RESUMO
Electrolytic lesions of the nucleus raphe dorsalis and medianus reduce the concentration of serotonin (5-hydroxytryptamine) within rat brain intraparenchymal blood vessels. The concentration of serotonin within these vessels increases or decreases after the administration of drugs that modify the biosynthesis and degradation of serotonin or destroy nerve terminals by an uptake-dependent mechanism. These studies provide evidence for the existence of a serotonin-containing pathway seemingly analogous to the neuronal projection that terminates on small parenchymal blood vessels from noradrenergic neurons of the locus coeruleus.
Assuntos
Tronco Encefálico/fisiologia , Encéfalo/irrigação sanguínea , Microcirculação/inervação , Núcleos da Rafe/fisiologia , Serotonina/fisiologia , Animais , Mapeamento Encefálico , Circulação Cerebrovascular , Núcleos da Rafe/citologia , Ratos , Triptofano Hidroxilase/metabolismoRESUMO
Taxol is a naturally occurring chemotherapeutic agent that is active against a variety of tumors. Taxol is believed to act by binding tightly to microtubules and preventing their disaggregation. Others have shown that depletion of cellular glutathione results in the disaggregation of microtubules, presumably by allowing the oxidation of some or all of the cysteine residues in tubulins. We studied the effect of glutathione (GSH) depletion by L-buthionine sulfoximine (L-BSO) on taxol cytotoxicity in two human tumor lines. After a 24-h incubation in 5 mM L-BSO, the breast adenocarcinoma line MCF-7 and the lung adenocarcinoma line A549 were exposed to varying concentrations of taxol for 24 h. GSH levels were undetectable in cells treated with L-BSO. At the highest concentrations of taxol (50 nM), control MCF-7 cells had 10% cell survival and control A549 cells had only 1% cell survival as assessed by clonogenic assay. Pretreatment with 5 mM L-BSO resulted in a 3-fold increase in survival of MCF-7 cells and a 10-fold increase in survival of A549 cells. Pretreatment with L-BSO had no effect on taxol uptake into A549 or MCF-7 cells, as assessed by measurement of binding of [3H]taxol to cells. Following exposure to 37 nM taxol for 24 h, both cell lines had over 80% of their population in G2/M and bromodeoxyuridine labeling showed that taxol markedly reduced the percentage of cells in S phase. L-BSO pretreatment had no effect on the cell cycle in either cell line in the absence of taxol. However, in cells treated with taxol, L-BSO increased the percentage of cells in S phase by 3-fold in both cell lines. We conclude that depletion of cellular GSH by L-BSO results in resistance to taxol in MCF-7 and A549 cells. Resistance to taxol mediated by GSH depletion is not due to alterations in cellular uptake of taxol by L-BSO. L-BSO increased the S-phase fraction of taxol-treated cells in both cell lines. These data suggest that GSH depletion interferes with cell cycle changes induced by taxol. The alteration in taxol-induced cell cycle effects may account for the resistance to taxol produced by L-BSO.
Assuntos
Glutationa/metabolismo , Metionina Sulfoximina/análogos & derivados , Paclitaxel/antagonistas & inibidores , Butionina Sulfoximina , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Imunofluorescência , Humanos , Técnicas In Vitro , Metionina Sulfoximina/farmacologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Células Tumorais CultivadasRESUMO
Orchidectomy results in increased LH and FSH levels by removal of negative feedback at the hypothalamus and pituitary gland. However, the precise central nervous system mechanisms involved in elevation of gonadotropins after castration are unclear. We tested the hypothesis that catecholamine neuronal activity mediates the rise in serum LH that occurs after withdrawal of testosterone (T) negative feedback. The effects of acute and selective T withdrawal on brain catecholamine and LHRH activity and serum LH levels were determined in adult male rats. At the time of orchidectomy, rats were given sc implants of both T-containing and empty Silastic capsules. After recovery from surgery, the T-containing capsule was atraumatically removed from half of the animals (T-withdrawn), while the empty capsule was removed from the remaining rats (T-replaced). Rats were killed before and 6, 12, and 24 h after capsule removal. Serum T and LH levels were determined by RIA. Catecholamine content in microdissected nuclei of the LHRH neuronal system [medial preoptic nucleus, suprachiasmatic nucleus, retrochiasmatic area, arcuate nucleus (ARC), and median eminence (ME)] was measured by HPLC with electrochemical detection. Norepinephrine turnover rate was also determined in these areas by measuring the rate of decline of NE levels after blockade of synthesis with diethyldithiocarbamate. Additionally, LHRH content was measured by RIA within the ARC and ME. In T-replaced rats, the T capsules maintained serum T and LH levels within the normal range for intact male rats. In T-withdrawn rats, T levels fell into castrate range by 6 h after removal of the T capsule [0.12 +/- 0.04 ng/ml (mean +/- SEM); P less than 0.01 vs. T-replaced], and LH levels increased significantly from 0.23 +/- 0.04 ng/ml before capsule removal to 1.31 +/- 0.14 and 2.80 +/- 0.20 ng/ml 12 and 24 after T withdrawal, respectively (both P less than 0.01 vs. T-replaced). Despite a marked increase in serum LH levels, no significant changes in catecholamine content or NE turnover rate were observed in any of the hypothalamic nuclei of the LHRH neuronal system at any time after T withdrawal. ARC and ME LHRH levels also did not change significantly at any point after T withdrawal. These results suggest that activation of hypothalamic catecholamine neuronal activity is not required for the rise in serum LH levels after acute withdrawal of T negative feedback.
