Human Astrocytoma Research Articles

ZNF545 suppresses human hepatocellular carcinoma growth by inhibiting NF-kB signaling.

Hepatocellular carcinoma (HCC) is one of the most common cancers and the second leading cause of cancer related death worldwide. ZNF545 is located in the chromosome 19q13.13, which is frequent loss of heterozygosity in human astrocytoma. Methylation of ZNF545 was found frequently in a few kinds of cancers. While the function of ZNF545 in human HCC remains unclear. The purpose of this study is to explore the function and mechanism of ZNF545 in human HCC. Restoration of ZNF545 expression suppressed cell proliferation, migration and invasion, induced G1/S arrest and apoptosis in SNU449 and Huh7 cells. Further study suggested that ZNF545 suppressed HCC cell growth by inhibiting NF-kB signaling. These results were further validated by siRNA knocking down technique in ZNF545 highly expressed HXBF344 cells. In vivo, ZNF545 suppressed tumor growth in SNU449 cell xenograft mice. In conclusion, ZNF545 suppresses human HCC growth by inhibiting NF-kB signaling.

Identification of novel selective P2Y6 receptor antagonists by high-throughput screening assay

AimsThe P2Y6 nucleotide receptor is widely involved in inflammatory responses, and is a promising molecular target for the treatment of inflammatory diseases. Although several P2Y6 receptor antagonists have been developed and evaluated thus far, none has successfully been developed into a therapeutic drug. In this study, we explored new promising compounds that inhibit the human P2Y6 receptor. Main methodsHigh-throughput screening (HTS) was used to study the effects of various compounds on human P2Y6 receptors expressed in 1321N1 human astrocytoma cells by monitoring intracellular Ca2+ concentration ([Ca2+]i) levels using an FDSS7000 real-time fluorescence detector. IL-8 concentration was measured by enzyme-linked immunosorbent assay. Key findingsAmong structurally diverse chemical libraries totalling 141,700 compounds, 43 compounds with an inhibitory activity against the P2Y6 receptor were identified. Further studies using a dose-response assay, receptor selectivity assay, and chemokine measurement assay revealed the selective P2Y6 receptor inhibitor TIM-38, which inhibited UDP-induced [Ca2+]i elevation in a dose-dependent manner. TIM-38 had an IC50 value of 4.3μM and inhibited P2Y6 without affecting the response induced by four other human P2Y or muscarinic receptors. In addition, TIM-38 inhibited UDP-induced interleukin-8 release in a dose-dependent manner without affecting releases caused by other stimulus such as interleukin-1β or tumour necrosis factor-α. Analyses of TIM-38 derivatives revealed that the nitro moiety is vital to P2Y6 receptor inhibition. SignificanceTIM-38 acts as a novel structural antagonist of P2Y6 receptor and may be a good lead compound for developing a P2Y6 receptor-targeted anti-inflammatory drug.

Anti‑inflammatory actions of gabapentin and pregabalin on the substance P‑induced mitogen‑activated protein kinase activation in U373 MG human glioblastoma astrocytoma cells.

Gabapentin (GBP) and pregabalin (PGB) exert antinociceptive effects on chronic nociceptive responses with neuropathic or inflammatory conditions. Furthermore, it is considered that GBP and PGB exhibit anti‑inflammatory effects by modulating the substance P (SP)‑mediated neurokinin‑1 receptor (NK1R; a SP receptor) response. Thus, in the present study, the effects of GBP and PGB on SP‑induced activation were investigated in the human glioblastoma astrocytoma U373 MG cell line, which expresses high levels of functional high‑affinity NK1R, and produces interleukin (IL)‑6 and IL‑8 in response to SP. The results indicated that GBP and PGB suppressed the SP‑induced production of IL‑6, and IL‑8 in U373 MG cells. Furthermore, GBP and PGB inhibited the SP‑induced phosphorylation of p38 mitogen‑activated protein kinase (MAPK) and nuclear factor (NF)‑κB, and the nuclear translocation of NF‑κB in U373 MG cells. Together, these observations suggest that GBP and PGB likely prevent SP‑induced IL‑6 and IL‑8 production in U373 MG cells via the inhibition of signaling molecules, including p38 MAPK and NF‑κB, thereby exhibiting antineuroinflammatory effects.

