Lactobacillus plantarum CBT LP3 ameliorates colitis via modulating T cells in mice.

Lactobacillus plantarum has been identified as a probiotic bacterium owing to its role in immune regulation and maintenance of intestinal permeability. Here, we investigated the anti-colitic effects and mechanism of L. plantarum CBT LP3 (LP3). This in vivo study was performed using dextran sodium sulfate (DSS) to induce colitis in mice. Mice were randomly divided into three groups: a control supplied with normal drinking water, a DSS-treated group followed by oral administration of vehicle, and a DSS-treated group gavaged with LP3 daily for 7 days following DSS administration. An analysis of macrophages and T cell subsets harvesting from peritonium cavity cells and splenocytes was performed using a flow cytometric assay. Gene expression and cytokine profiles were measured using quantitative reverse transcriptase polymerase chain reaction. The administration of LP3 significantly attenuated disease activity and histolopathology compared to control. LP3 had anti-inflammatory effects, with increased induction of regulatory T cells and type 2 helper T cells in splenocytes and restoration of goblet cells accompanied by suppression of proinflammatory cytokine expressions. These findings suggest that L. plantarum CBT LP3 can be used as a potent immunomodulator, which has significant implications for IBD treatment.

Precise differential diagnosis of acute bone marrow edema and hemorrhage and trabecular microfractures using naïve and gamma correction pinhole bone scans.

OBJECTIVE: To analyze the performance of sequential naïve pinhole bone scan (nPBS) and gamma correction pinhole bone scan (GCPBS), reinforced by ImageJ densitometry and pixelized microfracture measurement, for making specific diagnoses of bone marrow edema (BME), bone marrow hemorrhage (BMH), and trabecular microfractures (TMF). METHODS: We prospectively examined BME, BMH, TMF, and normal trabeculae in 10 patients using sequential nPBS and GCPBS. The intensity of 99mtechnetium-hydroxydiphosphonate (99mTc-HDP) uptake was measured using a pixelized method and calculated using ImageJ densitometry in terms of arbitrary units (AU). This overall method was termed a visuospatial-mathematic assay (VSMA). We analyzed the ability of the calculated AU values to discriminate between the four states using GraphPad Prism software, with reference to previous morphological data. RESULTS: The calculated values were categorized as ≤50 AU for normal trabecula, 51-100 AU for BME, 101-150 AU for BMH, and ≥151 AU for TMF. The difference in uptake between normal trabecula and BME was significant and the differences among BME, BMH, and TMF were highly significant. CONCLUSION: VSMA is a useful technique for refining objective individual diagnoses and for differentiating and quantitating BME, BMH, and TMF.

Synergistic Activity of Paclitaxel, Sorafenib, and Radiation Therapy in advanced Renal Cell Carcinoma and Breast Cancer.

Advanced cancer has been shown to be associated with a higher percentage of epigenetic changes than with genetic mutations. Preclinical models have shown that the combination of paclitaxel, sorafenib, and radiation therapy (RT) plays a crucial role in renal cell carcinoma (RCC) and breast cancer. This study aimed to investigate the involvement of mitochondrial cytochrome c-dependent apoptosis in the mechanism of action of a combination of paclitaxel, sorafenib, and RT in RCC and breast cancer. RCC and breast cancer cell lines were exposed to paclitaxel and sorafenib alone or combined in the presence of radiation, and cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The synergistic anticancer effects of the combination therapy on cell cycle and intracellular signaling pathways were estimated using flow cytometry and immunoblot analysis. RCC and breast cancer cell line xenograft models were used to examine the antitumor activity in vivo. Our results suggest that paclitaxel, sorafenib, and RT synergistically decreased the viability of RCC and breast cancer cells and significantly induced their apoptosis, as shown by caspase-3 cleavage. Paclitaxel, sorafenib, and radiation cotreatment reduced antiapoptotic factor levels in these cells and, thereby, significantly reduced the tumor volume of RCC and breast cancer cell xenografts. The current study suggests that paclitaxel, sorafenib, and radiation cotreatment was more effective than cotreatment with paclitaxel or sorafenib and radiation. These findings may offer a new therapeutic approach to RCC and breast cancer.

