多种胶质瘤细胞系中获取胶质瘤干细胞方法的研究

目的：进一步证明胶质瘤干细胞是广泛存在的,并寻找一种简洁的方法从不同胶质瘤细胞系中提取肿瘤干细胞。方法：将胶质瘤细胞以合适的密度接种于96孔板中,获取胶质瘤干细胞,并通过检测其自我更新能力、多向分化能力、成瘤能力及胶质瘤干细胞标记物的表达情况对其进行鉴定。结果：多种细胞系中均成功获取了胶质瘤干细胞。并且这细胞球表达神经干细胞的标志物,不袁达神经细胞分化标志物,同时又有多向分化的能力,仅5000个细胞就可以在裸鼠颅内成瘤。结论：我们的研究结果表明胶质瘤干细胞是广泛存在的,并为以后进一步研究胶质瘤干细胞的特性及靶向胶质瘤干细胞的药物做铺垫。     

Petasites japonicus Stimulates the Proliferation of Mouse Spermatogonial Stem Cells

Oriental natural plants have been used as medical herbs for the treatment of various diseases for over 2,000 years. In this study, we evaluated the effect of several natural plants on the preservation of male fertility by assessing the ability of plant extracts to stimulate spermatogonial stem cell (SSC) proliferation by using a serum-free culture method. In vitro assays showed that Petasites japonicus extracts, especially the butanol fraction, have a significant effect on germ cells proliferation including SSCs. The activity of SSCs cultured in the presence of the Petasites japonicus butanol fraction was confirmed by normal colony formation and spermatogenesis following germ cell transplantation of the treated SSCs. Our findings could lead to the discovery of novel factors that activate SSCs and could be useful for the development of technologies for the prevention of male infertility.     

The Roles of Hypoxia-Inducible Factors in Regulating Neural Stem Cells Migration to Glioma Stem Cells and Determinating Their Fates

The mortality of patients with malignant gliomas remains high despite the advancement in multi-modal therapy including surgery, radio- and chemotherapy. Glioma stem cells (GSCs), sharing some characteristics with normal neural stem cells (NSCs), contribute to the cellular origin for primary gliomas and the recurrence of malignant gliomas after current conventional therapy. Accordingly, targeting GSCs proves to be a promising avenue of therapeutic intervention. The specific tropism of NSCs to GSCs provides a novel platform for targeted delivery of therapeutic agents. Tropism and mobilization of NSCs are enhanced by hypoxia through upregulating chemotactic cytokines and activating several signaling pathways. Moreover, hypoxia-inducible factors (HIFs) produced under hypoxic microenvironment of the stem cell niche play critical roles in the growth and stemness phenotypes regulation of both NSCs and GSCs. However, the definite cellular and molecular mechanisms of HIFs involvement in the process remain obscure. In this review, we focus on the pivotal roles of HIFs in migration of NSCs to GSCs and potential roles of HIFs in dictating the fates of migrated NSCs and targeted GSCs.     

Mesenchymal Stem Cell Therapy Stimulates Endogenous Host Progenitor Cells to Improve Colonic Epithelial Regeneration

Patients who undergo pelvic radiotherapy may develop severe and chronic complications resulting from gastrointestinal alterations. The lack of curative treatment highlights the importance of novel and effective therapeutic strategies. We thus tested the therapeutic benefit of mesenchymal stem cells (MSC) treatment and proposed molecular mechanisms of action. MSC efficacy was tested in an experimental model of radiation-induced severe colonic ulceration histologically similar to that observed in patients. In this model, MSC from bone marrow were administered intravenously, immediately or three weeks (established lesions) after irradiation. MSC therapy reduces radiation-induced colonic ulceration and increases animal survival. MSC treatment induces therapeutic efficacy whatever the time of cell infusion. Infused-MSC engraft in the colon but also increase endogenous MSC mobilization in blood that have lasting benefits over time. In vitro analysis demonstrates that the MSC effect is mediated by paracrine mechanisms through the non-canonical WNT (Wingless integration site) pathway. In irradiated rat colons, MSC treatment increases the expression of the non-canonical WNT4 ligand by epithelial cells. The epithelial regenerative process is improved after MSC injection by stimulation of colonic epithelial cells positive for SOX9 (SRY-box containing gene 9) progenitor/stem cell markers. This study demonstrates that MSC treatment induces stimulation of endogenous host progenitor cells to improve the regenerative process and constitutes an initial approach to arguing in favor of the use of MSC to limit/reduce colorectal damage induced by radiation.     

c-Myc Is Required for Maintenance of Glioma Cancer Stem Cells

Background

Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells.

