Sami Khoshyomn

Inhibition of epidermal growth factor receptor-associated tyrosine kinase blocks glioblastoma invasion of the brain.

OBJECTIVE Glioblastoma multiforme is a malignant primary brain tumor associated with short patient survival despite aggressive treatment, in part because of its propensity to aggressively infiltrate into brain tissue. Glioblastoma multiforme is also unique because it is the only nonepithelial human tumor for which excessive activation of epidermal growth factor receptor (EGFR) has been consistently linked to tumor growth and patient survival, and EGFR activation promotes glioblastoma multiforme infiltration in vitro. METHODS Cocultures of human glioblastoma spheroids (derived from three separate patients) and fetal rat brain aggregates were examined for infiltration using confocal microscopy, in the presence of 0 to 100 mumol/L genistein, a tyrosine kinase (TK) inhibitor, and 3 mumol/L tyrphostin A25, a specific EGFR-TK inhibitor. RESULTS Infiltration (not attachment) was completely inhibited by genistein at 10 mumol/L, the IC20 for monolayer growth inhibition in two cell lines. Tyrphostin A25 at 3 mumol/L (the IC20 for monolayers) reduced invasion in a third cell line from 38.8 +/- 6.1% invasion-hour per hour (n = 5) to 2.9 +/- 1.2% invasion-hour per hour (n = 6) (P = 0.0002, two-tailed t test, 93% inhibition), and from 0.54 +/- 0.065% per hour (slope) to 0.028 +/- 0.018% per hour (P = 0.00001, 95% inhibition). Maximal percent invasion was reduced from 100 +/- 0 to 7.4 +/- 5.6% of the fetal rat brain aggregate. No change was detected in EGFR-associated tyrosine phosphorylation at those doses in monolayers by 32P immunolabeling, consistent with the known effects of low concentrations of TK inhibitors. An increase in expression of wild-type and truncated EGFR was demonstrated by Western blotting. Invasion was equally well inhibited by a monoclonal antibody to the high-affinity ligand binding domain of EGFR and not by antibody to an inactive domain. CONCLUSION Our observations support the role of EGFR activation as a determinant by which glioblastoma invades normal brain tissue, and we show that invasion can be effectively inhibited at much lower concentrations of TK inhibitors than are necessary for growth suppression.

Synergistic Action of Genistein and Cisplatin on Growth Inhibition and Cytotoxicity of Human Medulloblastoma Cells

Objective: Recent experimental data have shown that dietary soy isoflavones such as genistein can significantly suppress invasiveness and growth of a number of human malignancies. In this study we examined whether genistein, at a concentration typical of plasma levels following soy formula intake, in combination with cisplatin or vincristine exhibited an additive or synergistic inhibitory effect on the growth of medulloblastoma cells. Methods: Three human medulloblastomas cell lines (HTB-186, CRL-8805 and MED-1) were treated with genistein at 6 μM, the maximum reported dietary plasma level in children, combined with cisplatin (0–10 μM) or vincristine (0–1 μM). Monolayer cell growth and cytotoxicity, as measured by colonigenic survival in soft agarose, were then compared in control and drug-treated cultures. Presence of apoptosis, using the DNA ladder assay and laser scanning cytometry, was investigated in all cell lines at those concentrations at which an enhancement of antiproliferative effect of cisplatin and vincristine in presence of genistein was observed. Results: Genistein at 6 μM led to a 2.8-fold increase in the monolayer growth inhibitory effect of cisplatin (0.05 μM) in HTB-186 cells (p = 4.5 × 10–4 by one-tailed t test). Genistein increased colonigenic survival inhibition of HTB-186 2.6-fold at the same cisplatin concentration (p = 1.5 × 10–4). Genistein caused a 1.3-fold increase in antiproliferative effect of cisplatin (0.5 μM) in CRL-8805 cells (p = 3.1 × 10–4). Similarly the inhibition of colonigenic survival was enhanced 2.0-fold in CRL-8805 (p = 1.22 × 10–5). The addition of genistein to 0.5 μM cisplatin led to a 1.7-fold increase in monolayer growth inhibition and 2.4-fold increase in colonigenic survival inhibition of MED-1 cells (p = 8.3 × 10–4 and p = 1.1 × 10–4 respectively). These effects were primarily synergistic but also additive in nature. The combination of genistein and vincristine, as compared to vincristine alone, caused a minimal-to-modest increase in antiproliferative effect on medulloblastoma cells studied here. We were unable to detect apoptosis by two methodologies in any of the medulloblastoma lines when genistein was combined with cisplatin or vincristine. Conclusion: These results indicate that genistein at typical dietary plasma levels can significantly enhance the antiproliferative and cytotoxic action of cisplatin and, to a lesser extent, vincristine. The implication for treatment of medulloblastomas of early childhood may be a reduction in the chemotherapeutic dose recommendations of these agents and subsequently a decrease in the risk of treatment sequelae for these patients.

