A phase I and pharmacokinetic study of squalamine, a novel antiangiogenic agent, in patients with advanced cancers.
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PURPOSE: A Phase I study of squalamine, a novel antiangiogenic agent originally isolated from the dogfish shark Squalus acanthias, was conducted in patients with advanced cancers to: (a) determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT) and pharmacokinetics of squalamine lactate when given as a 120-h continuous i.v. infusion every two weeks; and (b) to obtain information on prolonged (>120-h) continuous i.v. infusions in patients who have tolerated 120-h infusions. EXPERIMENTAL DESIGN: A rapid dose escalation scheme was used that permitted intrapatient dose escalation. Three or more patients were treated at each dose, of which at least one patient started treatment de novo at that dose. Once DLT was encountered, the dose was decreased by one dose level, and the duration of infusion was prolonged from 10 up to 30 days in 5-day increments. RESULTS: Nineteen patients were treated at eight squalamine dose levels; the number of patients/dose level who received 120-h infusions were [expressed as dose in mg/m(2)/day (number of patients initiated de novo at that dose/total number of patients treated at that dose)]: 6 (3/3), 12 (3/6), 24 (1/5), 48 (2/6), 96 (4/10), 192 (2/6), 384 (3/8), and 538 (1/5). DLT was encountered at 384 mg/m(2)/day (1/3 de novo patients, 5/8 total patients) and 538 mg/m(2)/day (1/1 de novo patients, 4/5 total patients) and consisted of hepatotoxicity, characterized by grade 3 transaminase elevations that resolved 3-11 days after ceasing squalamine infusion. Three patients did not experience hepatotoxicity when first treated at 384 mg/m(2)/day but developed DLT at the same dose when de-escalated from 538 mg/m(2)/day. Other toxicities included grade 1-3 fatigue, grade 1-2 nausea, anorexia, and neuromuscular symptoms. The maximum duration of continuous i.v. infusion was 20 days at a dose rate of 192 mg/m(2)/day in one patient without adverse effects. Pharmacokinetic calculations revealed a linear relationship between area under the curve or Cmax and squalamine dose rate up to 384 mg/m(2)/day, with a prolonged terminal squalamine persistence in patient plasma (median t(1/2) = 18 h; range, 8-48 h). Transient tumor responses were observed in a patient with synovial cell sarcoma and a patient with breast carcinoma with cutaneous metastases. CONCLUSIONS: The best tolerated dose rate of squalamine when administered as a 120-h continuous i.v. infusion was 192 mg/m(2)/day; however, patients without prior exposure to squalamine appeared to tolerate a dose rate of 384 mg/m(2)/day without DLT. On the basis of preclinical evidence of synergy with cytotoxic agents and demonstration of human safety from this trial, additional clinical trials have been initiated with squalamine in combination with chemotherapy for patients with late stage lung cancer and ovarian cancer. (+info)
Brassinosteroid mutants uncover fine tuning of phytochrome signaling.
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Phytochromes (phy) A and B provide higher plants the ability to perceive divergent light signals. phyB mediates red/far-red light reversible, low fluence responses (LFR). phyA mediates both very-low-fluence responses (VLFR), which saturate with single or infrequent light pulses of very low fluence, and high irradiance responses (HIR), which require sustained activation with far-red light. We investigated whether VLFR, LFR, and HIR are genetically coregulated. The Arabidopsis enhanced very-low-fluence response1 mutant, obtained in a novel screening under hourly far-red light pulses, showed enhanced VLFR of hypocotyl growth inhibition, cotyledon unfolding, blocking of greening, and anthocyanin synthesis. However, eve1 showed reduced LFR and HIR. eve1 was found allelic to the brassinosteroid biosynthesis mutant dim/dwarf1. The analysis of both the brassinosteroid mutant det2 in the Columbia background (where VLFR are repressed) and the phyA eve1 double mutant indicates that the negative effect of brassinosteroid mutations on LFR requires phyA signaling in the VLFR mode but not the expression of the VLFR. Under sunlight, hypocotyl growth of eve1 showed little difference with the wild type but failed to respond to canopy shadelight. We propose that the opposite regulation of VLFR versus LFR and HIR could be part of a context-dependent mechanism of adjustment of sensitivity to light signals. (+info)
Squalamine and cisplatin block angiogenesis and growth of human ovarian cancer cells with or without HER-2 gene overexpression.
