Treatment Of Human Glioblastoma Multiforme Research Articles

Thiabendazole Inhibits Glioblastoma Cell Proliferation and Invasion Targeting Mini-chromosome Maintenance Protein 2

Thiabendazole (TBZ), approved by the US Food and Drug Administration (FDA) for human oral use, elicits a potential anticancer activity on cancer cells in vitro and in animal models. Here, we evaluated the efficacy of TBZ in the treatment of human glioblastoma multiforme (GBM). TBZ reduced the viability of GBM cells (P3, U251, LN229, A172, and U118MG) relative to controls in a dose- and time-dependent manner. However, normal human astrocytes (NHA) exhibited a greater IC50 than tumor cell lines and were thus more resistant to its cytotoxic effects. 5-Ethynyl-2'-deoxyuridine (EdU)-positive cells and the number of colonies formed were decreased in TBZ-treated cells (at 150 μM, P < 0.05 and at 150 μM, P < 0.001, respectively). This decrease in proliferation was associated with a G2/M arrest as assessed with flow cytometry, and the downregulation of G2/M check point proteins. In addition, TBZ suppressed GBM cell invasion. Analysis of RNA sequencing data comparing TBZ-treated cells with controls yielded a group of differentially expressed genes, the functions of which were associated with the cell cycle and DNA replication. The most significantly downregulated gene in TBZ-treated cells was mini-chromosome maintenance protein 2 (MCM2). SiRNA knockdown of MCM2 inhibited proliferation, causing a G2/M arrest in GBM cell lines and suppressed invasion. Taken together, our results demonstrated that TBZ inhibited proliferation and invasion in GBM cells through targeting of MCM2. SIGNIFICANCE STATEMENT: TBZ inhibits the proliferation and invasion of glioblastoma cells by downregulating the expression of MCM2. These results support the repurposing of TBZ as a possible therapeutic drug in the treatment of GBM.

2,5-Dimethyl Celecoxib Inhibits Proliferation and Cell Cycle and Induces Apoptosis in Glioblastoma by Suppressing CIP2A/PP2A/Akt Signaling Axis.

2,5-Dimethyl-celecoxib (DMC) is a close structural analog of the selective COX-2 inhibitor celecoxib that lacks COX-2-inhibitory function. Thus, DMC is a promising drug for anti-tumor. In this study, we evaluated the efficacy and the molecular basis of DMC in the treatment of human glioblastoma multiforme (GBM). DMC inhibited the growth and proliferation of GBM cell lines (LN229, A172, U251, and U87MG) in a dose-dependent manner (P < 0.001). In GBM cells treated with DMC, detection by flow cytometry showed cell cycle arrest, and proteins involved in cell cycle such as P21 were increased. Compared with control group, Annexin-V/PI-staining in DMC-treatment group was increased, indicating that DMC could induce apoptosis in GBM cells. Also, associated proteins including cleaved caspase 3 and cleaved PARP-1 were increased. It was further explored whether DMC blocked cell cycle and induced apoptosis in GBM cells through CIP2A/PP2A/AKT signaling pathway. After treatment of DMC, the phosphorylation of Akt was reduced while the total Akt level was not affected. DMC suppressed the expression of CIP2A in a time-dependent manner, while the CIP2A overexpression group reversed cell cycle and apoptotic protein expression led by DMC. Finally, in a xenograft model in nude mice using LN229 cells, DMC suppressed tumor growth. These findings proved that DMC could block cell cycle and induce apoptosis in GBM cells by suppressing CIP2A/PP2A/Akt signaling axis, which indicated that DMC could be an effective option for GBM treatment.

