RESUMEN
Rana pipiens oocytes contain two homologues of pancreatic ribonuclease A that are cytostatic and cytotoxic to human cancer cells. Extensively studied Onconase is in advanced Phase IIIb clinical trials against malignant mesothelioma, while Amphinase is a novel enzyme in pre-clinical development. Onconase is the smallest (104 amino acid residues) member of the ribonuclease A superfamily while Amphinase (114 residues) is the largest among amphibian ribonucleases. Both enzymes share the characteristic frog ribonucleases C-terminal disulfide bond but another signature of this group, the N-terminal pyroglutamate, an integral part of Onconase active site is not conserved in Amphinase. Although Onconase and Amphinase are weak catalysts their enzymatic activities are required for cytostatic and cytotoxic activity. While it was postulated that tRNA is the primary substrate of Onconase in vivo there is also extensive indirect evidence that suggests other RNA species, in particular micro RNAs, may actually be the critical target of these ribonucleases. The cytostatic effects of Onconase and Amphinase are manifested as cell arrest in the G(1) cell cycle phase. Apoptosis then follows involving activation of endonucleases(s), caspases, serine proteases and transglutaminase. Onconase was shown to be strongly synergistic when combined with numerous other antitumor modalities. Onconase and Amphinase are highly cationic molecules and their preferential toxicity towards cancer cells (having distinctly higher negative charge compared to normal cells) may depend on increased binding efficiency to the cell surface by electrostatic interactions.
Asunto(s)
Antineoplásicos/farmacología , Oocitos/enzimología , Rana pipiens , Ribonucleasas/farmacología , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Estabilidad de Enzimas , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , ARN de Transferencia/antagonistas & inhibidores , Ribonucleasas/química , Ribonucleasas/aislamiento & purificación , Alineación de SecuenciaRESUMEN
Onconase (ONC), an antitumor ribonuclease from oocytes of a frog Rana pipiens, capable of inducing apoptosis in many cell lines is synergistic with several other anticancer drugs. Since cytotoxic effects of numerous drugs are modulated by reactive oxygen intermediates (ROI), we have studied effects of ONC on the intracellular level of oxidants in several normal cell types as well as tumor cell lines. It is demonstrated for the first time that ONC substantially decreases the content of ROI in all cell lines studied. This effect depends on the ribonucleolytic activity of the enzyme and is due to both, decreased rate of ROI generation and accelerated rate of their degradation. Onconase decreases the mitochondrial transmembrane potential and consequently, generation of ATP. Simultaneously the enzyme decreases the expression of an antiapoptotic protein Bcl-2, and upregulates the proapoptotic Bax protein. These finding are consistent with the enzyme propensity to induce apoptosis. The observed antioxidant activity of ONC may be an important element of its cytotoxicity towards cancer cells. The enzyme seems to exert its biological activities by interfering with the redox system of cellular regulation.
Asunto(s)
Antineoplásicos/farmacología , Ribonucleasas/fisiología , Animales , Antineoplásicos/química , Apoptosis , Línea Celular Tumoral , Humanos , Células Jurkat , Oxidantes/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Rana pipiens , Especies Reactivas de Oxígeno , Ribonucleasas/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Onconase (Onc) is an amphibian ribonuclease of the pancreatic RNase family that is cytostatic and cytotoxic to several tumor lines. It also shows anti-tumor activity in mouse tumor models and is currently in phase III clinical trials. In animal tests and clinical trials Onc shows lesser toxicity and fewer side effects compared to most chemotherapeutic drugs. Intriguingly, repeated infusions of this protein do not cause apparent immunological reactions in patients. The aim of the present study was to investigate sensitivity to Onc of human lymphocytes during their mitogenic stimulation in response to the polyvalent mitogen phytohemagglutinin (PHA), and in mixed allogeneic lymphocyte cultures. Unexpectedly, we observed that frequency of cells undergoing activation-induced apoptosis was markedly increased in all cultures containing Onc. Apoptosis was measured