MG Human Glioma Cells Research Articles

Evaluation of 99mTc-CN5DG as a broad-spectrum SPECT probe for tumor imaging

Previously, we reported a [99mTc(ǀ)]+ labeled d-glucoamine derivative (99mTc-CN5DG) and evaluated it as a tumor imaging agent in mice bearing A549 tumor xenografts. In this paper, 99mTc-CN5DG was further studied in U87 MG (human glioma cells), HCT-116 (human colon cancer cells), PANC-1 (human pancreatic cancer cells) and TE-1 (human esophageal cancer cells) tumor xenografts models to verify its potential application for imaging of different kinds of tumors. The biodistribution data showed that 99mTc-CN5DG had a similar biodistribution pattern in four tumor models at 2 h post-injection with high accumulation in tumors and kidneys. The tumor/muscle ratios (from 4.08 ± 0.42 to 9.63 ± 3.53) and tumor/blood ratios (from 17.18 ± 7.40 to 53.17 ± 16.16) of 99mTc-CN5DG in four tumor models were high. All four kinds of tumors could be clearly seen on their corresponding SPECT/CT images. Pharmacokinetic study in healthy CD-1 mice demonstrated that 99mTc-CN5DG cleared fast from blood (2 min, 12.97 ± 0.88%ID/g; 60 min, 0.33 ± 0.06%ID/g) and the blood distribution, elimination half-life was 5.81 min and 21.16 min, respectively. No abnormality was observed through the abnormal toxicity study. All of the above results demonstrated that 99mTc-CN5DG could be a broad-spectrum SPECT probe for tumor imaging and its further clinical application is warranted.

Augmentation of invadopodia formation in temozolomide-resistant or adopted glioma is regulated by c-Jun terminal kinase–paxillin axis

Temozolomide (TMZ) is one of the few effective anticancer agents against gliomas. However, acquisition of TMZ resistance or adaptation by gliomas is currently a crucial problem, especially increased invasiveness which is critical for the determination of clinical prognosis. This study investigated the molecular regulatory mechanisms of TMZ resistance in gliomas involved in invasiveness, particularly invadopodia formation, a molecular complex formed at the invasive front to cause extracellular matrix degradation during cellular local invasion. The TMZ-resistant clone of the U343 MG human glioma cell line (U343-R cells) was established. U343-R cells demonstrated higher invadopodia formation compared with U343 cells without TMZ resistance (U343-Con cells). Immunoblot analysis of DNA damage-related mitogen-activated protein kinase signals found increased phosphorylation of c-Jun terminal kinase (JNK) and higher activation of its downstream signaling in U343-R cells compared with U343-Con cells. Treatment of U343-R cells with specific inhibitors of JNK or siRNA targeting JNK suppressed up-regulation of invadopodia formation. In addition, paxillin, one of the known JNK effectors which is phosphorylated and affects cell migration, was phosphorylated at serine 178 in JNK activity-dependent manner. Expression of paxillin with mutation of the serine 178 phosphorylation site in U343-R cells blocked invadopodia formation. The present findings suggest that increased formation of invadopodia in U343-R cells is mediated by hyperactivation of JNK-paxillin signaling, and both JNK and paxillin might become targets of novel therapies against TMZ-resistant gliomas.

Poly (ɛ-caprolactone) nanoparticles of carboplatin: Preparation, characterization and in vitro cytotoxicity evaluation in U-87 MG cell lines

Carboplatin is a platinum based drug used in the treatment of several malignancies. Due to poor cellular uptake, generally, a larger dose of drug is administered to achieve therapeutic levels, causing harmful side-effects such as hematologic toxicity. In order to enhance the cellular uptake of carboplatin, we have developed carboplatin loaded nanoparticles using the biodegradable polymer poly (ɛ-caprolactone) (PCL). Nanoparticles ranging from the size of 23.77±1.37 to 96.73±2.79nm with positive zeta potential and moderate entrapment efficiency (54.21±0.98%) were obtained. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) confirmed the spherical morphology and smooth surface of all nanoformulations. The concentrations of PCL and the stabilizer (DMAB) are found to play a role in determining the size and the entrapment efficiency of the nanoparticles. Drug release from nanoparticles followed a biphasic pattern with an initial burst release followed by a sustained release for 10h. Results of in vitro cellular uptake and cytotoxicity studies revealed that carboplatin in the form of PCL-nanoparticles were efficiently up taken and displayed profound cytotoxicity to U-87 MG (human glioma) cells than the free drug. Importantly, unlike the free carboplatin, carboplatin in the form of PCL nanoparticles did not present any haemolytic activity in rat erythrocytes, a major side effect of this chemotherapeutic drug. This suggests that poly (ɛ-caprolactone) nanoencapsulation of carboplatin might be an efficient approach to treat cancer, while reducing carboplatin induced haemolysis.