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Angiogenesis is important for growth and progression of ovarian cancers. Squalamine is a natural antiangiogenic sterol, and its potential role in treatment of ovarian cancers with or without standard cisplatin chemotherapy was assessed. Since HER-2 gene overexpression is associated with cisplatin resistance in vitro and promotion of tumor angiogenesis in vivo, the response of ovarian cancer cells with or without HER-2 gene overexpression to squalamine and cisplatin was evaluated both in tumor xenograft models and in tissue culture. Ovarian cancer cells with or without HER-2 overexpression were grown as subcutaneous xenografts in nude mice. Animals were treated by intraperitoneal injection with control vehicle, cisplatin, squalamine or cisplatin combined with squalamine. At the end of the experiment, tumors were assessed for tumor growth inhibition and for changes in microvessel density and apoptosis. Additional in vitro studies evaluated effects of squalamine on tumor and endothelial cell growth and on signaling pathways in human endothelial cells. Profound growth inhibition was elicited by squalamine alone and by combined treatment with squalamine and cisplatin for both parental and HER-2-overexpressing ovarian tumor xenografts. Immunohistochemical evaluation of tumors revealed decreased microvessel density and increased apoptosis. Although HER-2-overexpressing tumors had more angiogenic and less apoptotic activity than parental cancers, growth of both tumor types was similarly suppressed by treatment with squalamine combined with cisplatin. In in vitro studies, we found that squalamine does not directly affect proliferation of ovarian cells. However, squalamine significantly blocked VEGF-induced activation of MAP kinase and cell proliferation in human vascular endothelial cells. The results suggest that squalamine is anti-angiogenic for ovarian cancer xenografts and appears to enhance cytotoxic effects of cisplatin chemotherapy independent of HER-2 tumor status. (+info)
An Arabidopsis mitogen-activated protein kinase kinase kinase gene family encodes essential positive regulators of cytokinesis.
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The signal transduction pathways that control cytokinesis in plants are largely uncharacterized. Here, we provide genetic evidence that mitogen-activated protein kinase kinase kinases (MAPKKKs) play a role in the control of plant cell division. Using a reverse-genetic approach, we isolated plants carrying knockout alleles of the Arabidopsis MAPKKK genes ANP1, ANP2, and ANP3. The resulting single-mutant plants displayed no obvious abnormal phenotypes; two of the three double-mutant combinations displayed defects in cell division and growth; and the triple-mutant combination was not transmitted through either male or female gametes. The molecular and structural phenotypes displayed by the double mutants support a model in which the ANP family of MAPKKKs positively regulates cell division and growth and may negatively regulate stress responses. (+info)
Role of a heterotrimeric G protein in regulation of Arabidopsis seed germination.
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Seed germination is regulated by many signals. We investigated the possible involvement of a heterotrimeric G protein complex in this signal regulation. Seeds that carry a protein null mutation in the gene encoding the alpha subunit of the G protein in Arabidopsis (GPA1) are 100-fold less responsive to gibberellic acid (GA), have increased sensitivity to high levels of Glc, and have a near-wild-type germination response to abscisic acid and ethylene, indicating that GPA1 does not directly couple these signals in germination control. Seeds ectopically expressing GPA1 are at least a million-fold more responsive to GA, yet still require GA for germination. We conclude that the GPA1 indirectly operates on the GA pathway to control germination by potentiation. We propose that this potentiation is directly mediated by brassinosteroids (BR) because the BR response and synthesis mutants, bri1-5 and det2-1, respectively, share the same GA sensitivity as gpa1 seeds. Furthermore, gpa1 seeds are completely insensitive to brassinolide rescue of germination when the level of GA in seeds is reduced. A lack of BR responsiveness is also apparent in gpa1 roots and hypocotyls suggesting that BR signal transduction is likely coupled by a heterotrimeric G protein at various points in plant development. (+info)
Appetite suppression and weight reduction by a centrally active aminosterol.
