Paul L. Penar

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.

Nitric oxide and reactive oxygen species exert opposing effects on the stability of hypoxia-inducible factor-1α (HIF-1α) in explants of human pial arteries

Hypoxia induces angiogenesis, partly through stabilization of hypoxia‐inducible factor‐1α (HIF‐ 1α), leading to transcription of pro‐angiogenic factors. Here we examined the regulation of HIF‐ 1α by hypoxia and nitric oxide (NO) in explants of human cerebrovascular smooth muscle cells. Cells were treated with NO donors under normoxic or hypoxic (2% O2) conditions, followed by analysis of HIF‐1α protein levels. Treatment with the NO donor sodium nitroprusside reduced levels of HIF‐1α, whereas NO donors, NOC‐18 and S‐nitrosoglutathione, increased HIF‐1α levels. SIN‐1, which releases both NO and superoxide (O2•¯), reduced HIF‐1α levels, suggesting that inhibitory NO donors may elicit effects through peroxynitrite (ONOO•¯). O2•¯ generation by xanthine/xanthine oxidase also reduced HIF‐1α levels, confirming an inhibitory role for reactive oxygen species (ROS). Furthermore, superoxide dismutase increased HIF‐1α levels, and the NO scavenger carboxy‐PTIO reversed HIF‐1α stabilization by NO donors. Effects on HIF‐1α levels correlated with vascular endothelial growth factor transcription but did not affect HIF‐1α transcription, as measured by RT‐PCR and luciferase‐reporter assays. The results indicate that HIF‐1α is stabilized by agents that produce NO and reduce ROS but destabilized by agents that increase ROS, including O2•¯ and ONOO•¯. Thus we propose that the effect of NO on HIF‐1α signaling is critically dependent on the form of NO and the physiological environment of the responding cell.

Treatment of Single Brain Metastasis with Resection, Intracavity Carmustine Polymer Wafers, and Radiation Therapy Is Safe and Provides Excellent Local Control

Purpose: To define the safety and efficacy of carmustine polymer wafers when added to a regimen of surgery and external beam radiotherapy for treatment of a single brain metastasis. Experimental Design: Adult patients underwent craniotomy for a single brain metastasis, and carmustine polymer wafers were placed in the tumor resection cavity. Patients then received whole-brain radiotherapy and were followed for patterns of recurrence in the central nervous system, toxicity, and survival. Results: We enrolled 25 patients with solitary brain metastases from lung (13 patients), melanoma (4 patients), breast (3 patients), and renal carcinoma (3 patients). Two patients had severe adverse events thought to be related to wafer placement, one with seizures alone, and one with seizures and subsequent respiratory compromise. Both responded to medical therapy. There were no wound infections. The local recurrence rate was surprisingly low (0%). Four patients (16%) relapsed elsewhere in the brain, and two patients (8%) relapsed in the spinal cord. Median survival was 33 weeks; 33% of patients survived 1 year, and 25% survived 2 years. Conclusions: The addition of local chemotherapy delivered via carmustine polymer wafers to a regimen of surgical resection and external beam radiotherapy was well tolerated by patients undergoing surgery for a single brain metastasis. There were no local recurrences, suggesting that this treatment further reduced the risk of local relapse.

Protection against ischemia: a physiological function of the renin-angiotensin system.

