferation, migration chemotherapy and radiation response of ovarian cancer cell lines. O-ASC4 had more marked effects on migration and chemotherapy response on OVCA 429 and OVCA 433 cells than O-ASC1. Analysis of microarray data revealed that O-ASC4 and O-ASC5 have similar gene expression profiles, in contrast to O-ASC1, which was more similar to BM-MSCs and subcutaneous ASCs in hierarchical clustering. Human O-ASCs were detected in the stroma of human ovarian cancer murine xenografts but not uninvolved ovaries.

Conclusions

ASCs derived from the human omentum can promote ovarian cancer proliferation, migration, chemoresistance and radiation resistance in-vitro. Furthermore, clinical O-ASCs isolates demonstrate heterogenous effects on ovarian cancer in-vitro.     

Bone Mesenchymal Stem Cells Contributed to the Neointimal Formation after Arterial Injury

Objectives

Recent findings suggest that in response to repair-to-injury bone marrow mesenchymal stem cells (BMSCs) participate in the process of angiogenesis. It is unclear what role BMSCs play in the structure of the vessel wall. In present study, we aimed to determine whether BMSCs had the capacity of endothelial cells (ECs).

Methods

BMSCs were separated and cultured. FACS and RT-PCR analysis confirmed the gene expression phenotype. The capacity of migration and adhesion and the ultrastructure of BMSCs were examined. The effect of BMSCs transplantation on the vascular repair was investigated in a murine carotid artery-injured model.

Results

BMSCs could express some markers and form the tube-like structure. The migration and adhesion capacity of BMSCs increased significantly after stimulated. In addition, BMSCs had the intact cell junction. In vivo the local transfer of BMSCs differentiated into neo-endothelial cells in the injury model for carotid artery and contributed to the vascular remodeling.

Conclusion

These results showed that BMSCs could contribute to neointimal formation for vascular lesion and might be associated with the differentiation into ECs, which indicated the important therapeutic implications for vascular diseases.     

The role of <Emphasis Type="Italic">IFITM3</Emphasis> in the growth and migration of human glioma cells

Background

Interferon induced transmembrane protein 3 (IFITM3) is transcribed in most tissues and highly interferon-inducible. However, the role of IFITM3 in cancer is still poorly understood.

Methods

Expression levels ofIFITM3were analyzed in 60 glioma patients by immunohistochemistry (IHC). Following closely, we investigated the phenotype of IFITM3 knockdown on glioma cell growth and tumorigenesis in vitro using lentivirus-mediated loss-of-function strategy.

Results

Depletion of IFITM3in U251 cells dramatically inhibited cell proliferation and colony formation, which demonstrated that reduced IFITM3 protein levels could cause inhibition of tumorigenesis. Knockdown of IFITM3 also induced cell cycle arrest in G0/G1 phase, especially in the sub-G1 phase representing apoptotic cells. In addition, the migration of U251 cells was visibly weakened after IFITM3 knockdown, as determined by Transwell assay.

Conclusions

Our findings provide new evidence that IFITM3 plays an important role in glioma cell growth and migration, suggesting that silencing of IFITM3 by RNA interference (RNAi) may be a potential approach to suppress glioma growth.

     

Extracellular HSP70/HSP70-PCs Promote Epithelial-Mesenchymal Transition of Hepatocarcinoma Cells

Background

Extracellular heat shock protein 70 and peptide complexes (eHSP70/HSP70-PCs) regulate a variety of biological behaviors in tumor cells. Whether eHSP70/HSP70-PCs are involved in the epithelial-mesenchymal transition (EMT) of tumor cells remains unclear.

Aims

To determine the effects of eHSP70/HSP70-PCs on EMT of hepatocarcinoma cells.

Methods

The expressions of E-cadherin, HSP70, α-smooth muscle actin protein (α-SMA) and p-p38 were detected immunohistochemically in liver cancer samples. Immunofluorescence, western blotting and real-time RT-PCR methods were used to analyze the effects of eHSP70/HSP70-PCs on the expressions of E-cadherin, α-SMA and p38/MAPK in vivo.

Results

HSP70, E-cadherin, α-SMA and p-p38 were elevated in hepatocellular carcinoma tissues. The expression of HSP70 was positively correlated with malignant differentiated liver carcinoma. The expressions of HSP70, α-SMA and p-p38 correlated with recurrence-free survival after resection. eHSP70/HSP70-PCs significantly promoted the expressions of α-SMA and p-p38 and reduced the expressions of E-cadherin in vivo. The effect was inhibited by SB203580.

Conclusion

The expressions of HSP70, E-cadherin, α-SMA and p-p38 may represent indicators of malignant potential and could discriminate the malignant degree of liver cancer. eHSP70/HSP70-PCs play an important role in the EMT of hepatocellular carcinoma via the p38/MAPK pathway.     

microRNA-124 Inhibits Migration and Invasion by Down-Regulating ROCK1 in Glioma

Background

The extraordinary invasive capability is a major cause of treatment failure and tumor recurrence in glioma, however, the molecular and cellular mechanisms governing glioma invasion remain poorly understood. Evidence in other cell systems has implicated the regulatory role of microRNA in cell motility and invasion, which promotes us to investigate the biological functions of miR-124 in glioma in this regard.

