The inhibition of BDNF/TrkB/PI3K/Akt signal mediated by AG1601 promotes apoptosis in malignant glioma

Malignant glioma is the most aggressive primary brain tumor and has a poor survival rate. Even if extensive methods are preformed to treat glioma, the mortality rate is still very high. It is necessary for discovering and developing new drugs for malignant glioma treatment. AG1601 is one of AG-series drugs, including AG1031 and AG1503, and it has been optimized on the original basis. In our study, we found that AG1601 markedly inhibited proliferation and promoted C6 glioma cell apoptosis in vitro. AG1601 also reduced the size and weight of glioma in vivo. The growth ability of glioma was significantly inhibited after treatment with AG1601. It also showed that the expression levels of BDNF/TrkB/PI3K/Akt signal related proteins were obviously decreased in C6 glioma cells after treatment with AG1601 in vivo and in vitro. We also found that BDNF, as the activator of BDNF/TrkB/PI3K/Akt signal, reversed the anti-proliferation and pro-apoptosis of C6 glioma cells caused by AG1601. K252a, a specific inhibitor of TrkB, and AG1601 in combination aggravated C6 glioma cell apoptosis. These results indicate that AG1601 has good effects on the anti-proliferation and pro-apoptosis of malignant glioma via BDNF/TrkB/PI3K/Akt signal and could be considered as a potential drug in treating malignant glioma.     

Detection of apoptosis and phagocytosis in vitro in C6 rat glioma cells treated with tiazofurin

Tiazofurin, an anticancer drug which inhibits IMP dehydrogenase activity and decreases GTP concentration in various malignant cells, induced inhibition of growth and apoptosis in C6 rat glioma in vitro. The effects of tiazofurin were significantly blocked by addition of exogenous guanosine, suggesting the role of decreased GTP in triggering specific signal transduction pathways involved in apoptosis of C6 cells. The most interesting result of this study was the evidence of phagocytosis of apoptotic cells in vitro by neighbouring cells, a phenomenon considered to occur only in apoptosis in vivo. The possibility of observing phagocytosis in C6 glioma cells suggests that this cell system could be a good model for studying mechanisms of phagocytosis in vitro.     

Enhanced Anti-Tumor Effect of Zoledronic Acid Combined with Temozolomide against Human Malignant Glioma Cell Expressing O6-Methylguanine DNA Methyltransferase

Temozolomide (TMZ), a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O⁶-methylguanine-DNA methyltranferase (MGMT), a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL), which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance.     

Suppression of Autophagy Enhanced Growth Inhibition and Apoptosis of Interferon-β in Human Glioma Cells

Interferon-beta (IFN-β) is a cytokine with anti-viral, anti-proliferative, and immunomodulatory effects. In this study, we investigated the effects of IFN-β on the induction of autophagy and the relationships among autophagy, growth inhibition, and apoptosis induced by IFN-β in human glioma cells. We found that IFN-β induced autophagosome formation and conversion of microtubule associated protein 1 light chain 3 (LC3) protein, whereas it inhibited cell growth through caspase-dependent cell apoptosis. The Akt/mTOR signaling pathway was involved in autophagy induced by IFN-β. A dose- and time-dependent increase of p-ERK 1/2 expression was also observed in human glioma cells treated with IFN-β. Autophagy induced by IFN-β was suppressed when p-ERK1/2 was impaired by treatment with U0126. We also demonstrated that suppression of autophagy significantly enhanced growth inhibition and cell apoptosis induced by IFN-β, whereas inhibition of caspase-dependent cell apoptosis impaired autophagy induced by IFN-β. Collectively, these findings indicated that autophagy induced by IFN-β was associated with the Akt/mTOR and ERK 1/2 signaling pathways, and inhibition of autophagy could enhance the growth inhibitory effects of IFN-β and increase apoptosis in human glioma cells. Together, these findings support the possibility that autophagy inhibitors may improve IFN-β therapy for gliomas.     

The influence of simulated microgravity on proliferation and apoptosis in U251 glioma cells

Several studies have indicated that microgravity can influence cellular progression, proliferation, and apoptosis in tumor cell lines. In this study, we observed that simulated microgravity inhibited proliferation and induced apoptosis in U251 malignant glioma (U251MG) cells. Furthermore, expression of the apoptosis-associated proteins, p21 and insulin-like growth factor binding protein-2 (IGFBP-2), was upregulated and downregulated, respectively, following exposure to simulated microgravity. These findings indicate that simulated microgravity inhibits proliferation while inducing apoptosis of U251MG cells. The associated effects appear to be mediated by inhibition of IGFBP-2 expression and stimulation of p21 expression. This suggests that simulated microgravity might represent a promising method to discover new targets for glioma therapeutic strategy.     

