Human Glioblastoma Multiforme Cells Research Articles

Tricetin suppresses the migration/invasion of human glioblastoma multiforme cells by inhibiting matrix metalloproteinase-2 through modulation of the expression and transcriptional activity of specificity protein 1

Objective: Glioblastoma multiforme (GBM) is a severely invasive tumor that can be fatal because it is difficult to treat. Tricetin, a natural flavonoid, was demonstrated to inhibit the growth of various cancers, but the effect of tricetin on cancer motility is largely unknown.Research design and methods: In the present study, we examined the anti-invasive properties of tricetin in huwman GBM cells.Results: Our results showed that tricetin inhibited the migration/invasion of two GBM cell lines. We found that tricetin inhibited MMP-2 expression in the GBM cells. Real-time polymerase chain reaction and promoter activity assays indicated that tricetin inhibited MMP-2 expression at the transcriptional level. Such inhibitory effects were associated with the suppression of specificity protein-1 (SP-1) DNA-binding activity. An examination of clinical samples revealed a positive correlation between SP-1 and MMP-2 in glioma specimens, and higher expression levels were correlated with a worse probability of survival. Moreover, blocking the extracellular signal-regulated kinase (ERK) pathway also inhibited MMP-2-mediated cell motility, and further enhanced the anti-invasive ability of tricetin in GBM cells.Conclusions: SP-1 is an important target of tricetin for suppressing MMP-2-mediated cell motility in GBM cells, and a combination of tricetin and an ERK inhibitor may be a good strategy for preventing GBM invasion.

Synthesis, characterization, and anticancer activities of lipophilic pyridinecarboxaldimine platinum(II) complexes

We have prepared eleven pyridinecarboxaldimines (N-N’R) from the condensation of 2-pyridinecarboxaldehyde and the corresponding lipophilic primary amines. Addition of these ligands to [PtCl2(η2-coe)]2 (coe=cis-cyclooctene) gave complexes of the type cis-PtCl2(N-N’R) (2a-k) in moderate to high yields. The molecular structure of the N-butylphenyl derivative (2h) has been confirmed by an X-ray diffraction study. Crystals of 2h were monoclinic with a=8.5414(9)Å, b=11.6774(12)Å, c=16.1235(16)Å, β=92.7610(10)° in the space group P2(1)/c. All platinum complexes were examined for their in vitro cytotoxic properties against two human glioblastoma multiforme cell lines LN18 and LN405. The DNA binding properties of one of the most cytotoxic compounds was compared with that of cisplatin, but no effect was observed under the conditions tested.

Physical and Electrochemical Properties of PEDOT:PSS as a Tool for Controlling Cell Growth.

PSS), are prepared using two different techniques, spin coating and electrochemical polymerization, and their oxidation state is subsequently changed electrochemically with the application of an external bias. The electrochemical properties of these different types of PSS are studied through cyclic voltammetry and spectrophotometry to assess the effectiveness of the oxidation process and its stability over time. Their surface physical properties and their dependence on the redox state of PSS are investigated using atomic force microscopy (AFM), water contact angle goniometry and sheet resistance measurements. Finally, human glioblastoma multiforme cells (T98G) and primary human dermal fibroblasts (hDF) are cultured on PSS films with different oxidation states, finding that the effect of the substrate on the cell growth rate is strongly cell-dependent: T98G growth is enhanced by the reduced samples, while hDF growth is more effective only on the oxidized substrates that show a strong chemical interaction with the cell culture medium.