by flow cytometry using markers that detect activation of caspases, the in situ presence of DNA strand breaks, and loss of fragmented DNA ('sub-G1' cell subpopulation). The enhancement of frequency of activation-induced apoptosis (up to 244%) was observed at 4.2-83 nM Onc concentration, which is at least an order magnitude lower than its minimal concentration reported to affect proliferation or induce apoptosis of leukemic and solid tumor cell lines. The cell cycle progression of lymphocytes that responded to PHA mitogenically was not affected at 8.3 or 83 nM Onc concentration. Because activation-induced apoptosis is the key mechanism regulating several in vivo immunological functions including induction of tolerance, the observed effects of Onc may explain the apparent lack of immune reactions to this protein in treated patients. The propensity of Onc to potentiate the activation-induced apoptosis suggests that this drug may have clinical utility as immunomodulating agent, e.g., to suppress transplant rejection or treat autoimmune diseases.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas del Huevo/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos/patología , Ribonucleasas/farmacología , Animales , Inhibidores de Caspasas , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Humanos , Etiquetado Corte-Fin in Situ , Linfocitos/enzimología , Fitohemaglutininas/farmacología , Propidio/metabolismo , Rana pipiensRESUMEN
Onconase (Onc) is a ribonuclease from amphibian oocytes that is cytostatic and cytotoxic to many tumour lines. It shows in vivo antitumour activity in mouse tumour models and is currently in Phase III clinical trials. The present study was designed to test whether cytotoxic effects of ONC can be modulated by differentiating agents. Human leukaemic HL-60 and prostate cancer LNCaP and JCA-1 cells were treated with Onc in the absence and presence of several inducers of differentiation and frequency of apoptosis was assessed using three different cytometric methods and confirmed by analysis of cell morphology. A moderate degree of apoptosis observed after 48-72 h incubation of HL-60 cells in the presence of 0.42 microM Onc alone was markedly potentiated by administration of retinoic acid (all trans), sodium butyrate or dimethylsulfoxide at concentrations known to induce differentiation but be minimally cytotoxic. Likewise, the frequency of apoptosis of LNCaP and JCA-1 cells treated with Onc was increased in the cultures to which phenylbutyrate was added. Although cell treatment with Onc alone, with each of the differentiating agents alone or with Onc in combination with the differentiating agents led to an increase in the proportion of G1 cells, no specific cell cycle phase preference in induction of apoptosis was observed. The data suggest that cells undergoing differentiation are particularly vulnerable to Onc; a combination of Onc and differentiating agents should be considered for further in vivo tests to assess its possible usefulness in the clinic.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas del Huevo/farmacología , Ribonucleasas/farmacología , Butiratos/farmacología , Diferenciación Celular , ADN/metabolismo , Dimetilsulfóxido/farmacología , Sinergismo Farmacológico , Células HL-60 , Humanos , Masculino , Fenilbutiratos/farmacología , Neoplasias de la Próstata , ARN/metabolismo , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
The RNase-like onconase, isolated from amphibian oocytes, showed increases in median tumor pO2 in solid tumors (1). This led us to consider if onconase could decrease cellular O2 consumption (QO2) on 9L rat glioma as well as DU145 human prostate adenocarcinoma cells. Using a Clark-type electrode chamber, we observed that onconase significantly inhibited QO2 in both tumors we tested. Since onconase-induced reduction in QO2 could lead to increases in radiation sensitivity, due to the diffusion of O2 to previously hypoxic tumor cells, we used androgen-insensitive DU145 cells to study onconase-induced changes in radiation sensitivity in vitro. Radiation sensitization was achieved with > 5 micrograms/ml of onconase, regardless of the p53 status of tumor cells. Data presented here suggested that onconase-induced enhancement in radiation sensitization in vitro of androgen-insensitive prostate cancer cells warranted further studies of radiation responses in vivo, prior to clinical settings for the advanced-stages of prostate cancer.