Survival of Cancer Stem-Like Cells Under Metabolic Stress via CaMK2α-mediated Upregulation of Sarco/Endoplasmic Reticulum Calcium ATPase Expression.

Purpose: Cancer cells grow in an unfavorable metabolic milieu in the tumor microenvironment and are constantly exposed to metabolic stress such as chronic nutrient depletion. Cancer stem-like cells (CSC) are intrinsically resistant to metabolic stress, thereby surviving nutrient insufficiency and driving more malignant tumor progression. In this study, we aimed to demonstrate the potential mechanisms by which CSCs avoid Ca2+-dependent apoptosis during glucose deprivation.Experimental Design: We investigated cell viability and apoptosis under glucose deprivation, performed genome-wide transcriptional profiling of paired CSCs and parental cells, studied the effect of calcium/calmodulin-dependent protein kinase 2 alpha (CaMK2α) gene knockdown, and investigated the role of nuclear factor kappa B (NFκB) in CSCs during time-dependent Ca2+-mediated and glucose deprivation-induced apoptosis. We also observed the effect of combined treatment with 2-deoxy-d-glucose, a metabolic inhibitor that mimics glucose deprivation conditions in mouse xenograft models, and thapsigargin, a specific inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA).Results: We demonstrated the coordinated upregulation of SERCA in CSCs. SERCA, in turn, is transcriptionally regulated by CaMK2α via NFκB activation. Combined treatment with 2-deoxy-d-glucose and thapsigargin, a specific inhibitor of SERCA, significantly reduced tumor growth compared with that in untreated control animals or those treated with the metabolic inhibitor alone.Conclusions: The current study provides compelling evidence that CaMK2α acts as a key antiapoptosis regulator in metabolic stress-resistant CSCs by activating NFκB. The latter induces expression of SERCA, allowing survival in glucose-deprived conditions. Importantly, our combination therapeutic strategy provides a novel approach for the clinical application of CSC treatment. Clin Cancer Res; 24(7); 1677-90. ©2017 AACR.

Regulation of Acetate Utilization by Monocarboxylate Transporter 1 (MCT1) in Hepatocellular Carcinoma (HCC).

Altered energy metabolism is a biochemical fingerprint of cancer cells. Hepatocellular carcinoma (HCC) shows reciprocal [18F]fluorodeoxyglucose (FDG) and [11C]acetate uptake, as revealed by positron emission tomography/computed tomography (PET/CT). Previous studies have focused on the role of FDG uptake in cancer cells. In this study, we evaluated the mechanism and roles of [11C]acetate uptake in human HCCs and cell lines. The expression of monocarboxylate transporters (MCTs) was assessed to determine the transporters of [11C]acetate uptake in HCC cell lines and human HCCs with different [11C]acetate uptake. Using two representative cell lines with widely different [11C]acetate uptake (HepG2 for high uptake and Hep3B for low uptake), changes in [11C]acetate uptake were measured after treatment with an MCT1 inhibitor or MCT1-targeted siRNA. To verify the roles of MCT1 in cells, oxygen consumption rate and the amount of lipid synthesis were measured. HepG2 cells with high [11C]acetate uptake showed higher MCT1 expression than other HCC cell lines with low [11C]acetate uptake. MCT1 expression was elevated in human HCCs with high [11C]acetate uptake compared to those with low [11C]acetate uptake. After blocking MCT1 with AR-C155858 or MCT1 knockdown, [11C]acetate uptake in HepG2 cells was significantly reduced. Additionally, inhibition of MCT1 suppressed mitochondrial oxidative phosphorylation, lipid synthesis, and cellular proliferation in HCC cells with high [11C]acetate uptake. MCT1 may be a new therapeutic target for acetate-dependent HCCs with high [11C]acetate uptake, which can be selected by [11C]acetate PET/CT imaging in clinical practice.