Abstract 4548: Simultaneous hyperthermic-chemotherapy for glioblastoma using a single anti-cancer compound with intrinsic magnetism

Abstract Background: Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor in humans. In spite of advances in treatment strategies, survival rate of GBM remains poor. We have recently reported a novel anti-cancer compound with intrinsic magnetism(EI236). In addition to its anti-cancer effect, EI236 generates a large amount of heat upon an alternating magnetic field (AMF) application because of its magnetism, i.e., hyperthermic effect. In this study, we have examined the effect of EI236 on human GBM in vitroand vivo. Materials and Methods: Human GBM cell lines, U251MG, YKG-1, and U87, were used in this study. Cell proliferation was assessed by methyl thiazolyl tetrazorium (MTT) assays in the presence of EI236, in comparison to temozolomide (TMZ) and calmustine (BCNU), i.e. the clinical standard drugs for GBM. Apoptosis was analyzed by fluorescence activated cells sorting. In addition, we have examined whether EI236 inhibited the tumor growth with/without AMF application in animal models. Tumors were induced by implantation of U251MG cells into the hip of female Balb-c mice. Seven days after the implantation of GBM cells, EI236, BCNU or normal saline (control) was injected respectively into the tumors and mice were exposed to an AMF application(330.8A, 280kHz) for 30 minutes. Therefore, we calculated the tumor volume and the regression rate of tumors twice a week. Result: EI236 inhibited the proliferation of all cell lines and increased apoptosis in a dose dependent manner. Moreover, we found that EI236 had much greater anti-cancer effect than the clinical standard drugs. Mice bearing GBM cancer cells in their hip were then subjected to a double combination therapy, consisting of local injection of EI236 and hyperthermic treatment by AMF application. Tumor sizes in both drug stimulation groups were reduced, especially that in EI236 with AMF application group was highly reduced, while that in control group was increased. Conclusion: EI236 has greater anti-cancer effect than TMZ and BCNU, i.e. the clinical standard drugs. Besides this strong anti-cancer effect, EI236 has hyperthermic effect upon an AMF application because of heat generation. Accordingly, EI236 may enable us to develop novel strategies in GBM treatment, i.e. hyperthermic-chemotherapy with a single-drug compound. Note: This abstract was not presented at the meeting. Citation Format: Makoto Ohtake, Masanari Umemura, Itaru Sato, Kayoko Oda, Akane Nagasako, Ayako Makino, Haruki Aoyama, Mayumi Katsumata, Haruki Eguchi, Nobutaka Kawahara, Yoshihiro Ishikawa. Simultaneous hyperthermic-chemotherapy for glioblastoma using a single anti-cancer compound with intrinsic magnetism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4548. doi:10.1158/1538-7445.AM2015-4548

Control of the Survival and Growth of Human Glioblastoma Grafted Into the Spinal Cord of Mice by Taking Advantage of Immunorejection.

Recent studies have demonstrated that transplantation of induced pluripotent stem cell-derived neurospheres can promote functional recovery after spinal cord injury in rodents, as well as in nonhuman primates. However, the potential tumorigenicity of the transplanted cells remains a matter of apprehension prior to clinical applications. As a first step to overcome this concern, this study established a glioblastoma multiforme xenograft model mouse. The feasibility of controlling immune suppression to ablate the grafted cells was then investigated. The human glioblastoma multiforme cell line U251 MG was transplanted into the intact spinal cords of immunodeficient NOD/SCID mice or into those of immunocompetent C57BL/6J H-2kb mice treated with or without immunosuppressants [FK506 plus anticluster of differentiation (CD) 4 antibody (Ab), or FK506 alone]. In vivo bioluminescent imaging was used to evaluate the chronological survival of the transplanted cells. The graft survival rate was 100% (n = 9/9) in NOD/SCID mice, 0% (n = 6/6) in C57BL/6J mice without immunosuppressant treatment, and 100% (n = 37/37) in C57BL6/J mice with immunosuppressant treatment. After confirming the growth of the grafted cells in the C57/BL6J mice treated with immunosuppressants, immune suppression was discontinued. The grafted cells were subsequently rejected within 3 days in C57BL/6J mice treated with FK506 alone, as opposed to 26 days in C57BL/6J mice treated with FK506 plus anti-CD4 Ab. Histological evaluation confirmed the ablation of the grafted cells. Although this work describes a xenograft setting, the results suggest that this immunomodulatory strategy could provide a safety lock against tumor formation stemming from transplanted cells.