Methodology/Principal Findings

Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G₀/G₁ phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice.

Conclusions/Significance

These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.     

Topoisomerase I Inhibitors,Shikonin and Topotecan,Inhibit Growth and Induce Apoptosis of Glioma Cells and Glioma Stem Cells

Gliomas, the most malignant form of brain tumors, contain a small subpopulation of glioma stem cells (GSCs) that are implicated in therapeutic resistance and tumor recurrence. Topoisomerase I inhibitors, shikonin and topotecan, play a crucial role in anti-cancer therapies. After isolated and identified the GSCs from glioma cells successfully, U251, U87, GSCs-U251 and GSCs-U87 cells were administrated with various concentrations of shikonin or topotecan at different time points to seek for the optimal administration concentration and time point. The cell viability, cell cycle and apoptosis were detected using cell counting kit-8 and flow cytometer to observe the inhibitory effects on glioma cells and GSCs. We demonstrated that shikonin and topotecan obviously inhibited proliferation of not only human glioma cells but also GSCs in a dose- and time-dependent manner. According to the IC50 values at 24 h, 2 μmol/L of shikonin and 3 μmol/L of topotecan were selected as the optimal administration concentration. In addition, shikonin and topotecan induced cell cycle arrest in G0/G1 and S phases and promoted apoptosis. The down-regulation of Bcl-2 expression with the activation of caspase 9/3-dependent pathway was involved in the apoptosis process. Therefore, the above results showed that topoisomerase I inhibitors, shikonin and topotecan, inhibited growth and induced apoptosis of GSCs as well as glioma cells, which suggested that they might be the potential anticancer agents targeting gliomas to provide a novel therapeutic strategy.     

MicroRNA-107 Inhibits U87 Glioma Stem Cells Growth and Invasion

Glioma stem cells (GSCs) are thought to be critical for resistance to radiotherapy and chemotherapy and for tumor recurrence after surgery in glioma patients. Identification of new therapeutic strategies that can target GSCs may thus be critical for improving patient survival. MicroRNAs (miRNAs) are small non-coding RNAs that function as tumor suppressors or oncogenes. In this study, we confirmed that miR-107 was down-regulated in GSCs. To investigate the role of miR-107 in tumorigenesis of GSCs, a lentiviral vector over-expressing miR-107 in U87GSCs was constructed. We found that over-expression of miR-107 suppressed proliferation and down-regulated Notch2 protein and stem cell marker (CD133 and Nestin) expression in U87GSCs. Furthermore, enhanced miR-107 expression significantly inhibited U87GSC invasion and reduced matrix metalloproteinase-12 expression. miR-107 also suppressed U87GSCs xenograft growth in vivo. These findings suggest that miR-107 is involved in U87GSCs growth and invasion and may provide a potential therapeutic target for glioma treatment.     

Extracellular ATP Stimulates Calcium Influx in Neuroblastoma Glioma Hybrid NG108–15 Cells