Overexpression of N-myc downstream-regulated gene 1 inhibits human glioma proliferation and invasion via phosphoinositide 3-kinase/AKT pathways.

N-myc downstream-regulated gene 1 (NDRG1) was previously shown to exhibit low expression in glioma tissue as compared with that in normal brain tissue; however, the role of NDRG1 in human glioma cells has remained to be elucidated. The present study used the U87 MG and SHG-44 human glioma cell lines as well as the normal human astrocyte cell line 1800, which are known to have differential NDRG1 expression. Small interfering (si)RNA targeting NDRG1, and NDRG1 overexpression vectors were transfected into the SHG-44 and U87 MG glioma cells, respectively. Cell proliferation, invasion, apoptosis and cell cycle arrest were subsequently examined by MTT assay, transwell chamber assay, flow cytometry and western blot analysis, respectively. Furthermore, a subcutaneous tumor mouse model was used to investigate the effects of NDRG1 on the growth of glioma cells in vivo. Overexpression of NDRG1 was shown to inhibit cell proliferation and invasion, and induce apoptosis in the U87 MG glioma cells, whereas NDRG1 downregulation increased proliferation, suppressed apoptosis and promoted invasion of the SHG-44 glioma cells. In addition, in the subcutaneous tumor mouse model, overexpression of NDRG1 in U-87 MG cells suppressed tumorigenicity in vivo. The findings of the present study indicated that NDRG1 is required for the inhibition of gliomagenesis; therefore, targeting NDRG1 and its downstream targets may represent novel therapies for the treatment of glioma.

The positive correlation between DJ‐1 and β‐catenin expression shows prognostic value for patients with glioma

The relationship between DJ-1 and β-catenin, and its impact on the prognosis for glioma patients has not been fully understood. This study determined the effect of DJ-1 on β-catenin and the prognostic significance of this interaction in glioma patients. We collected tumor specimens from 88 glioma patients and determined the expression of DJ-1, β-catenin and PTEN by using immunohistochemical staining. The involvement of DJ-1 and β-catenin in glioma cell lines was evaluated by immunohistochemistry and Western blotting. High DJ-1 expression (37.5%) and high β-catenin expression (34.1%) in glioma specimens were significantly associated with high grade and poor prognosis in glioma patients. However, only high levels of DJ-1 (P = 0.014) was a strong independent prognostic factor, correlated with a reduced overall survival time. In vitro DJ-1 expression was positively correlated with the expression levels of β-catenin and p-Akt, and negatively correlated with PTEN expression in U87, U251 MG, SWO-38 and SHG44 human glioma cell lines. After the knockdown of DJ-1, Akt, p-Akt or β-catenin expression levels were not affected in the PTEN-null cell lines (U87 and U251 MG). However, in the SWO-38 cell line, which has wild-type PTEN protein, the level of PTEN increased while Akt/p-Akt and β-catenin levels were reduced. Furthermore, β-catenin staining weakened in SWO-38 cells after DJ-1 levels decreased according to immunocytochemical analysis. In conclusion, DJ-1 and β-catenin may contribute to the development and recurrence of glioma and are valuable prognostic factors for glioma patients. DJ-1 may regulate β-catenin expression via PTEN and p-Akt.