Asunto(s)
Antineoplásicos/toxicidad , División Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Proteínas del Huevo/toxicidad , Consumo de Oxígeno/efectos de los fármacos , Tolerancia a Radiación/efectos de los fármacos , Ribonucleasas/toxicidad , Adenocarcinoma , Animales , Neoplasias Encefálicas , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Glioma , Humanos , Masculino , Neoplasias de la Próstata , Ratas , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/análisisRESUMEN
BACKGROUND AND OBJECTIVES: The effects of Onconase (Onc) on the tumor growth in vitro and in vivo were examined. Because elevated tumor interstitial fluid pressure (TIFP) is one of the major causes of inadequate drug delivery into solid tumors, we tested if Onc could lower TIFP in solid tumors. METHODS: We used several assays including a clonogenic assay and a growth delay assay for the determination of anti-tumoricidal effects of Onc. We also measured Onc-induced changes in several tumor physiological parameters. RESULTS: Onc demonstrated cytotoxic effects in all eight exponentially growing cell lines in vitro. It effectively inhibited the growth of all four transplanted tumors in vivo, and significantly reduced TIFP in all four tumors. Onc also induced increases in tumor blood flow (TBF) as well as increases in median tumor oxygen partial pressure (pO(2)) in solid tumors. CONCLUSIONS: Onc showed anti-tumoral effects on various tumor cells in vitro as well as in vivo. We also gained some insight regarding the potential physiological benefit of Onc as a new therapeutic agent in cancer treatment. Due to increases in both TBF and tumor pO(2), Onc could be a potential candidate as a novel radiation enhancer; therefore, the study of the radiation response in vivo is warranted.
Asunto(s)
Antineoplásicos/farmacología , Proteínas del Huevo/farmacología , Neoplasias/patología , Ribonucleasas/farmacología , Animales , Circulación Sanguínea , Femenino , Humanos , Flujometría por Láser-Doppler , Ratones , Ratones Endogámicos C3H , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/fisiopatología , Ratas , Azul de Tripano , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
Onconase is a 12 kDa protein homologous to pancreatic RNase A isolated from amphibian oocytes which shows cytostatic and cytotoxic activity in vitro, inhibits growth of tumors in mice and is in phase III clinical trials. The present study was aimed to reveal mechanisms by which onconase perturbs the cell cycle progression. Human histiocytic lymphoma U937 cells were treated with onconase and expression of cyclins D3 and E, as well as of the cyclin-dependent kinase inhibitors (CKIs) p16INK4A, p21WAF1/CIP1 and p27KIP1 (all detected immunocytochemically) was measured by multiparameter flow cytometry, in relation to the cell cycle position. Also monitored was the status of phosphorylation of retinoblastoma protein (pRb) by a novel method utilizing mAb which specifically detects underphosphorylated pRb in individual cells. Cell incubation with 170 nM onconase for 24 h and longer led to their arrest in G1 which was accompanied by a decrease in expression of cyclin D3, no change in cyclin E, and enhanced expression of all three CKIs. pRb was underphosphorylated in the onconase arrested G1 cells but was phosphorylated in the cells that were still progressing through S and G2/M in the presence of onconase. The cytostatic effect of onconase thus appears to be mediated by downregulation of cyclin D3 combined with upregulation of p27KIP1, p16INK4A and p21WAF1/CIP1, the events which may prevent phosphorylation of pRb during G0/1 and result in cell arrest at the restriction point controlled by Cdk4/6 and D type cyclins.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Ciclo Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Ciclinas/metabolismo , Proteínas del Huevo/farmacología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína de Retinoblastoma/metabolismo , Ribonucleasas/farmacología , Proteínas Supresoras de Tumor , Animales , Ciclina D3 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Fase G1 , Genes Supresores de Tumor , Humanos , Linfoma de Células B Grandes Difuso/metabolismo , Ratones , Fosforilación , Células Tumorales CultivadasRESUMEN
