RESUMEN
BACKGROUND: In early-stage breast cancer, adjuvant chemotherapy is associated with significant systemic toxicity with only a modest survival benefit. Therefore, there is considerable interest in identifying predictive markers of response to therapy. Doxorubicin, one of the most common drugs used to treat breast cancer, is an anthracycline chemotherapeutic agent, a class of drugs known to be affected by hypoxia. Accordingly, we examined whether expression of the endogenous hypoxia marker carbonic anhydrase IX (CA IX) is predictive of outcome in early-stage breast cancer patients treated with doxorubicin. METHODS: We obtained 209 early-stage pre-treatment surgically-resected breast tumours from patients, who received doxorubicin in their chemotherapeutic regimen and had >10 years of follow-up. Immunohistochemistry was used to detect CA IX, and we used fluorescence in situ hybridisation to detect both human epidermal growth factor receptor (HER2) and DNA topoisomerase II-alpha (TOP2A) gene amplification. RESULTS: Carbonic anhydrase IX intensity was significantly correlated with progression-free survival (PFS) and overall survival (OS) in patients receiving 300 mg m(-2) of doxorubicin (HR=1.82 and 3.77; P=0.0014 and 0.010, respectively). There was a significant, inverse correlation between CA IX score and oestrogen receptor expression, but no significant correlations were seen with either HER2 or TOP2A ratio. CONCLUSION: We demonstrate that CA IX expression is correlated with worse PFS and OS for breast cancer patients treated with doxorubicin, independent of HER2 or TOP2A gene amplification. This study provides evidence that using CA IX to detect hypoxia in surgically-resected breast tumours may be of clinical use in choosing an appropriate chemotherapy regimen.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Anhidrasas Carbónicas/metabolismo , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos , Adulto , Anciano , Antígenos de Neoplasias/genética , Biomarcadores de Tumor , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Anhidrasa Carbónica IX , Hipoxia de la Célula , Quimioterapia Adyuvante , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Supervivencia sin Enfermedad , Femenino , Amplificación de Genes , Genes erbB-2 , Humanos , Persona de Mediana Edad , Proteínas de Unión a Poli-ADP-RibosaRESUMEN
BACKGROUND: In selected patients with early-stage and low-risk breast cancer, an MRI-linac based treatment might enable a radiosurgical, non-invasive alternative for current standard breast conserving therapy. AIM: To investigate whether single dose accelerated partial breast (APBI) to the intact tumor in both the prone and supine radiotherapy positions on the MRI-linac is dosimetrically feasible with respect to predefined coverage and organs at risk (OAR) constraints. MATERIAL & METHODS: For 20 patients with cTis or low-risk cT1N0M0 non-lobular breast carcinoma, previously treated with single dose preoperative APBI in the supine (nâ¯=â¯10) or prone (nâ¯=â¯10) position, additional intensity modulated radiotherapy plans with 7 coplanar beams in the presence of a 1.5T magnetic field were generated. A 20â¯Gy and 15â¯Gy dose was prescribed to the gross tumor and clinical target volume, respectively. The percentage of plans achieving predefined organ at risk (OAR) constraints, currently used in clinical practice, was assessed. Dosimetry differences between the prone versus supine approach and the MRI-linac versus clinically delivered plans were evaluated. RESULTS: All MRI-linac plans met the coverage and predefined OAR constraints. The prone approach appeared to be more favorable with respect to the chest wall, and ipsilateral lung dose compared to the supine position. No dosimetric differences were observed for the ipsilateral breast. No treatment position was clearly more beneficial for the skin or heart, since dosimetry varied among parameters. Overall, the MRI-linac and clinical plans were comparable, with minor absolute dosimetric differences. CONCLUSION: MRI-linac based single dose APBI to the intact tumor is a promising and a dosimetrically feasible strategy in patients with low-risk breast cancer. Preliminary OAR dosimetry favored the prone radiotherapy position.
