Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells: a model of glioblastoma multiforme pathophysiology.

Hypervascularity, focal necrosis, persistent cerebral edema, and rapid cellular proliferation are key histopathologic features of glioblastoma multiforme (GBM), the most common and malignant of human brain tumors. By immunoperoxidase and immunofluorescence, we definitively have demonstrated the presence of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFr) in five out of five human glioma cell lines (U-251MG, U-105MG, D-65MG, D-54MG, and CH-235MG) and in eight human GBM tumor surgical specimens. In vitro experiments with glioma cell lines revealed a consistent and reliable relation between EGFr activation and VEGF production; namely, EGF (1-20 ng/ml) stimulation of glioma cells resulted in a 25-125% increase in secretion of bioactive VEGF. Conditioned media (CM) prepared from EGF-stimulated glioma cell lines produced significant increases in cytosolic free intracellular concentrations of Ca2+ ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs). Neither EGF alone or CM from glioma cultures prepared in the absence of EGF induced [Ca2+]i increases in HUVECs. Preincubation of glioma CM with A4.6.1, a monoclonal antibody to VEGF, completely abolished VEGF-mediated [Ca2+]i transients in HUVECs. Likewise, induction by glioma-derived CM of von Willebrand factor release from HUVECs was completely blocked by A4.6.1 pretreatment. These observations provide a key link in understanding the basic cellular pathophysiology of GBM tumor angiogenesis, increased vascular permeability, and cellular proliferation. Specifically, EGF activation of EGFr expressed on glioma cells leads to enhanced secretion of VEGF by glioma cells. VEGF released by glioma cells in situ most likely accounts for pathognomonic histopathologic and clinical features of GBM tumors in patients, including striking tumor angiogenesis, increased cerebral edema and hypercoagulability manifesting as focal tumor necrosis, deep vein thrombosis, or pulmonary embolism.     

Targeting glioblastoma multiforme with an IL-13/diphtheria toxin fusion protein in vitro and in vivo in nude mice

Fusion proteins composed of tumor binding agents and potent catalytic toxins show promise for intracranial therapy of brain cancer and an advantage over systemic therapy. Glioblastoma multiforme (GBM) is the most common form of brain cancer and overexpresses IL-13R. Thus, we developed an interleukin-13 receptor targeting fusion protein, DT(390)IL13, composed of human interleukin-13 and the first 389 amino acids of diphtheria toxin. To measure its ability to inhibit GBM, DT(390)IL13 was tested in vitro and found to inhibit selectively the U373 MG GBM cell line with an IC(50) around 12 pmol/l. Cytotoxicity was neutralized by anti-human-interleukin-13 antibody, but not by control antibodies. In vivo, small U373 MG glioblastoma xenografts in nude mice completely regressed in most animals after five intratumoral injections of 1 microg of DT(390)IL13 q.o.d., but not by the control fusion protein DT(390)IL-2. DT(390)IL13 was also tested against primary explant GBM cells of a patient's excised tumor and the IC(50) was similar to that measured for U373 MG. Further studies showed a therapeutic window for DT(390)IL13 of 1-30 microg/injection and histology studies and enzyme measurements showed that the maximum tolerated dose of DT(390)IL13 had little effect on kidney, liver, spleen, lung and heart in non-tumor-bearing immunocompetent mice. Together, these data suggest that DT(390)IL13 may provide an important, alternative therapy for brain cancer.     

Inhibition of cell growth and tumorigenesis of human glioblastoma cells by a neutralizing antibody against human basic fibroblast growth factor.

We report here that a neutralizing mouse monoclonal antibody against basic FGF inhibited both anchorage-dependent and anchorage-independent growth of U-87MG and T98G human glioblastoma cells and HeLa cells, all of which express both the basic FGF and the FGF receptor genes. In addition, the subcutaneous administration of this antibody significantly suppressed the tumor development of these tumor cells in nude mice. Therefore, basic FGF plays an important role in neoplastic growth of these cells. The neutralization of basic FGF will be effective in controlling the growth of tumors, such as glioblastoma and other cancer cells which bear basic FGF and FGF receptors.     

Equine herpesvirus type 1-mediated oncolysis of human glioblastoma multiforme cells

The cytolytic animal virus equine herpesvirus type 1 (EHV-1) was evaluated for its oncolytic potential against five human glioblastoma cell lines. EHV-1 productively infected four of these cell lines, and the degree of infection was positively correlated with glioma cell death. No human major histocompatibility complex class 1 (MHC-I) was detected in the resistant glioma line, while infection of the susceptible glioma cell lines, which expressed human MHC-I, were blocked with antibody to MHC-I, indicating that human MHC-I acts as an EHV-1 entry receptor on glioma cells.     

Isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion.     

Phorbol 12-myristate 13-acetate induces epidermal growth factor receptor transactivation via protein kinase Cdelta/c-Src pathways in glioblastoma cells

Both the epidermal growth factor receptor (EGFR) and protein kinase C (PKC) play important roles in glioblastoma invasive growth; however, the interaction between the EGFR and PKC is not well characterized in glioblastomas. Treatment with EGF stimulated global phosphorylation of the EGFR at Tyr(845), Tyr(992), Tyr(1068), and Tyr(1045) in glioblastoma cell lines (U-1242 MG and U-87 MG). Interestingly, phorbol 12-myristate 13-acetate (PMA) stimulated phosphorylation of the EGFR only at Tyr(1068) in the two glioblastoma cell lines. Phosphorylation of the EGFR at Tyr(1068) was not detected in normal human astrocytes treated with the phorbol ester. PMA-induced phosphorylation of the EGFR at Tyr(1068) was blocked by bisindolylmaleimide (BIM), a PKC inhibitor, and rottlerin, a PKCdelta-specific inhibitor. In contrast, Go 6976, an inhibitor of classical PKC isozymes, had no effect on PMA-induced EGFR phosphorylation. Furthermore, gene silencing with PKCdelta small interfering RNA (siRNA), siRNA against c-Src, and mutant c-Src(S12C/S48A) and treatment with a c-Src inhibitor (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine) abrogated PMA-induced EGFR phosphorylation at Tyr(1068). PMA induced serine/threonine phosphorylation of Src, which was blocked by both BIM and rottlerin. Inhibition of the EGFR with AG 1478 did not significantly alter PMA-induced EGFR Tyr(1068) phosphorylation, but completely blocked EGF-induced phosphorylation of the EGFR. The effects of PMA on MAPK phosphorylation and glioblastoma cell proliferation were reduced by BIM, rottlerin, the MEK inhibitor U0126, and PKCdelta and c-Src siRNAs. Taken together, our data demonstrate that PMA transactivates the EGFR and increases cell proliferation by activating the PKCdelta/c-Src pathway in glioblastomas.     

Hepatocyte growth factor increases mitochondrial mass in glioblastoma cells

Hepatocyte growth factor/scatter factor (HGF) is a multifunctional growth factor that is linked to the initiation and/or progression of numerous malignancies. HGF also alters cancer cell responses to DNA damaging cytotoxic agents. Many cell responses to Met activation require alterations in metabolic activity but how the metabolic machinery responds to Met activation remains poorly defined. Treating human glioblastoma cells with HGF followed by the topoisomerase inhibitor camptothecin was found to increase the activity per cell of the mitochondrial respiratory chain enzyme succinate-tetrazolium reductase (>80% increase, p < 0.05) and the tricarboxylic acid cycle enzyme succinate dehydrogenase (>25% increase, p < 0.05). Treatment with either HGF or camptothecin alone had no effect on enzyme activity. The mitochondrial enzymatic response to HGF was dose- and time-dependent with the maximum increase occurring in cells pre-treated with 30 ng/ml HGF for 48h prior to camptothecin exposure. This enzymatic response was associated with a concurrent increase in mitochondrial mass of comparable magnitude (approximately 56%, p < 0.05) as measured by fluorescent mitochondrial staining and flow cytometry. The mitochondrial mass response to HGF was prevented by the MAP-kinase pathway inhibitor PD98059 and was unaffected by the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin. These findings suggest that HGF influences cell responses to chemotherapeutic stress, in part, by altering mitochondrial functions through a MAP-kinase dependent increase in mitochondrial mass.     

T cell suppressor factor from human glioblastoma cells is a 12.5-kd protein closely related to transforming growth factor-beta.

T cell suppressor factor produced by human glioblastoma cells inhibits T cell proliferation in vitro and more specifically interferes with interleukin-2 (IL-2)-dependent T cell growth. Here we report the purification of this factor from conditioned medium of the human glioblastoma cell line 308. Amino-terminal sequence analysis of the 12.5-kd protein demonstrates that eight out of the first 20 amino acids are identical to human transforming growth factor-beta. Purified glioblastoma-derived T cell suppressor factor and transforming growth factor-beta from porcine platelets inhibit both IL-2-induced proliferation of ovalbumin-specific T helper cells and lectin-induced thymocyte proliferation with similar specific activities. If released by glioblastoma cells in vivo, the factor may contribute to impaired immunosurveillance and to the cellular immunodeficiency state detected in the patients.     