AXL Promotes Zika Virus Infection in Astrocytes by Antagonizing Type I Interferon Signaling Pathway

Abstract Zika virus (ZIKV) is known to be associated with neonate microcephaly and Guillain-Barré syndrome in human adults. Despite the progress on understanding the causal link between ZIKV infection and microcephaly, the life cycle and pathogenesis of ZIKV remains elusive. In particular, recent studies have revealed a controversial role of AXL, a TAM family kinase receptor previously deemed as the entry receptor of ZIKV. In this study, we show that though genetic ablation of AXL protects primary human astrocytes and U-251MG astrocytoma cells from ZIKV infection, AXL knockout (KO) does not block the entry of ZIKV, instead it facilitates the up-regulation of type I interferon (IFN) signaling genes, including interferon regulatory factor 7 (IRF7), a key interferon transcriptional factor, and a number of type I interferon and interferon stimulating genes (ISGs). Knockout of type I IFN receptor a chain (IFNAR1) rescues the vulnerability of AXL KO astrocytes to ZIKV infection. In addition, AXL KO in astrocytes results in the down-regulation of SOCS1 in primary astrocytes and the failure to retain the homeostasis of SOCS1 in astrocyte cell lines, a known inhibitor of type I IFN signaling pathway, thus revealing important roles of AXL in maintaining the homeostasis of SOCS1 and thereby in containing type I interferon signaling pathway. Taken together, our study demonstrates that AXL does not function as the sole, if at all, entry receptor of ZIKV, but instead promotes ZIKV infection in astrocytes by SOCS1-mediated antagonism of type I interferon (IFN) signaling.

Ultrastructural characterization of the Mitochondria-associated membranes abnormalities in human astrocytomas: Functional and therapeutics implications

ABSTRACTMitochondria-associated membranes (MAMs) are currently considered an intracellular organelle “hot spot” for the intracellular signaling. MAMs are thought to function in cellular energy homeostasis, apoptosis, and calcium signaling. MAM ultrastructure in surgical specimens from human astrocytic neoplasms was studied. Abnormalities in respect to density, length, and width were found. Poorly differentiated glioma like-stem cells deficient in MAM and well-differentiated glioma cells abundant in MAM were observed. This finding could be the structural basis of functional role of MAM linked to some metabolic abnormalities in astrocytic tumors associated to mitochondrial dysfunction and the Warburg effect and their therapeutics implications.

Mevalonate Cascade Inhibition by Simvastatin Induces the Intrinsic Apoptosis Pathway via Depletion of Isoprenoids in Tumor Cells

The mevalonate (MEV) cascade is responsible for cholesterol biosynthesis and the formation of the intermediate metabolites geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) used in the prenylation of proteins. Here we show that the MEV cascade inhibitor simvastatin induced significant cell death in a wide range of human tumor cell lines, including glioblastoma, astrocytoma, neuroblastoma, lung adenocarcinoma, and breast cancer. Simvastatin induced apoptotic cell death via the intrinsic apoptotic pathway. In all cancer cell types tested, simvastatin-induced cell death was not rescued by cholesterol, but was dependent on GGPP- and FPP-depletion. We confirmed that simvastatin caused the translocation of the small Rho GTPases RhoA, Cdc42, and Rac1/2/3 from cell membranes to the cytosol in U251 (glioblastoma), A549 (lung adenocarcinoma) and MDA-MB-231(breast cancer). Simvastatin-induced Rho-GTP loading significantly increased in U251 cells which were reversed with MEV, FPP, GGPP. In contrast, simvastatin did not change Rho-GTP loading in A549 and MDA-MB-231. Inhibition of geranylgeranyltransferase I by GGTi-298, but not farnesyltransferase by FTi-277, induced significant cell death in U251, A549, and MDA-MB-231. These results indicate that MEV cascade inhibition by simvastatin induced the intrinsic apoptosis pathway via inhibition of Rho family prenylation and depletion of GGPP, in a variety of different human cancer cell lines.