18F-Fluorodeoxyglucose uptake on positron emission tomography/computed tomography is associated with metastasis and epithelial-mesenchymal transition in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer mortality worldwide. Several studies have investigated the relationship between 18F-fluorodeoxyglucose (18F-FDG) uptake on positron emission tomography and the prognosis of patients with HCC, although the relationship between 18F-FDG uptake and expression of EMT-related proteins in these patients remains unclear. We retrospectively enrolled 116 patients with HCC treated by curative surgical resection and who underwent 18F-FDG positron emission tomography/computed tomography (PET/CT) for preoperative staging. The relationship between the tumor-to-liver standardized uptake value ratio (TLR) and the presence of metastasis was determined. By using HCC cell lines with different 18F-FDG uptake, we assessed the effect of 18F-FDG uptake on in vitro cell proliferation and migration on the inhibition of glucose uptake. Ten (29.4%) of 34 patients with high TLRs had extrahepatic metastases, whereas six (7.3%) of 82 patients with low TLRs had extrahepatic metastases (p = 0.002). Hepatocellular carcinomas with high TLRs showed higher expression of glucose transporter isoform 1 and EMT markers than did HCCs with low TLRs. After treatment with a glucose uptake inhibitor, HCC cells with high 18F-FDG uptake showed decreased cell proliferation and migration and a reversal in the expression of EMT markers. High 18F-FDG uptake on PET/CT is associated with frequent extrahepatic metastasis and EMT in patients with HCC. Inhibition of glucose uptake reduced cell proliferation, reversed EMT-related protein expression, and decreased cellular migration. Glycolytic regulation could be a new therapeutic target to reduce tumor growth and metastatic potential in HCCs with a high glycolytic phenotype.

Synergistic Activity of N-hydroxy-7-(2-naphthylthio) Heptanomide and Sorafenib Against Cancer Stem Cells, Anaplastic Thyroid Cancer.

Anaplastic thyroid carcinoma (ATC) although rare is the most deadly form of thyroid cancer. The fatality rate for ATC is high-pitched, the survival rate at 1 year after diagnosis is <20%. Control of ATC is severely hard and widespread with unpredictability. We Previous proved that histone gene reviser and epigenetic changes role significant parts in papillary and anaplastic thyroid cancer tumorigenesis. Herein, the goal of this study was to investigate the anti-tumor activities of a HDAC inhibitor, HNHA alone and in combination with sorafenib in ATC cells in vitro and in vivo and to explore its effects on apoptotic cell death pathways. Three ATC cell lines were exposed to sorafenib in the presence or absence of HNHA, and cell viability was determined by MTT assay. Effects of combined treatment on cell cycle and intracellular signaling pathways were assessed by flow cytometry and western blot analysis. The ATC cell lines xenograft model was used to examine the anti-tumor activity in vivo. Our data showed that HNHA and sorafenib synergistically decreased cell viability in ATC cells, and also significantly increased apoptotic cell death in these cells, as proved by the cleavage of caspase-3 and DNA fragmentation. HNHA and sorafenib combination was reduced anti-apoptotic factor in ATC. Thus, combination therapy with HNHA and sorafenib significantly decreased vessel density, and most significantly reduced tumor volume and increased survival in ATC xenografts. These results propose that HNHA in combination with sorafenib has significant anti-cancer activity in preclinical models, potentially suggesting a new clinical approach for patients of advanced thyroid cancer type.

Inhibition of glioblastoma tumorspheres by combined treatment with 2-deoxyglucose and metformin.