Glioma-astrocyte interactions on white matter tract-mimetic aligned electrospun nanofibers.

Gliomas are highly invasive forms of brain cancer comprising more than 50% of brain tumor cases in adults, and astrocytomas account for ∼60-70% of all gliomas. As a result of multiple factors, including enhanced migratory properties and extracellular matrix remodeling, even with current standards of care, mean survival time for patients is only ∼12 months. Because glioblastoma multiforme (GBM) cells arise from astrocytes, there is great interest in elucidating the interactions of these two cell types in vivo. Previous work performed on two-dimensional assays (i.e., tissue culture plastic and Boyden chamber assays) utilizes substrates that lack the complexities of the natural microenvironment. Here, we employed a three-dimensional, electrospun poly-(caprolactone) (PCL) nanofiber system (NFS) to mimic some features of topographical properties evidenced in vivo. Co-cultures of human GBM cells and rat astrocytes, as performed on the NFS, showed a significant increase in astrocyte GFAP expression, particularly in the presence of extracellular matrix (ECM) deposited by GBM cells. In addition, GBM migration increased in the presence of astrocytes or soluble factors (i.e., conditioned media). However, the presence of fixed astrocytes acted as an antagonist, lowering GBM migration rates. This data suggests that astrocytes and GBM cells interact through a multitude of pathways, including soluble factors and direct contact. This work demonstrates the potential of the NFS to duplicate some topographical features of the GBM tumor microenvironment, permitting analysis of topographical effects in GBM migration.

The Synergistic Effect of Combination Progesterone and Temozolomide on Human Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most common and most aggressive malignant brain tumor. Despite optimal treatment and evolving standard of care, the median survival of patients diagnosed with GBM is only 12–15 months. In this study, we combined progesterone (PROG) and temozolomide (TMZ), a standard chemotherapeutic agent for human GBM, to test whether PROG enhances the antitumor effects of TMZ and reduces its side effects. Two WHO grade IV human GBM cells lines (U87MG and U118MG) and primary human dermal fibroblasts (HDFs) were repeatedly exposed to PROG and TMZ either alone or in combination for 3 and 6 days. Cell death was measured by MTT reduction assay. PROG and TMZ individually induced tumor cell death in a dose-dependent manner. PROG at high doses produced more cell death than TMZ alone. When combined, PROG enhanced the cell death-inducing effect of TMZ. In HDFs, PROG did not reduce viability even at the same high cytotoxic doses, but TMZ did so in a dose-dependent manner. In combination, PROG reduced TMZ toxicity in HDFs. PROG alone and in combination with TMZ suppressed the EGFR/PI3K/Akt/mTOR signaling pathway and MGMT expression in U87MG cells, thus suppressing cell proliferation. PROG and TMZ individually reduced cell migration in U87MG cells but did so more effectively in combination. PROG enhances the cytotoxic effects of TMZ in GBM cells and reduces its toxic side effects in healthy primary cells.

MiR-29b attenuates tumorigenicity and stemness maintenance in human glioblastoma multiforme by directly targeting BCL2L2.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor and exhibits aggressive and invasive behavior. We previously identified four miRNAs-miR-29b, 494, 193a-3p, and 30e-with enhanced expression in GBM following treatment of ionizing radiation by miRNA microarray analysis. In this study, we found that only miR-29b inhibited tumor cell migration and invasion by reducing MMP-2 activity via phospho-AKT/β-catenin signaling, and stimulated a more epithelial-like morphology. Moreover, miR-29b inhibits angiogenesis by attenuating tube formation and the expression of VEGF and Ang-2, and stemness maintenance in GBM cells, as demonstrated by decreasing neurosphere formation and cancer stem cell marker protein expression. These findings support the anti-tumor properties of miR-29b in human GBM cells. Furthermore, miR-29b expression was inversely proportional to that of BCL2L2 mRNA or protein in various cancer cell types. Interestingly, BCL2L2 mRNA is highly expressed in the mesenchymal type of GBM. To further elucidate the relationship between miR-29b and BCL2L2 in GBM, we performed co-transfection reporter assays and determined that miR-29b downregulates BCL2L2 expression by directly binding its 3'UTR. Finally, we confirmed that BCL2L2 repression is of central importance to miR-29b anti-tumor activity using functional assays to examine cell migration, invasion, angiogenesis, and stemness. From these data, we propose that miR-29b may be a useful therapeutic agent in GBM.