Synergistic effect of genistein and BCNU on growth inhibition and cytotoxicity of glioblastoma cells

AbstractObjective: Recent experiments have shown that dietary soy isoflavones such as genistein can significantly suppress invasiveness and growth of a number of human malignancies. This study examined whether genistein, at a concentration typical of plasma levels following soy diet intake, in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, carmustine) exhibited an additive or synergistic inhibitory effect on the growth of glioma cells. Methods: The human glioblastoma multiforme (GBM) cell line U87 and the rodent C6 glioma were treated with genistein at 4 μM, combined with BCNU (0–50 μM). Monolayer cell growth and cytotoxicity, as measured by colonigenic survival in soft agarose, were then compared in control and drug-treated cultures. Presence of apoptosis, using the DNA ladder assay and laser scanning cytometry (LSC), was investigated in all cell lines at those concentrations where an enhancement of antiproliferative effect of BCNU in presence of genistein was observed. Results: A 32–41% increase in monolayer growth inhibition and a 28–42% increase in colony cytotoxicity in the U87 cell line were observed when genistein (4 μM) was added to BCNU in the 0–10 μM dose range. In the C6 cell line, a 30–36% increase in monolayer growth inhibition and a 39–54% increase in colony cytotoxicity were observed with the BCNU dose range of 0–50 μM. All experiments showed a significant increase in growth inhibition and a decrease in colonogenic survival (P<0.05). We were unable to detect apoptosis in any of the lines when genistein was combined with BCNU. Conclusion: These results indicate that genistein at typical adult dietary plasma levels can significantly enhance the antiproliferative and cytotoxic action of BCNU. The implication for treatment of GBM may be a reduction in the chemotherapeutic dose recommendations of these agents and subsequently a decrease in the risk of treatment sequelae for these patients.

Brain tumor invasion rate measured in vitro does not correlate with Ki-67 expression.

The need for more accurate prediction of the biological behavior of brain tumors has lead to the use of immunohistochemical methods for assessment of proliferating cell nuclear antigens such as Ki-67. There is a variable association of glioma Ki-67 labeling index with patient survival. Brain invasion by individual tumor cells also defines biological aggressiveness, and can be assessed in vitro. Further, proliferation and migration seem to be mutually exclusive behaviors for a given cell at a point in time. We studied the relationship between Ki-67 labeling index and invasion rate in a group of 10 gliomas, and 2 meningiomas. Human tumor spheroids obtained from operative speciments were co-cultured with fetal rat brain aggregates, and invasion rate was measured by confocal microscopic observation. There was no correlation between two measures of invasion and Ki-67 labeling. This finding supports the dichotomous nature of glioma proliferation and invasion, and may in part explain the limited usefulness of proliferation marker labeling.

Four-dimensional analysis of human brain tumor spheroid invasion into fetal rat brain aggregates using confocal scanning laser microscopy

The advent of confocal microscopy and fluorescence probes has made possible the routine visualization of the complex three-dimensional structures of thick fixed or live specimens. Four-dimensional (4-D) imaging of biological specimens (three-dimensional image reconstruction of the same living sample at different time points), remains a seldom-used application of confocal microscopy. In the present study we used 4-D imaging techniques to quantitate the invasion of human brain tumor spheroids into fetal rat brain aggregates (FRBAs), using the vital fluorescence membrane dyes, 3,3′-Dioctadecyloxacarbocyanine perchlorate (DiO) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) as visualization probes. We found invasion patterns similar to the in vivo behavior of these tumors in the brain. Glioblastoma spheroids showed diffuse and circumscribed infiltration accompanied by cystic degeneration or necrosis of FRBAs. Spheroids from cerebral metastasis, however, showed a sharp delimitation of the invasive margin, and did not penetrate the FRBA beyond a depth of 55 µm. Measured rates of glioblastoma invasion varied with the tumor specimens examined. The slopes of the mid-portions of plots of % infiltration vs. time (hours) for four glioblastoma cell lines were 1.7 ± 0.21 (SD), 0.67 ± 0.11, 1.4 ± 0.22 and 1.3 ± 0.18. We conclude that confocal microscopy with vital fluorescence probes is a practical method that allows for close monitoring and quantitation of the process of invasion in live tissue preparations, and may be used for assessing the in vitro effects of various tumor treatments.