Infective capacity of Cryptococcus neoformans and Cryptococcus gattii in a human astrocytoma cell line.

Pathogenesis of cryptococcosis in the central nervous system (CNS) is a topic of ongoing research, including the mechanisms by which this fungus invades and infects the brain. Astrocytes, the most common CNS cells, play a fundamental role in the local immune response. Astrocytes might participate in cryptococcosis either as a host or by responding to fungal antigens. To determine the infectivity of Cryptococcusneoformans var. grubii and Cryptococcusgattii in a human astrocytoma cell line and the induction of major histocompatibility complex (MHC) molecules. A glioblastoma cell line was infected with C.neoformans var. grubii and C.gattii blastoconidia labelled with FUN-1 fluorescent stain. The percentage of infection and expression of HLA class I and II molecules were determined by flow cytometry. The interactions between the fungi and cells were observed by fluorescence microscopy. There was no difference between C.neoformans var. grubii and C.gattii in the percentage infection, but C.neoformans var. grubii induced higher expression of HLA class II than C.gattii. More blastoconidia were recovered from C.neoformans-infected cells than from C. gattii infected cells. Cryptococcus neoformans var. grubii may have different virulence mechanisms that allow its survival in human glia-derived cells.

Serum amyloid A1 is upregulated in human glioblastoma.

Serum amyloid A1 (SAA1) is a sensitive acute phase reactant primarily produced by the liver in response to acute inflammation. We have recently shown that SAA affects proliferation, migration, and invasion of glioblastoma cell lines, which suggest its participation in the malignant process. Consistently, levels of SAA have been used as a non-invasive biomarker for the prognosis of many cancers. In this study, we aimed to investigate SAA serum levels and expression of SAA genes in human astrocytomas tissues. Serum and tissue samples were obtained from patients with astrocytoma grades I to III and glioblastoma (GBM or grade IV). Levels of circulating SAA were significantly higher in the serum of patients with AGII-IV when compared to non-neoplastic samples derived from non-neoplastic patients (NN) (p > 0.0001). Quantitative real time PCR (qRT-PCR) of 148 astrocytomas samples (grades I-IV) showed that SAA1 mRNA was significantly higher in GBM when compared to AGI-III and NN samples (p < 0.0001). Immunohistochemistry analysis revealed cytoplasmic positivity for SAA in GBM. There was no correlation of SAA1 with clinical end-point of overall survival among GBM patients. However, it was found a positive correlation between SAA1 and genes involved in tumor progression, such as: HIF1A (r = 0.50; p < 0.00001), CD163 (r = 0.52; p < 0.00001), CXCR4 (r = 0.42; p < 0.00001) and CXCR7 (r = 0.33; p = 0.002). In conclusions, we show that astrocytoma patients have increased levels of serum SAA and SAA1 is expressed and secreted in GBM, and its co-expression with tumor-related genes supports its involvement in GBM angiogenesis and progression.

Cadmium-induced IL-6 and IL-8 expression and release from astrocytes are mediated by MAPK and NF-κB pathways

Chronic exposure to cadmium has been linked to brain cancers, learning disabilities and memory deficits. Previous studies of cadmium toxicity in the central nervous system report cadmium induces oxidative stress in neurons and astrocytes. In the peripheral system, cadmium promotes interleukin-6 (IL-6) and IL-8 production and release. Elevation of IL-6 expression is linked to the pathogenesis of neurodegenerative diseases and astrogliosis. IL-8 plays a role in angiogenesis of gliomas and neurodegenerative diseases. Herein, the effects of non-toxic concentrations of cadmium on the production of IL-6 and IL-8 and the underlying mechanisms were investigated. U-87 MG human astrocytoma cells and primary human astrocytes were exposed to cadmium chloride. At 24h post-exposure to 1 and 10μM, levels of intracellular cadmium in U-87 MG cells were 11.89±3.59 and 53.08±7.59μg/g wet weight, respectively. These concentrations had minimal effects on cell morphology and viability. IL-6 and IL-8 mRNA levels and secretion increased in dose- and time-dependent manners post cadmium exposure. Acute exposure to cadmium increased phosphorylation of ERK1/2, p38 MAPK, and p65 NF-κB. Pretreatment with U0126–an inhibitor of MEK1 and MEK2 kinases–SB203580–a p38 MAPK inhibitor–and SC-514–an IKKβ inhibitor–suppressed cadmium-induced IL-8 expression and release. Upregulation of cadmium-induced IL-6 was inhibited by U0126 and SC-514, but not SB203580. On the other hand, SP600125–a JNK inhibitor–and celecoxib–a selective COX-2 inhibitor–had no effect on production of both cytokines. In conclusion, non-toxic concentrations of cadmium can stimulate IL-6 and IL-8 release through MAPK phosphorylation and NF-κB activation. Suppressing IL-6 and IL-8 production could be novel approaches to prevent cadmium-induced angiogenesis in gliomas and inflammation in the brain.