Assuntos
Catecolaminas/fisiologia , Hipotálamo/fisiologia , Hormônio Luteinizante/metabolismo , Orquiectomia , Testosterona/fisiologia , Animais , Catecolaminas/metabolismo , Retroalimentação , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/citologia , Hipotálamo/metabolismo , Hormônio Luteinizante/sangue , Masculino , Neurônios/metabolismo , Neurônios/fisiologia , Norepinefrina/metabolismo , Ratos , Ratos Endogâmicos , Testosterona/sangueRESUMO
We have used clonogenic survival assays and flow cytometry of human lung A549, breast MCF7 and pancreas adenocarcinoma P-SW cell lines to examine the effects of gemcitabine (2'-deoxy-2', 2'-difluorocytidine) in combination with cisplatin, paclitaxel or radiation. Additive cell killing was observed for all cell lines when they were treated with cisplatin for 1 h followed by varying concentrations of gemcitabine for 24 h. Likewise, additive cell killing was noted in all cell lines when treated with gemcitabine for 24 h followed by varying doses of radiation. When A549 cells were exposed to gemcitabine for 24 h followed by a 1 h exposure to cisplatin, synergistic effects were noted. Using the latter regimen, MCF7 cells demonstrated additive cell kill, while the P-SW cells showed a more complex relationship with additive killing below 50 nM gemcitabine and less than additive effect above 50 nM gemcitabine. All three cell lines were also tested with various gemcitabine/paclitaxel combinations. When gemcitabine and paclitaxel were incubated concurrently, gemcitabine antagonized the cell kill produced by paclitaxel. All cell lines showed less than additive killing when either gemcitabine incubation preceded the paclitaxel incubation or the paclitaxel incubation preceded the gemcitabine incubation. Our results show that gemcitabine acts as a radiation sensitizer to increase the effects of radiation. Likewise, we demonstrate that the only uniformly beneficial drug combination schedule in all three cell lines was when cisplatin incubation preceded gemcitabine incubation. The gemcitabine/paclitaxel combinations were much more disturbing with respect to potential clinical trials. Our results would caution any planned clinical trials combining paclitaxel with gemcitabine to be reconsidered because of the potential for less than additive and even antagonistic effects of the combination.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cisplatino/administração & dosagem , Desoxicitidina/análogos & derivados , Paclitaxel/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/administração & dosagem , Humanos , Células Tumorais Cultivadas , GencitabinaRESUMO
Clinical trials combining paclitaxel with other active chemotherapy agents are currently underway. In vitro preclinical studies may assist in the selection of appropriate drug combinations or sequences for clinical investigation. We have used clonogenic cell survival assays and DNA flow cytometry to examine the effect of paclitaxel combined with melphalan, thiotepa, or cisplatin on the survival and cell-cycle parameters of human lung A549 and breast MCF-7 adenocarcinoma cells. A549 and MCF-7 cells were incubated with paclitaxel for 24 h, followed by a 1 h exposure to cisplatin, melphalan, or thiotepa. Both cell types were also incubated with cisplatin or an alkylator for 1 h followed by a 24 h paclitaxel exposure. When the paclitaxel exposure preceded either melphalan, thiotepa, or cisplatin, the cytotoxicity was additive for both cell lines tested. When cisplatin or alkylator exposure was given prior to the paclitaxel, cytotoxicity was also additive in MCF-7 cells. However, cisplatin or alkylators were antagonistic to paclitaxel cytotoxicity when they preceded the paclitaxel exposure in A549 cells (e.g., 2% survival with 100 nM paclitaxel vs. 7% survival with cisplatin and paclitaxel). Cell-cycle analysis revealed that exposure to 100 nM paclitaxel for 24 h blocked a majority of the cells into the G2/M phase (> or = 80% for A549 cells, 60-65% for MCF-7 cells). However, exposure to alkylators before incubation in paclitaxel reduced the proportion of A549 but not MCF-7 cells in G2/M--e.g., exposure to 30 micrograms/ml cisplatin prior to paclitaxel exposure caused only 25% of A549 and 55% of MCF-7 cells to block in G2/M.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , DNA de Neoplasias/análise , Esquema de Medicação , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/patologia , Melfalan/administração & dosagem , Paclitaxel/administração & dosagem , Tiotepa/administração & dosagem , Células Tumorais Cultivadas/efeitos dos fármacosAssuntos