Induction of apoptosis in human and rat glioma by agonists of the nuclear receptor PPARgamma

Malignant astrocytomas are among the most common brain tumours and few therapeutic options exist. It has recently been recognized that the ligand-activated nuclear receptor PPARgamma can regulate cellular proliferation and induce apoptosis in different malignant cells. We report the effect of three structurally different PPARgamma agonists inducing apoptosis in human (U87MG and A172) and rat (C6) glioma cells. The PPARgamma agonists ciglitazone, LY171 833 and prostaglandin-J2, but not the PPARalpha agonist WY14643, inhibited proliferation and induced cell death. PPARgamma agonist-induced cell death was characterized by DNA fragmentation and nuclear condensation, as well as inhibited by the synthetic receptor-antagonist bisphenol A diglycidyl ether (BADGE). In contrast, primary murine astrocytes were not affected by PPARgamma agonist treatment. The apoptotic death in the glioma cell lines treated with PPARgamma agonists was correlated with the transient up-regulation of Bax and Bad protein levels. Furthermore, inhibition of Bax expression by specific antisense oligonucleotides protected glioma cells against PPARgamma-mediated apoptosis, indicating an essential role of Bax in PPARgamma-induced apoptosis. However, PPARgamma agonists not only induced apoptosis but also caused redifferentiation as indicated by outgrowth of long processes and expression of the redifferentiation marker N-cadherin in response to PPARgamma agonists. Taken together, treatment of glioma cells with PPARgamma agonists may hold therapeutic potential for the treatment of gliomas.     

Effects of chlorogenic acid,caffeine, and coffee on behavioral and biochemical parameters of diabetic rats

Mesenchymal stem cells (MSCs) represent a potential therapeutic target for glioma. We determined the molecular mechanism of inhibitory effect of human umbilical cord-derived MSCs (hUC-MSCs) on the growth of C6 glioma cells. We demonstrated that hUC-MSCs inhibited C6 cell growth and modulated the cell cycle to G0/G1 phase. The expression of β-catenin and c-Myc was downregulated in C6 cells by conditioned media from hUC-MSCs, and the levels of secreted DKK1 were positively correlated with concentrations of hUCMSCs-CM. The inhibitory effect of hUC-MSCs on C6 cell proliferation was enhanced as the concentration of DKK1 in hUCMSCs-CM increased. When DKK1 was neutralized by anti-DKK1 antibody, the inhibitory effect of hUC-MSCs on C6 cells was attenuated. Furthermore, we found that conditioned media from hUC-MSCs transfection with siRNA targeting DKK1 mRNA or pEGFPN1-DKK1 plasmid lost or enhanced the abilities to regulate the Wnt signaling in C6 cells. Therefore, hUC-MSCs inhibited C6 glioma cell growth via secreting DKK1, an inhibitor of Wnt pathway, may represent a novel therapeutic strategy for malignant glioma.     

CDK4/6 inhibition suppresses tumour growth and enhances the effect of temozolomide in glioma cells

In adults, glioma is the most commonly occurring and invasive brain tumour. For malignant gliomas, the current advanced chemotherapy includes TMZ (temozolomide). However, a sizeable number of gliomas are unyielding to TMZ, hence, giving rise to an urgent need for more efficient treatment choices. Here, we report that cyclin‐dependent kinases 4 (CDK4) is expressed at significantly high levels in glioma cell lines and tissues. CDK4 overexpression enhances colony formation and proliferation of glioma cells and extends resistance to inhibition of TMZ‐mediated cell proliferation and induction of apoptosis. However, CDK4 knockdown impedes colony formation and cell proliferation, and enhances sensitivity of glioma cells to TMZ. The selective inhibition of CDK4/6 impedes glioma cell proliferation and induces apoptotic induction. The selective inhibitors of CDK4/6 may enhance glioma cell sensitivity to TMZ. We further showed the possible role of RB phosphorylation mediated by CDK4 for its oncogenic function in glioma. The growth of glioma xenografts was inhibited in vivo, through combination treatment, and corresponded to enhanced p‐RB levels, reduced staining of Ki‐67 and enhanced activation of caspase 3. Therefore, CDK4 inhibition may be a favourable strategy for glioma treatment and overcomes TMZ resistance.     

