We established a cancer stem (CS) cell line, U87CS, by means of spheroid culture of U87MG cells derived from glioblastoma (GBM) in neuronal stem cell medium. U87CS cells presented positive immunohistochemical staining for multidrug resistance (MDR)1 and CD133, a marker for a subset of leukemia and GBM CS cells. The gene expression of MDR1 and CD133 on U87CS cells increased by an average of 8.51 and 47.18 times, respectively, compared to the levels on U87MG cells by real-time quantitative RT-PCR. U87CS cells possessed stronger drug-resistance to conventional anti-cancer drugs, such as doxorubicin (Dox), etoposide (VP-16), carboplastin, and BCNU than U87MG cells. Double immunofluoresence staining showed co-expression of MDR1 and CD133 on U87CS cells transplanted into nude mice brains. In addition, we identified the crossreactivity of CD133 and MDR1 in a surgical specimen of GBM. Our results suggest that CS cells may be resistant to current chemotherapy and represent a novel target for GBM therapeutics.

BackgroundOvarian cancer has the highest mortality rate and is among the most common of female malignancies. Ovarian cancer cells are known to develop resistance to standard chemotherapeutic treatments.Gene silencing via RNA interferences (RNAi) mediated by short interfering RNA (siRNA) has enormous therapeutic potential for the treatment of cancer.Hypoxic inducible factor-1α (HIF-1α) is often overexpressed in cancers including ovarian cancer and it is associated with tumor aggressiveness, angiogenesis, cell migration, proliferation, survival, glucose metabolism, metastasis and drug resistance. It is predicted that HIF-1α suppression via siRNA technique would provide effective tumoricidal outcome in human ovarian cancer cells.The siRNA gene therapy is hindered because achieving sufficient concentration of siRNA at the tumor site(s) is difficult. siRNA has a high degradation rate in serum due to its physical characteristics, a rapid elimination by the renal pathway and a low permeability across cellular membranes. One way to enhance the delivery of siRNA to the site of action is a development of a suitable delivery platform with characteristics that enables biocompatibility, a high loading capacity, protection of siRNA during transport and a high targeting ability.Recently the delivery system for gene therapy has moved from viral vectors to synthetic and natural cationic polymers because viral vectors have potential to evoke immunogenic responses and can be hazardous during preparation. A representative cationic polysaccharide is a natural substance, chitosan. Nano-metric particles are easily formed by crosslinking chitosan with a counter ion such as tripolyphosphates (TPP), which particle provides a protection against degradation of loaded siRNA.There are problems in the use of chitosan for gene delivery. First, excessive positive charge that remains on the surface of the nanoparticles after formulation brings about interaction with red blood cells (RBCs), opsonization and activation of immune system resulting in elimination of them. Second, achieving a sufficient concentration of siRNA at the tumor site in a timely manner is difficult, as systemically administrated chitosan nanoparticles are only passively delivered there via the enhanced permeability and retention (EPR) effect. Third, uptake of chitosan nanoparticles by nonspecific endocytosis in tumor cells results in a low siRNA transfection efficiency. To address these problems, an active targeting system that can also aid the uptake of nanoparticles is required.In the present study we have emplyed folic acid (FA) as a targeting ligand because FA is harmless on normal cells, little immunogenic, inexpensive and stable under both in-vitro and in-vivo conditions. The expression level of the FA receptor is high in ovarian cancer cells, which promotes the receptor-mediated endocytosis of nanoparticles. The present study shows the potential utility of an siRNA delivery system with FA-PEG-COL nanoparticles encapsulating an HIF-1α siRNA for a targeted ovarian cance...

Extracellular zinc, which is released from hippocampal neurons in response to brain ischaemia, triggers morphological changes in microglia. Under ischaemic conditions, microglia exhibit two opposite activation states (M1 and M2 activation), which may be further regulated by the microenvironment. We examined the role of extracellular zinc on M1 activation of microglia. Pre-treatment of microglia with 30–60 μM ZnCl2 resulted in dose-dependent increases in interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNFα) secretion when M1 activation was induced by lipopolysaccharide administration. In contrast, the cell-permeable zinc chelator TPEN, the radical scavenger Trolox, and the P2X7 receptor antagonist A438079 suppressed the effects of zinc pre-treatment on microglia. Furthermore, endogenous zinc release was induced by cerebral ischaemia–reperfusion, resulting in increased expression of IL-1β, IL-6, TNFα, and the microglial M1 surface marker CD16/32, without hippocampal neuronal cell loss, in addition to impairments in object recognition memory. However, these effects were suppressed by the zinc chelator CaEDTA. These findings suggest that extracellular zinc may prime microglia to enhance production of pro-inflammatory cytokines via P2X7 receptor activation followed by reactive oxygen species generation in response to stimuli that trigger M1 activation, and that these inflammatory processes may result in deficits in object recognition memory.

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Cancer stem cells are an important target for effective therapy, since they show tumorigenicity, chemoresistance, and radioresistance. We isolated cancer stem cells from glioma cell lines and tissues and examined the expression of cancer testis antigen (CTA) genes as potential target molecules for cancer vaccine therapy. CTA genes were highly and frequently expressed in cancer stem cells compared with differentiated cells. In addition, histone acetylation levels in the promoter regions of CTA genes were high in cancer stem cells and low in differentiated cells, while DNA methylation analysis of the promoter regions revealed hypomethylation in cancer stem cells. This epigenetic difference between cells leads to heterogeneous expression of CTA genes in the tumor mass, which consists of cells at various levels of differentiation. Moreover, the expression level of HLA class I antigens was not affected by the differentiation status, suggesting that CTA genes may present as surface antigens in cancer stem cells. Taken together, these findings suggest that CTA genes may be attractive candidates for targeted vaccine therapy against cancer stem cells in glioma patients.