Background: Deprivation of tumor bioenergetics by inhibition of multiple energy pathways has been suggested as an effective therapeutic approach for various human tumors. However, this idea has not been evaluated in glioblastoma (GBM). We hypothesized that dual inhibition of glycolysis and oxidative phosphorylation could effectively suppress GBM tumorspheres (TS). Methods: Effects of 2-deoxyglucose (2DG) and metformin, alone and in combination, on GBM-TS were evaluated. Viability, cellular energy metabolism status, stemness, invasive properties, and GBM-TS transcriptomes were examined. In vivo efficacy was tested in a mouse orthotopic xenograft model. Results: GBM-TS viability was decreased by the combination of 2DG and metformin. ATP assay and PET showed that cellular energy metabolism was also decreased by this combination. Sphere formation, expression of stemness-related proteins, and invasive capacity of GBM-TS were also significantly suppressed by combined treatment with 2DG and metformin. A transcriptome analysis showed that the expression levels of stemness- and epithelial mesenchymal transition-related genes were also significantly downregulated by combination of 2DG and metformin. Combination treatment also prolonged survival of tumor-bearing mice and decreased invasiveness of GBM-TS. Conclusion: The combination of 2DG and metformin effectively decreased the stemness and invasive properties of GBM-TS and showed a potential survival benefit in a mouse orthotopic xenograft model. Our findings suggest that targeting TS-forming cells by this dual inhibition of cellular bioenergetics warrants expedited clinical evaluation for the treatment of GBM.

Inhibiting stemness and invasive properties of glioblastoma tumorsphere by combined treatment with temozolomide and a newly designed biguanide (HL156A).

Studies have investigated biguanide-derived agents for the treatment of cancers and have reported their effects against tumorspheres (TSs). The purpose of this study was determining the effects of HL156A, a newly designed biguanide with improved pharmacokinetics, on glioblastoma TSs (GMB TSs) and assess the feasibility of this drug as a new line of therapy against glioblastoma, alone or combined with a conventional therapeutic agent, temozolomide(TMZ). The effects of HL156A, alone and combined with TMZ, on the stemness and invasive properties of GBM TSs and survival of orthotopic xenograft animals were assessed. HL156A, combined with TMZ, inhibited the stemness of GBM TSs, proven by neurosphere formation assay and marker expression. Three-dimensional collagen matrix invasion assays provided evidence that combined treatment inhibited invasive properties, compared with control and TMZ-alone treatment groups. TMZ alone and combined treatment repressed the expression of epithelial-mesenchymal transition-related genes. A gene ontology comparison of TMZ and combination-treatment groups revealed altered expression of genes encoding proteins involved in cellular adhesion and migration. Combined treatment with HL156A and TMZ showed survival benefits in an orthotopic xenograft mouse model. The inhibitory effect of combination treatment on the stemness and invasive properties of GBM TSs suggest the potential usage of this regimen as a novel strategy for the treatment of GBM.

Potential anti-cancer effect of N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA), a novel histone deacetylase inhibitor, for the treatment of thyroid cancer.

BACKGROUND: Thyroid cancer has been indicated to have a higher global proportion of DNA methylation and a decreased level of histone acetylation. Previous studies showed that histone gene reviser and epigenetic changes role significant parts in papillary and anaplastic thyroid cancer tumorigenesis. The goal of this research was to study the endoplasmic reticulum (ER) stress-mediated actions of the dominant histone deacetylase (HDAC) inhibitor, N-hydroxy-7-(2-naphthylthio) hepatonomide (HNHA), in thyroid cancer and to explore its effects on apoptotic cell death pathways. METHODS: Experiments were achieved to conclude the effects of HNHA in papillary thyroid cancer (PTC) and anaplastic thyroid cancer (ATC) cell lines and xenografts, as compared with two other established HDAC inhibitors (SAHA; suberoylanilide hydroxamic acid and TSA; trichostatin A). RESULTS: Apoptosis, which was induced by all HDAC inhibitors, was particularly significant in HNHA-treated cells, where noticeable B-cell lymphoma-2 (Bcl-2) suppression and caspase activation were observed both in vitro and in vivo. HNHA increased Ca(2+) release from the ER to the cytoplasm. ER stress-dependent apoptosis was induced by HNHA, suggesting that it induced caspase-dependent apoptotic cell death in PTC and ATC. PTC and ATC xenograft studies demonstrated that the antitumor and pro-apoptotic effects of HNHA were greater than those of the established HDAC inhibitors. These HNHA activities reflected its induction of caspase-dependent and ER stress-dependent apoptosis on thyroid cancer cells. CONCLUSIONS: The present study indicated that HNHA possibly provide a new clinical approach to thyroid cancers, including ATC.