Purinergic transmission in brain tumors and its impact on drug development.

Extracellular nucleoside and nucleotides acting via adenosine/P1 and nucleotide P2 (P2X/P2Y) purinoceptors are fundamental signaling molecules controlling the survival and proliferation of astrocytes and oligodendrocytes (Ceruti & Abbracchio, Adv Exp Med Biol 986:13, 2013). The malignant transformation of these cells to progressively more aggressive tumors (respectively, astrocytomas and anaplastic glioblastoma, and glioblastoma multiforme, containing proliferating cells resembling Oligodendrocytes Precursor Cells, OPCs) confers growth advantage and chemoresistance. Characterization of the specific P1 and P2 receptors on these tumors may unveil new strategies to reduce cancer growth and/or promote differentiation to non-cancerous glial phenotypes. The adenosine A3 receptor (A3AR) has emerged as a potential target. Under hypoxia, a condition typical of gliomas’ core, A3AR mediates chemoresistance via the PKB/Akt pathway (leading to inactivation of the pro-apopototic Bad protein) and by upregulating matrix metalloproteinase-9, that degrades extracellular matrix and promotes migration of glioma cells towards healthy brain regions (Ceruti & Abbracchio and ref therein). Thus, inhibition of A3AR with selective antagonists could represent an appealing therapeutic approach. More recently, the P2X7 receptor has been recently found to be over-expressed in grade IV human gliomas (Monif et al., J Inflammation, 11:25, 2014) and its blockade with the synthetic antagonist Brilliant Blue G decreased tumour cell number. Finally, treatment of human glioblastoma multiforme cells with UDP, UDP-glucose or LTD4, that act as agonists at the new P2Y-like GPR17 receptor, reduced the formation of glioma spheres, suggesting that GPR17 stimulation on highly proliferating tumor OPCs may drive their differentiation to the oligodendroglial fate, negatively affecting both tumor proliferation and self-renewal (Dougherty et al., Cancer Res 72:4856, 2012).

β-elemene enhances both radiosensitivity and chemosensitivity of glioblastoma cells through the inhibition of the ATM signaling pathway.

Glioblastoma multiforme (GBM), a tumor associated with poor prognosis, is known to be resistant to radiotherapy and alkylating agents such as temozolomide (TMZ). β-elemene, a monomer found in Chinese traditional herbs extracted from Curcuma wenyujin, is currently being used as an antitumor drug for different types of tumors including GBM. In the present study, we investigated the roles of β-elemene in the radiosensitivity and chemosensitivity of GBM cells. Human GBM cell lines U87-MG, T98G, U251, LN229 and rat C6 cells were treated with β-elemene combined with radiation or TMZ. We used MTT and colony forming assays to evaluate the proliferation and survival of the cells, and the comet assay to observe DNA damage. Expression of proteins was analyzed by immunoblotting. In the present study, we found that β-elemene inhibited the proliferation and survival of different GBM cell lines when combined with radiotherapy or TMZ via inhibition of DNA damage repair. Treatment of GBM cells with β-elemene decreased the phosphorylation of ataxia telangiectasia mutated (ATM), AKT and ERK following radiotherapy or chemotherapy. These results revealed that β-elemene could significantly increase the radiosensitivity and chemosensitivity of GBM. β-elemene may be used as a potential drug in combination with the radiotherapy and chemotherapy of GBM.