Experimental and clinical studies on radiation and curcumin in human glioma

PurposeThere is progressing evidence for the anti-cancer potential of the natural compound and dietary spice curcumin. Curcumin has been ascribed to be cytotoxic for various tumour cell types, to inhibit cell proliferation and to interfere with the cellular oxidant status. The compound has been notified as a therapeutic agent with radiosensitizing potential in brain tumour therapy. We considered the rationale to combine curcumin with radiation in the treatment of human glioblastoma multiforme (GBM).MethodDetermination of clonogenic cell survival following exposure of U251 human glioma cells to single dose (1–6 Gy) and fractionated irradiation (5 daily fractions of 2 Gy) without and with curcumin. Additional literature search focused on the interaction between curcumin and radiotherapy in experimental and clinical studies on human glioma.ResultsNo interaction was found on the survival of U251 human glioma cells after irradiation in combination with curcumin at clinically achievable concentrations. Experimental in vitro and in vivo data together with clinical bioavailability data from the literature do not give evidence for a radiosensitizing effect of curcumin. Reported GBM intratumoural curcumin concentrations are too low to either exert an own cytotoxic effect or to synergistically interact with radiation. Novel approaches are being explored to increase the bioavailability of curcumin and to facilitate transport over the blood–brain barrier, aimed to reach therapeutic curcumin levels at the tumour site.ConclusionThere is neither a biological nor clinical rationale for using curcumin as radiosensitizer in the therapy of GBM patients.

Triazole Modified Tetraiodothyroacetic Acid Conjugated to Polyethylene Glycol: High Affinity Thyrointegrin αvβ3 Antagonist with Potent Anticancer Activities in Glioblastoma Multiforme.

Discovery of bioactive molecules that target integrins has implicated their role in tumor angiogenesis, tumor growth, metastasis, and other pathological angiogenesis processes. Integrins are members of a family of cell surface receptors that play a critical role in the angiogenesis process. Tetraiodothyroacetic acid (tetrac), a deaminated derivative of l-thyroxine (T4), is a "thyrointegrin" antagonist that blocks the actions of l-triiodothyronine (T3) and T4 with an interaction site that is located at or near the RGD recognition site identified on integrin αvβ3's binding pocket (thyrointegrin αvβ3 receptors). We have enhanced the biological activity of a tetrac-based inhibitor via significantly improving its αvβ3 receptor binding affinity by introducing a triazole ring on the outer ring of tetrac and covalently conjugating to polymer to increase the product's hydrophilicity via PEGylation. The product, P-bi-TAT, was restricted from nuclear translocation and demonstrated high blood brain barrier permeability and retention in contrast to the non-PEG conjugated derivative. Results of biological activity indicated that this macromolecule new chemical entity P-bi-TAT has greater than 400-fold potent integrin αvβ3 affinity versus the parent compound tetrac and has potent anticancer/anti-angiogenesis efficacy against glioblastoma multiforme (GBM). P-bi-TAT administered subcutaneously once daily for 21 days at 1-10 mg/kg mouse body weight resulted in a dose-dependent suppression of GBM tumor growth and viability as monitored with IVIS imaging (P < 0.001). GBM tumors had >95% volume loss and maximal loss of GBM cell viability during the 21 days ON-treatment experiment as well as in the 21 days ON followed by 21 days OFF-treatment experiment (P < 0.001). In conclusion, P-bi-TAT is a promising lead clinical candidate effective in the treatment of human GBM.

The Natural Flavonoid Galangin Elicits Apoptosis, Pyroptosis, and Autophagy in Glioblastoma.

Galangin (GG), a flavonoid, elicits a potent antitumor activity in diverse cancers. Here, we evaluated the efficacy of GG in the treatment of human glioblastoma multiforme (GBM) and investigated the molecular basis for its inhibitory effects in the disease. GG inhibited viability and proliferation of GBM cells (U251, U87MG, and A172) in a dose-dependent manner (IC50 = 221.8, 262.5, 273.9 μM, respectively; P < 0.001; EdU, ~40% decrease at 150 μM, P < 0.001), and the number of colonies formed was significantly reduced (at 50 μM, P < 0.001). However, normal human astrocytes were more resistant to its cytotoxic effects (IC50 >450 μM). Annexin-V/PI staining was increased indicating that GG induced apoptosis in GBM cells (26.67 and 30.42%, U87MG and U251, respectively) and associated proteins including BAX and cleaved PARP-1 were increased (~3×). Cells also underwent pyroptosis as determined under phase-contrast microscopy. Knockdown of gasdermin E (GSDME), a protein involved in pyroptosis, alleviated pyroptosis induced by GG through aggravating nuclear DNA damage in GBM cells. Meanwhile, fluorescent GFP-RFP-MAP1LC3B puncta associated with autophagy increased under GG treatment, and transmission electron microscopy confirmed the formation of autophagic vesicles. Inhibition of autophagy enhanced GG-induced apoptosis and pyroptosis in GBM cells. Finally, in an orthotopic xenograft model in nude mice derived from U87MG cells, treatment with GG in combination with an inhibitor of autophagy, chloroquine, suppressed tumor growth, and enhanced survival compared to GG monotherapy (P < 0.05). Our results demonstrated that GG simultaneously induces apoptosis, pytoptosis, and protective autophagy in GBM cells, indicating that combination treatment of GG with autophagy inhibitors may be an effective therapeutic strategy for GBM.