IL-17 induces reactive astrocytes and up-regulation of vascular endothelial growth factor (VEGF) through JAK/STAT signaling

Spinal cord injury is a grave neurological disability resulting in neuron degeneration and permanent paralysis. The inflammation triggered by the injury would promote the spinal cord lesion in turn. Activated astrocytes during inflammatory response could promote glial scar formation and contribute to the progression of the spinal cord injury. Interleukin 17 (IL-17) was upregulated in inflammatory responses to contusion or compression of the spinal cord. in this study, IL-17 could induce reactive astrocytes which was indicated by a well-known hallmark glial fibrillary acidic protein (GFAP) in vitro and in vivo. Moreover, we demonstrated that the upregulation of VEGF was induced by IL-17 human astrocytoma cells. In our further investigation, IL-17 induced the expression of VEGF in spinal cord injury by activating JAK/STAT signaling pathway both in vitro and in vivo. In addition, we also found that IL-17 significantly changed tissue preservation and residual urine volumes and blood-spinal cord-barrier integrity in vivo. This newly found IL-17-JAK/STAT-VEGF axis improves our understanding of the molecular mechanism of spinal cord injury during inflammatory response and provides another potential target of spinal cord injury.

A dangerous liaison: Leptin and sPLA2-IIA join forces to induce proliferation and migration of astrocytoma cells.

Glioblastoma, the most aggressive type of primary brain tumour, shows worse prognosis linked to diabetes or obesity persistence. These pathologies are chronic inflammatory conditions characterized by altered profiles of inflammatory mediators, including leptin and secreted phospholipase A2-IIA (sPLA2-IIA). Both proteins, in turn, display diverse pro-cancer properties in different cell types, including astrocytes. Herein, to understand the underlying relationship between obesity and brain tumors, we investigated the effect of leptin, alone or in combination with sPLA2-IIA on astrocytoma cell functions. sPLA2-IIA induced up-regulation of leptin receptors in 1321N1 human astrocytoma cells. Leptin, as well as sPLA2-IIA, increased growth and migration in these cells, through activation/phosphorylation of key proteins of survival cascades. Leptin, at concentrations with minimal or no activating effects on astrocytoma cells, enhanced growth and migration promoted by low doses of sPLA2-IIA. sPLA2-IIA alone induced a transient phosphorylation pattern in the Src/ERK/Akt/mTOR/p70S6K/rS6 pathway through EGFR transactivation, and co-addition of leptin resulted in a sustained phosphorylation of these signaling regulators. Mechanistically, EGFR transactivation and tyrosine- and serine/threonine-protein phosphatases revealed a key role in this leptin-sPLA2-IIA cross-talk. This cooperative partnership between both proteins was also found in primary astrocytes. These findings thus indicate that the adipokine leptin, by increasing the susceptibility of cells to inflammatory mediators, could contribute to worsen the prognosis of tumoral and neurodegenerative processes, being a potential mediator of some obesity-related medical complications.