Roles of the Akt/mTOR/p70S6K and ERK1/2 signaling pathways in curcumin-induced autophagy

Curcumin has a potent anticancer effect and is a promising new therapeutic strategy. We previously demonstrated that curcumin induced non-apoptotic autophagic cell death in malignant glioma cells in vitro and in vivo. This compound inhibited the Akt/mammalian target of rapamycin/p70 ribosomal protein S6 kinase pathway and activated the extracellular signal-regulated kinases 1/2 thereby inducing autophagy. Interestingly, activation of the first pathway inhibited curcumin-induced autophagy and cytotoxicity, whereas inhibition of the latter pathway inhibited curcumin-induced autophagy and induced apoptosis, thus augmenting the cytotoxicity of curcumin. These results imply that these two autophagic pathways have opposite effects on curcumin's cytotoxicity. However, inhibition of nuclear factor kappaB, which is the main target of curcumin for its anticancer effect, was not observed in malignant glioma cells. These results suggest that autophagy but not nuclear factor kappaB plays a central role in curcumin anticancer therapy and warrant further investigation toward application in patients with malignant gliomas. Here, we discuss the therapeutic role of two autophagic pathways influenced by curcumin.     

Panaxydol inhibits the proliferation and induces the differentiation of human hepatocarcinoma cell line HepG2

Panaxydol, a polyacetylene compound isolated from Panax ginseng, exerts anti-proliferative effects against malignant cells. No previous study, however, has been reported on its effects on hepatocellular carcinoma cells. Here, we investigated the effects of panaxydol on the proliferation and differentiation of human hepatocarcinoma cell line HepG2. We studied by electronic microscopy of morphological and ultrastructural changes induced by panaxydol. We also examined the cytotoxicities of panaxydol against HepG2 cells using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and the effect of panaxydol on cell cycle distributions by flow cytometry. We investigated the production of liver proteins in panaxydol-treated cells including alpha-fetoprotein and albumin and measured the specific activity of alkaline phosphatase and gamma-glutamyl transferase. We further investigated the effects of panaxydol on the expression of Id-1, Id-2, p21 and pRb by RT-PCR or immunoblotting analysis. We found that panaxydol inhibited the proliferation of HepG2 cells and caused morphological and ultrastructural changes in HepG2 cells resembling more mature forms of hepatocytes. Moreover, panaxydol induced a cell cycle arrest at the G₁ to S transition in HepG2 cells. It also significantly decreased the secretion of alpha-fetoprotein and the activity of gamma-glutamyl transferase. By contrast, panaxydol remarkably increased the secretion of albumin and the alkaline phosphatase activity. Furthermore, panaxydol increased the mRNA content of p21 while reducing that of Id-1 and Id-2. Panaxydol also increased the protein levels of p21, pRb and the hypophosphorylated pRb in a dose-dependent manner. These findings suggest that panaxydol is of value for further exploration as a potential anti-cancer agent.     

Mimosine-Induced Apoptosis in C6 Glioma Cells Requires the Release of Mitochondria-Derived Reactive Oxygen Species and p38, JNK Activation

Growth-inhibitory effects of mimosine, a plant amino acid, on rat C6 glioma cells were analyzed. Mimosine markedly inhibited proliferation and induced apoptosis of C6 glioma cells in a dose- and time-dependent manner. Mimosine-mediated apoptosis was accompanied by promoting reactive oxygen species (ROS) generation in mitochondria, and by decreased mitochondrial membrane potential (Δψ), and release of cytochrome c from mitochondria, followed by caspase 3 activation. Furthermore, mimosine increased the phosphorylation level of c-Jun-N-terminal protein kinase and p38, which was the downstream effect of ROS accumulation. Mimosine was confirmed to show profound effects on apoptosis of C6 glioma cells by ROS-regulated mitochondria pathway, and these results bear on the hypothesized potential for mimosine as promising agents in the treatment of malignant gliomas.     