Abstract 242: Analysis of temozolomide-loaded CLENs for treatment of human glioblastoma multiforme in vitro

Abstract Introduction: Glioblastoma multiforme (GBM) is the most prevalent neoplasm of the central nervous system, and has a 100 percent mortality rate despite current standard of care. We previously reported the use of cell membrane lipid-extracted nanoliposomes (CLENs) for tumor targeting. The approach involved the extraction of lipids directly from breast cancer cell membranes to develop nanoliposomes to efficiently target the same cell population from which the lipids were derived. This current study involves the development of CLENs for treatment of drug resistance in GBM, based on optimized formulation properties from our research involving breast cancer. Methods: Three liposome preparations were formulated with either U87-MG (human GBM) cell line membrane lipid extract (LE), DSPE-PEG5000 (PEG), or both, in addition to conventional components. The specific formulations contain DOPC, Cholesterol, LE, and PEG at ratios of 60/25/10/5 (Prep 1), 65/25/10/0 (Prep 2), and 70/25/0/5 (Prep 3), respectively. Temozolomide (TMZ) drug loading studies were determined by UV-spectroscopy. Cellular uptake of liposome preparations by U87-MG was determined with fluorescence intensity values using rhodamine-labeled liposomes in vitro. Cytotoxicity analysis of TMZ-loaded liposome preparations was evaluated using a sulforhodamine B (SRB) cytotoxicity assay. Results: Liposome preparations were characterized for mean particle size (nm) and surface charge (mv) in the presence and absence of incorporated TMZ. Average particle size for formulations loaded with TMZ were 148 nm (Prep 1), 221 nm (Prep 2), and 153 nm (Prep 3), which were not significantly different from preparations without incorporated drug, 149 nm, 217 nm, and 148 nm, respectively. Average zeta potential values for formulations loaded with TMZ were 3.3 mV (Prep 1), -2.7 mV (Prep 2), and -7.9 mV (Prep 3). For preparations without TMZ, values were -5.1 mV (Prep 1), -3.1 mV (Prep 2), and -5.8 mV (Prep 3). Cellular uptake studies suggested that the inclusion of LE had no effect on uptake of the liposomes by U87-MG target cells, with minimal effect on cytotoxicity. Moreover, the cytotoxicity conferred by the preparation containing PEG in the absence of LE (Prep 3) was consistent with a greater drug-incorporation efficiency than Prep 1. Conclusion: The role of LE incorporation for selective tumor targeting involving CLENs developed for GBM remains unclear, and seems to confer diminished cytotoxicity and formulation properties, when compared to favorable findings involving use of breast cancer cell lines published elsewhere. It is thus possible that the inclusion of LE in nanoliposomes for brain cancer may require more personalized optimization in relation to the brain tissue environment over the use of more standard or shared formulation considerations studied in other organ tissues. Optimization studies involving CLENs to test the theory are currently underway. Citation Format: Alexander Arau, Ilia Pidroutchniak, Woong Jae Song, Robert Campbell. Analysis of temozolomide-loaded CLENs for treatment of human glioblastoma multiforme in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 242.

Abstract 1300: Novel thyrointegrin αvβ3 antagonist in the treatment of glioblastoma multiforme