Small molecule inhibition of Axl receptor tyrosine kinase potently suppresses multiple malignant properties of glioma cells.

Glioblastoma multiforme (GBM) often features a combination of tumour suppressor gene inactivation and multiple oncogene overactivation. The Axl receptor tyrosine kinase is found overexpressed in GBM and thought to contribute to invasiveness, chemoresistance and poor survival. Here, we have evaluated the effect of BGB324, a clinical candidate Axl-specific small molecule inhibitor, on the invasive behaviour of human GBM cells in vitro, as an indicator of its potential in GBM therapy and also to elucidate the role of Axl in GBM pathogenesis.Two cultured adult GBM cell lines, SNB-19 and UP007, were treated with Gas6 and/or BGB324, and analysed in assays for survival, 3D colony growth, motility, migration and invasion. Western blot was used to detect protein expression and signal protein phosphorylation. In both cell lines, BGB324 inhibited specifically phosphorylation of Axl as well as Akt kinase further downstream. BGB324 also inhibited survival and proliferation of both cell lines in a concentration-dependent manner, as well as completely suppressing migration and invasion. Furthermore, our results indicate co-operative activation between the Axl and Tyro3 receptors, as well as ligand-independent Axl signalling, to take place in GBM cells. In conclusion, small molecule inhibitor-led targeting of Axl may be a promising therapy for GBM progression.

Identification of the proliferative effect of Smad2 and 3 in the TGF β2/Smad signaling pathway using RNA interference in a glioma cell line.

Gliomas are the most frequently occurring primary tumor in the brain. The most malignant form of glioma, glioblastoma multiforme (GBM), is characterized by rapid and invasive growth and is restricted to the central nervous system (CNS). The transforming growth factor β2 (TGFβ2)/small mothers against decapentaplegic (Smad) signaling pathway is important, not only in GBM cell metabolism and invasion, but also in GBM cell proliferation. However, the functions of the downstream mediators of the TGFβ2/Smads signaling pathway remain to be fully elucidated. In the present study, short hairpin (sh)RNA interference was used to specifically inhibit the expression of Smad2 and Smad3 in the TGFβ2/Smad signaling pathway to investigate the effects of shRNA on the proliferation of human GBM cells. The results demonstrated that knockdown of either Smad2 or Smad3 enhanced cellular proliferation. Additionally, the key target genes involved in GBM cell proliferation, induced by TGFβ2, were found to be dependent on Smad3, but not Smad2.

Imaging chemical exchange saturation transfer (CEST) effects following tumor-selective acidification using lonidamine.

Increased lactate production through glycolysis in aerobic conditions is a hallmark of cancer. Some anticancer drugs have been designed to exploit elevated glycolysis in cancer cells. For example, lonidamine (LND) inhibits lactate transport, leading to intracellular acidification in cancer cells. Chemical exchange saturation transfer (CEST) is a novel MRI contrast mechanism that is dependent on intracellular pH. Amine and amide concentration-independent detection (AACID) and apparent amide proton transfer (APT*) represent two recently developed CEST contrast parameters that are sensitive to pH. The goal of this study was to compare the sensitivity of AACID and APT* for the detection of tumor-selective acidification after LND injection. Using a 9.4-T MRI scanner, CEST data were acquired in mice approximately 14 days after the implantation of 10(5) U87 human glioblastoma multiforme (GBM) cells in the brain, before and after the administration of LND (dose, 50 or 100 mg/kg). Significant dose-dependent LND-induced changes in the measured CEST parameters were detected in brain regions spatially correlated with implanted tumors. Importantly, no changes were observed in T1- and T2-weighted images acquired before and after LND treatment. The AACID and APT* contrast measured before and after LND injection exhibited similar pH sensitivity. Interestingly, LND-induced contrast maps showed increased heterogeneity compared with pre-injection CEST maps. These results demonstrate that CEST contrast changes after the administration of LND could help to localize brain cancer and monitor tumor response to chemotherapy within 1 h of treatment. The LND CEST experiment uses an anticancer drug to induce a metabolic change detectable by endogenous MRI contrast, and therefore represents a unique cancer detection paradigm which differs from other current molecular imaging techniques that require the injection of an imaging contrast agent or tracer.