RESUMEN
Primary resistance to vincristine (VCR) has been selected in rhabdomyosarcoma xenograft HxRh12 by sequential administration of VCR at 1.5 and subsequently 3 mg/kg/passage. The resistant tumor (HxRh12/VCR-3) was approximately 4-fold resistant to VCR and resistance was stable in the absence of selecting pressure (greater than 2 yr). HxRh12/VCR-3 was 2- to 3-fold cross-resistant to L-phenylalanine mustard (L-PAM) but only slightly cross-resistant to ifosfamide. To determine whether selection for primary resistance to L-PAM conferred cross-resistance to VCR we selected an L-PAM-resistant subline of rhabdomyosarcoma xenograft HxRh28 (HxRh28/L-PAM-13). This tumor was 2- to 3-fold resistant to L-PAM and 3-(p-fluorophenyl)-L-alanyl-3-[m-bis-(2-chloroethyl)-aminophenyl]-L- alanyl-L-methionine ethoxyhydrochloride, cross-resistant to cyclophosphamide and ifosfamide, and completely resistant to VCR under in vivo conditions. Pharmacokinetic studies in HxRh12/VCR-3 showed decreased retention of [G-3H]VCR but not alteration in metabolism. Expression of mdr1, a gene that encodes P-glycoprotein, associated with the multiple drug resistance phenotype, was examined. Expression of mdr1 was detected in both HxRh12 and HxRh28 tumors, sensitive to VCR, but there was no increase in expression in tumors selected for primary resistance to VCR or L-PAM. Data suggest that mechanisms other than those associated with "classical" multiple drug resistance confer resistance in these tumors. In clinical evaluation against childhood rhabdomyosarcoma, L-PAM has demonstrated only slight activity in patients relapsing on conventional therapy (including VCR) but demonstrated marked activity in patients with advanced previously untreated disease. It appears likely, therefore, that cross-resistance between VCR and L-PAM as demonstrated in this model may have clinical significance.
Asunto(s)
Melfalán/uso terapéutico , Rabdomiosarcoma/tratamiento farmacológico , Vincristina/uso terapéutico , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Animales , Línea Celular , Resistencia a Medicamentos , Femenino , Regulación de la Expresión Génica , Humanos , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos CBA , Trasplante de Neoplasias , Rabdomiosarcoma/genética , Trasplante HeterólogoRESUMEN
Benzo(a)pyrene metabolite: deoxyribonucleoside adducts were analyzed in hepatic and pulmonary cells isolated from rabbits 24 h after i.v. administration of [3H]BP (1 mg/kg; 50 mCi/kg). The major adduct in each of the cell types analyzed was (+)-r-7, t-8-dihydroxy-t-9, 10-oxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene: deoxyguanosine, but (+/-)-r-7, t-8-dihydroxy-c-9, 10-oxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene: deoxyguanosine and very low levels of (-)-r-7, t-8-dihydroxy-t-9, 10-oxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene -deoxyguanosine and an unidentified adduct were also observed. The level of the major adduct was similar in each of the isolated cell types and was at least as high in cells with very low cytochrome P-450-dependent monooxygenase activity (hepatic nonparenchymal cells and alveolar macrophages) as in those with higher activity (hepatocytes, alveolar type II cells, and Clara cells). The binding of benzo(a)pyrene metabolites to proteins was also determined, and again binding levels did not correlate with differences in cytochrome P-450 activity.
Asunto(s)
Benzo(a)pireno/metabolismo , ADN/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido , Animales , Benzopirenos/metabolismo , Sistema Enzimático del Citocromo P-450/fisiología , Desoxiguanosina/metabolismo , Masculino , Unión Proteica , ConejosRESUMEN
We have previously described a mitoxantrone-resistant human breast carcinoma cell line, MCF7/MX, in which resistance was associated with a defect in the energy-dependent accumulation of mitoxantrone in the absence of P-glycoprotein overexpression (M. Nakagawa et al., Cancer Res. 52: 6175-6181, 1992). We now report that this cell line is highly cross-resistant to the camptothecin analogues topotecan (180-fold), 9-aminocamptothecin (120-fold), CPT-11 (56-fold), and SN38 (101-fold), but is only mildly cross-resistant to the parent compound camptothecin (3.2-fold) and 10,11-methylenedioxy-camptothecin (2.9-fold). Topotecan accumulation was decreased in MCF7/MX cells compared to parental MCF7/WT cells, and there was a corresponding reduction in topotecan-mediated stimulation of the enzyme/DNA complex formation in MCF7/MX cells compared to MCF7/WT cells. No overexpression of the multidrug resistance-associated protein was detected compared to parental MCF7/WT cells. Furthermore, both sensitive MCF7/WT and mitoxantrone-resistant MCF7/MX cells contain equal amounts of DNA topoisomerase I protein, and DNA relaxation activities were equal in both cell lines and inhibited to the same extent by topotecan and camptothecin. Thus, these results suggest a novel mechanism of resistance to topoisomerase I inhibitors in cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Camptotecina/análogos & derivados , Mitoxantrona/farmacología , Inhibidores de Topoisomerasa I , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Camptotecina/metabolismo , Camptotecina/farmacología , Supervivencia Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/metabolismo , Resistencia a Medicamentos , Femenino , Humanos , Topotecan , Células Tumorales CultivadasRESUMEN