Inhibition of rat glioma cell migration and proliferation by a calix[8]arene scaffold exposing multiple GlcNAc and ureido functionalities

Beta1,4-Galactosyltransferases (beta1,4-GalTase) exposed on the cell surface are involved in cell migration. Specifically, beta1,4-GalTase V is highly expressed in glioma and promotes invasion, growth, and survival of glioma cells. A glycocalix[8]arene exposing N-acetylglucosamine (GlcNAc) residues (compound 1) inhibited rat C6 glioma cell migration as assessed in a scratch wound model. This effect was related to inhibition of focal adhesion kinase phosphorylation, measured by western blot analysis, and specifically observed in the area bordering the scratch wound. Compound 1 inhibited also C6 cell proliferation, an effect unrelated to its ability to interact with GalTase as it was mimicked by different calix[8]arene derivatives, all characterized by multivalency and ureido groups. Compound 1 did not induce apoptotic death, but caused a different distribution of C6 cells within the cell cycle. The results here reported identify compound 1 as a molecule able to exert inhibitory effects on C6 cell migration and proliferation, independently, because of distinct components in its structure.     

Co-inhibition of epidermal growth factor receptor and type 1 insulin-like growth factor receptor synergistically sensitizes human malignant glioma cells to CD95L-induced apoptosis

Inhibition of epidermal growth factor receptor (EGFR) signaling sensitizes human malignant glioma cells to death ligand-induced apoptosis. However, tumor cells may compensate the loss of EGFR signaling by activation of the type 1 insulin-like growth factor receptor (IGF-1R). We here report that antagonism of the IGF-1R with the small-molecule inhibitor AG1024 in combination with inhibitors of the EGFR synergistically sensitizes human malignant glioma cells to CD95L-induced apoptosis. This cell death is p53-independent, but requires caspase 8 activity. The levels of the receptor, CD95, are not altered by the inhibitors alone or in combination. Analysis of the downstream signaling pathways reveals synergistic inhibition of ribosomal protein S6 phosphorylation by inhibitor co-treatment, suggesting an involvement of the mammalian target of rapamycin pathway. These findings suggest that adding inhibitors of IGF-1R may be a strategy to overcome escape from the anti-apoptotic effects of EGFR inhibition in malignant gliomas.     

Cord Blood Stem Cell-Mediated Induction of Apoptosis in Glioma Downregulates X-Linked Inhibitor of Apoptosis Protein (XIAP)

Background

XIAP (X-linked inhibitor of apoptosis protein) is one of the most important members of the apoptosis inhibitor family. XIAP is upregulated in various malignancies, including human glioblastoma. It promotes invasion, metastasis, growth and survival of malignant cells. We hypothesized that downregulation of XIAP by human umbilical cord blood mesenchymal stem cells (hUCBSC) in glioma cells would cause them to undergo apoptotic death.

Methodology/Principal Findings

We observed the effect of hUCBSC on two malignant glioma cell lines (SNB19 and U251) and two glioma xenograft cell lines (4910 and 5310). In co-cultures of glioma cells with hUCBSC, proliferation of glioma cells was significantly inhibited. This is associated with increased cytotoxicity of glioma cells, which led to glioma cell death. Stem cells induced apoptosis in glioma cells, which was evaluated by TUNEL assay, FACS analyses and immunoblotting. The induction of apoptosis is associated with inhibition of XIAP in co-cultures of hUCBSC. Similar results were obtained by the treatment of glioma cells with shRNA to downregulate XIAP (siXIAP). Downregulation of XIAP resulted in activation of caspase-3 and caspase-9 to trigger apoptosis in glioma cells. Apoptosis is characterized by the loss of mitochondrial membrane potential and upregulation of mitochondrial apoptotic proteins Bax and Bad. Cell death of glioma cells was marked by downregulation of Akt and phospho-Akt molecules. We observed similar results under in vivo conditions in U251- and 5310-injected nude mice brains, which were treated with hUCBSC. Under in vivo conditions, Smac/DIABLO was found to be colocalized in the nucleus, showing that hUCBSC induced apoptosis is mediated by inhibition of XIAP and activation of Smac/DIABLO.

Conclusions/Significance

Our results indicate that downregulation of XIAP by hUCBSC treatment induces apoptosis, which led to the death of the glioma cells and xenograft cells. This study demonstrates the therapeutic potential of XIAP and hUCBSC to treat malignant gliomas.     