Abstract Background: The proliferative and pro-angiogenic actions mediated by L-thyroxine, pro-inflammatory and all known growth factors in glioblastoma multiforme (GBM) are initiated at cell surface thyrointegrin αvβ3 receptors for thyroid hormone on the extracellular domain of integrin αvβ3. Thyrointegrin αvβ3 receptors are over-expressed on cancer and rapidly dividing blood vessel cells, but quiescent on normal cells. A macromolecule Polyethylene glycol-conjugated bi-TriAzole Tetraiodothyroacetic acid (P-bi-TAT) acts with high affinity (Ki 3.1 nM) and specificity for the thyrointegrin αvβ3 receptors without any significant nuclear translocation. Methods: In the present studies, three primary human GBM cells and U87 glioma cell line were implanted orthotopically or subcutaneously (s.c.) into nude mice that were treated daily for up to 21 days with P-bi-TAT at different doses. Additionally, preclinical Pharmacokinetic and Safety assessments were carried out in multiple species. Results: Pharmacokinetic profiles indicated once-a-day dosing, and safety assessment studies demonstrated high safety and tolerability at 100-fold over the anticipated human equivalent dose. Fluorescence labeled P-bi-TAT administered s.c. demonstrated high biodistribution to GBM tumor in the brain that was comparable to peripherally implanted tumor. P-bi-TAT administered daily for 10-21 days s.c. at 0.3-10 mg/kg resulted in a dose-dependent suppression of GBM tumor growth and viability monitored with IVIS imaging (p &amp;lt;0.001). Histopathological analysis of tumors revealed 95% loss of the vascularity of treated tumors at 10 days (p &amp;lt;0.001) along with extensive cellular necrosis and apoptosis, without intratumoral hemorrhage. GBM tumors had a 97% volume loss and maximal loss of GBM cell viability during a subsequent 22 days off-treatment period (p &amp;lt; 0.001). Genomic micro-array studies with human primary GBM revealed that over 30 pathways relevant to the progression of GBM are modulated by P-bi-TAT. Conclusions: P-bi-TAT is a promising lead clinical candidate effective in the treatment of human GBM. Citation Format: Shaker A. Mousa, Thangirala Sudha, Kavitha Godugu, Mehdi Rajabi, Stewart Sell, Paul J. Davis. Novel thyrointegrin αvβ3 antagonist in the treatment of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1300.

Inhibition of glioma growth by flavokawain B is mediated through endoplasmic reticulum stress induced autophagy

ABSTRACT Flavokawain B (FKB), a natural kava chalcone, displays potent antitumor activity in various types of cancer. The mechanism of action, however, remains unclear. Here, we evaluated the efficacy of FKB in the treatment of human glioblastoma multiforme (GBM) as well as the molecular basis for its inhibitory effects in cancer. Approximately 60% of GBM cells became senescent after treatment with FKB as assessed in the senescence-associated (SA)-GLB1/SA-β-galactosidase assay. The cellular process of autophagy potentially contributed to the establishment of senescence. Transmission electron microscopy revealed the formation of autophagic vesicles under FKB treatment, and MAP1LC3B (microtubule associated protein 1 light chain 3 beta)-II was increased. Transfection of ATG5 or ATG7 small interfering RNAs (siRNAs) inhibited FKB-induced autophagy in U251 cells. Western blot revealed that molecular components of the endoplasmic reticulum stress pathway were activated, including ATF4 (activating transcription factor 4) and DDIT3 (DNA damage inducible transcript 3), while levels of TRIB3 (tribbles pseudokinase 3) increased. In addition, based on the phosphorylation status, the AKT-MTOR-RPS6KB1 pathway was inhibited, which induced autophagy in GBM cells. Inhibition of autophagy by autophagy inhibitors 3-methyladenine and chloroquine or knockdown of ATG5 or ATG7 caused FKB-treated U251 cells to switch from senescence to apoptosis. Finally, knockdown of ATG5 or treatment with chloroquine in combination with FKB, significantly inhibited tumor growth in vivo. Our results demonstrated that FKB induced protective autophagy through the ATF4-DDIT3-TRIB3-AKT-MTOR-RPS6KB1 signaling pathway in GBM cells, indicating that the combination treatment of FKB with autophagy inhibitors may potentially be an effective therapeutic strategy for GBM. Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATF4: activating transcription factor 4; ATG: autophagy related; CASP3: caspase 3; CCK-8: cell counting kit-8; CDKN1A: cyclin-dependent kinase inhibitor 1A; CQ: chloroquine; DDIT3: DNA damage inducible transcript 3; DMEM: Dulbecco’s modified Eagle’s medium; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; FKB: flavokawain B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBM: glioblastoma multiforme; GFP: green fluorescent protein; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase; 1RPS6KB1: ribosomal protein S6 kinase B1; SA-GLB1: senescence-associated galactosidase beta 1; siRNA: short interfering RNA; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TRIB3: tribbles pseudokinase 3; TUNEL: deoxynucleotidyl transferase-mediated dUTP nick-end labeling

Nitidine chloride inhibits the malignant behavior of human glioblastoma cells by targeting the PI3K/AKT/mTOR signaling pathway.