A human breast cancer cell line (MCF7/WT) was selected for resistance to etoposide (VP-16) by stepwise exposure to 2-fold increasing concentrations of this agent. The resulting cell line (MCF7/VP) was 28-, 21-, and 9-fold resistant to VP-16, VM-26, and doxorubicin, respectively. MCF7/VP cells also exhibited low-level cross-resistance to 4'-(9-acridinylamino)-methanesulfon-m-anisidide, mitoxantrone, and vincristine and no cross-resistance to genistein and camptothecin. Furthermore, these cells were collaterally sensitive to the alkylating agents melphalan and chlorambucil. DNA topoisomerase II levels were similar in both wild-type MCF7/WT and drug-resistant MCF7/VP cells. In contrast, topoisomerase II from MCF7/VP cells appeared to be 7-fold less sensitive to drug-induced cleavable complex formation in whole cells and 3-fold less sensitive in nuclear extracts than topoisomerase II from MCF7/WT cells. Although this suggested that the resistant cells may contain a qualitatively altered topoisomerase II, no mutations were detected in either the ATP-binding nor the putative breakage/resealing regions of either DNA topoisomerase II alpha or II beta. In addition, the steady-state intracellular VP-16 concentration was reduced by 2-fold in the resistant cells, in the absence of detectable mdr1/P-gp expression and without any change in drug efflux. In contrast, expression of the gene encoding the MRP was increased at least 10-fold in resistant MCF7/VP cells as compared to sensitive MCF7/WT cells. These results suggest that resistance to epipodophyllotoxins in MCF7/VP cells is multifactorial, involving a reduction in intracellular drug concentration, possibly as a consequence of MRP overexpression, and an altered DNA topoisomerase II drug sensitivity.
Asunto(s)
Neoplasias de la Mama/enzimología , ADN-Topoisomerasas de Tipo II/metabolismo , Etopósido/metabolismo , Tenipósido/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Proteínas Portadoras/análisis , ADN-Topoisomerasas de Tipo I/análisis , ADN-Topoisomerasas de Tipo II/análisis , ADN-Topoisomerasas de Tipo II/genética , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos/genética , Etopósido/farmacología , Femenino , Humanos , Glicoproteínas de Membrana/análisis , ARN Mensajero/análisis , Tenipósido/farmacología , Células Tumorales CultivadasRESUMEN
A melphalan-resistant human rhabdomyosarcoma xenograft, TE-671 MR, was established in athymic mice by serial melphalan treatment of the parent xenograft, TE-671, at the 10% lethal dosage (LD10); significant resistance was evident after ten passages of the tumor. TE-671 MR demonstrated a doubling time of 3.5 days and a latency period to 1000-mm3 tumors of 27.5 days. The glutathione level of TE-671 MR was 2.36 mumol/g tumor, wet weight, 2-fold higher than the parent line. The glutathione S-transferase activity of TE-671 MR was 117.8 mumol/min/mg protein, essentially unchanged from the parent line. Although TE-671 MR demonstrated cross-resistance to vincristine, dot blot analysis did not reveal an elevated expression of mdr1 mRNA in the resistant line. TE-671 MR demonstrated a 9.7-day growth delay following treatment with melphalan at the LD10 (compared to 20.9 days for the parent line). Treatment with L-buthionine-SR-sulfoximine (BSO) resulted in increased sensitivity to melphalan subsequently administered at 50% of the LD10 (melphalan alone, growth delays of 3.7 and 4.6 days in duplicate trials; melphalan plus BSO, growth delays of 7.2 and 9.8 days). Sensitivity to melphalan equal to that of the parent line TE-671 was not achieved, however. Treatment with BSO did not result in significantly enhanced sensitivity to subsequently administered vincristine (50% of the LD10) (vincristine alone, growth delays of 6.8 and 6.9 days in duplicate trials; vincristine plus BSO, growth delays of 10.9 and 7.5 days). These results suggest that generation of melphalan resistance may be associated with development of cross-resistance to vincristine; this resistance may be associated with (although not necessarily mediated by) glutathione elevation; this resistance may be partially overcome by BSO-mediated depletion of glutathione.