The bifunctional autophagic flux by 2-deoxyglucose to control survival or growth of prostate cancer cells.

BACKGROUND: Recent reports using metabolism regulating drugs showed that nutrient deprivation was an efficient tool to suppress cancer progression. In addition, autophagy control is emerging to prevent cancer cell survival. Autophagy breaks down the unnecessary cytoplasmic components into anabolic units and energy sources, which are the most important sources for making the ATP that maintains homeostasis in cancer cell growth and survival. Therefore, the glucose analog 2-deoxyglucose (2DG) has been used as an anticancer reagent due to its inhibition of glycolysis. METHODS: Prostate cancer cells (PC3) were treated with 2DG for 6 h or 48 h to analyze the changing of cell cycle and autophagic flux. Rapamycin and LC3B overexpressing vectors were administered to PC3 cells for autophagy induction and chloroquine and shBeclin1 plasmid were used to inhibit autophagy in PC3 cells to analyze PC3 cells growth and survival. The samples for western blotting were prepared in each culture condition to confirm the expression level of autophagy related and regulating proteins. RESULTS: We demonstrated that 2DG inhibits PC3 cells growth and had discriminating effects on autophagy regulation based on the different time period of 2DG treatment to control cell survival. Short-term treatment of 2DG induced autophagic flux, which increased microtubule associated protein 1 light chain 3B (LC3B) conversion rates and reduced p62 levels. However, 2DG induced autophagic flux is remarkably reduced over an extended time period of 2DG treatment for 48 h despite autophagy inducing internal signaling being maintained. The relationship between cell growth and autophagy was proved. Increased autophagic flux by rapamycin or LC3B overexpression powerfully reduced cell growth, while autophagy inhibition with shBeclin1 plasmid or chloroquine had no significant effect on regulating cell growth. CONCLUSION: Given these results, maintaining increased autophagic flux was more effective at inhibiting cancer cell progression than inhibition of autophagic flux, which is necessary for the survival of PC3 cells. Autophagic flux should be tightly regulated to maintain metabolic homeostasis for cancer cell growth and survival in PC3 cells and is a suitable target for cancer therapy.

The regulation of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase by autophagy in low-glycolytic hepatocellular carcinoma cells.

The glycolytic phenotype is a dominant metabolic phenomenon in cancer and is reflected in becoming aggressive. Certain hepatocellular carcinoma lack increased glycolysis and prefer to uptake acetate than glucose for metabolism. Autophagy plays a role in preserving energies and nutrients when there is limited external nutrient supply and maintains glucose level of blood though supporting gluconeogenesis in the liver. As the role of autophagy and gluconeogenesis in HCC following the glycolic activity was not clear, we cultured HCC cells with different glycolytic levels in Hank's balanced salt solution (HBSS) to induce autophagy and conducted the activity of gluconeogenesis. Both autophagy and gluconeogenesis were induced in low glycolytic HCC cells (HepG2). In glycolytic Hep3B cells, only autophagy without gluconeogenesis was induced upon starvation. When autophagy was blocked, the level of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) was reduced in HepG2 cells and not in Hep3B. Altogether, we investigated contribution of hepatic gluconeogenesis to the metabolic phenotype of HCC cells and the role of autophagy as a potential mechanism regulating gluconeogenesis in low glycolytic HCC.

The novel histone deacetylase inhibitor, N-hydroxy-7-(2-naphthylthio) hepatonomide, exhibits potent antitumor activity due to cytochrome-c-release-mediated apoptosis in renal cell carcinoma cells.