BCL-2 Proapoptotic Proteins Distribution in U-87 MG Glioma Cells before and after Hypericin Photodynamic Action

Apoptosis is a key process in the development and maintenance of tissue homeostasis. This process of controlled cell death is tightly regulated by a balance between cell survival and damage signals. We focused our attention towards the intrinsic mitochondrial apoptotic pathway, where Bcl-2 family of proteins plays the major role. We were particularly interested in two pro-apoptotic players Bak and Bax. Here we investigated their role in apoptosis triggered by photodynamic action. We show the localization of Bax and Bak in U-87 MG human glioma cells incubated with photosensitizer hypericin (Hyp) before and after photodynamic action. Apoptotic stimulus by Hyp photodynamic action caused Bax translocation into mitochondria. However our results suggest that under these conditions there are two populations of mitochondria: one which contains Bax and Bak simultaneously, and is almost exclusively localized near the plasma membrane; the other which contains Bax only and is distributed throughout the cell. The different protein content and spatial distribution of these two populations suggest that they can play different roles in response to apoptotic stimuli.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Bcl-2 proapoptotic proteins distribution in U-87 MG glioma cells before and after hypericin photodynamic action

Apoptosis is a key process in the development and maintenance of tissue homeostasis. This process of controlled cell death is tightly regulated by a balance between cell survival and damage signals. We focused our attention towards one apoptotic pathway, the intrinsic mitochondrial one where Bcl-2 family of proteins plays the major role. We are particularly interested in two pro-apoptotic players Bak and Bax from this family. Here we investigated their role in apoptosis triggered by photodynamic action. Targeted photodynamic therapy (PDT) is a promising approach to diagnose and treat different types of cancer. We show the localization of Bax and Bak in U-87 MG human glioma cells incubated with photosensitizer hypericin (Hyp) before and after photodynamic action. Apoptotic stimulus by Hyp photodynamic action causes Bax translocation into mitochondria. However our results suggest that under these conditions there are two populations of mitochondria: one which contains Bax and Bak simultaneously, and is almost exclusively localized near the plasma membrane; the other which contains Bax only and is distributed throughout the cell. The different protein content and spatial distribution of these two populations suggest that they can play different roles in response to apoptotic stimuli.

Menadione : sodium orthovanadate combination eliminates and inhibits migration of detached cancer cells.

Exposure of cancer cells to anticancer agents in cultures induces detachment of cells that are usually considered dead. These drug-induced detached cells (D-IDCs) may represent a clinical problem for chemotherapy since they may survive anoikis, enter the circulation, invade other tissues and resume proliferation, creating a metastasis, especially in tissues where the bioavailability of anticancer agents is not enough to eliminate all cancer cells. In this study we evaluated the antiproliferative effect of menadione : sodium orthovanadate (M : SO) combination on A549 lung cancer cells as well as the ability of M : SO to induce cell detachment. In addition, we followed the fate and chemosensitivity of M : SO-induced detached cells. Using transwell chambers, we found that a fraction of the M : SO-induced detached cells were viable and, furthermore, were able to migrate, re-attach, and resume proliferation when re-incubated in drug-free media. The total elimination of A549 detachment-resistant cells and M : SO-induced detached cells were successfully eliminated by equivalent M : SO concentration (17.5 μM : 17.5 μM). Thus, M : SO prevented cell migration. Similar results were obtained on DBTRG.05MG human glioma cells. Our data guarantee further studies to evaluate the in vivo occurrence of D-IDCs, their implications for invasiveness and metastasis and their sensitivity to anticancer drugs.

Abstract 2447: Measurement of tumor vascular integrity changes in an orthotopic glioma model in rats induced by antiangiogenic treatment using DCE-MRI