Adenoviral-mediated interleukin-18 expression in mesenchymal stem cells effectively suppresses the growth of glioma in rats

Glioma is the most common primary intracranial malignant tumor. Despite advances in surgical techniques and adjuvant radio- and chemotherapies, the prognosis for patients with glioma remains poor. We have explored the effects of using genetically modified mesenchymal stem cells (MSCs) to treat malignant glioma in rats. Mesenchymal stem cells isolated from Sprague-Dawley rats can directly suppress the growth of C6 cells in vitro. MSCs transplanted intratumorally can also significantly inhibit the growth of glioma and prolong survival in C6 glioma-bearing models. MSCs producing Interleukin-18 infected by adenoviral vector inhibited glioma growth and prolonged the survival of glioma-bearing rats. Transplantation of IL-18 secreting MSCs was associated with enhanced T cell infiltration and long-term anti-tumor immunity. Thus, IL-18 may be an effective adoptive immunotherapy for malignant glioma. When used in conjunction with MSCs as targeting vehicles in vivo, IL-18 may offer a promising new treatment option for malignant glioma.     

藏红花素对C6胶质瘤细胞生长及Survivin和Livin表达的影响

目的:研究藏红花素对大鼠C6胶质瘤细胞生长及凋亡蛋白抑制因子Survivin和Livin表达的影响。方法:体外培养C6胶质瘤细胞,加入不同浓度的藏红花素培养液,并于不同时间点进行观测,采用四甲基偶氮唑蓝(MTT)比色法绘制细胞生长曲线,观察C6细胞的生长活性;通过相差显微镜和Hoechst荧光染色法观察C6细胞的形态学变化;采用Western blot法检测Survivin和Livin蛋白的表达水平。结果:C6胶质瘤细胞经藏红花素作用后细胞生长受到明显抑制,用含2、4和8 mg/ml藏红花素的培养液作用48h后各组C6细胞的OD值分别为0.732±0.013、0.421±0.010和0.289±0.017,细胞生长抑制率分别为26.8±0.01%、58.0±0.02%和71.1±0.02%,其中4 mg/ml和8 mg/ml藏红花素实验组细胞生长抑制率与阴性对照组均有显著性差异(P均0.05);相差显微镜和Hoechst荧光染色法观察显示实验组C6细胞出现典型的凋亡形态学改变;Western blot检测显示实验组C6细胞Survivin和Livin蛋白表达明显下调。结论:藏红花素能明显抑制C6胶质瘤细胞的体外生长,其抑制作用与诱导C6细胞发生凋亡和下调凋亡蛋白抑制因子Survivin和Livin的表达有关。     

RNA interference-mediated silencing of focal adhesion kinase inhibits growth of human malignant glioma xenograft in nude mice

FAK (focal adhesion kinase), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human malignant glioma. The expression of FAK increases with the advance of tumour grade and stage. Based on these observations, we hypothesized that attenuation of FAK expression may have inhibitory effects on the growth of malignant glioma. In the present study, human glioma cell line U251 was transfected with plasmids containing U6 promoter-driven shRNAs (small-hairpin RNAs) against human FAK using cationic liposome. The effects of FAK knockdown in U251 cells in vitro were analysed by using flow cytometry and PI (propidium iodide)-staining assays. Based on the encouraging in vitro results with FAK silencing, plasmids encoding FAK-targeted shRNA were encapsulated by DOTAP (dioleoyltrimethylammonium propane):Chol (cholesterol) cationic liposome and injected via tail vein to evaluate its therapeutic efficiency on suppressing tumour growth in a human glioma xenograft model. PCNA (proliferating-cell nuclear antigen), CD34 immunostaining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay were used to assess the changes in tumour angiogenesis, apoptosis and proliferation respectively. The results indicated that DOTAP:Chol cationic liposome could deliver therapeutic plasmids systemically to tumour xenografts, resulting in suppression of tumour growth. Treatment with plasmid encoding FAK-targeted shRNA reduced mean tumour volume by approx. 70% compared with control groups (P<0.05), accompanied with angiogenesis inhibition (P<0.05), tumour cell proliferation suppression (P<0.05) and apoptosis induction (P<0.05). Taken together, our results demonstrated that shRNA-mediated silencing of FAK might be a potential therapeutic approach against human malignant glioma.     