BACKGROUND: Epigenetic modifications play a critical role in the regulation of all DNA-based processes, such as transcription, repair, and replication. Inappropriate histone modifications can result in dysregulation of cell growth, leading to neoplastic transformation and cell death. Renal tumors have been shown to have a higher global methylation percentage and reduced histone acetylation. Preclinical models have revealed that histone gene modifiers and epigenetic alterations play important roles in renal cell carcinoma (RCC) tumorigenesis. Recently, a novel HDAC inhibitor, N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA), has been introduced as an example of a new class of anti-cancer agents. The anti-cancer activity of HNHA and the underlying mechanisms of action remain to be clarified. METHODS: The MTS assay using a panel of RCC cells was used to evaluate the anti-proliferative effects of HNHA. The established HDAC inhibitors, SAHA and TSA, were used for comparison. Western blotting analysis was performed to investigate the acetylation of histone H3 and the expression of apoptotic markers in vitro and in vivo. Subcellular fractionation was performed to evaluate expression of Bax and cytochrome c in the cytosol and mitochondria, and also translocation of cytochrome c from the cytoplasm to the nucleus. A confocal microscopic evaluation was performed to confirm inhibition of cell proliferation, induction of apoptosis, and the nuclear translocation of cytochrome c in RCC cells. RESULTS: In this study, we investigated the apoptosis-inducing activity of HNHA in cultured kidney cancer cells. Apoptosis in the HNHA-treated group was induced significantly, with marked caspase activation and Bcl-2 suppression in RCC cells in vitro and in vivo. HNHA treatment caused cytochrome c release from mitochondria, which was mediated by increased Bax expression and caspase activation. HNHA also induced nuclear translocation of cytochrome c, suggesting that HNHA can induce caspase-independent nuclear apoptosis in RCC cells. An in vivo study showed that HNHA had greater anti-tumor and pro-apoptotic effects on RCC xenografts than the established HDAC inhibitors. CONCLUSIONS: HNHA has more potent anti-tumor activity than established HDAC inhibitors. Its activities are mediated by caspase-dependent and cytochrome-c-mediated apoptosis in RCC cells. These results suggest that HNHA may offer a new therapeutic approach to RCC.

TIMP-1 modulates chemotaxis of human neural stem cells through CD63 and integrin signalling.

We recently reported that hNSCs (human neural stem cells) have the interesting characteristic of migration towards an intracranial glioma. However, the molecules and mechanisms responsible for tumour tropism are unclear. In the present study, we used microarray and proteomics analyses to identify a novel chemoattractant molecule, TIMP-1 (tissue inhibitor of metalloproteinase-1), secreted from human brain tumour tissues. We demonstrate that TIMP-1 significantly enhances hNSC adhesion and migration in a cell culture system. These effects were critically dependent on CD63, as shRNA-mediated ablation of CD63 expression attenuated the response. TIMP-1 significantly increased the number of FAs (focal adhesions) and cytoskeletal reorganization for cell migration in hNSCs, whereas knockdown of CD63 resulted in decreased hNSC spreading, FAs and migration, even after TIMP-1 treatment. In addition, TIMP-1 binding to CD63 activated ß1 integrin-mediated signalling through Akt and FAK phosphorylation, leading to pattern changes in distribution of vinculin and F-actin (filamentous actin). Furthermore, inactivation of ß1 integrin by use of a blocking antibody or inhibition of PI3K (phosphoinositide 3-kinase) signalling impaired the migration of hNSCs towards TIMP-1. Collectively, our results underline TIMP-1 as a novel and effective key regulator of CD63 and ß1 integrin-mediated signalling, which regulates hNSC adhesion and migration.

A new histone deacetylase inhibitor improves liver fibrosis in BDL rats through suppression of hepatic stellate cells.