Asunto(s)
Glutatión/antagonistas & inhibidores , Melfalán/uso terapéutico , Rabdomiosarcoma/tratamiento farmacológico , Vincristina/uso terapéutico , Animales , Butionina Sulfoximina , Interacciones Farmacológicas , Resistencia a Medicamentos , Femenino , Glutatión Transferasa/análisis , Humanos , Masculino , Metionina Sulfoximina/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Trasplante HeterólogoRESUMEN
In a systematic study to elucidate the involvement of pro- and anti-apoptotic proteins in alkylating drug resistance of tumor cells, we utilized the A2780(100) line, that was selected by repeated exposure of A2780 cell line (human ovarian carcinoma line) to chlorambucil (CBL). A2780(100) was 5 - 10-fold more resistant to nitrogen mustards (IC50 of 50 - 60 microM) and other DNA crosslinking agents, e.g., cisplatin, and also to DNA topoisomerase inhibitor etoposide (ETO) than A2780. CBL (125 microM) induced extensive apoptosis in A2780 associated with mitochondrial damage but not in A2780(100). No significant differences were observed between A2780 and A2780(100) cells in the basal levels, or the enhanced levels in some cases after CBL treatment, of DNA repair proteins involved in repair of alkyl base adducts or in repair of DNA crosslinks or double strand break repair. However, the basal levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were 4 - 8-fold higher in A2780(100) than in A2780 neither of which expressed Bcl-2. In contrast, the levels of pro-apoptotic Bax and Bak were 3 - 5-fold higher in the CBL-treated A2780 but not in A2780(100). ETO (5 microM) induced apoptosis in A2780 without altering the levels of Bax and Bak in these cells. At the same time, neither overexpression of Bcl-xL in A2780, nor its antisense expression in A2780(100), and nor overexpression of Bax in A2780(100), significantly affected drug sensitivity of either line. Our results suggest that a change in an early step in DNA damage processing which affects intracellular signaling, such as enhanced DNA double-strand break repair, could be the primary cause for development of resistance in A2780(100) cells to drugs which induce DNA crosslinks or double strand-breaks.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Apoptosis , Neoplasias Ováricas/tratamiento farmacológico , Clorambucilo/farmacología , Daño del ADN , Resistencia a Antineoplásicos , Etopósido/farmacología , Femenino , Humanos , Etiquetado Corte-Fin in Situ , Mitocondrias/efectos de los fármacos , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
A new and highly selective procedure is described for the rapid selection and cloning of antibody-secreting hybridomas with antigen-coated magnetic beads. Immune splenocytes were fused to Sp2 myeloma cells with a PEG/DMSO mixture. Cells were cultured in HAT and screened for the presence of specific antibody after 7-10 days. Hybridomas from the positive colonies were mixed with antigen-coated magnetic polymer beads and antigen-specific cells separated on a magnet. Cloning was carried out directly by limiting dilution with the magnetic beads still bound on the cells. No obvious toxic effects were observed. The antibodies established by this technique were of a high affinity (greater than 10(9) l/mol) and were generated in 50% of the usual process time. The procedure described here should greatly facilitate the process of obtaining hybridomas and expand the range of monoclonal antibodies available.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Separación Celular/métodos , Epítopos , Hibridomas , Magnetismo , Microesferas , Animales , Fusión Celular , Gonadotropina Coriónica/inmunología , Femenino , Hibridomas/inmunología , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos BALB C , Formación de RosetaRESUMEN
A sensitive chemiluminescence based immunoassay is described for measuring antibody to staphylococcal peptidoglycan in blood and dialysates from patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Peptidoglycan was isolated from a strain of S. epidermidis obtained from the dialysate of a CAPD patient with peritonitis and after sonication used to coat polystyrene beads. The coated beads were incubated with standard or sample and bound IgG was detected by the addition of affinity-purified goat anti-human IgG labelled with acridinium ester. After a wash stage 0.1 M nitric acid containing 0.1% hydrogen peroxide was added to the beads. Subsequently the chemiluminescence produced following the addition of 0.3 M sodium hydroxide was measured over a 2 s time interval with an automatic luminescence analyser. Using this technique the optimum dilution of serum for detecting antibodies to peptidoglycan was found to be 1/800 and for overnight effluent from CAPD patients the dilution was 1/8. Initial values of serum and dialysate antibody levels from 34 subjects are presented. This method has the advantage that it will detect concentrations of anti-peptidoglycan which are less than 1% of those in sera, the reagents remain stable for long periods and large numbers of samples can be processed on the same day.