Recent studies have demonstrated that nitidine chloride (NC), a natural bioactive alkaloid, displays potent antitumor activity in various types of cancer. In the present study, NC was examined for efficacy in the treatment of human glioblastoma multiforme (GBM) as well as the molecular basis for its more general inhibitory effects in cancer. U251 and U87 GBM cell lines were exposed to three concentrations of NC (5, 25 and 50µM) invitro, and tumor cell growth was assessed on the basis of proliferation, migration and energy metabolism. Decreases in viability and proliferation reached ~50% for both cell lines with 50µM NC at 24h as assessed by cell viability Cell Counting Kit-8 (CCK-8) and EdU assays. In wound closure and Transwell assays, migration and invasion were inhibited at 50µM after 24h (~20 and 80%, respectively; P<0.05). ATP and L-lactate levels were also decreased after treatment with NC (50µM, 24h; P<0.05 and P<0.01, respectively). Finally, in western blot analysis, phosphorylation of Akt and mTOR was suppressed by NC, but partially restored when cells were treated simultaneously with a novel Akt activator, SC79. Partial restoration was also observed in viability/proliferation (U251 and U87, ~15 vs. 40%; NC vs. NC+SC79; P<0.05) and invasion (U251 and U87, ~30 vs. 60%; NC vs. NC+SC79; P<0.05). Our results demonstrated that NC inhibits development of GBM by targeting the PI3K/Akt/mTOR signaling pathway and provides a potential therapeutic agent for the treatment of GBM.

Imatinib and Nilotinib increase glioblastoma cell invasion via Abl-independent stimulation of p130Cas and FAK signalling.

Imatinib was the first targeted tyrosine kinase inhibitor to be approved for clinical use, and remains first-line therapy for Philadelphia chromosome (Ph+)-positive chronic myelogenous leukaemia. We show that treatment of human glioblastoma multiforme (GBM) tumour cells with imatinib and the closely-related drug, nilotinib, strikingly increases tyrosine phosphorylation of p130Cas, focal adhesion kinase (FAK) and the downstream adaptor protein paxillin (PXN), resulting in enhanced cell migration and invasion. Imatinib and nilotinib-induced tyrosine phosphorylation was dependent on expression of p130Cas and FAK activity and was independent of known imatinib targets including Abl, platelet derived growth factor receptor beta (PDGFRβ) and the collagen receptor DDR1. Imatinib and nilotinib treatment increased two dimensional cell migration and three dimensional radial spheroid invasion in collagen. In addition, silencing of p130Cas and inhibition of FAK activity both strongly reduced imatinib and nilotinib stimulated invasion. Importantly, imatinib and nilotinib increased tyrosine phosphorylation of p130Cas, FAK, PXN and radial spheroid invasion in stem cell lines isolated from human glioma biopsies. These findings identify a novel mechanism of action in GBM cells for two well established front line therapies for cancer resulting in enhanced tumour cell motility.

ET-34 * HIGHLY SELECTIVE HSV VIROTHERAPY FOR TREATMENT OF GLIOBLASTOMA

Glioblastoma Multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSV) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals. Although proven safe for treatment of GBM in patients, oHSVs efficacy has been limited, a consequence of poor intra-tumoral virus replication and spread. To counter these limitations, we have developed oHSVs whose selective replication in GBM cells does not rely on defective genes. This was accomplished by (i) full retargeting of oHSV to utilize the epidermal growth factor receptor (EGFR) for infection of human GBM tumor cells and (ii) further vector engineering to modify the essential HSV immediate early gene (ICP4) for sensitivity to repression by the microRNA mir-124. Mir-124 is highly expressed in neurons but virtually absent in GBM and highly conserved among different species. The mir-124-regulated vector was unable to replicate in nude mice following intracranial inoculation supporting vector safety and was shown to be effective (50-60% long term survival) in the treatment of human GBM in nude mice. To enhance vector intra-tumor vector spread, our EGFR retargeted-mir-124 controlled vector was further modified by vector arming with the matrix metalloproteinase gene encoding MMP9. MMP9 degrades collagen type IV, a major component of the extracellular matrix (ECM) and basement membranes of glioblastomas but absent in normal brain tissue. Studies are ongoing to determine whether MMP9 expression enhances vector spread in GBM neurospheres and as a therapeutic agent for enhanced treatment of human GBM in animals.