The renin-angiotensin system (RAS) is involved in a complex mechanism that serves to preserve the blood supply to organs so that they can maintain cellular function. Angiotensin II exerts this effect, independently of the blood pressure generated, through two time-related events: a fast opening of the reserve collateral circulation and a much slower response of new vessel formation or angiogenesis. This effect is observed in rats with ligation of the abdominal aorta and in gerbils with abrupt or progressive unilateral carotid artery ligation. Inhibition of the angiotensin-converting enzyme (ACE) or the angiotensin II receptor represses this effect, and it appears that it is mediated through a non-AT1 receptor site of angiotensin II. Many tumors, both benign and malignant, express renin and angiotensin. It seems that the stimulating action of angiotensin II on angiogenesis could also be involved in preserving the blood supply to tumor cells. Administration of converting enzyme inhibitors increases survival and decreases tumor size in tumor-bearing rats. These observations support the hypothesis that the RAS, directly or indirectly, is involved in situations in which the restoration of blood supply is critical for the viability of cells and that it is present not only in normal but also in pathological conditions such as tumors. In view of the ubiquitous presence of renins and angiotensins, it is also likely to be involved in other conditions, such as inflammation, arthritis, diabetic retinopathy, and retrolental fibroplasia, among others in which angiogenesis is prominent. In addition, angiotensin II could be involved, through the counterbalance of the AT1 and AT2 receptors, in the rarefaction of blood vessels as an etiologic component of essential hypertension.

Oxyhemoglobin-Induced Suppression of Voltage-Dependent K+ Channels in Cerebral Arteries by Enhanced Tyrosine Kinase Activity

Cerebral vasospasm following aneurysmal subarachnoid hemorrhage (SAH) has devastating consequences. Oxyhemoglobin (oxyhb) has been implicated in SAH-induced cerebral vasospasm as it causes cerebral artery constriction and increases tyrosine kinase activity. Voltage-dependent, Ca2+-selective and K+-selective ion channels play an important role in the regulation of cerebral artery diameter and represent potential targets of oxyhb. Here we provide novel evidence that oxyhb selectively decreases 4-aminopyridine sensitive, voltage-dependent K+ channel (Kv) currents by ≈30% in myocytes isolated from rabbit cerebral arteries but did not directly alter the activity of voltage-dependent Ca2+ channels or large conductance Ca2+-activated (BK) channels. A combination of tyrosine kinase inhibitors (tyrphostin AG1478, tyrphostin A23, tyrphostin A25, genistein) abolished both oxyhb-induced suppression of Kv channel currents and oxyhb-induced constriction of isolated cerebral arteries. The Kv channel blocker 4-aminopyridine also inhibited oxyhb-induced cerebral artery constriction. The observed oxyhb-induced decrease in Kv channel activity could represent either channel block, or a decrease in Kv channel density on the plasma membrane. To explore whether oxyhb altered trafficking of Kv channels to the plasma membrane, we used an antibody generated against an extracellular epitope of Kv1.5 channels. In the presence of oxyhb, staining of Kv1.5 on the plasma membrane surface was markedly reduced. Furthermore, oxyhb caused a loss of spatial distinction between staining with Kv1.5 and the general anti-phosphotyrosine antibody PY-102. We propose that oxyhb-induced suppression of Kv currents occurs via a mechanism involving enhanced tyrosine kinase activity and channel endocytosis. This novel mechanism may contribute to oxyhb-induced cerebral artery constriction following SAH.

α-Thrombin Stimulates Urokinase Production and DNA Synthesis in Cultured Human Cerebral Microvascular Endothelial Cells

Abstract α-Thrombin regulation of endothelial cell (EC) fibrinolysis has been documented by using endothelia derived from a number of anatomic locations but not with those derived from the human cerebral vasculature. In the present study, the fibrinolytic properties of human cerebral microvascular ECs and their regulation by α-thrombin are delineated and contrasted with those of human umbilical vein and foreskin microvascular ECs. In cerebral ECs, α-thrombin elicited a unique dose-dependent increase in urokinase production and DNA synthesis. Maximal stimulation, observed with 10 nmol/L α-thrombin, resulted in a 30- to 50-fold increase in urokinase production and a concomitant fourfold increase in DNA synthesis; the increase in urokinase was reflected in higher steady-state levels of urokinase mRNA. The major urokinase product secreted is the single-chain form of the enzyme. No effect was observed with the addition of other proteases or catalytically inactive variants of α-thrombin. A thrombin receptor agonist peptide upregulated urokinase production but had no effect on DNA synthesis, suggesting that fibrinolysis is mediated by the thrombin receptor but that proliferation is regulated by a different pathway. These findings suggest the possibility that the cerebral microvasculature may be a specialized region of the vascular system in which urokinase-type plasminogen activator, not tissue-type plasminogen activator, is the key catalyst of fibrin lysis when the brain responds to thrombotic events and that α-thrombin may regulate repair of the cerebral microvascular system.