Onconase (ONC) a ribonuclease from amphibian oocytes is cytostatic and cytotoxic to many human tumor lines, shows in vivo antitumor activity in mouse tumor models and is in Phase III clinical trials. The mechanism of antitumor activity of ONC is presumed to be due to its internalization, degradation of intracellular RNA and suppression of protein synthesis. Since apoptosis triggered by TNF-alpha is known to be potentiated by inhibitors of protein synthesis, we have hypothesized that it also may be potentiated by ONC. Indeed, preincubation of U-937 or HL-60 leukemic cells with 0.17 microM ONC rendered them more sensitive to induction of apoptosis by TNF-alpha or antibody to CD95 (Fas). The mechanism by which ONC amplifies the effect of TNF-alpha may involve suppression of induction of the survival genes whose expression is triggered by activation of NFkB by this factor.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas del Huevo/metabolismo , Ribonucleasas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Antígenos CD/metabolismo , Sinergismo Farmacológico , Proteína Ligando Fas , Células HL-60 , Humanos , Glicoproteínas de Membrana/farmacología , Ratones , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral , Células Tumorales Cultivadas , Receptor fas/metabolismoRESUMEN
Ribonucleases appear to have physiologic roles in host defense against cancer, viruses, and other parasites. Previously it was shown that select ribonucleases added to cells concurrently with virions blocked human immunodeficiency virus, type I (HIV-1) infection of H9 cells. We now report that a ribonuclease homologous to RNase A, named onconase, inhibits virus replication in chronically HIV-1-infected human cells without killing the virally infected cell. Examining the mechanism of this inhibition shows that onconase enters the infected cells and degrades HIV-1 RNA without degrading ribosomal RNA or the three different cellular messenger RNAs analyzed. The homologous human pancreatic RNase lacks anti-viral activity. Comparing recombinant forms of onconase and a onconase-human RNase chimera shows that the N-terminal 9 amino acids and the pyroglutamyl residue of onconase are required for full anti-viral activity. Thus extracellular ribonucleases can enter cells, metabolize select RNAs, and inhibit HIV virion production within viable replicating cells.
Asunto(s)
Antivirales , Proteínas del Huevo/metabolismo , VIH-1/crecimiento & desarrollo , ARN Viral/metabolismo , Ribonucleasas/metabolismo , Proteínas del Huevo/farmacología , Espacio Extracelular/enzimología , Proteína p24 del Núcleo del VIH/análisis , Humanos , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , Proteínas Recombinantes , Ribonucleasas/farmacología , Relación Estructura-Actividad , Especificidad por Sustrato , Células Tumorales Cultivadas , Replicación Viral/efectos de los fármacosRESUMEN
The role of systemic cytotoxic therapy for the treatment of advanced non-small cell lung cancer (NSCLC) remains controversial. The response rate (RR) and the median survival time (MST) are the two most frequently used parameters for the assessment of efficacy of the anti-cancer therapies. The relationship between the previously reported RRs and MSTs from published chemotherapy trials in patients with advanced NSCLC was examined using linear regression analysis. The MST of the thirty patients with advanced NSCLC treated with ONCONASE (ONC) as a single agent was 7.7 months which compared favorably with the MSTs of patients treated with a variety of chemotherapeutic regimens either as single agents or combinations, as well as placebo and supportive care only. Moreover, the toxicity profile of ONC compared favorably to the profiles of other chemotherapy regimens. ONC had a favorable impact on the overall MST, including patients with stage IV disease, patients with poor performance status, and patients previously treated with radiotherapy and chemotherapy. The MST of 5 patients who had a stabilization of previously progressive disease was 9.3 months. Based on its positive impact on the MST, ONC appears to have a single agent activity in patients with advanced NSCLC, and it should be further investigated, particularly in combination with synergistic drugs, in concurrently controlled and prospectively randomized clinical trials. The duration and the quality of survival should be considered as the most meaningful parameters in assessing clinical efficacy of anti-cancer agents.
RESUMEN
Onconase and bovine seminal RNase, two members of the RNase A superfamily, inhibit human immunodeficiency virus type 1 replication in H9 leukemia cells 90-99.9% over a 4-day incubation at concentrations not toxic to uninfected H9 cells. Two other members of the same protein family, bovine pancreatic RNase A and human eosinophil-derived neurotoxin, have no detectable antiviral activity, demonstrating a strikingly selective antiviral activity among homologous ribonucleases. The antiviral RNases do not appear to affect viral particles directly but inhibit replication in host cell cultures. Onconase, already in clinical trials for cancer therapy, and bovine seminal RNase have potential as antiviral therapeutics.