Decitabine nanoconjugate sensitizes human glioblastoma cells to temozolomide.

In this study, we developed and characterized a delivery system for the epigenetic demethylating drug, decitabine, to sensitize temozolomide-resistant human glioblastoma multiforme (GBM) cells to alkylating chemotherapy. A poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) based nanoconjugate was fabricated to encapsulate decitabine and achieved a better therapeutic response in GBM cells than that with the free drug. After synthesis, the highly efficient uptake process and intracellular dynamics of this nanoconjugate were monitored by single-molecule fluorescence tools. Our experiments demonstrated that, under an acidic pH due to active glycolysis in cancer cells, the PLGA-PEG nanovector could release the conjugated decitabine at a faster rate, after which the hydrolyzed lactic acid and glycolic acid would further acidify the intracellular microenvironment, thus providing positive feedback to increase the effective drug concentration and realize growth inhibition. In temozolomide-resistant GBM cells, decitabine can potentiate the cytotoxic DNA alkylation by counteracting cytosine methylation and reactivating tumor suppressor genes, such as p53 and p21. Owing to the excellent internalization and endolysosomal escape enabled by the PLGA-PEG backbone, the encapsulated decitabine exhibited a better anti-GBM potential than that of free drug molecules. Hence, the synthesized nanoconjugate and temozolomide could act in synergy to deliver a more potent and long-term antiproliferative effect against malignant GBM cells.

NFATc1 activation promotes the invasion of U251 human glioblastoma multiforme cells through COX-2.

Recent studies have revealed that the nuclear factor of activated T-cells (NFAT) is a transcription factor that is highly expressed in aggressive cancer cells and tissues, and mediates invasion through the transcriptional induction of pro-invasion and pro-migration genes. However, the mechanisms through which nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), in particular, translocates to the nucleus and regulates the invasion of human glioblastoma multiforme (GBM) cells have not yet been fully elucidated. In the present study, to investigate the role of NFATc1 in GBM cells, we established a U251 cell line expressing a constitutively active form of NFATc1 (CA-NFATc1). On the other hand, RNA interference was used to knock down NFATc1 expression in the U251 cell line. Our results demonstrated that the expression of CA-NFATc1 promoted cancer cell invasion, while small interfering RNA (siRNA) against NFATc1 successfully inhibited the invasion ability of the U251 cell line. Moreover, we demonstrated that NFATc1 promoted U251 cell invasion through the induction of cyclooxygenase-2 (COX‑2). NFAT transcriptionally regulates the induction of COX-2 induction in U251 cells and binds to the promoter. We also demonstrated that a large proportion of GBM specimens expressed NFATc1. NFATc1 expression increased according to the histopathological grade of the glioma. However, no NFATc1 staining was observed in the non-neoplastic brain tissues. These findings suggest that the inhibition of the activation of the NFATc1 pathway is an effective therapeutic strategy for the clinical management of GBM.

The effect of silver nanoparticles (AgNPs) on proliferation and apoptosis of in ovo cultured glioblastoma multiforme (GBM) cells.