Abstract— ATP-induced changes in the intracellular Ca²⁺concentration ([Ca²⁺]_i) in neuroblastoma glioma hybrid NG108–15 cells were studied. Using the fluorescent Ca²⁺indicator fura-2, we have shown that the [Ca²⁺]_i increased in response to ATP. ATP at 3 mM caused the greatest increase in [Ca^z+]_i, whereas at higher concentrations of ATP the response became smaller. Two nonhydrolyzable ATP analogues, adenosine 5′-thiotriphosphate and 5′-adenylyl-β, γ-imidodiphosphate, could not trigger significant [Ca²⁺]_i change, but they could block the ATP effect. Other adenine nucleotides, including ADP, AMP, α,β-methylene-ATP, β,γ-methylene-ATP, and 2-methylthio-ATP, as well as UTP and adenosine, all had no effect on [Ca²⁺]_i at 3 mM. In the absence of extracellular Ca²⁺, the effect of ATP was inhibited totally, but could be restored by the addition of Ca²⁺ to the cells. Upon removal of Mg²⁺, the maximum increase in [Ca²⁺]_i induced by ATP was enhanced by about 42%. Ca²⁺-channel blockers partially inhibited the ATP-induced [Ca²⁺]_i rise. The ATP-induced [Ca²⁺]_i rise was not affected by thapsigargin pretreatment, though such pretreatment blocked bradykinin-induced [Ca²⁺]_i rise completely. No heterologous desensitization of [Ca²⁺]_i rise was observed between ATP and bradykinin. The magnitude of the [Ca²⁺]_i rise induced by ATP increased between 1.5 and 3.1 times when external Na⁺was replaced with Tris, N-methyl-d -glucamine, choline, or Li⁺. The addition of EGTA or verapamil to cells after their maximum response to ATP immediately lowered the [Ca²⁺]_i to the basal level in Na⁺-containing or Na⁺-free Tris solution. Our results suggest that ATP stimulates Ca²⁺influx via at least two pathways: ion channels that are permeable to Ca²⁺ and Na⁺, and pores formed by ATP^4-.     

Turning Cancer Stem Cells Inside Out: An Exploration of Glioma Stem Cell Signaling Pathways

Tumors are complex collections of heterogeneous cells with recruited vasculature, inflammatory cells, and stromal elements. Neoplastic cells frequently display a hierarchy in differentiation status. Recent studies suggest that brain tumors have a limited population of neoplastic cells called cancer stem cells with the capacity for sustained self-renewal and tumor propagation. Brain tumor stem cells contribute to therapeutic resistance and tumor angiogenesis. In this minireview, we summarize recent data regarding critical signaling pathways involved in brain tumor stem cell biology and discuss how targeting these molecules may contribute to the development of novel anti-glioma therapies.Cancers can be considered organ systems with aberrant activation of developmental and wound response pathways. Recent evidence suggests that within some tumors there is a cell subpopulation with the special capacity for sustained self-renewal and tumor propagation in vivo. Cells fulfilling these criteria were originally reported in acute myeloid leukemia (1), but similar populations were soon successively identified within various solid tumors (2). The proper terminology regarding these cells remains unsettled, with most groups using terms such as CSCs,² tumor-initiating/propagating cells, and stem-like cancer cells. Although CSCs are a source of controversy, the concept recognizes the well described heterogeneity of tumor cells. Many critics contest the hypothesis on the grounds of a potential stem cell origin, challenge of current markers, or CSC frequency, none of which are implicit requirements of the CSC hypothesis (3).Malignant gliomas are essentially universally lethal despite conventional therapy, with surgical resection and chemoradiation limited to palliation. Glioma CSCs were among the first solid tumor CSCs described (4) and remain one of the most widely used CSC models. Glioma CSCs share significant similarities with normal NSCs, including the expression of stem cell markers (CD133, Nestin, Musashi, and Sox2) and the capacity to differentiate into multiple lineages (5), but the overlap is incomplete. Notably, glioma CSCs are also highly resistant to chemoradiotherapies (5, 6), underscoring the importance of developing more efficient therapies against CSCs and prompting researchers to elucidate the molecular mechanisms regulating CSCs. Here, we summarize recent findings regarding the signaling pathways that are critical to glioma CSC biology.     

Glypican 1 Stimulates S Phase Entry and DNA Replication in Human Glioma Cells and Normal Astrocytes