Surveying Mutagenic and Anti-mutagenic Effects of Citrus Limon

Background and objective: Cancer is one of the main causes of mortality in the world and countless mutagenic chemical compound kill millions of cancer patients every year. Scientists are searching for natural foodstuff to prevent cancer. The present paper examines anti-mutagenic and anti-carcinogenic effects of citrus lemon.Methodology: Human astrocytoma cancerous cell line was cultured in DMEM (Gibco) with FBS10% (fetal bovine serum), L-glutamine, penicillin, and streptomycin at 37ºC for two days. Then the samples were treated with citrus lemon juice and biotic potential of cells was examined through the MTT method. Anti-mutagenic and anti-carcinogenic effects of citrus lemon were studied through back mutation assessment standard method (Ames test). The test was done using Salmonella typhimurium strain (TA100) with the mutation in histidine operon that needs external histidine source. The strain creates the reverse colony in presence of carcinogenic agents (sodium azide). Findings: Based on the MTT method, human astrocytoma cancerous cell line demonstrated significant cellular death compared with the control groups (p&lt;0.01). Ames test showed that the juice prevented back mutation and rate of inhibition, based on the anti-mutagenic effect, of half-ripen and ripen citrus lemon was 71.7% and 34.3% respectively. In addition, the anti-carcinogenic effect of half-ripen and ripen citrus lemon were 83.3% and 50% respectively. Conclusion: Anti-mutagenic and anti-carcinogenic effects of citrus lemon were examined for the first time. The results showed that in this regard half-ripen citrus lemon was more effective than ripening citrus lemon.&#x0D;

Ketamine suppresses the substance P-induced production of IL-6 and IL-8 by human U373MG glioblastoma/astrocytoma cells.

The neuropeptide substanceP(SP) is an important mediator of neurogenic inflammation within the central and peripheral nervous systems. SP has been shown to induce the expression of pro-inflammatory cytokines implicated in the pathogenesis of several disorders of the human brain via the neurokinin-1receptor(NK-1R). Ketamine, an intravenous anesthetic agent, functions as a competitive antagonist of the excitatory neurotransmission N-methyl-D‑aspartate(NMDA) receptor, and also antagonizes the NK-1R by interfering with the binding of SP. In the present study, we investigated the anti-inflammatory effects of ketamine on the SP-induced activation of a human astrocytoma cell line, U373MG, which expresses high levels of NK-1R. The results from our experiments indicated that ketamine suppressed the production of interleukin(IL)-6 and IL-8 by the U373MGcells. Furthermore, ketamine inhibited the SP-induced activation of extracellular signal‑regulated kinase(ERK)1/2, p38mitogen-activated protein kinase(MAPK) and nuclear factor-κB(NF-κB). Taken together, these observations suggest that ketamine may suppress the SP-induced activation(IL-6 and IL-8 production) of U373MGcells by inhibiting the phosphorylation of signaling molecules(namely ERK1/2, p38MAPK and NF-κB), thereby exerting anti‑inflammatory effects. Thus, ketamine may modulate SP-induced inflammatory responses by NK-1R‑expressing cells through the suppression of signaling molecules(such as ERK1/2, p38MAPK and NF-κB).

Tbx2 confers poor prognosis in glioblastoma and promotes temozolomide resistance with change of mitochondrial dynamics

Tbx2 is a cancer-related protein that was found to be overexpressed in several human malignancies. The present study aims to investigate the clinical significance and biological role of Tbx2 in human astrocytoma. We examined its protein expression in 102 cases of astrocytoma tissues using immunohistochemical staining. Negative Tbx2 staining was observed in normal astrocytes, and positive nuclear staining was found in 41 out of 102 astrocytoma specimens. The rate of Tbx2 overexpression in pylocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, and glioblastoma multiform (GBM) were 0%, 26.1%, 40%, and 52%, respectively. Tbx2 overexpression correlated with poor prognosis in patients with astrocytoma or GBM. Tbx2 plasmid transfection was performed in A172 cells, and Tbx2 siRNA knockdown was carried out in U251 cells. Cell Counting Kit-8, cell cycle analysis, and matrigel invasion assay showed that Tbx2 overexpression upregulated cell proliferation, G1-S transition, and invasion, with corresponding change of cyclin D1, p21, and MMP 2 and 9. Importantly, we demonstrated that Tbx2 reduced apoptosis and conferred resistance to temozolomide in GBM cell lines. Further experiments showed that Tbx2 could regulate mitochondrial fission/fusion balance. Western blot showed that Tbx2 overexpression reduced caspase 3 cleavage, while it induced Bcl-2 and p-Drp1 upregulation. In conclusion, our results indicated that Tbx2 might serve as an indicator for poor prognosis and also be useful as an important therapeutic in human GBM, which inhibits apoptosis through regulation of mitochondrial function.