Abstract Background Glioblastoma is the most aggressive subtype of brain tumors. Antiangiogenic compounds are being tested in clinical trials, for this pathology, mostly in combination with other molecules. The availability of an imaging biomarker of efficacy could help optimize the dosing and scheduling of these combinations. The aim of this study was to validate DCE-MRI, using two contrast agents of different molecular weight, as an imaging biomarker of the efficacy of sorafenib (Nexavar®, BAY 43-9006) in an orthotopic glioma model in Nude rats. Methods U-87 MG human glioma cells were inoculated by stereotactic injection in the right caudate nucleus of 24 Nude rats. All rats were first imaged 10 days after cell injection to measure their baseline tumor volume (TV) using T2-weighted MRI (Bruker Pharmascan, 4.7T). Based on their TV, 20 rats were selected and imaged at D-1 (14 days after cell injection). The rats were then randomized into 2 groups of 8 rats according to their tumor vascular status following a full DCE-MRI protocol using either Gd-DTPA (Magnevist®) or P846 (two parameters: Ktrans from pharmaco-kinetic modeling of the contrast agent uptake curve, and iAUC60, the initial area under the gadolinium concentration time curve after 60 seconds). P846 is a medium-molecular weight, gadolinium-based contrast agent provided by Dr P. Robert (Guerbet). In both subgroups of 8 rats, each animal received a daily per os administration of either sorafenib (100 mg/kg/adm) or its vehicle. All 16 rats were then imaged at D1, D3 and D7. At each time point, voxelwise maps of DCE-MRI parameters were derived from imaging data and their distribution analyzed within regions of interest within the tumor and the contralateral lesion-free tissue. Results Sorafenib was well tolerated and it increased the median survival of tumor-bearing rats by 20%. At D7, despite no difference in tumor growth kinetics, DCE-MRI parameters (Ktrans and iUAC60) showed significant differences between sorafenib-treated and control groups using either Magnevist® or P846. Visual assessment showed that the heterogeneity of contrast agent uptake was reduced by antiangiogenic treatment with sorafenib. The quantification of this phenomenon by statistical analysis of the parameter distributions within the ROIs is pending. Conclusions In conclusion, we have shown that DCE-MRI, with either Magnevist® or P846 contrast agents, is a suitable non-invasive imaging technique to show changes of tumor vasculature integrity induced by antiangiogenic treatment such as sorafenib in orthotopically-implanted glioma in Nude rats. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2447. doi:1538-7445.AM2012-2447

Overexpression of the long non‐coding RNA MEG3 impairs in vitro glioma cell proliferation

Gliomas are the most common type of primary brain tumor in the central nervous system of adults. Maternally Expressed Gene 3 (MEG3) is an imprinted gene located at 14q32 that encodes a non-coding RNA (ncRNA) associated with tumorigenesis. However, little is known about whether and how MEG3 regulates glioma development. In the present study we assayed the expression of MEG3 in glioma tissue samples by real-time polymerase chain reaction assay, and defined the biological functions and target genes by CCK-8 assay, flow cytometry, and RNA immunoprecipitation. We first demonstrated that MEG3 expression was markedly decreased in glioma tissues compared with adjacent normal tissues. Moreover, ectopic expression of MEG3 inhibited cell proliferation and promoted cell apoptosis in U251 and U87 MG human glioma cell lines. We further verified that MEG3 was associated with p53 and that this association was required for p53 activation. These data suggest an important role of MEG3 in the molecular etiology of glioma and implicate the potential application of MEG3 in glioma therapy.

Cytotoxic effect of menadione and sodium orthovanadate in combination on human glioma cells

Gliomas are the most common primary brain tumor, and their treatment is still a challenge. Here, we evaluated the antiproliferative effect of a novel combination of two potent oxidative stress enhancers: menadione (M) and sodium orthovanadate (SO). We observed both short-term and prolonged growth inhibitory effects of M or SO alone as well as in combination (M:SO) on DBTRG.05MG human glioma cells. A stronger antiproliferative effect was observed in the short-term proliferation assay with the M:SO combination compared to either investigated agent alone. In the long-term proliferation assay, a 10-day exposure to M:SO at concentrations of 10 μM:17.5 μM or 17.5 μM:10 μM was enough to kill 100% of the cells; no cell regrowth was observed after re-incubation in drug-free media. When used in combination, the single concentration of M and SO could be decreased by 2.5- to 5-fold of those used for each experimental drug alone and still obtain a similar antiproliferative effect. The underlying molecular mechanism was investigated by co-incubating M:SO with dithiothreitol (DTT) and genistein. Both substances partially neutralized the effects of the M:SO combination, showing additive effects. This observation suggests a role of oxidative stress and tyrosine kinase stimulation in the M:SO cytotoxic effect. Our results indicate that M:SO combination is an attractive alternative for glioma treatment that encourages further study. The neutralizing effects of genistein and DTT reveal a possibility for their use in the minimization of potential M:SO systemic toxicity.