Tronco Encefálico/metabolismo , Plexo Corióideo/metabolismo , Núcleos da Rafe/metabolismo , Serotonina/metabolismo , Animais , Córtex Cerebral/metabolismo , Fenclonina/farmacologia , Masculino , Inibidores da Monoaminoxidase/farmacologia , Vias Neurais/metabolismo , Neurônios/metabolismo , Ratos , Receptores de Serotonina/metabolismo , Receptores de Serotonina/fisiologia , Triptofano/farmacologia , p-Cloroanfetamina/farmacologiaRESUMO
The cytotoxicity of paclitaxel against eight human tumour cell lines has been studied with in vitro clonogenic assays. The fraction of surviving cells fell sharply after exposure for 24 h to paclitaxel concentrations ranging from 2 to 20 nM; the paclitaxel IC50 was found to range between 2.5 and 7.5 nM. Increasing the paclitaxel concentration above 50 nM, however, resulted in no additional cytotoxicity after a 24 h drug exposure. Cells incubated in very high concentrations of paclitaxel (10,000 nM) had an increase in survival compared with cells treated with lower concentrations of the drug. Prolonging the time of exposure of cells to paclitaxel from 24 to 72 h increased cytotoxicity from 5 to 200 fold in different cell lines. Exponentially growing cells were more sensitive to paclitaxel than were cells in the plateau phase of growth. Cremophor EL, the diluent in which the clinical preparation of paclitaxel is formulated, antagonised paclitaxel at concentrations of 0.135% (v/v). These data suggest that paclitaxel will be most effective clinically when there is prolonged exposure of tumour to the drug. Further, it appears that modest concentrations (i.e., 50 nM) should be as effective as higher concentrations of paclitaxel. Finally, we have noted that Cremophor EL is a biologically active diluent and, at high concentrations (0.135% v/v), can antagonise paclitaxel cytotoxicity.
Assuntos
Paclitaxel/toxicidade , Células Tumorais Cultivadas/efeitos dos fármacos , Adenocarcinoma/metabolismo , Astrocitoma/metabolismo , Neoplasias da Mama/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Relação Dose-Resposta a Droga , Feminino , Glicerol/análogos & derivados , Glicerol/farmacologia , Células HeLa/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Ovarianas/metabolismo , Paclitaxel/administração & dosagem , Paclitaxel/antagonistas & inibidores , Neoplasias Pancreáticas/metabolismo , Neoplasias do Colo do Útero/metabolismoRESUMO
BACKGROUND: Taxol is a novel chemotherapeutic agent that promotes microtubule assembly and stabilizes tubulin polymer formation. Clinical evaluation of its antineoplastic activity as a single agent and in combination with other chemotherapeutic drugs is in progress. METHODS: To evaluate the effect of combining taxol with other commonly used antineoplastic agents, clonogenic survival of human breast cancer MCF7 cells, human lung adenocarcinoma A549 cells, and human ovarian cancer OVG1 cells were assayed after an initial exposure to taxol for 24 hours (approximately LD90 for taxol), followed by a 1-hour incubation with varying concentrations of doxorubicin or etoposide (total taxol incubation time, 25 hours). RESULTS: When corrected for taxol-induced cytotoxicity, doxorubicin and etoposide caused less cell killing in the presence of taxol compared with control incubations of doxorubicin and etoposide alone. To determine if a different schedule of drug application resulted in a similar finding, MCF7, A549, and OVG1 cells were exposed to doxorubicin for 1 hour, followed by incubation with varying concentrations of taxol for 24 hours. Less-than-additive cytotoxicity for the combination of taxol and doxorubicin was found. Flow cytometry studies in MCF7 cells showed that taxol caused a G2/M cell cycle block. Fewer cells were found to be in S-phase, which is the most doxorubicin-sensitive phase of the cell cycle. The application of doxorubicin or etoposide to MCF7 cells for 1 hour resulted in partial G1 and G2/M cell cycle blocks. Fewer cells were found to be moving through the cell cycle, which is likely required for taxol cytotoxicity. CONCLUSION: Although direct antagonism of the cytotoxicity of doxorubicin or etoposide by taxol has not been proven, there is less-than-additive in vitro cytotoxicity when taxol is combined with these chemotherapeutic agents. The clinical implications of these findings are unknown; however, these findings generate concern about the combination of these agents in clinical trials and suggest that additional studies to determine optimal scheduling are needed.