Asunto(s)
Antivirales/toxicidad , Proteínas del Huevo/toxicidad , VIH-1/fisiología , Ribonucleasa Pancreática/toxicidad , Ribonucleasas/toxicidad , Replicación Viral/efectos de los fármacos , Animales , Antineoplásicos/toxicidad , Bovinos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , VIH-1/efectos de los fármacos , Humanos , Cinética , Leucemia , Masculino , Semen/enzimología , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Purkinje cell toxicity is one of the characteristic features of the Gordon phenomenon, a syndrome manifested by ataxia, muscular rigidity, paralysis, and tremor that may lead to death (Gordon, 1933). Two members of the RNase superfamily found in humans, EDN (eosinophil-derived neurotoxin) and ECP (eosinophil cationic protein), cause the Gordon phenomenon when injected intraventricularly into guinea pigs or rabbits. We have found that another member of the RNase superfamily, an antitumor protein called onconase, isolated from Rana pipiens oocytes and early embryos, will also cause the Gordon phenomenon when injected into the cerebrospinal fluid of guinea pigs at a dose similar to that of EDN (LD50, 3-4 micrograms). Neurologic abnormalities of onconase-treated animals were indistinguishable from those of EDN-treated animals, and histology showed dramatic Purkinje cell loss in the brains of onconase-treated animals. The neurotoxic activity of onconase correlates with ribonuclease activity. Onconase modified by iodoacetic acid to eliminate 70% and 98% of the ribonuclease activity of the native enzyme displays a similar decrease in ability to cause the Gordon phenomenon. In contrast, the homologous bovine pancreatic RNase A injected intraventricularly at a dose 5000 times greater than the LD50 dose of EDN or onconase is not toxic and does not cause the Gordon phenomenon. A comparison of the RNase activities of EDN, onconase, and bovine pancreatic RNase A using three pancreatic RNA substrates demonstrates that onconase is orders of magnitude less active enzymatically than EDN and RNase A. Thus, another member of the RNase superfamily in addition to EDN and ECP can cause the Gordon phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Antineoplásicos/toxicidad , Proteínas Sanguíneas/toxicidad , Proteínas del Huevo/toxicidad , Neurotoxinas/toxicidad , Células de Purkinje/efectos de los fármacos , Ribonucleasa Pancreática/toxicidad , Ribonucleasas/toxicidad , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/administración & dosificación , Proteínas Sanguíneas/química , Ventrículos Cerebrales/efectos de los fármacos , Ventrículos Cerebrales/fisiología , Proteínas del Huevo/administración & dosificación , Proteínas del Huevo/química , Proteínas en los Gránulos del Eosinófilo , Neurotoxina Derivada del Eosinófilo , Femenino , Cobayas , Humanos , Inyecciones Intraventriculares , Inyecciones Espinales , Datos de Secuencia Molecular , Neurotoxinas/administración & dosificación , Neurotoxinas/química , Células de Purkinje/patología , Conejos , Ribonucleasa Pancreática/administración & dosificación , Ribonucleasa Pancreática/química , Ribonucleasas/administración & dosificación , Ribonucleasas/química , Homología de Secuencia de Aminoácido , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiologíaRESUMEN
ONCONASE(R) (ONC), previously known as P-30 Protein, is a novel amphibian protein isolated from Rana pipiens eggs/early embryos (1) which demonstrates cytostatic and cytotoxic activity against several human tumor cell lines in vitro, as well as anti-tumor activity in vivo. Animal toxicology studies in rats and dogs revealed dose-dependent weight loss, some skeletal muscle and myocardial degenerative changes, a decrease in albumin and bilirubin levels in rats, and a dose-related elevation of serum transaminases and alkaline phosphatase in both species. A human weekly schedule Phase I study of intravenous bolus ONC was initiated, with dose levels ranging from 60 mug/m2 (anticipated human dose) to 960 mug/m2. Five patients were treated per dose level, without dose escalations within the same patients. Dose levels were doubled in new groups of patients with a variety of relapsing and resistant tumors. A correlation was noted between the dose level and the number of doses (cumulative effect), and the toxicities observed. The dose limiting toxicity was renal as manifested by proteinuria with edema, +/- azotemia and fatigue. Other side effects included flushing, myalgias, transient dizziness, and decreased appetite. Two patients, one at 480 mug/m2 and another at 960 mug/m2 levels, developed reversible hypotensive reactions preceded by flushing. The maximum tolerated dose (MTD) appears to be 960 mug/m2. Incidental findings included some objective responses in non-small cell lung, esophageal, and colorectal carcinomas. It has been concluded that ONCONASE was well tolerated by the majority of patients, demonstrated a consistent and reversible clinical toxicity patterns, did not induce most of the toxicities (such as, e.g., myelosuppression and alopecia) associated with most of the chemotherapeutic agents and, in view of its demonstrated objective clinical activity observed in patients harboring resistant solid tumors, the Phase II clinical trials have been initiated and are currently ongoing.