Pankiller effect of prolonged exposure to menadione on glioma cells: potentiation by vitamin C

SummaryMenadione (Vitamin K3) has anti-tumoral effects against a wide range of cancer cells. Its potential toxicity to normal cells and narrow therapeutic range limit its use as single agent but in combination with radiation or other anti-neoplastic agents can be of therapeutic use. In this paper, we first evaluated the early (within 3 h) effect of menadione on ongoing DNA replication. In normal rat cerebral cortex mini-units menadione showed an age dependent anti-proliferative effect. In tissue mini-units prepared from newborn rats, menadione inhibited ongoing DNA replication with an IC 50 of approximately 10 μM but 50 μM had no effect on mini-units from prepared adult rat tissue. The effect of short (72 h) and prolonged exposure (1–2 weeks) to menadione alone in the DBTRG.05MG human glioma cells line and in combination with vitamin C was studied. After short period of exposure data show that menadione alone or in combination with vitamin C provided similar concentration-response curves (and IC50 values). Prolonged exposure to these drugs was evaluated by their ability to kill 100% of glioma cells and prevent regrowth when cells are re-incubated in drug-free media. In this long-term assay, menadione:vitamin C at a ratio 1:100 showed higher anti-proliferative activity when compared to each drug alone and allowed to reduce each drug concentration between 2.5 to 5-fold. Similar anti-proliferative effect was demonstrated in 8 patient derived glioblastoma cell cultures. Our data should be able to encourage further advanced studies on animal models to evaluate the potential use of this combination therapy for glioma treatment.

Radiosensitization of glioma cells by modulation of Met signalling with the hepatocyte growth factor neutralizing antibody, AMG102

The hepatocyte growth factor (HGF)/Met signalling pathway is up-regulated in many cancers, with downstream mediators playing a role in DNA double strand break repair. Previous studies have shown increased radiosensitization of tumours through modulation of Met signalling by genetic methods. We investigated the effects of the anti-HGF monoclonal antibody, AMG102, on the response to ionizing radiation in a model of glioblastoma multiforme in vitro and in vivo. Radiosensitivity was evaluated in vitro in the U-87 MG human glioma cell line. Met activation was measured by Western blot, and the effect on survival following radiation was evaluated by clonogenic assay. Mechanism of cell death was evaluated by apoptosis and mitotic catastrophe assays. DNA damage was quantitated by γH2AX foci and neutral comet assay. Growth kinetics of subcutaneous tumours was used to assess the effects of AMG102 on in vivo tumour radiosensitivity. AMG102 inhibited Met activation after irradiation. An enhancement of radiation cell killing was shown with no toxicity using drug alone. Retention of γH2AX foci at 6 and 24 hrs following the drug/radiation combination indicated an inhibition of DNA repair following radiation, and comet assay confirmed DNA damage persisting over the same duration. At 48 and 72 hrs following radiation, a significant increase of cells undergoing mitotic catastrophe was seen in the drug/radiation treated cells. Growth of subcutaneous tumours was slowed in combination treated mice, with an effect that was greater than additive for each modality individually. Modulation of Met signalling with AMG102 may prove a novel radiation sensitizing strategy. Our data indicate that DNA repair processes downstream of Met are impaired leading to increased cell death through mitotic catastrophe.

IL-6 augments the invasiveness of U87MG human glioblastoma multiforme cells via up-regulation of MMP-2 and fascin-1

Invasion into adjacent brain parenchyma is the key cause of recurrent human glioblastoma multiforme (GBM). The role of interleukin 6 (IL-6) in the malignant progression of glioma remains undefined. Here, we found that IL-6 promotes the invasion of U87 MG human glioma cells but not in U343 cells. An advanced level of STAT3 activity, and increased expression and secretion of MMP-2, induced by IL-6 in U87 MG cells were observed. Blocking the STAT3 pathway with specific inhibitors of STAT3, JSI-124 or small interfering RNA (siRNA), inhibited the invasion of U87MG promoted by IL-6 with concomitant down-regulation of MMP-2. Furthermore, rapid up-regulation of fascin-1 (a cell motility-related protein) induced by IL-6 was apparent in U87MG cells. However, this up-regulation of fascin-1 was not inhibited by JSI-124 or siRNA. These results suggest that the STAT3 pathway and fascin-1 may play crucial roles in the invasiveness of U87MG cells promoted by IL-6.