Induction of cellular proliferation in a human astrocytoma cell line by a Trypanosoma cruzi-derived antigen: a mechanism of pathogenesis?

Trypanosoma cruzi can compromise the human central nervous system (CNS) during acute infection or reactivation in immune-suppressed hosts. Astrocytes have been identified as targets of T. cruzi's CNS infection in humans. Despite a high degree of parasitism and cellular lysis by T. cruzi in vitro the number of astrocytoma cells did not change when compared to uninfected cultures. This work evaluated cellular proliferation, changes in Major Histocompatibility Complex (MHC) expression as a reflection of antigen processing, and cytokine (IL-6 &amp; IL-8) secretion in a human astrocytoma cell line exposed to a trypomastigote-derived antigen. Light microscopy was used to evaluate the number of cells; MHC molecule expression, cell cycle and cytokine secretion were assessed by flow cytometry. The number of astrocytoma cells increased proportional to the amount of antigen used and the percentage of cells in G2/M phase was higher when compared to control cultures. Antigen exposure increased expression of MHC class II, but not MHC class I in comparison to cultures incubated without antigen. Astrocytoma cell secretion of IL-6 and IL-8 was unaffected by antigen exposure. These results suggest the participation of a trypomastigote-derived mediator that induces astrocytoma cell proliferation without an inflammatory response; which may contribute to the pathogenesis of neurologic Chagas disease.

Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

Interferon (IFN)-induced protein 35 (IFI35) negatively regulates IFN-β-phosphorylated STAT1-RIG-I-CXCL10/CCL5 axis in U373MG astrocytoma cells treated with polyinosinic-polycytidylic acid

Interferon (IFN)-stimulated genes (ISGs) exert multiple functions in immune system. IFN-induced protein 35 (IFI35) is a member of ISGs, and has been suggested to regulate innate immune reaction. However, the physiological functions and pathological roles of IFI35 in the central nervous system are not characterized well. In the present study, we found that the expression of IFI35 was induced by a Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid (poly IC) in U373MG human astrocytoma cells. Knockdown of IFI35 using RNA interference resulted in increased expression of IFN-β, phosphorylated STAT1 (P-STAT1), retinoic acid-inducible gene-I (RIG-I), CXCL10 and CCL5 induced by poly IC. Poly IC-induced expression of CXCL10 and CCL5 was decreased by knockdown of RIG-I. These results suggest that IFI35 may negatively regulate the TLR3-IFN-β-P-STAT1-RIG-I-CXCL10/CCL5 axis in U373MG cells, and IFI35 may play a role at least partially in the regulation of innate immune reactions in astrocytes.

Proliferative and Invasive Effects of Progesterone-Induced Blocking Factor in Human Glioblastoma Cells.

Progesterone-induced blocking factor (PIBF) is a progesterone (P4) regulated protein expressed in different types of high proliferative cells including astrocytomas, the most frequent and aggressive brain tumors. It has been shown that PIBF increases the number of human astrocytoma cells. In this work, we evaluated PIBF regulation by P4 and the effects of PIBF on proliferation, migration, and invasion of U87 and U251 cells, both derived from human glioblastomas. PIBF mRNA expression was upregulated by P4 (10 nM) from 12 to 24 h. Glioblastoma cells expressed two PIBF isoforms, 90 and 57 kDa. The content of the shorter isoform was increased by P4 at 24 h, while progesterone receptor antagonist RU486 (10 μM) blocked this effect. PIBF (100 ng/mL) increased the number of U87 cells on days 4 and 5 of treatment and induced cell proliferation on day 4. Wound-healing assays showed that PIBF increased the migration of U87 (12–48 h) and U251 (24 and 48 h) cells. Transwell invasion assays showed that PIBF augmented the number of invasive cells in both cell lines at 24 h. These data suggest that PIBF promotes proliferation, migration, and invasion of human glioblastoma cells.