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Paclitaxel/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , DNA de Neoplasias/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Doxorrubicina/antagonistas & inibidores , Interações Medicamentosas , Ensaios de Seleção de Medicamentos Antitumorais , Etoposídeo/administração & dosagem , Etoposídeo/antagonistas & inibidores , Feminino , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/administração & dosagem , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Tiron (1,2-dihydroxybenzene-3,5-disulfonate), a nontoxic chelator of a variety of metals, is used to alleviate acute metal overload in animals. It is also oxidized to the EPR-detectable semiquinone radical by various biologically relevant oxidants, such as .OH, O2-., alkyl, and alkoxyl radicals. Since Tiron reacts with potentially toxic intracellular species and is also a metal chelator, we evaluated its protective effects in V79 cells subjected to various types of oxidative damage and attempted to distinguish the protection due to direct detoxification of intracellular radicals from that resulting from chelation of redox-active transition metals. We found that Tiron protects Chinese hamster V79 cells against both O2.(-)-induced (and H2O2 via dismutation of O2.-) and H2O2-induced cytotoxicity as measured by clonogenic assays. In experiments where Tiron was incubated with V79 cells and rinsed prior to exposure to HX/XO or H2O2, cytoprotection was observed, indicating that it protects against intracellular oxidative damage. On the other hand, Tiron did not protect V79 cells against the damage caused by ionizing radiation under aerobic conditions, which is predominantly mediated by H., .OH, and hydrated electrons in a metal-independent fashion. We demonstrate also that in in vitro studies, Tiron protects supercoiled DNA from metal-mediated superoxide-dependent strand breaks. We conclude that Tiron is a potentially useful protecting agent against the lethal effects of oxidative stress and suggest that it offers protection by chelating redox-active transition metal ions, in contrast to earlier reports where the protection by this compound in cellular systems subjected to oxidative damage has been interpreted as due to radical scavenging alone.
Assuntos
Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Animais , Linhagem Celular , Quelantes , Cricetinae , Óxidos N-Cíclicos , Dano ao DNA/efeitos dos fármacos , Espectroscopia de Ressonância de Spin Eletrônica , Sequestradores de Radicais Livres , Radicais Livres , Peróxido de Hidrogênio/farmacologia , Ferro/metabolismo , Oxirredução , Marcadores de Spin , Superóxidos/farmacologiaRESUMO
BACKGROUND: Most patients with esophageal carcinoma present with locally advanced disease and a poor prognosis. Surgery or radiation provides palliation for locally advanced esophageal carcinoma. The role of neoadjuvant therapy remains to be defined. We administered neoadjuvant chemotherapy consisting of 5-fluorouracil (5-FU), leucovorin, interferon-alpha, and cisplatin to 11 patients with locally advanced disease. METHODS: Eleven patients with squamous cell or adenocarcinoma of the esophagus were treated peroperatively with two to three cycles of combination chemotherapy. Nine patients underwent resection with curative intent. RESULTS: Six patients received three cycles of chemotherapy, and five received two. Dose reduction was necessary for two patients. One patient achieved a pathologic complete response, histologically confirmed. Of the eleven patients, two did not undergo surgery because of progressive disease during chemotherapy. Seven of the 9 patients relapsed after surgery and 2 have been disease free for 27 months. CONCLUSIONS: The combination 5-FU leucovorin, interferon-alpha-2a, and cisplatin administered in a neoadjuvant setting resulted in a median survival of 11.8 months with a median time to relapse of 7 months.