Experimental study in vivo of the inhibitory effect of SEPI7 gene therapy for U251 MG human glioblastoma xenograft in nude mice

Objective To investigate the growth - inhibitory and anti - invasion effects of SEPT7 gene on U251 MG human glioma cells in nude mice in vivo. Methods Nude mice bearing subcutaneous U251 MG glioma xenograft tumor model was set up and treated with SEPT7 recombinant adenovirus. The general status of the mice was monitored and the tumor volume was measured every three days. After treatment for 4 weeks, all the tumors were removed and the expression of SEPT7 and other invasion - related proteins ( MMP2 ,MMP9 ,MT1 - MMP.TIMP1 .TIMP2) and integrin αvβ3 were examined. Results SEPT7 could inhibit tumor growth. The average tumor volume of SEPT7 treatment group was smaller than those of the control group and empty vector group significantly. In SEPT7 treatment group, the SEPT7 expression was up - regulated. The expression of MMP2 ,MMP9,MT1 - MMP and integrinαvβ3 was lowered, while TIMP1 and TIMP2 was activated. Conclusion SEPT7 gene therapy has inhibitory effect on the invasion and growth of human gliomas. Key words: Gliomas; SEPT7; Invasion; Gene therapy; MMPs, TIMPs, Integrin

Antitumor activity of cytokine-induced killer cells in nude mouse xenograft model

Malignant glioma is the most common primary brain tumor in adults and the median survival for patients is less than a year. Despite aggressive treatments including surgical resection, radiotherapy, and chemotherapy, only modest improvement has been achieved in the survival of patients with glioma. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against human glioma cancer was evaluated in vitro and in vivo. Human peripheral blood mononuclear cells were cultured with IL-2-containing medium in anti-CD3 antibody-coated flasks for 5 days, followed by incubation in IL-2-containing medium for 9 days. The number of cells increased more than 200-fold and the viability was >90%. The resulting populations were consisted of 96% CD3(+), 2% CD3(-)CD56(+), 68% CD3(+)CD56(+), 2% CD4(+), <1% CD4(+)CD56(+), 80% CD8(+), and 49% CD8(+)CD56(+). This heterogeneous cell population was called as CIK cells. At an effector-target cell ratio of 30:1, CIK cells destroyed 43% of U-87 MG human glioma cells, as measured by the (51)Cr-release assay. In addition, CIK cells at doses of 0.3, 1, and 3 million cells per mouse inhibited 23%, 40%, and 50% of U-87 MG tumor growth in nude mouse xenograft assays, respectively. This study suggests that CIK cells may be used as an adoptive immunotherapy for glioma cancer patients.

Characterization of the Interaction of Hypericin with Protein Kinase C in U‐87 MG Human Glioma Cells

A fluorescence imaging technique was used to monitor intracellular localization of protein kinase C (PKC) in U-87 MG human glioma cells in the presence of hypericin (Hyp) and phorbol 12-myristate-13-acetate (PMA). It is shown that PKC localization, which reflects its activity, is influenced by Hyp and this influence is different from that observed for PMA which acts as PKC activator. Fluorescence binding experiments were used to determine the binding constants of Hyp to several isoforms of PKC. The obtained values of K(d)s (approximately 100 nM) suggest that Hyp binds with high affinity to PKC. Finally, molecular modeling was used to compare structural models of the interaction of C1B domain of PKC (PKC isoforms alpha, delta, gamma) with Hyp and our previously published model of the (C1B domain PKCgamma)/PMA complex. The influence of Hyp on PKC translocation in U-87 MG cells in comparison with PMA, colocalization fluorescence pattern of Hyp and PKC, the higher binding affinity of Hyp to PKC in comparison with known binding constants of phorbol esters, as well as the binding mode of Hyp and PMA to the C1B domain of PKC suggested by molecular modeling, support the idea that Hyp and PMA might competitively bind to the regulatory domain of PKC.

509 POSTER In vitro evaluation of glioma cell lines and primary glioma cell cultures chemosensitivities: the effect of pharmacological modulation of peripheral benzodiazepine receptors

