Investigating interaction of ligands with voltage dependent anion channel through noise analyses.

Ion channel-protein complexes inserted in the membrane act as molecular gates for transport across the membrane. The opening and closing of these gates can be controlled by one or more variables like ligands (small molecules, proteins, etc.), transmembrane voltage, and the concentration gradient of a chemical across the membrane. We have shown how current noise profile of voltage dependent anion channel can be used to monitor change in the gating of the channel after its modulation by various ligands. This is being proposed as a novel method to probe the interaction of ion channels with ligands.

Effect of angiostatin on liver metastasis of pancreatic cancer in hamsters.

The liver is the most common site of metastasis in pancreatic cancer, and there are no promising strategies to treat it. Angiostatin, a kringle-containing fragment of plasminogen, is a potent inhibitor of angiogenesis. The effect of angiostatin on liver metastasis in pancreatic cancer was investigated by using our established hamster model of liver metastasis. Pancreatic cancer cells (PGHAM-1, 1 x 10(6)) derived from N-nitrosobis(2-oxopropyl)amine (BOP)-induced pancreatic tumor in Syrian golden hamsters were transplanted into the spleen of female hamsters, and the animals were subcutaneously injected with angiostatin and saline. Subsequently, the macroscopic appearance of liver surface metastases was evaluated. In addition, histological sections of the liver metastases were analyzed for neovascularization, proliferation, and apoptosis on the basis of von Willebrand factor, argyrophilic nucleolar organizer region (Ag-NOR), and TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining, respectively. The results showed significant tumor growth retardation and inhibition of angiogenesis in metastatic liver tumors in response to treatment with angiostatin. Moreover, the metastases remained in a nearly dormant state due to a balance between apoptosis and proliferation of the tumor, with no detectable side effects. This is the first experimental trial of angiostatin on pancreatic cancer and liver metastasis. The results suggest that angiostatin therapy could be effective against liver metastases of pancreatic cancer.

The efficacy of plasminogen-urokinase combination in inducing posterior vitreous detachment.

PURPOSE: To investigate the toxicity of intravitreal plasminogen, urokinase, and their combination, and to evaluate their efficacy in the production of posterior vitreous detachment (PVD) in the rabbit eye. METHODS: Fifty-six albino New Zealand rabbits were examined before and after injection using the indirect ophthalmoscope, slit-lamp biomicroscopy, and electroretinography. Various concentrations of urokinase or recombinant plasminogen or a combination were injected intravitreally into the right eyes of four rabbits for each concentration. The left eyes of the animals served as controls and received 0.1 mL balanced salt solution. Group 1 was injected with pure urokinase (1,000, 5,000, or 10,000 IU); Group 2 with recombinant plasminogen (0.1, 0.4, 1.0, 2.0, 4.0, 8.0, or 16.0 caseinolytic units [CU]); and Group 3 with a combination of 1,000 IU urokinase (highest nontoxic dose) and nontoxic concentrations of plasminogen (0.1, 0.4, 1.0, or 2.0 CU). The animals were killed and the eyes enucleated 15 days after injection. Electron and light microscopy were performed. RESULTS: A concentration of 1,000 IU of urokinase was found to be nontoxic to the retina. Plasminogen concentrations of 2.0 CU or less did not produce retinal toxicity, whereas 4.0, 8.0, and 16.0 CU of plasminogen caused minimal-to-severe inflammatory response in the vitreous without histologic or electroretinographic changes. Neither plasminogen nor urokinase alone was successful in producing PVD. The combination of 1,000 IU of urokinase and 1.0 to 2.0 CU of plasminogen was effective without causing retinal toxicity. CONCLUSION: Posterior vitreous detachment can be produced in the rabbit eye using a combination of plasminogen and urokinase.

Angiostatin and endostatin: endogenous inhibitors of tumor growth.