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The rise in obesity and its complications has generated enormous interest in the regulation of feeding and body weight. We show that a spermine metabolite of cholesterol (MSI-1436) decreases body weight, specifically fat, by suppressing feeding and preventing the reduction in energy expenditure, hormonal changes, and patterns of neuropeptide expression normally associated with weight loss. MSI-1436 enters the brain after peripheral injection and is more potent when injected into the cerebral ventricle (intracerebroventricular [ICV]). Systemic or ICV MSI-1436 administration induced similar patterns of Fos immunoreactivity in the brain, especially the paraventricular hypothalamic nucleus (PVN). This brain region integrates neural signals from hypothalamic and brain stem nuclei and regulates feeding behavior, autonomic function, and neuroendocrine function. Microinjection of MSI-1436 into the PVN potently suppressed feeding and reduced body weight for several days. Unlike caloric restriction, MSI-1436 decreased mRNA levels of agouti-related peptide and neuropeptide Y in the hypothalamus. These findings indicate that MSI-1436 acts in the brain to regulate food intake and energy expenditure, likely through suppression of orexigenic hypothalamic pathways. (+info)
Major brassinosteroid (BR) effects such as BR-induced growth are mediated through genomic pathways because RNA synthesis inhibitors and protein synthesis inhibitors interfere with these processes. A limited number of BR-regulated genes have been identified hitherto. The majority of genes (such as BRU1, CycD3, Lin6, OPR3, and TRIP-1) were identified by comparisons of BR-treated versus control-treated plants. However, altered transcript levels after BR application may not reflect normal physiological events. A complementary approach is the comparison of BR-deficient plants versus wild-type plants. No artificial treatments interfere with endogenous signaling pathways, but a subset of phenotypic alterations of phytohormone-deficient plants most probably is secondary. To identify genes that are subject to direct BR regulation, we analyzed CPD antisense and dwf1-6 (cbb1) mutant plants. Both show a mild phenotype in comparison with BR-deficient mutants such as cpd/cbb3, det2, and dwf4. Plants were grown under two different environments to filter out BR deficiency effects that occur only at certain environmental conditions. Finally, we established expression patterns after BR treatment of wild-type and dwf1-6 (cbb1) plants. Ideally, a BR-regulated gene displays a dose-response relationship in such a way that a gene with decreased transcript levels in BR-deficient plants is BR inducible and vice versa. Expression profile analysis of above ground part of plants was performed by means of Affymetrix Arabidopsis Genome Arrays. (+info)
BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling.
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Brassinosteroids regulate plant growth and development through a protein complex that includes the leucine-rich repeat receptor-like protein kinase (LRR-RLK) brassinosteroid-insensitive 1 (BRI1). Activation tagging was used to identify a dominant genetic suppressor of bri1, bak1-1D (bri1-associated receptor kinase 1-1Dominant), which encodes an LRR-RLK, distinct from BRI1. Overexpression of BAK1 results in elongated organ phenotypes, while a null allele of BAK1 displays a semidwarfed phenotype and has reduced sensitivity to brassinosteroids (BRs). BAK1 is a serine/threonine protein kinase, and BRI1 and BAK1 interact in vitro and in vivo. Expression of a dominant-negative mutant allele of BAK1 causes a severe dwarf phenotype, resembling the phenotype of null bri1 alleles. These results indicate BAK1 is a component of BR signaling. (+info)