Enhancing the Efficacy of Drug-loaded Nanocarriers against Brain Tumors by Targeted Radiation Therapy

Glioblastoma multiforme (GBM) is a common, usually lethal disease with a median survival of only ~15 months. It has proven resistant in clinical trials to chemotherapeutic agents such as paclitaxel that are highly effective in vitro, presumably because of impaired drug delivery across the tumor's blood-brain barrier (BBB). In an effort to increase paclitaxel delivery across the tumor BBB, we linked the drug to a novel filomicelle nanocarrier made with biodegradable poly(ethylene-glycol)-block-poly(ε-caprolactone-r-D,L-lactide) and used precisely collimated radiation therapy (RT) to disrupt the tumor BBB's permeability in an orthotopic mouse model of GBM. Using a non-invasive bioluminescent imaging technique to assess tumor burden and response to therapy in our model, we demonstrated that the drug-loaded nanocarrier (DLN) alone was ineffective against stereotactically implanted intracranial tumors yet was highly effective against GBM cells in culture and in tumors implanted into the flanks of mice. When targeted cranial RT was used to modulate the tumor BBB, the paclitaxel-loaded nanocarriers became effective against the intracranial tumors. Focused cranial RT improved DLN delivery into the intracranial tumors, significantly improving therapeutic outcomes. Tumor growth was delayed or halted, and survival was extended by >50% (p less than 0.05) compared to the results obtained with either RT or the DLN alone. Combinations of RT and chemotherapeutic agents linked to nanocarriers would appear to be an area for future investigations that could enhance outcomes in the treatment of human GBM.

Pharmacologic blockade of chloride channel synergistically enhances apoptosis of chemotherapeutic drug-resistant cancer stem cells

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is the most commonly used chemotherapeutic agent in the treatment of human glioblastoma multiforme (GBM). However, BCNU chemotherapy fails due to subpopulations of intrinsic resistant-cells within the tumor mass. In our previous study, we dissociated BCNU-resistant cancer stem cells (CSCs) and observed the over-expression of multiple ion channel genes related to drug efflux. In the present study, we identified chloride intracellular channel 1 (CLIC1) in dissociated-BCNU-resistant CSCs using 2-DE and MALDI-TOF/MS analysis. To develop the specific target therapy of BCNU-resistant CSCs, we evaluated the drug-sensitivity of these CSCs using the chloride channel blocker, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). When combined with BCNU, DIDS synergistically increased the apoptotic events of BCNU-resistant CSCs in vitro and augmented BCNU sensitivity ex vivo. These findings suggest that CLIC1 is involved in the resistance of BCNU-resistant CSCs and BCNU/DIDS combined-therapy can provide valuable insight for promoting apoptosis or sensitizing glioblastomas to BCNU chemotherapy.

147. Effective Gene Transfer to Human Glioma Cells Using High Capacity Adenoviral Vectors: Human Glioma Cells Express Substantial Levels of CAR and Integrin Adenoviral Co-Receptors

Glioblastoma Multiforme (GBM) is the most common subtype of primary brain tumor and, to date, it has no cure. Although adenoviral vectors have been used for gene delivery to glioma tumors in clinical trials, the infectivity of adenoviral vectors in human glioma cells as well as their expression of adenoviral receptors remains controversial. In the present work we report on the expression of two adenoviral receptors, i.e., Coxsackie adenoviral receptor (CAR) and Integrin |[alpha]|V (INT) in murine and human glioma cells. Further, we also report that novel, gutless high-capacity adenoviral (HC- Ad) vectors efficiently infect and express their respective therapeutic transgenes in glioma cell lines derived from murine and human GBM. We constructed and purified HC-Ad vectors expressing b-Galactosidase, HSV1-thymidine kinase (TK) or human soluble Flt3L (hsFlt3L). These were used to infect mouse GL26 cells, rat CNS-1 cells, and the established human glioma cell lines, U251 and U87, and the primary glioma cell lines, derived from surgical biopsies, i.e., IN859 and IN 2045. All cells tested expressed variable amounts of both, CAR and INT, as detected by immunocytochemistry and Western blots. The infectivity and expression of HC-Ad vectors encoding b-Gal was determined by immunocytochemistry and enzymatic activity. High transgene expression was achieved in all the human glioma cells, and in rat CNS-1 cells, while the expression in GL26 cells was lower. The HC-Ad vector encoding TK was successfully expressed in all the cell lines under study, as detected by immunocytochemistry. In the presence of the prodrug ganciclovir, this vector induced cell death in all the murine and human glioma cells, as measured by flow cytometric analysis of cell death. The HC-Ad vector expressing Flt3L also infected efficiently the different cell lines tested as assessed by immunocytochemistry. Finally, high levels of Flt3L were detected, by ELISA, in the supernatant of infected cultures. Our data show that adenoviral receptors are expressed not only in murine glioma cell lines used in preclinical GBM models, but also in human glioma cell lines and primary GBM cell cultures. Importantly, adenoviral vectors successfully infected human glioma cells and elicited high expression levels of the therapeutic transgenes. Taken together, our data strongly support the use of adenoviral vectors in gene therapy approaches for the treatment of human GBM.