Recently, it has been shown that silver nanoparticles (AgNPs) provide a unique approach to the treatment of tumors, especially those of neuroepithelial origin. Thus, the aim of this study was to evaluate the impact of AgNPs on proliferation and activation of the intrinsic apoptotic pathway of glioblastoma multiforme (GBM) cells cultured in an in ovo model. Human GBM cells, line U-87, were placed on chicken embryo chorioallantoic membrane. After 8 days, the tumors were divided into three groups: control (non-treated), treated with colloidal AgNPs (40 μg/ml), and placebo (tumors supplemented with vehicle only). At the end of the experiment, all tumors were isolated. Assessment of cell proliferation and cell apoptosis was estimated by histological, immunohistochemical, and Western blot analyses. The results show that AgNPs can influence GBM growth. AgNPs inhibit proliferation of GBM cells and seem to have proapoptotic properties. Although there were statistically significant differences between control and AgNP groups in the AI and the levels of active caspase 9 and active caspase 3, the level of these proteins in GBM cells treated with AgNPs seems to be on the border between the spontaneous apoptosis and the induced. Our results indicate that the antiproliferative properties of silver nanoparticles overwhelm proapoptotic ones. Further research focused on the cytotoxic effect of AgNPs on tumor and normal cells should be conducted.Electronic supplementary materialThe online version of this article (doi:10.1186/s11671-015-0823-5) contains supplementary material, which is available to authorized users.

Podocalyxin promotes glioblastoma multiforme cell invasion and proliferation by inhibiting angiotensin-(1-7)/Mas signaling.

Podocalyxin (PODX) reportedly enhances invasion in many human cancers including glioblastoma multiforme (GBM). Recent studies have shown that the local renin-angiotensin system (RAS) in tumor environment contributes significantly to tumor progression. As a counter-regulatory axis in RAS, angiotensin (Ang)-(1-7)/Mas signaling has been shown to inhibit the growth and invasiveness of several human cancers including GBM. In the present study, we examined the crosstalk between PODX and Ang-(1-7)/Mas signaling in GBM cells, and assessed its impact on GBM cell invasion and proliferation. A strong negative correlation between the expression of PODX and Mas in GBM tumor tissues from 10 consecutive patients (r=-0.768, p<0.01) was observed. The stable overexpression of PODX in LN-229 and U-118 MG human GBM cells decreased the expression of Mas at the mRNA and protein levels, which led to decreased density of Ang-(1-7)-binding Mas on the cell membrane. This effect was completely abolished by selective phosphatidylinositol 3-kinase (PI3K) inhibitor BKM120. By contrast, the stable knockdown of PODX in LN-229 and U-118 MG cells increased the expression of Mas and the density of Ang-(1-7)-binding Mas on the cell membrane. Overexpression and knockdown of PODX respectively reversed and enhanced the inhibitory effects of Ang-(1-7) on the expression/activity of matrix metalloproteinase-9 and cell invasion and proliferation in GBM cells. Although the overexpression of Mas showed no significant effect on the promoting effect of PODX on GBM cell invasion and proliferation in the absence of Ang-(1-7), it completely eliminated the effect of PODX in the presence of Ang-(1-7). In conclusion, to the best of our knowledge, the present study provided the first evidence that PODX inhibits Ang-(1-7)/Mas signaling by downregulating the expression of Mas through a PI3K-dependent mechanism in GBM cells. This effect led to enhanced GBM cell invasion and proliferation. The results of this study add new insight into the biological functions of PODX and the molecular mechanisms underlying GBM progression.

MiR‑27a suppresses the clonogenic growth and migration of human glioblastoma multiforme cells by targeting BTG2.

miR-27a and BTG2 are implicated in gliomagenesis and glioma progression. However, hitherto, a link between miR-27a and BTG2 in glioma has not been reported. In the present study, we investigated the effects of miR-27a on the proliferation and invasiveness of glioblastoma cells in vitro and in a mouse xenograft model and further studied the relation between miR‑27a expression and its target gene BTG2, which was identified by computation prediction algorithms. Our MTT and clonogenic assays showed that miR-27a overexpression significantly increased the clonogenic growth of glioblastoma U87MG and U251MG cells. The Transwell assays further revealed that miR-27a overexpression markedly increased the number of migrated U87MG and U251MG cells. TargetScan and other prediction algorithms identified BTG2 as a target gene of miR-27a, which was confirmed by EGFP reporter and immunoblotting assays showing an inverse relation between miR-27a expression and endogenous BTG2 expression. BTG2 overexpression also increased the proliferation and invasiveness of glioblastoma cells and BTG2 functioned downstream of miR-27a in modulating the proliferation and migration of glioblastoma cells. In conclusion, miR-27a modulates human glioblastoma growth and invasion by targeting BTG2.