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Inmunoensayo , Mediciones Luminiscentes , Peptidoglicano/inmunología , Staphylococcus epidermidis/inmunología , Soluciones para Diálisis/análisis , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoensayo/métodos , Inmunoglobulina G/análisis , Peptidoglicano/aislamiento & purificación , Diálisis Peritoneal Ambulatoria ContinuaRESUMEN
Simple, rapid and sensitive competitive enzyme immunoassays for the estimation of adenosine 3',5' cyclic monophosphate (cAMP) and guanosine 3',5' cyclic monophosphate (cGMP) in human plasma and urine are described. Specific antisera to each nucleotide were raised in rabbits by immunization with succinyl cyclic nucleotide--human serum albumin conjugates. For the assay, specific antibodies were incubated with a mixture of succinyl cyclic nucleotide labelled with horseradish peroxidase together with unlabelled standard or sample. The antibody-bound enzyme conjugate was separated from free hapten by anti-rabbit (IgG) sera immobilized to a microtitre plate. Activity of the bound enzyme conjugate was determined with tetramethylbenzidine. The assays were capable of detecting levels as low as 2 fmol of cAMP and cGMP. Good correlations were obtained between values generated by enzyme immunoassay and radioimmunoassay.
Asunto(s)
AMP Cíclico/análisis , GMP Cíclico/análisis , Técnicas para Inmunoenzimas , Animales , AMP Cíclico/sangre , AMP Cíclico/orina , GMP Cíclico/sangre , GMP Cíclico/orina , Peroxidasa de Rábano Silvestre , Humanos , Conejos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A series of 21 N-substituted phenoxazines has been synthesized in an effort to find more specific and less toxic modulators of multidrug resistance (MDR) in cancer chemotherapy. Thus, N-(omega-chloroalkyl)- and N-(chloroacyl)phenoxazines were found to undergo iodide-catalyzed nucleophilic substitution on reaction with various secondary amines, including N,N-diethylamine, N,N-diethanolamine, morpholine, piperidine, pyrrolidine and (beta-hydroxyethyl)piperazine. Products were characterized by UV, IR, 1H-, and 13C-NMR, mass spectral data, and elemental analyses. All of the compounds were examined for cytotoxicity and for their ability to increase the accumulation of the vinca alkaloids, vincristine (VCR) and vinblastine (VLB) in multidrug-resistant GC3/Cl (human colon adenocarcinoma) and KBChR-8-5 (HeLa variant) cell lines. Compounds were compared to the standard modulator verapamil (VRP). Substitutions on the phenoxazine ring at position 10 were associated with an increase in antiproliferative and anti-MDR activities. Modification of the length of the alkyl bridge and the type of amino side chain also influenced the potency of these effects. From among the compounds examined, 10 derivatives were found to increase the accumulation of VCR and VLB in GC3/Cl and KBChR-8-5 cells relative to the effect of VRP, suggesting that with the exception of pyrrolidinyl, the tertiary amine attachments to the phenoxazine nucleus linked through a three- or four-carbon alkyl chain resulted in enhanced anti-MDR activity. On the basis of their 50% growth inhibitory (IC50) values, five of the ten compounds, namely, 10-(3'-chloropropyl)phenoxazine, 10-[3'-[N-bis(hydroxyethyl)- amino]propyl]phenoxazine, 10-(3'-N-morpholinopropyl)phenoxazine, 10-(4'-N-morpholinobutyl)phenoxazine and 10-(N-piperidinoacetyl)phenoxazine were selected as relatively nontoxic chemosensitizers. These modulators, at nontoxic concentrations, potentiated the cytotoxicity of VCR and VLB in GC3/Cl and KBChR-8-5 cells. Further, two compounds 10-(3'-N-morpholinopropyl)phenoxazine, and the butyl derivative, enhanced accumulation of VLB in GC3/Cl, KBChR8-5 and highly resistant KB-V1 cells to a level significantly greater than the maximal level achieved with VRP. Additional experiments to understand the mechanism of action of these agents in modulating MDR are in progress.