BACKGROUND AND PURPOSE: Activation of hepatic stellate cells (HSCs) is a crucial step in the pathogenesis of hepatic fibrosis. Histone deacetylase (HDAC) is an attractive target in liver fibrosis because it plays a key role in gene expression and cell differentiation. We have developed a HDAC inhibitor, N-hydroxy-7-(2-naphthylthio)heptanomide (HNHA), and investigated the anti-fibrotic activity of HNHA in vitro and in vivo. EXPERIMENTAL APPROACH: We investigated the anti-fibrotic effect of HNHA on mouse and human HSC activation in vitro and in the liver of bile duct-ligated (BDL) rats in vivo using cell proliferation assays, cell cycle analysis, biochemical assay, immunohistochemistry and Western blots. Liver pathology was assessed with histochemical techniques. KEY RESULTS: HNHA inhibited proliferation and arrested the cell cycle via p21 induction in HSCs. In addition, HNHA induced apoptosis of HSCs, which was correlated with reduced COX-2 expression, NF-κB activation and cell death signals. HNHA restored liver function and decreased the accumulation of extracellular matrix in the liver via suppression of HSC activation in BDL rats in vivo. HNHA administration also increased survival in BDL rats. CONCLUSIONS AND IMPLICATIONS: HNHA improved liver function, suppressed liver fibrosis and increased survival of BDL rats, accompanied by reduction of cell growth, activation and survival of HSCs. These findings show that HNHA may be a potent anti-fibrosis agent against hepatic fibrosis because of its multi-targeted inhibition of HSC activity in vivo and in vitro.

Identification of gliotropic factors that induce human stem cell migration to malignant tumor.

Neural stem cells are mobile, are attracted to regions of brain damage, and can migrate a considerable distance to reach a glioma site. However, the molecular basis of the progression of gliotropism to malignant gliomas remains poorly understood. With the use of clinically and histologically assessed glioma cells, we have assessed their protein and gene profiles via proteomics and microarray approaches, and have identified candidate genes from human glioma tissues. This research is expected to provide clues to the molecular mechanisms underlying the migration of neural stem cells (F3 cell) to glioma sites. The expression of 16 proteins was shown to have increased commonly in human glioma tissues. Among them, the expression of annexin A2, TIMP-1, COL11A1, bax, CD74, TNFSF8, and SPTLC2 were all increased in human glioma cells, as confirmed by Western blotting and immunohistochemical staining. In particular, annexin A2 effects an increase in migration toward F3 and glioblastoma cells (U87 cell) in a Boyden chamber migration assay. An ERK inhibitor (PD98057) and a CDK5 inhibitor (rescovitine) inhibited 50% and 90% of annexin A2-induced migration in F3 cells, respectively. A similar chemotactic migration was noted in F3 and U87 cells. These results demonstrated that 7 candidate proteins may harbor a potential glioma tropism factor relevant to the pathology of malignant glioma. These results reveal that this novel molecular approach to the monitoring of glioma may provide clinically relevant information regarding tumor malignancy, and should also prove appropriate for high-throughput clinical screening applications.

Migration of human neural stem cells toward an intracranial glioma.

Many in vivo and in vitro studies have demonstrated the targeted migration of neural stem cells (NSC) to infiltrating brain tumors, including malignant glioma, highlighting a potential therapeutic approach. However, there is not enough information to apply this approach to clinical therapy. The most important things in stem cell therapy for brain tumors involve selecting the appropriate neural progenitor type and optimizing the efficiency of the cell engraftment. By histological analysis using two different live-dyes, human NSCs were shown to migrate away from the transplanted site in the direction of the expanding C6 glioma and to intermix with the tumor bed, especially with the tumor core. This intermixing occurred within 7 days when NSCs were implanted into glioma model. The time course of migratory HB1.F5 with the greatest mobility of three NSC lines was as follows. As early as 3 days after transplantation, several NSCs were found leaving the implant site, primarily approaching microsatellites and frontier cells located near the site of NSC implantation. Through 7 days post-transplantation, massive numbers of NSCs continued to be attracted to and interspersed with C6 glioma, and were finally distributed extensively throughout the whole tumor bed, including the core and penumbra of the tumor mass. However, NSCs appeared to penetrate into the tumor mass very well, whereas normal fibroblast cells could not migrate. These findings strengthen the potential for human NSCs as attractive vehicles to improve therapeutic gene delivery to cancer or glioma if they are optimized to selectively kill neoplastic cells.