Results

We have found that miR-124 is dramatically downregulated in clinical specimen of glioma and is negatively correlated with the tumor pathological grading in the current study. The cells transfected by miR-124 expression vector have demonstrated retarded cell mobility. Using a bioinformatics analysis approach, rho-associated coiled-coil containing protein kinase 1 (ROCK1), a well-known cell mobility-related gene, has been identified as the target of miR-124. A dual-luciferase reporter assay was used to confirm that miR-124 targeted directly the 3′UTR of ROCK1 gene and repressed the ROCK1 expression in U87MG human glioma cell line. Furthermore, experiments have shown that the decreased cell mobility was due to the actin cytoskeleton rearrangements and the reduced cell surface ruffle in U87MG glioma cells. These results are similar to the cellular responses of U87MG glioma cells to the treatment of Y-27632, an inhibitor of ROCK protein. Moreover, a constitutively active ROCK1 in miR-124 over-expressed glioma cells reversed the effects of miR-124. Our results revealed a novel mechanism that miR-124 inhibits glioma cells migration and invasion via ROCK1 downregulation.

Conclusions

These results suggest that miR-124 may function as anti-migration and anti-invasion influence in glioma and provides a potential approach for developing miR-124-based therapeutic strategies for malignant glioma therapy.     

Expression and Functional Relevance of Cannabinoid Receptor 1 in Hodgkin Lymphoma

Background

Cannabinoid receptor 1 (CB₁) is expressed in certain types of malignancies. An analysis of CB₁ expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date.

Design and Methods

We examined the distribution of CB₁ protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB₁ signaling on cell survival was investigated.

Results

A predominant expression of CB₁ was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB₁-abundance and displayed a dose-dependent decline of viability under CB₁ inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells.

Conclusions

The present study identifies CB₁ as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.     

Poly(ADP-Ribose) Polymerase 1 (PARP1) Overexpression in Human Breast Cancer Stem Cells and Resistance to Olaparib

Background

Breast cancer stem cells (BCSCs) have been recognized as playing a major role in various aspects of breast cancer biology. To identify specific biomarkers of BCSCs, we have performed comparative proteomics of BCSC-enriched and mature cancer cell populations from the human breast cancer cell line (BCL), BrCA-MZ-01.

Methods

ALDEFLUOR assay was used to sort BCSC-enriched (ALDH+) and mature cancer (ALDH−) cell populations. Total proteins were extracted from both fractions and subjected to 2-Dimensional Difference In-Gel Electrophoresis (2-D DIGE). Differentially-expressed spots were excised and proteins were gel-extracted, digested and identified using MALDI-TOF MS.

Results

2-D DIGE identified poly(ADP-ribose) polymerase 1 (PARP1) as overexpressed in ALDH+ cells from BrCA-MZ-01. This observation was confirmed by western blot and extended to four additional human BCLs. ALDH+ cells from BRCA1-mutated HCC1937, which had the highest level of PARP1 overexpression, displayed resistance to olaparib, a specific PARP1 inhibitor.

Conclusion

An unbiased proteomic approach identified PARP1 as upregulated in ALDH+, BCSC-enriched cells from various human BCLs, which may contribute to clinical resistance to PARP inhibitors.     

Long Non-Coding RNA MALAT1 Mediates Transforming Growth Factor Beta1-Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells

Purpose

To study the role of long non-coding RNA (lncRNA) MALAT1 in transforming growth factor beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells.

Methods

ARPE-19 cells were cultured and exposed to TGF-β1. The EMT of APRE-19 cells is confirmed by morphological change, as well as the increased expression of alpha-smooth muscle actin (αSMA) and fibronectin, and the down-regulation of E-cadherin and Zona occludin-1(ZO-1) at both mRNA and protein levels. The expression of lncRNA MALAT1 in RPE cells were detected by quantitative real-time PCR. Knockdown of MALAT1 was achieved by transfecting a small interfering RNA (SiRNA). The effect of inhibition of MALAT1 on EMT, migration, proliferation, and TGFβ signalings were observed. MALAT1 expression was also detected in primary RPE cells incubated with proliferative vitreoretinopathy (PVR) vitreous samples.

Results

The expression of MALAT1 is significantly increased in RPE cells incubated with TGFβ1. MALAT1 silencing attenuates TGFβ1-induced EMT, migration, and proliferation of RPE cells, at least partially through activating Smad2/3 signaling. MALAT1 is also significantly increased in primary RPE cells incubated with PVR vitreous samples.

Conclusion

LncRNA MALAT1 is involved in TGFβ1-induced EMT of human RPE cells and provides new understandings for the pathogenesis of PVR.