Malignant gliomas are highly lethal neoplasms with limited treatment options. We previously found that the heparan sulfate proteoglycan glypican 1 (GPC1) is universally and highly expressed in human gliomas. In this study, we investigated the biological activity of GPC1 expression in both human glioma cells and normal astrocytes in vitro. Expression of GPC1 inactivates the G₁/S checkpoint and strongly stimulates DNA replication. Constitutive expression of GPC1 causes DNA rereplication and DNA damage, suggesting a mutagenic activity for GPC1. GPC1 expression leads to a significant downregulation of the tumor suppressors pRb, Cip/Kip cyclin-dependent kinase inhibitors (CKIs), and CDH1, and upregulation of the pro-oncogenic proteins cyclin E, cyclin-dependent kinase 2 (CDK2), Skp2, and Cdt1. These GPC1-induced changes are accompanied by a significant reduction in all types of D cyclins, which is independent of serum supplementation. It is likely that GPC1 stimulates the so-called Skp2 autoinduction loop, independent of cyclin D-CDK4/6. Knockdown of Skp2, CDK2, or cyclin E, three key elements within the network modulated by GPC1, results in a reduction of the S phase and aneuploid fractions, implying a functional role for these regulators in GPC1-induced S phase entry and DNA rereplication. In addition, a significant activation of both the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways by GPC1 is seen in normal human astrocytes even in the presence of growth factor supplement. Both pathways are constitutively activated in human gliomas. The surprising magnitude and the mitogenic and mutagenic nature of the effect exerted by GPC1 on the cell cycle imply that GPC1 may play an important role in both glioma tumorigenesis and growth.     

Increased Notch Signaling Enhances Radioresistance of Malignant Stromal Cells Induced by Glioma Stem/ Progenitor Cells

Background

Host malignant stromal cells induced by glioma stem/progenitor cells were revealed to be more radiation-resistant than the glioma stem/progenitor cells themselves after malignant transformation in nude mice. However, the mechanism underlying this phenomenon remains unclear.

Methods

Malignant stromal cells induced by glioma stem/progenitor cell 2 (GSC-induced host brain tumor cells, ihBTC2) were isolated and identified from the double color-coded orthotopic glioma nude mouse model. The survival fraction at 2 Gy (SF2) was used to evaluate the radiation resistance of ihBTC2, the human glioma stem/progenitor cell line SU3 and its radiation-resistant sub-strain SU3-5R and the rat C6 glioma cell line. The mRNA of Notch 1 and Hes1 from ihBTC2 cells were detected using qPCR before and after 4 Gy radiation. The expression of the Notch 1, pAkt and Bcl-2 proteins were investigated by Western blot. To confirm the role of the Notch pathway in the radiation resistance of ihBTC2, Notch signaling blocker gamma secretase inhibitors (GSIs) were used.

Results

The ihBTC2 cells had malignant phenotypes, such as infinite proliferation, hyperpentaploid karyotype, tumorigenesis in nude mice and expression of protein markers of oligodendroglia cells. The SF2 of ihBTC2 cells was significantly higher than that of any other cell line (P<0.05, n = 3). The expression of Notch 1 and Hes1 mRNAs from ihBTC2 cells was significantly increased after radiation. Moreover, the Notch 1, pAkt and Bcl-2 proteins were significantly increased after radiation (P<0.05, n = 3). Inhibition of Notch signaling markedly enhanced the radiosensitivity of ihBTC2 cells.

Conclusions

In an orthotopic glioma model, the malignant transformation of host stromal cells was induced by glioma stem/progenitor cells. IhBTC2 cells are more radiation-resistant than the glioma stem/progenitor cells, which may be mediated by activation of the Notch signaling pathway.     

Inhibition of Fatty Acid Synthase Decreases Expression of Stemness Markers in Glioma Stem Cells

Cellular metabolic changes, especially to lipid metabolism, have recently been recognized as a hallmark of various cancer cells. However, little is known about the significance of cellular lipid metabolism in the regulation of biological activity of glioma stem cells (GSCs). In this study, we examined the expression and role of fatty acid synthase (FASN), a key lipogenic enzyme, in GSCs. In the de novo lipid synthesis assay, GSCs exhibited higher lipogenesis than differentiated non-GSCs. Western blot and immunocytochemical analyses revealed that FASN is strongly expressed in multiple lines of patient-derived GSCs (G144 and Y10), but its expression was markedly reduced upon differentiation. When GSCs were treated with 20 μM cerulenin, a pharmacological inhibitor of FASN, their proliferation and migration were significantly suppressed and de novo lipogenesis decreased. Furthermore, following cerulenin treatment, expression of the GSC markers nestin, Sox2 and fatty acid binding protein (FABP7), markers of GCSs, decreased while that of glial fibrillary acidic protein (GFAP) expression increased. Taken together, our results indicate that FASN plays a pivotal role in the maintenance of GSC stemness, and FASN-mediated de novo lipid biosynthesis is closely associated with tumor growth and invasion in glioblastoma.