Carboplatin is a platinum based drug used in the treatment of several malignancies. Due to poor cellular uptake, generally, a larger dose of drug is administered to achieve therapeutic levels, causing harmful side-effects such as hematologic toxicity. In order to enhance the cellular uptake of carboplatin, we have developed carboplatin loaded nanoparticles using the biodegradable polymer poly (ɛ-caprolactone) (PCL). Nanoparticles ranging from the size of 23.77 ± 1.37 to 96.73 ± 2.79 nm with positive zeta potential and moderate entrapment efficiency (54.21 ± 0.98%) were obtained. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) confirmed the spherical morphology and smooth surface of all nanoformulations. The concentrations of PCL and the stabilizer (DMAB) are found to play a role in determining the size and the entrapment efficiency of the nanoparticles. Drug release from nanoparticles followed a biphasic pattern with an initial burst release followed by a sustained release for 10 h. Results of in vitro cellular uptake and cytotoxicity studies revealed that carboplatin in the form of PCL-nanoparticles were efficiently up taken and displayed profound cytotoxicity to U-87 MG (human glioma) cells than the free drug. Importantly, unlike the free carboplatin, carboplatin in the form of PCL nanoparticles did not present any haemolytic activity in rat erythrocytes, a major side effect of this chemotherapeutic drug. This suggests that poly (ɛ-caprolactone) nanoencapsulation of carboplatin might be an efficient approach to treat cancer, while reducing carboplatin induced haemolysis.

Targeting the c-Met Pathway Potentiates Glioblastoma Responses to γ-Radiation

Resistance to current cytotoxic therapies limits the treatment of most solid malignancies. This results, in part, from the overactivation of receptor tyrosine kinases and their downstream pathways in tumor cells and their associated vasculature. In this report, we ask if targeting the multifunctional mitogenic, cytoprotective, and angiogenic scatter factor/hepatocyte growth factor (SF/HGF)/c-Met pathway potentiates antitumor responses to gamma-radiation. Endogenous expression of SF/HGF and c-Met was targeted in U87 MG human malignant glioma cells and xenografts using chimeric U1/ribozymes. The effects of U1/ribozymes +/- gamma-radiation on glioma cell proliferation, apoptosis, xenograft growth, and animal survival were examined. U1/ribozymes knocked down SF/HGF and c-Met mRNA and protein levels, sensitized cells to gamma-radiation (P < 0.005), and enhanced radiation-induced caspase-dependent cytotoxicity in vitro (P < 0.005). Intravenous U1/ribozyme therapy as liposome/DNA complexes or radiation alone modestly and transiently inhibited the growth of s.c. U87 xenografts. Combining the therapies caused tumor regression and a 40% tumor cure rate. In animals bearing intracranial xenografts, long-term survival was 0% in response to radiation, 20% in response to intratumoral adenoviral-based U1/ribozyme delivery, and 80% (P < 0.0005) in response to combining U1/ribozymes with radiation. This apparent synergistic antitumor response was associated with a approximately 70% decrease in cell proliferation (P < 0.001) and a approximately 14- to 40-fold increase in apoptosis (P < 0.0001) within xenografts. Targeting the SF/HGF/c-Met pathway markedly potentiates the anti-glioma response to gamma-radiation. Clinical trials using novel SF/HGF/c-Met pathway inhibitors in glioma and other malignancies associated with c-Met activation should ultimate include concurrent radiation and potentially other cytotoxic therapeutics.

A novel E1A-E1B mutant adenovirus induces glioma regression in vivo.

Malignant gliomas are the most frequently occurring primary brain tumors and are resistant to conventional therapy. Conditionally replicating adenoviruses are a novel strategy in glioma treatment. Clinical trials using E1B mutant adenoviruses have been reported recently and E1A mutant replication-competent adenoviruses are in advanced preclinical testing. Here we constructed a novel replication-selective adenovirus (CB1) incorporating a double deletion of a 24 bp Rb-binding region in the E1a gene, and a 903 bp deleted region in the E1b gene that abrogates the expression of a p53-binding E1B-55 kDa protein. CB1 exerted a potent anticancer effect in vitro in U-251 MG, U-373 MG, and D-54 MG human glioma cell lines, as assessed by qualitative and quantitative viability assays. Replication analyses demonstrated that CB1 replicates in vitro in human glioma cells. Importantly, CB1 acquired a highly attenuated replicative phenotype in both serum-starved and proliferating normal human astrocytes. In vivo experiments using intracranially implanted D-54 MG glioma xenografts in nude mice showed that a single dose of CB1 (1.5 x 10(8) PFU/tumor) significantly improved survival. Immunohistochemical analyses of expressed adenoviral proteins confirmed adenoviral replication within the tumors. The CB1 oncolytic adenovirus induces a potent antiglioma effect and could ultimately demonstrate clinical relevance and therapeutic utility.