RESUMEN
A novel amphibian oocyte RNase, ONCONASE(R) (ONC), has been previously shown to have synergistic tumor cell growth inhibitory activity when combined with tamoxifen (TMX) and/or trifluoperazine (TFP) in human ASPC-1 pancreatic and A-549 lung carcinoma cells, respectively. It has recently been reported that several drugs known to bind to the intracellular antiestrogen binding site (AEBS)/histamine (H(IC)) receptors, including tricyclic (amitriptyline, AMT) and non-tricyclic (fluoxetine, FLX) antidepressants, TMX, phenothiazines and the prototype H(IC)-binder DPPE, can stimulate the in vitro and in vivo growth of rodent tumor cells, while having a normal cell growth inhibitory activity, as reflected by the inhibition of the DNA synthesis. It has been presently shown that while at the clinically relevant concentrations some of these H(IC)-binding drugs mildly stimulated (up to 15%) the cell growth in the human lines studied when used as single agents, in most instances this stimulation did not exceed 10% above the control values. When used in combination with ONC, neither of these H(IC)-binding drugs demonstrated any apparent synergistic activity as judged from the ED50 values. However, the combinations of DPPE+TFP and AMT+TFP, in both the ASPC-1 and COLO 320DM lines, demonstrated a significant cell growth inhibition, while there was no difference between the effects of AMT alone and the AMT+TFP combination in the U87MG line. The most effective cell growth inhibition was obtained when ONC was combined with DPPE+TFP and/or AMT+TFP, as reflected by the significantly decreased ED50 values.
RESUMEN
The P-30 protein (Onconase) of Rana pipiens oocytes and early embryos is homologous to members of the pancreatic ribonuclease superfamily and exhibits an antitumor activity in vitro and in vivo. It appears that the ribonucleolytic activity of P-30 protein may be required for its antitumor effects. A comparative molecular model of P-30 protein has been constructed based upon the known three-dimensional structure of bovine pancreatic RNase A in order to provide structural information. Functionally, these enzymes hydrolyze oligoribonucleotides to pyrimidine-3'-phosphate monoesters and 5'-OH ribonucleotides. In the modeling procedure, automated sequence alignments were revised based upon the inspection of the RNase A structure before the amino acids of the P-30 protein were assigned the coordinates of the RNase A template. The inevitable intermolecular steric clashes that result were relieved on an interactive graphics device through the adjustment of side chain torsion angles. This process was followed by energy minimization of the model, which served to optimize stereochemical geometry and to relieve any remaining unacceptably close contacts. The resulting model retains the essential features of RNase A as sequence insertions and deletions are almost exclusively found in exposed surface loops. The all atom superposition of active site residues of the P-30 protein model and an identically minimized RNase A structure has a root mean square deviation of 0.52 A. Though tentative, the model is consistent with a pyrimidine specificity.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Antineoplásicos/química , Proteínas del Huevo/química , Rana pipiens/embriología , Ribonucleasa Pancreática/química , Ribonucleasas/química , Secuencia de Aminoácidos , Animales , Bovinos , Cristalización , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
A novel anti-tumour amphibian oocyte RNase, ONCONASER (ONC), previously known as P-30 Protein, is in the clinical trials. The effect of ONC alone and in combination with lovastatin (LVT), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme of mevalonate (MVA) and cholesterol synthesis pathway, in three human tumour cell lines ASPC-1 pancreatic, A-549 lung, and HT-520 lung carcinomas, has been presently studied. A synergism between ONC and LVT in inducing the cytostatic and cytotoxic effects was observed. The cytostatic effect, seen during the early phase of the treatment with this combination of drugs was manifested as prolongation of the cell cycle duration, especially of the G1 phase; cell death was apparent after 72 h of treatment. The synergistic effect of ONC and LVT was also evident in the clonogenicity assays. Both LVT lactone and its in vitro activated beta-hydroxy acid form, alone and in respective combinations with ONC, exerted similar degree of growth suppression. The effects of both forms of LVT (used alone or in combination with ONC) were reversed by MVA, which suggests that HMG-CoA reductase inhibition is a primary mechanism of LVT action. The data indicate that the LVT lactone can be activated intracellularly by tumour cells studied, and that the combination of ONC with LVT can produce significantly enhanced anti-tumour activities.