Aspirin inhibits cancer stem cells properties and growth of glioblastoma multiforme through Rb1 pathway modulation

Several clinical studies indicated that the daily use of aspirin or acetylsalicylic acid reduces the cancer risk via cyclooxygenases (Cox-1 and Cox-2) inhibition. In addition, aspirin-induced Cox-dependent and -independent antitumor effects have also been described. Here we report, for the first time, that aspirin treatment of human glioblastoma cancer (GBM) stem cells, a small population responsible for tumor progression and recurrence, is associated with reduced cell proliferation and motility. Aspirin did not interfere with cell viability but induced cell-cycle arrest. Exogenous prostaglandin E₂ significantly increased cell proliferation but did not abrogate the aspirin-mediated growth inhibition, suggesting a Cox-independent mechanism. These effects appear to be mediated by the increase of p21 ^waf1 and p27 ^Kip1, associated with a reduction of Cyclin D1 and Rb1 protein phosphorylation, and involve the downregulation of key molecules responsible for tumor development, that is, Notch1, Sox2, Stat3, and Survivin. Our results support a possible role of aspirin as adjunctive therapy in the clinical management of GBM patients.     

Hepatocyte growth factor stimulates the growth and activates mitogen-activated protein kinase in human hepatoma cells

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and various epithelial cells. Unexpectedly, it has been reported to inhibit the growth of hepatoma cells in vitro. To clarify this phenomenon, we examined the effects of recombinant baculovirus-expressed HGF on the growth of 6 human hepatoma cell lines. The growth of Hep3B and HepG2 cells was markedly stimulated to 1.8- and 1.7-fold, respectively, PLC/PRF/5 to 1.4-fold, and SK-Hep-1 to 1.2-fold in a dose-dependent manner under HGF concentrations below 20 ng/ml. Neither HuH-7 nor HCC36 were affected. None of these cells were inhibited. All these cells expressed c-Met, the membrane receptor for HGF, and their c-Met would be activated to be phosphorylated upon addition of HGF. They also contained the ERK2 subgroup of mitogen-activated protein kinases (MAPKs). When HGF was added, their ERK2 would also be phosphorylated. The extent of ERK2 phosphorylation was partially correlated to their growth response to HGF. In conclusion, HGF could stimulate the growth of certain human hepatoma cells, probably through activation of c-Met and MAPKs.     

Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoptosis

Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-kappaB, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-kappaB, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease.     

Bone morphogenetic protein receptor inhibitors suppress the growth of glioblastoma cells

Molecular and Cellular Biochemistry - Glioblastomas (GBMs) are aggressive brain tumors that are resistant to chemotherapy and radiation. Bone morphogenetic protein (BMP) ligand BMP4 is being...     

Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction

U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit ³H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A₁, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A₁ pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.     

Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction

U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit ³H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A₁, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A₁ pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.     

The protein tyrosine phosphatase TCPTP suppresses the tumorigenicity of glioblastoma cells expressing a mutant epidermal growth factor receptor

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and GBMs frequently contain amplifications or mutations of the EGFR gene. The most common mutation results in a truncated receptor tyrosine kinase known as Delta EGFR that signals constitutively and promotes GBM growth. Here, we report that the 45-kDa variant of the protein tyrosine phosphatase TCPTP (TC45) can recognize Delta EGFR as a cellular substrate. TC45 dephosphorylated Delta EGFR in U87MG glioblastoma cells and inhibited mitogen-activated protein kinase ERK2 and phosphatidylinositol 3-kinase signaling. In contrast, the substrate-trapping TC45-D182A mutant, which is capable of forming stable complexes with TC45 substrates, suppressed the activation of ERK2 but not phosphatidylinositol 3-kinase. TC45 inhibited the proliferation and anchorage-independent growth of Delta EGFR cells but TC45-D182A only inhibited cellular proliferation. Notably, neither TC45 nor TC45-D182A inhibited the proliferation of U87MG cells that did not express Delta EGFR. Delta EGFR activity was necessary for the activation of ERK2, and pharmacological inhibition of ERK2 inhibited the proliferation of Delta EGFR-expressing U87MG cells. Expression of either TC45 or TC45-D182A also suppressed the growth of Delta EGFR-expressing U87MG cells in vivo and prolonged the survival of mice implanted intracerebrally with these tumor cells. These results indicate that TC45 can inhibit the Delta EGFR-mediated activation of ERK2 and suppress the tumorigenicity of Delta EGFR-expressing glioblastoma cells in vivo.