Weakness of Sympathetic Neural Control of Human Pial Compared With Superficial Temporal Arteries Reflects Low Innervation Density and Poor Sympathetic Responsiveness

BACKGROUND AND PURPOSE The primary goal of these studies was to understand and investigate the capacity of perivascular nerves to influence the tone of human pial arteries and to compare them with other human cephalic arteries, the superficial temporal and middle meningeal. METHODS Responses to electrical activation of intramural nerves and related features of fresh segments of human cephalic arteries-the pial (PA; 478+/-34 microm ID), middle meningeal (MMA; 540+/-41 microm ID), and superficial temporal (STA; 639+/-49 microm ID)-obtained from patients aged 15 to 82 years during surgical procedures were studied on a resistance artery myograph. RESULTS The PA segment responses to electrical nerve activation and to norepinephrine (NE; 10[-5] mol/L) were 1% and 21% of tissue maximum, respectively, compared with 6% and 34% for the MMA and 14% and 90% for the STA. Tissue maximum was defined as the force increase to 127 mmol/L KCl plus arginine vasopressin (1 microm). All arteries dilated well to acetylcholine. Possible explanations for the PA marginal neurogenic responses were assessed. NE ED50 was 5.4+/-2.2 X 10(-7) mol/L and did not vary with age or diameter. NE responsiveness did not increase in vessels with spontaneous or raised potassium-induced tone. Relaxation to isoproterenol was variable and propranolol did not increase the neurogenic response. Neither N(G)-monomethyl-L-arginine, N(G)-nitro-L-arginine methyl ester, endothelium removal, nor indomethacin consistently influenced the contractions to NE or neurogenic reactivity. The weak PA neurogenic response is in keeping with its poor innervation. As determined by catecholamine histofluorescence, innervation in the PA is sparse, with density increasing in the order PA, MMA, and STA. The incidence of nerve structures in the PA adventitio-medial junction was only 3% of those in the STA, and these were situated more than 3 microm from the closest smooth muscle cell. CONCLUSIONS We conclude that the weak neurogenic response of adult human pial artery reflects its poor innervation and responsiveness to NE, implying that these features are not important in the regulation of its diameter.

Role of Ca2+-Activated K+ Channels in the Regulation of Membrane Potential and Tone of Smooth Muscle in Human Pial Arteries

Smooth muscle cells (SMCs) in 58% of human pial arteries obtained during surgery showed no spontaneous contractions and displayed a stable resting membrane potential (MP) of -54.7 +/- 1.5 mV. Those that exhibited periodic spontaneous contractions associated with periodic depolarization and generation of spontaneous action potentials (APs) had a less negative MP of -43.1 +/- 0.5 mV (42%). Inhibition of calcium-activated potassium (KCa) channels in the silent arteries by charybdotoxin (CTX) and tetraethylammonium ions (TEA) induced dose-dependent depolarization, AP generation, and contraction. TEA and CTX enhanced the spontaneous depolarization and force in arteries that exhibited spontaneous activity. They also prolonged the spontaneous APs up to several times and increased their upstroke amplitude. Both TEA and CTX failed to produce significant depolarization in arteries treated with nifedipine. It is concluded that KCa channels are important regulators of human pial artery SMC resting MP and tone. They are also involved in the control of AP amplitude and duration and the associated contractions. These data suggest that alterations in the activity of SMC KCa channels could be responsible for the appearance of spontaneous activity in human pial arteries in vitro and that impaired function of these channels might be related to vasospastic phenomena in human cerebral circulation.

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.