Asunto(s)
Resistencia a Medicamentos , Oxazinas/síntesis química , Alcaloides de la Vinca/farmacología , Humanos , Oxazinas/farmacología , Oxazinas/toxicidad , Relación Estructura-Actividad , Células Tumorales Cultivadas/efectos de los fármacos , Alcaloides de la Vinca/metabolismoRESUMEN
1-Aryl-3-(hydroxymethyl)-3-alkyltriazenes [ArN = NN(CH3)CH2OH] have been synthesized by diazonium coupling to the carbinolamine (RNHCH2OH), generated in situ from the alkylamine and formaldehyde mixtures. The (hydroxymethyl)triazene structure has been confirmed by IR, NMR, and mass spectral analysis and also by the preparation of a crystalline benzoate derivative. The mass spectra of the (hydroxymethyl)triazenes suggest that they fragment by loss of formaldehyde to give the methyltriazene, which is also the product of hydrolysis in solution. The degradation of the (hydroxymethyl)triazenes in solution has been followed by UV spectroscopy and by HPLC analysis, and the half-lives were determined under a variety of conditions. The half-lives of the corresponding methyl- and (hydroxymethyl)triazenes are very similar. Both methyl- and (hydroxymethyl)triazenes decompose on silica plates during TLC analysis to give products consistent with known diazo-migration reactions. The (hydroxymethyl)triazenes have pronounced antitumor activity against the TLX5 tumor in vivo; in vivo-in vitro bioassay experiments suggest that the (hydroxymethyl)triazenes exert their in vivo antitumor activity via the degradation product, the alkyltriazene.
Asunto(s)
Antineoplásicos/síntesis química , Triazenos/uso terapéutico , Animales , Cromatografía Líquida de Alta Presión , Femenino , Linfoma/tratamiento farmacológico , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Espectrofotometría InfrarrojaRESUMEN
Tumor responsiveness to vincristine (VCR) was determined in xenografts of human rhabdomyosarcoma (RMS), in sublines of RMS selected in vivo for VCR resistance, in a KB line (KB-ChR8-5) selected in vitro for colchicine resistance, and in a colon adenocarcinoma (GC3). Sensitivity to VCR was associated with prolonged retention of VCR by the tumors after a single i.p. injection, whereas in tumors with acquired or intrinsic VCR resistance the drug was eliminated more rapidly. The sensitive tumors with prolonged retention of drug also showed increased levels of mitotic accumulation for up to 72 hr following VCR administration. There were good correlations between VCR sensitivity, VCR retention and the proposed mechanism of VCR cytotoxicity-mitotic arrest. A model has been developed consistent with data obtained that can explain the responsiveness to VCR of a series of human tumor xenografts irrespective of their tissue of origin.
Asunto(s)
Mitosis/efectos de los fármacos , Neoplasias Experimentales/tratamiento farmacológico , Vincristina/farmacocinética , Animales , Resistencia a Medicamentos , Femenino , Humanos , Ratones , Ratones Endogámicos CBA , Trasplante de Neoplasias , Neoplasias Experimentales/metabolismo , Trasplante Heterólogo , Vincristina/uso terapéuticoRESUMEN
The effect of the calcium channel blocker verapamil (VRP) on the accumulation and retention of vincristine (VCR) has been examined in mice bearing xenografts of human rhabdomyosarcomas. The tumors were Rh18, moderately sensitive to VCR, and its subline, Rh18/VCR3, selected in vivo for primary resistance to VCR. Administration of VRP by i.p. bolus at dose levels above 75 mg/kg was limited by acute lethality. At this dose, the maximal concentration in plasma was 24 microM, with rapid elimination such that plasma concentrations reported to modulate resistance in vitro (approximately 5-10 microM) were maintained for less than 60 min. To sustain a 10 microM plasma concentration, mice were infused with VRP at 6.25 mg/kg/hr (150 mg/kg/day) for up to 7 days using osmotic pumps implanted in the peritoneal cavity. Steady-state plasma levels were greater than or equal to 10 microM for at least 96 hr, and this schedule demonstrated minimal toxicity. Administration of VCR 20 hr after the start of VRP infusion produced significant lethality, requiring an 8-fold reduction in the VCR dose. Pharmacokinetic studies showed that VRP markedly increased the uptake and retention of VCR in small intestine, liver and kidney of mice. In small intestine, 8-fold greater levels of VCR were determined 24 hr after VCR administration, and this was associated with in increase in T1/2 for elimination from 350 to 913 min. HPLC analysis of extracts from small intestine showed that greater than 90% of the radiolabel eluted with VCR or 4-desacetyl-VCR. Modulation of VCR retention was also related to the dose of VCR administered. The VRP-sensitive efflux pathway appeared more effective in certain tissues only at higher concentrations of VCR. In contrast, VRP did not alter significantly the uptake and retention of VCR in either the parent or VCR-resistant human xenografts. The data demonstrated that, in the mouse, VRP modulates the uptake and retention of VCR in several tissues, and this may indicate that drug efflux mediated by a VRP-sensitive mechanism (e.g. GP-170, associated with the multiple drug resistance phenotype) has a protective function against xenobiotics in these tissues.