Modulation of Sonic hedgehog signaling and WW domain containing oxidoreductase WOX1 expression enhances radiosensitivity of human glioblastoma cells.

WW domain containing oxidoreductase, designated WWOX, FOR or WOX1, is a known pro-apoptotic factor when ectopically expressed in various types of cancer cells, including glioblastoma multiforme (GBM). The activation of sonic hedgehog (Shh) signaling, especially paracrine Shh secretion in response to radiation, is associated with impairing the effective irradiation of cancer cells. Here, we examined the role of Shh signaling and WOX1 overexpression in the radiosensitivity of human GBM cells. Our results showed that ionizing irradiation (IR) increased the cytoplasmic Shh and nuclear Gli-1 content in GBM U373MG and U87MG cells. GBM cells with exogenous Shh treatment exhibited similar results. Pretreatment with Shh peptides protected U373MG and U87MG cells against IR in a dose-dependent manner. Cyclopamine, a Hedgehog/Smoothened (SMO) inhibitor, reversed the protective effect of Shh in U87MG cells. Cyclopamine increased Shh plus IR-induced H2AX, a marker of DNA double-strand breaks, in these cells. To verify the role of Shh signaling in the radiosensitivity of GBM cells, we tested the effect of the Gli family zinc finger 1 (Gli-1) inhibitor zerumbone and found that it could sensitize GBM cells to IR. We next examined the role of WOX1 in radiosensitivity. Overexpression of WOX1 enhanced the radiosensitivity of U87MG (possessing wild type p53 or WTp53) but not U373MG (harboring mutant p53 or MTp53) cells. Pretreatment with Shh peptides protected both WOX1-overexpressed U373MG and U87MG cells against IR and increased the cytoplasmic Shh and nuclear Gli-1 content. Zerumbone enhanced the radiosensitivity of WOX1-overexpressed U373MG and U87MG cells. In conclusion, overexpression of WOX1 preferentially sensitized human GBM cells possessing wild type p53 to radiation therapy. Blocking of Shh signaling may enhance radiosensitivity independently of the expression of p53 and WOX1. The crosstalk between Shh signaling and WOX1 expression in human glioblastoma warrants further investigation.

Identification of a novel cell-penetrating peptide targeting human glioblastoma cell lines as a cancer-homing transporter

Cell-penetrating peptides (CPPs) as a novel biomedical delivery system have been highly anticipated, since they can translocate across biological membranes and are capable of transporting their cargo inside live cells with minimal invasiveness. However, non-selective internalization in various cell types remains a challenge in the clinical application of CPPs, especially in cancer treatment. In this study, we attempted to identify novel cancer-homing CPPs to target glioblastoma multiforme (GBM), which is often refractory and resistant to treatment. We screened for CPPs showing affinity for the human GBM cell line, U87MG, from an mRNA display random peptide library. One of the candidate peptides which amino-acid sequence was obtained from the screening showed selective cell-penetrating activity in U87MG cells. Conjugation of the p16INK4a functional peptide to the GBM-selective CPP induced cellular apoptosis and reduced phosphorylated retinoblastoma protein levels. This indicates that the CPP was capable of delivering a therapeutic molecule into U87MG cells inducing apoptosis. These results suggest that the novel CPP identified in this study permeates with high affinity into GBM cells, revealing it to be a promising imaging and therapeutic tool in the treatment of glioblastoma.