Correlative analyses of the expression levels of PIAS3, p-SHP2, SOCS1 and SOCS3 with STAT3 activation in human astrocytomas.

The importance of signal transducer and activator of transcription3 (STAT3) signaling in the growth and survival of glioblastoma cells has been well documented, while the reasons leading to STAT3 activation remains to be elucidated. Suppressors of cytokine signaling (SOCS)1 and SOCS3, SH2 domain‑containing phosphatase (SHP2) and protein inhibitors of activated STAT3 (PIAS3) are known to inhibit STAT3 signal transduction, while their expression statuses in the four grades of astrocytomas and relevance with STAT3 activation remain to be described. The present study aimed to address these issues by tissue microarray‑based immunohistochemical profiling the expression levels of phosphorylated (p)‑STAT3, SOCS1, SOCS3, PIAS3 and p‑SHP2. The results revealed that p‑STAT3 nuclear translocation was rarely observed in non‑cancerous brain tissues and its frequencies were increased in a tumor grade‑associated manner (65.2, 77.1, 81.8 and 85.7% for gradeI‑IV, respectively). PIAS3, p‑SHP2, SOCS1 and SOCS3 were expressed in higher levels (++ and +++) in 63.6, 90, 87.5 and 81.8% of tumor surrounding brain tissues, which reduced to 13.1, 47.8, 33.3 and 50% in gradeI, 11.4, 65.7, 58.3 and 77.1% in gradeII, 9.1, 63.6, 38.1 and 31.8% in gradeIII and 7.1, 66.7, 30.8 and 7.1% in gradeIV astrocytomas. The above results revealed that although the expression levels of SOCS1, SOCS3 and, in particular, p‑SHP2, tend to decrease in the four types of astrocytomas, PIAS3 downregulation is more negatively correlated with STAT3 activation in the stepwise progress of astrocytomas and would indicate an unfavorable outcome.

Region-Specific Dok2 Overexpression Associates with Poor Prognosis in Human Astrocytoma.

Astrocytoma is the most frequent malignancies of the brain. Despite present clinical advancements, median survival time in malignant forms remains poor. Downstream of kinase protein 2 (Dok2) is adaptor protein known to modulate the effect of tyrosine kinase. Previously, Dok2 is shown to be marker of poor prognosis in colorectal and gastric cancer, and reduced levels of Dok2 were reported in lung adenocarcinoma and gastric cancer. The aim of the present study was to evaluate prognostic significance of pDok2 expression in surgically resected astrocytoma tissue samples. In the present study, 47 numbers of tissue samples were collected from patients who underwent surgery for astrocytoma. Temporal lobe epilepsy tissues were used as control. Real-time PCR was used to study transcript expression while protein expression was studied by western blotting and immunohistochemistry. The pDok2 expression was categorized as pDok2 positive and pDok2 negative on the basis of intensity of protein expression. This observation was confirmed by two independent pathologists. Control and few GII tissues were used as reference on account for low expression of pDok2 protein. Basic information of patients as anatomic origin of tumor and follow-up details were retrieved from hospital registry. Kaplan-Meier test was used to analyze the association of pDok2 expression and survival outcome in clinical cases. Real-time PCR signifies pDok2 is overexpressed in high-grade (GIII+GIV) tissue samples compared with low-grade (GII) and control brain tissue samples (p<0.005). Western blotting and immunohistochemistry analysis signifies overexpression of pDok2 protein expression in tumor tissue samples as compared with control brain tissues. Clinico-pathological analysis reveals 83% of high-grade astrocytoma (GIII+GIV) and 30% of low-grade (GII) tissue samples which were detected with pDok2 expression. Tumor location was found to be predominant at the frontal and temporal lobes. Survival studies underline prognostic importance of pDok2 protein. Median survival of 20months was reported with patients with positive pDok2 expression (95% CI 0.083 to 0.49). Taken together, pDok2 protein overexpression is associated with poor prognosis in astrocytoma clinical cases and appears to be an attractive target for therapeutic intervention. Noticeable anatomic origin at the frontal and temporal lobe suggests site-specific role of developmental factors in tumor occurrence.