Asunto(s)
Rabdomiosarcoma/metabolismo , Verapamilo/farmacología , Vincristina/farmacocinética , Vincristina/toxicidad , Animales , Biotransformación , Resistencia a Medicamentos , Humanos , Ratones , Trasplante de Neoplasias , Distribución Tisular , Verapamilo/farmacocinética , Verapamilo/toxicidadRESUMEN
The antitumoral activity and metabolism of 1-(4-acetylphenyl)-3,3-dimethyltriazene [pAc-(CH3)2] and 1-(4-acetylphenyl)-3,3-diethyltriazene [pAc-(C2H5)2] were studied in mice. pAc-(CH3)2 showed significant antitumoral activity against M5076 ovarian reticular cell sarcoma, L1210 leukemia, EL 4 lymphoma in mice, but not against Lewis lung carcinoma. pAc-(C2H5)2 was inactive in all these murine tumors and was much more toxic than pAc-(CH3)2. pAc-(CH3)2 and pAc-(C2H5)2 were rapidly metabolized in vitro and in vivo to their respective monoalkyltriazenes and to 4-aminoacetophenone (pAc-NH2). In vitro, 79% of the dimethyltriazene was metabolized to its monomethyl analogue, but only 27% of the diethyltriazene was metabolized to the monoethyltriazene. The monoalkytriazenes were almost completely biotransformed to pAc-NH2 by a 9000 g liver fraction. The metabolic pattern in the in vitro study was comparable to that found in vivo.
Asunto(s)
Antineoplásicos , Neoplasias Experimentales/tratamiento farmacológico , Triazinas/metabolismo , Animales , Femenino , Leucemia L1210/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Masculino , Tasa de Depuración Metabólica , Ratones , Metástasis de la Neoplasia , Sarcoma Experimental/tratamiento farmacológico , Relación Estructura-Actividad , Triazinas/uso terapéutico , Triazinas/toxicidadRESUMEN
P-glycoprotein is a membrane ATPase that transports drugs out of cells and confers resistance to a variety of chemically unrelated drugs (multidrug resistance). P-glycoprotein is phosphorylated by protein kinase C (PKC), and PKC blockers reduce P-glycoprotein phosphorylation and increase drug accumulation. These observations suggest that phosphorylation of P-glycoprotein stimulates drug transport. However, there is evidence that PKC inhibitors directly interact with P-glycoprotein, and therefore the mechanism of their effects on P-glycoprotein-mediated drug transport and the possible role of phosphorylation in the regulation of P-glycoprotein function remain unclear. In the present work, we studied the effects of different kinds of PKC inhibitors on drug transport in cells expressing wild-type human P-glycoprotein and a PKC phosphorylation-defective mutant. We demonstrated that PKC blockers inhibit drug transport hy mechanisms independent of P-glycoprotein phosphorylation. Inhibition by the blockers occurs by (i) direct competition with transported drugs for binding to P-glycoprotein, and (ii) indirect inhibition through a pathway that involves PKC inhibition, but is independent of P-glycoprotein phosphorylation. The effects of the blockers on P-glycoprotein phosphorylation do not seem to play an important role, but the PKC-signaling pathway regulates P-glycoprotein-mediated drug transport.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Inhibidores Enzimáticos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Células 3T3 , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Marcadores de Afinidad/farmacología , Animales , Azidas/farmacología , Transporte Biológico/efectos de los fármacos , Dihidropiridinas/farmacología , Humanos , Ratones , Fosforilación , Proteína Quinasa C/genética , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/farmacología , Células Tumorales CultivadasRESUMEN
The metabolism of 1-(4-acetylphenyl)-3,3-dimethyltriazene has been studied in vivo and in vitro in mice. This dimethyltriazene was extensively metabolised in vivo and HPLC analysis of the plasma revealed the presence of two metabolites, the monomethyltriazene, 1-(4-acetylphenyl)-3-methyltriazene, and the arylamine, 4-aminoacetophenone. The dimethyltriazene was also biotransformed in vitro by a 9000 g fraction of mouse liver homogenate to products which were selectively toxic to TLX5 lymphoma cells. HPLC analysis of the products of in vitro metabolism under these conditions showed the presence of the monomethyltriazene but in an amount insufficient to account for the observed cytotoxicity. The monomethyltriazene was itself rapidly biotransformed by a 9000 g fraction of mouse liver homogenate, and by isolated mouse hepatocytes.