Modulation of major histocompatibility complex Class I molecules and major histocompatibility complex-bound immunogenic peptides induced by interferon-alpha and interferon-gamma treatment of human glioblastoma multiforme.

Little is known about the quantitative modulation of major histocompatibility complex (MHC) Class I expression on human gliomas that is effected by interferons; even less is known about the immunogenic peptides that are accommodated in the peptide-binding motifs of MHC Class I alleles in these brain tumors. In this article the authors investigated the ability of interferon (IFN)alpha and IFNgamma to upregulate MHC Class I expression and to modulate acid-eluted Class I-bound peptides on human glioblastoma multiforme (GBM) cells in vitro. Early-passage primary human GBM cell cultures and U87MG GBM cells were incubated with varying doses of INFalpha or IFNgamma ranging between 0 and 2000 U/ml. Upregulation of MHC Class I expression was assayed by immunocytochemical analysis, flow cytometry, and Western blot analysis. Modulation of acid-eluted MHC Class I-bound peptides from the IFN-treated GBM cells was examined with the aid of mass spectroscopy. The in vitro expression of the MHC Class I molecule was upregulated by both IFNalpha and IFNgamma in a dose-dependent manner. Interferon-gamma exhibited a more potent effect on MHC Class I upregulation, peaking at 10 U/ml; whereas the effect of IFNalpha was less marked, reaching a plateau at 500 U/ml. In addition, a native peptide eluted from MHC Class I molecules of human GBM cells was identified and found to be consistently upregulated by IFN treatment. Interferon-alpha and IFN-gamma can significantly upregulate the MHC Class I molecules that are expressed on the cell surface of human GBM cells as well as the potentially immunogenic peptides bound to the MHC. These results may help explain the molecular basis for increased immunogenicity with IFN treatment of human GBMs and might provide added insight into the design of future antitumor vaccines for human brain tumors.

The hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor genes reduces the tumorigenicity and growth of human glioblastoma multiforme.

We investigated whether the hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor (EGFR) genes would lead to a loss of tumorigenicity of a glioblastoma multiforme cell line. Glioblastoma multiforme cells were treated in vitro with 0 (HU0) or 100 micromol/L (HU100) hydroxyurea and then injected into the flanks of nude mice. Survival and tumor volumes were evaluated. Pulsed-field gel electrophoresis, Southern blot hybridization, and slot-blot analysis were used to determine EGFR amplification levels. Flow cytometry and immunofluorescent staining were used for cell-cycle analysis and EGFR protein expression. Prior to injection, HU100 cells lost 95% of their amplified EGFR genes and developed into tumors 6 weeks after injection versus 3 weeks for HU0 cells. Mice with HU100 tumors had a median survival of 62 days versus 43 days for control mice with HU0 tumors. Pulse-field gel electrophoresis analysis showed that HU100 tumors had reamplified the EGFR gene as double-minute chromosomes of the same size as those originally present before hydroxyurea treatment. When HU100 cells were cultured in the absence of hydroxyurea, the EGFR gene also reamplified. HU100 cells grew at less than half the rate of untreated HU0 control cells in culture and showed a decreased number of cells entering the cell cycle. Immunofluorescent staining of HU150 (150 micromol/L) cells showed decreased EGFR protein expression. The EGFR gene is important for tumorigenicity in mice and growth in culture. Hydroxyurea induces the loss of double-minute chromosome-amplified EGFR genes against a selection gradient and significantly delays the onset of tumors. These results support the potential use of low-dose hydroxyurea for the treatment of human glioblastoma multiforme.