Caudatin induces caspase-dependent apoptosis in human glioma cells with involvement of mitochondrial dysfunction and reactive oxygen species generation

Caudatin as one species of C-21 steroidal from Cynanchum bungei decne displays potential anticancer activity. However, the underlying mechanisms remain elusive. In the present study, the growth suppressive effect and mechanism of caudatin on human glioma U251 and U87 cells were evaluated in vitro. The results indicated that caudatin significantly inhibited U251 and U87 cell growth in both a time- and dose-dependent manner. Flow cytometry analysis revealed that caudatin-induced cell growth inhibition was achieved by induction of cell apoptosis, as convinced by the increase of Sub-G1 peak, PARP cleavage and activation of caspase-3, caspase-7 and caspase-9. Caudatin treatment also resulted in mitochondrial dysfunction which correlated with an imbalance of Bcl-2 family members. Further investigation revealed that caudatin triggered U251 cell apoptosis by inducing reactive oxygen species (ROS) generation through disturbing the redox homeostasis. Moreover, pretreatment of caspase inhibitors apparently weakens caudatin-induced cell killing, PARP cleavage and caspase activation and eventually reverses caudatin-mediated apoptosis. Importantly, caudatin significantly inhibited U251 tumour xenografts in vivo through induction of cell apoptosis involving the inhibition of cell proliferation and angiogenesis, which further validate its value in combating human glioma in vivo. Taken together, the results described above all suggest that caudatin inhibited human glioma cell growth by induction of caspase-dependent apoptosis with involvement of mitochondrial dysfunction and ROS generation.     

The expression of the phosphotyrosine phosphatase DEP-1/PTPeta dictates the responsivity of glioma cells to somatostatin inhibition of cell proliferation

Here we characterize the intracellular effectors of the antiproliferative activity of somatostatin in glioma cell lines and post-surgical specimens. The responsiveness to somatostatin correlated with the expression of the phosphotyrosine phosphatase DEP-1/PTPeta, identified in C6 and U87MG cells, in which somatostatin inhibited cell growth. The expression of a dominant negative mutant of DEP-1/PTPeta in C6 cells abolished somatostatin effects, confirming the involvement of this phosphotyrosine phosphatase in such effects. Somatostatin treatment increased the activity of DEP-1/PTPeta and inhibited ERK1/2 activation. Conversely, basic fibroblast growth factor-dependent MEK phosphorylation was not affected, suggesting a direct effect on ERK1/2. In vitro experiments showed that PTPeta was able to interact and dephosphorylate ERK1/2 activated by basic fibroblast growth factor. Furthermore, by transfecting PTPeta in the somatostatin-unresponsive, DEP-1/PTPeta-deficient U373MG cells, the somatostatin-dependent control of cell proliferation was recovered. Finally we evaluated the requirement for DEP-1/PTPeta in somatostatin inhibition of cell proliferation in post-surgical specimens derived from different grade human gliomas. Although all of the glioma analyzed expressed somatostatin receptor mRNA, DEP-1/PTPeta expression was limited to 8 of 22 of the tumors. Culturing seven gliomas, a correlation between the expression of DEP-1/PTPeta and the somatostatin antiproliferative effects was identified. In conclusion we propose that the expression and activation of DEP-1/PTPeta is required for somatostatin inhibition of glioma proliferation.     

Ivermectin inhibits the growth of glioma cells by inducing cell cycle arrest and apoptosis in vitro and in vivo

Glioma, the most predominant primary malignant brain tumor, remains uncured due to the absence of effective treatments. Hence, it is imperative to develop successful therapeutic agents. This study aimed to explore the antitumor effects and mechanisms of ivermectin (IVM) in glioma cells in vitro and in vivo. The effects of IVM on cell viability, cell cycle arrest, apoptosis rate, and morphological characteristics were determined respectively by MTT assay/colony formation assay, flow cytometry, and transmission electron microscope. In addition, the expression levels of cycle-related and apoptosis-associated proteins were individually examined by Western blot analysis. Moreover, cell proliferation and apoptosis analyses were carried out by TUNEL, Ki-67, cleaved caspase-3, and cleaved caspase-9 immunostaining assay. Our results demonstrated that IVM has a potential dosage-dependent inhibition effect on the apoptosis rate of glioma cells. Meanwhile, the results also revealed that IVM induced apoptosis by increasing caspase-3 and caspase-9 activity, upregulating the expressions of p53 and Bax, downregulating Bcl-2, activating cleaved caspase-3 and cleaved caspase-9, and blocking cell cycle in G0/G1 phase by downregulating levels of CDK2, CDK4, CDK6, cyclin D1, and cyclin E. These findings suggest that IVM has an inhibition effect on the proliferation of glioma cells by triggering cell cycle arrest and inducing cell apoptosis in vitro and in vivo, and probably represents promising agent for treating glioma.