Considerable progress has been made in the understanding of the molecular structure and mechanistic aspects of Angiostatin and Endostatin, endogenous angiogenesis inhibitors that have been shown to regress tumors in murine models. The growing body of literature surrounding these molecules and on the efficacy of these proteins is in part due to the ability to generate these proteins in recombinant systems as well characterized molecules. Recombinant human Angiostatin and Endostatin are in Phase I trials, following the manufacture of clinical grade material at large scale. This review highlights the recent advances made on understanding the structure and function of Angiostatin and Endostatin.

Simplified production of a recombinant human angiostatin derivative that suppresses intracerebral glial tumor growth.

Angiostatin is an endogenous inhibitor of tumor neovascularization that inhibits the proliferation of endothelial cells. Production of sufficient quantities of biologically active angiostatin by the enzymatic cleavage of plasminogen has proven difficult in that it has delayed clinical testing. We have cloned, expressed, and purified a recombinant human angiostatin derivative (K1-3) using a mammalian expression system. Through the addition of a secretory signal and polyhistidine sequence tag, K1-3 can be purified from post-culture medium by simple column chromatography. Purified K1-3 protein is apparently folded in an active conformation, as evidenced by its ability to bind to lysine-Sepharose. In vitro, recombinant K1-3 significantly suppressed endothelial cell proliferation in a dose-dependent manner with an IC50 of 50 nM. Using an animal model of intracranial brain tumors in immune-competent rats, systemic administration of purified recombinant K1-3 resulted in up to 85% suppression of tumor growth (P = 0.011). Growth suppression was accompanied by a 32% decrease (P = 0.01) in tumor neovascularization. This study demonstrates a simple method to produce a biologically active recombinant angiostatin derivative. The ability to suppress intracerebral tumor growth after systemic administration suggests that K1-3 is likely to have therapeutic value in the treatment of malignant glial tumors.

Angiostatin induces and sustains dormancy of human primary tumors in mice.

There is now considerable direct evidence that tumor growth is angiogenesis-dependent. The most compelling evidence is based on the discovery of angiostatin, an angiogenesis inhibitor that selectively instructs endothelium to become refractory to angiogenic stimuli. Angiostatin, which specifically inhibits endothelial proliferation, induced dormancy of metastases defined by a balance of apoptosis and proliferation. We now show that systemic administration of human angiostatin potently inhibits the growth of three human and three murine primary carcinomas in mice. An almost complete inhibition of tumor growth was observed without detectable toxicity or resistance. The human carcinomas regressed to microscopic dormant foci in which tumor cell proliferation was balanced by apoptosis in the presence of blocked angiogenesis. This regression of primary tumors without toxicity has not been previously described. This is also the first demonstration of dormancy therapy, a novel anticancer strategy in which malignant tumors are regressed by prolonged blockade of angiogenesis.

The preclinical toxicology of anisoylated plasminogen streptokinase activator complex.

The preclinical toxicological evaluation of anisoylated plasminogen streptokinase activator complex (APSAC) was designed with specific regard to the potential for immunogenic effects in animals arising from the high molecular weight of the complex. Animals were treated with multiples of the human dose by use of a dose regimen spanning that proposed clinically and in conventional repeat dose toxicity studies employing the maximum practicable dose level and duration. Few adverse effects were noted, despite the large doses administered with respect to the clinical dose. The thrombolytic activity of APSAC resulted in pronounced acute effects on blood coagulation times and fibrinogen levels in rats and dogs but there was little evidence of clinically relevant systemic toxicity in either species. Evidence of a possible effect on the liver was seen 24 hours after single doses much higher than the proposed human dose in the rat (40-fold higher) and dog (9-fold higher). No hepatic effects were apparent following repeated administration. The main adverse effect was focal acute myocarditis, which was seen only in rats of the Sprague Dawley strain. Administration of human plasminogen alone or in combination with streptokinase also produced this lesion, suggesting that plasminogen may play a central role in its appearance. Experiments in anaesthetised dogs showed APSAC to be devoid of undesirable haemodynamic effects. An intravenous acute toxicity study in rats with p-anisic acid, which is released on deacylation of APSAC, showed the levels of p-anisic acid which occur in humans to be of no toxicological significance. Finally, in a series of tests designed to investigate potential genetic toxicity, no mutagenic activity was detected.