Localization of CD44 at the Invasive Margin of Glioblastomas by Immunoelectron Microscopy

Glioblastoma multiforme is a highly invasive primary brain tumor, which is known to strongly express the CD44 cell adhesion receptor. A number of experimental studies suggest that the interaction of this receptor with extracellular matrix (ECM) proteins such as hyaluronic acid may in part mediate human glioma cell adhesion and invasion of brain tissue. Although the expression of CD44 and its spliced variants in brain tumors have been extensively studied, there have been no reports localizing its expression to the invasive margin of the tumor. The authors used immunoelectron microscopy to investigate the expression patterns of CD44 in an in vitro organotypic invasion assay. Tumor spheroids initiated from the U373 MG human glioblastoma line were confronted with fetal rat brain aggregates in a spheroid coculture system. The CD44 expression appeared at the interface between glioblastoma tumor spheroids and brain tissue, as well as in the spheroid itself. CD44 immunoreactivity was not detectable in mature 21-day fetal brain aggregates. The findings provide direct evidence that CD44 is expressed at the confrontational invasive border between glioblastomas and brain tissue, further supporting its role in glioma cell-ECM recognition and attachment.

Tumefactive multiple sclerosis plaque

We present a case of a large solitary demyelinating plaque in the brain masquerading as tumour. A 34 year old healthy woman presented with sudden onset of left hemiparesis. Subsequent MRI of the brain showed a single large 2.5 cm right sided enhancing lesion within the white matter of the parietal lobe …

Idiopathic Lymphocytic Cerebellitis

Accessible online at: www.karger.com/journals/pne A 16-year-old girl presented with a 3-month history of worsening right temporal and retroauricular headaches. She had previously been treated for otitis media with antibiotics, but her headache and pain continued to increase. Later, she developed an ataxic gait, increasing somnolence, vomiting and diplopia that prompted admission to hospital. On examination, she was also noticed to have right third-division trigeminal neuralgic pain and rightsided dysmetria on finger to nose testing.

Aneurysmal Bone Cyst of the Cervical Spine

Accessible online at: www.karger.com/journals/pne The patient was a 4-year-old white female presenting with a 3-month history of neck pain. Neurological exam was normal. Physical examination of the neck was significant for a palpable and firm right-sided posterior cervical mass. Plain X-rays of the neck revealed a destructive lesion of the C4 vertebra (fig. 1A). Flexion and extension lateral X-rays of the cervical spine did not reveal any gross instability. CT of the cervical spine showed a large cystic mass of the posterior elements of the C4 vertebra with involvement of the vertebral body. This lesion appeared to engulf the right vertebral artery and had a calcified rim (fig. 1B). MRI of the cervical spine showed an enhancing tumor (fig. 1C–E) without spinal cord compression (not shown). The differential diagnosis included osteoblastoma, aneurysmal bone cyst and giant cell tumor. Open biopsy of the lesion was performed. The tumor appeared to have a thin osseous wall with a blood-filled sponge-like content. Subsequent pathology demonstrated cavernous blood-filled spaces lined by fibrous walls consistent with an aneurysmal bone cyst.

High signal in subarachnoid spaces on FLAIR MR images in an adult with propofol sedation

Increased signal intensity in subarachnoid spaces and basal cisterns on T2 fluid attenuated inversion recovery (FLAIR) MR images occurred in children sedated with propofol.1 These MRI changes may be the result of increased CSF protein content or change in arterial oxygenation following propofol sedation. However, these findings have not been reported in adults sedated with propofol. A 47-year-old white man presenting with ataxia and myoclonic seizures underwent an MRI …