Asunto(s)
Antineoplásicos/metabolismo , Triazenos/metabolismo , Animales , Antineoplásicos/farmacología , Biotransformación , Supervivencia Celular/efectos de los fármacos , Femenino , Técnicas In Vitro , Hígado/metabolismo , Linfoma/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/tratamiento farmacológicoRESUMEN
Ovarian carcinoma cells 10-fold resistant to the alkylating agent chlorambucil (CBL) were isolated after repeated exposure of the parent cells to gradually escalating concentrations of the drug. The resistant variant, A2780(100), was highly cross-resistant (9-fold) to melphalan and showed lower-level resistance to other cross-linking agents. The resistant A2780(100) cells had almost 5-fold higher glutathione S-transferase (GST) activity than the parental A2780 cells with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The pi-class GST(s) was the major isoform(s) in both cell lines. However, the resistant A2780(100) cells had at least 11-fold higher GST mu as compared with the parental cells, in which this isoform was barely detectable. A significant induction of GST mu was observed in A2780 cells, but not in the resistant cells, 18 hr after a single exposure to 100 microM CBL. The induction of GST mu by CBL was both time- and concentration-dependent. Assays of the conjugation of CBL with GSH showed that the human mu-class GST had 3.6- and 5.2-fold higher catalytic efficiency relative to the pi- and alpha-class GSTs, respectively. This difference was reflected in the relatively higher (about 6-fold) efficiency of CBL conjugation in A2780(100) cells as compared with the parental cells. These results have demonstrated for the first time a near-linear correlation between CBL resistance and overexpression of mu-class GSTs and suggest that this overexpression maybe responsible, at least in part, for the acquired resistance of ovarian carcinoma cells to CBL, and possibly the other bifunctional alkylating agents. Consistent with this hypothesis, we found evidence for decreased formation of DNA lesions in A2780(100) compared with the drug-sensitive A2780 cells after exposure to CBL.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Clorambucilo/farmacología , Glutatión Transferasa/metabolismo , Neoplasias Ováricas/enzimología , Antineoplásicos Alquilantes/metabolismo , Catálisis , División Celular/efectos de los fármacos , Clorambucilo/metabolismo , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Inducción Enzimática , Femenino , Glutatión Transferasa/biosíntesis , Humanos , Inactivación Metabólica , Isoenzimas/biosíntesis , Isoenzimas/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The long-term treatment of essential hypertension with terazosin, a new once-a-day alpha 1-adrenergic blocking agent, was evaluated in 364 hypertensive patients who received total daily doses of 1 to 40 mg for 3 weeks to 56 months. Consistent mean decreases in supine and standing systolic and diastolic blood pressures were observed throughout the study for patients treated with terazosin as monotherapy (supine, 9 to 12/10 to 13 mm Hg; and standing, 12 to 18/11 to 14 mm Hg) or in combination with other antihypertensive agents (supine, 12 to 16/12 to 15 mm Hg; and standing, 16 to 22/13 to 19 mm Hg). The most commonly reported adverse experiences were dizziness, headache, asthenia, cold symptoms, and nasal congestion. Adverse effects and metabolic disorders often associated with diuretics and beta blockers such as sexual dysfunction, hyperglycemia, hyperuricemia, hypokalemia, or adverse lipid effects were seen infrequently during long-term treatment with terazosin as monotherapy. Overall, terazosin was shown to be effective, safe, and well tolerated by most patients.