Asunto(s)
Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Proteínas del Huevo/farmacología , Lovastatina/farmacología , Ribonucleasas/farmacología , Adenocarcinoma , Línea Celular , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Cinética , Neoplasias Pulmonares , Ácido Mevalónico/farmacología , Oocitos/enzimología , Neoplasias PancreáticasRESUMEN
In vitro tumor cell growth inhibitory and cytotoxic synergisms between a novel amphibian oocytic ribonuclease (ONCONASE)(ONC) and tamoxifen (TMX), lovastatin (LVT) and cisplatin (CDDP), have been observed in the human, estrogen and androgen receptor positive, chemotherapy-resistant NIH-OVCAR-3 ovarian carcinoma cell line. In view of the fact that the resistance to the available systemic chemotherapy represents one of the most important causes of treatment failure in patients with advanced ovarian cancer, the observed various forms of combination therapy synergisms suggest that these regimens could be tested in vivo, including human clinical trials. Particularly important are findings of significant synergistic tumor cell growth inhibitory and cytotoxic activities exerted by the combination of ONC with CDDP; NIH-OVCAR-3 cells are reportedly resistant to the latter. Of specific interest are the observed synergisms between ONC and TMX and between ONC and LVT, an inhibitor of the 3-hydroxy-3- methyl-glutaryl-CoA reductase which is a rate-limiting enzyme in the mevalonate/cholesterol synthesis pathway. A facilitation of apoptosis-induction by the drug combinations presently studied is discussed as a possible mechanism of the observed synergisms.
RESUMEN
Magnetic resonance (MR) imaging of the parotid glands were performed in 13 cases of Sjögren syndrome to assess if there are any typical MR features. The MR findings were compared with sialography and pathology results. Signal intensity ratios of parotid (minus background noise) to skeletal muscle (minus background noise) were measured in these 13 cases as well as in 10 normal controls. Both T1- and T2-weighted images showed multiple hypointense mixed with hyperintense foci (salt-and-pepper appearance) throughout the glands in the six Sjögren cases of intermediate severity (46%), inhomogeneous glands in five cases with early or advanced disease, and homogeneous glands in the remaining two cases with the earliest stage of disease. Mean intensity ratios in T2-weighted pulse sequences in patients with salt-and-pepper appearance and inhomogeneous glands were significantly smaller than those in normal controls (p less than 0.05 and p less than 0.001, respectively). Pathologic studies tend to indicate that focal lymphocytic aggregates associated with increased interlobular fibrosis are probably responsible for the hypointense foci and the decrease in intensity ratios. We think that the salt-and-pepper appearance is suggestive of Sjögren syndrome and that the decreased intensity ratios combined with a typical clinical picture may lead to a highly probable diagnosis of this disorder.
Asunto(s)
Imagen por Resonancia Magnética , Síndrome de Sjögren/diagnóstico , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Glándula Parótida/anatomía & histología , Glándula Parótida/patología , Valores de Referencia , Sialografía , Síndrome de Sjögren/patologíaRESUMEN
MR imaging and CT were performed prospectively in 35 patients with esophageal carcinoma to determine the resectability of the primary tumors, because at our institution patients with resectable tumors have surgery regardless of the presence of distant metastases. Tumors with evidence of aortic or tracheobronchial invasion on MR or CT were considered to be unresectable. Tracheobronchial invasion was diagnosed when the tumor extended into the lumen of the airway, and aortic invasion was diagnosed when the triangular fat space between the esophagus, aorta, and spine adjacent to the primary tumor was obliterated. Two patients were excluded because of suboptimal MR images produced by motion artifacts. Pathologic proof was obtained from either surgery or autopsy in 31 patients. Of these, six patients (19%) had proved unresectable tumors (three aortic invasion and three tracheobronchial invasion). In all six cases, these features were correctly detected with both MR and CT. One patient had false-positive findings on MR and CT. An indeterminate diagnosis was obtained with MR in three patients and with CT in four patients. These incorrect or indeterminate results were all related to the diagnosis of aortic invasion. No patient had a false-negative result. When indeterminate diagnoses were considered false-positive, sensitivity, specificity, and accuracy for resectability were 100%, 84%, and 87%, respectively, for MR and 100%, 80%, and 84%, respectively, for CT. We conclude that MR and CT have nearly the same accuracy in predicting resectability of tumors in patients with esophageal carcinoma.
Asunto(s)
Neoplasias Esofágicas/patología , Imagen por Resonancia Magnética , Tomografía Computarizada por Rayos X , Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/epidemiología , Adenocarcinoma/patología , Adulto , Anciano , Carcinoma de Células Escamosas/diagnóstico por imagen , Carcinoma de Células Escamosas/epidemiología , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/diagnóstico por imagen , Neoplasias Esofágicas/epidemiología , Humanos , Japón/epidemiología , Persona de Mediana Edad , Estadificación de Neoplasias/métodos , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
Rana pipiens oocytes and early embryos contain large amounts of a basic protein with antiproliferative/cytotoxic activity against several tumor cell lines in vitro (Darzynkiewicz, Z., Carter, S. P., Mikulski, S. M., Ardelt, W., and Shogen, K. (1988) Cell Tissue Kinet. 21, 169-182; Mikulski, S.M., Viera, A., Ardelt, W., Menduke, H., and Shogen, K. (1990) Cell Tissue Kinet. 23, 237-246), as well as antitumor activity in vivo (Mikulski, S. M., Ardelt, W., Shogen, K., Bernstein, E. H., and Menduke, H. (1990) J. Natl. Cancer Inst. 82, 151-153). The protein, provisionally named P-30 Protein, was purified to homogeneity from early embryos and characterized. It is a single-chain protein consisting of 104 amino acid residues in the following sequence: less than Glu1-Asp-Trp-Leu-Thr-Phe-Gln-Lys-Lys-His-Ile-Thr-Asn-Thr- Arg15-Asp-Val-Asp-Cys-Asp-Ans-Ile-Met-Ser-Thr-Asn-Leu-Phe-His-C ys30-Lys-Asp-Lys - Asn-Thr-Phe-Ile-Tyr-Ser-Arg-Pro-Glu-Pro-Val-Lys45-Ala-Ile-Cys-Lys- Gly-Ile-Ile- Ala-Ser-Lys-Asn-Val-Leu-Thr-Thr60-Ser-Glu-Phe-Tyr-Leu-Ser-Asp -Cys-Asn-Val-Thr-Ser-Arg-Por-Cys75-Lys-Tyr-Lys-Leu-Lys-Lys-Ser-Thr -Asn-Lys-Phe- Cys-Val-Thr-Cys90-Glu-Asn-Gln-Ala-Pro-Val-His-Phe-Val-Gly-Val-Gly- Ser-Cys104-OH . Its molecular weight calculated from the sequence is 11,819. The sequence homology clearly indicates that the protein belongs to the superfamily of pancreatic ribonuclease. It is also demonstrated that it indeed exhibits a ribonucleolytic activity against highly polymerized RNA and that this activity seems to be essential for its antiproliferative/cytotoxic effects.