RESUMEN
UNLABELLED: Preclinical animal models are useful tools to better understand tumor initiation and progression and to predict the activity of an anticancer agent in the clinic. Ideally, these models should recapitulate the biological characteristics of the tumor and of the related tumor microenvironment (e.g. vasculature, immune cells) in patients. Even if several examples of translational success have been reported it is a matter of fact that clinical trials in oncology often fail to meet their primary endpoints despite encouraging preclinical data. For this reason, there is an increasing need of improved and more predictive models. This review aims to give an overview on existing mouse models for preclinical evaluation of cancer therapeutics and their applicability. Different types of mouse models commonly used for the evaluation of cancer therapeutics are described and considerations for a "fit-for purpose" application of these models for the evaluation of different cancer therapeutics dependent on their mode of action are outlined. Furthermore, considerations for study design and data interpretation to translatability of findings into the clinics are given. CONCLUSION: Detailed knowledge of the molecular/biological properties of the respective model, diligent experimental setup, and awareness of its limitations are indispensable prerequisites for the successful translational use of animal models.
Asunto(s)
Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Neoplasias/tratamiento farmacológico , Animales , Ingeniería Genética , Humanos , Factores Inmunológicos/uso terapéutico , Ratones , Ratones Transgénicos , Proyectos de Investigación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Insights into tumour biology of breast cancer have led the path towards the introduction of targeted treatment approaches; still, breast cancer-related mortality remains relatively high. Efforts in the field of basic research revealed new druggable targets which now await validation within the context of clinical trials. Therefore, questions concerning the optimal design of future studies are becoming even more pertinent. Aspects such as the ideal end point, availability of predictive markers to identify the optimal cohort for drug testing, or potential mechanisms of resistance need to be resolved. An expert panel representing the academic community, the pharmaceutical industry, as well as European Regulatory Authorities met in Vienna, Austria, in November 2012, in order to discuss breast cancer biology, identification of novel biological targets and optimal drug development with the aim of treatment individualization. This article summarizes statements and perspectives provided by the meeting participants.
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Receptor ErbB-2/genética , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/terapia , Ensayos Clínicos como Asunto , Femenino , Humanos , Terapia Molecular Dirigida , Transducción de Señal , Neoplasias de la Mama Triple Negativas/clasificación , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
BACKGROUND: Trastuzumab and pertuzumab target the Human Epidermal growth factor Receptor 2 (HER2). Combination therapy has been shown to provide enhanced antitumour activity; however, the downstream signalling to explain how these drugs mediate their response is not clearly understood. METHODS: Transcriptome profiling was performed after 4 days of trastuzumab, pertuzumab and combination treatment in human ovarian cancer in vivo. Signalling pathways identified were validated and investigated in primary ovarian xenografts at the protein level and across a timeseries. RESULTS: A greater number and variety of genes were differentially expressed by the combination of antibody therapies compared with either treatment alone. Protein levels of cyclin-dependent kinase inhibitors p21 and p27 were increased in response to both agents and further by the combination; pERK signalling was inhibited by all treatments; but only pertuzumab inhibited pAkt signalling. The expression of proliferation, apoptosis, cell division and cell-cycle markers was distinct in a panel of primary ovarian cancer xenografts, suggesting the heterogeneity of response in ovarian cancer and a need to establish predictive biomarkers. CONCLUSION: This first comprehensive study of the molecular response to trastuzumab, pertuzumab and combined therapy in vivo highlights both common and distinct downstream effects to agents used alone or in combination, suggesting that complementary pathways may be involved.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Expresión Génica/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Western Blotting , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trastuzumab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Increasing the efficacy of targeted cancer therapies requires the identification of robust biomarkers suitable for patient stratification. This study focused on the identification of molecular mechanisms causing resistance against the anti-ERBB2-directed therapeutic antibodies trastuzumab and pertuzumab presently used to treat patients with ERBB2-amplified breast cancer. Immunohistochemistry and clinical data were evaluated and yielded evidence for the existence of ERBB2-amplified breast cancer with high-level epidermal growth-factor receptor (EGFR) expression as a separate tumor entity. Because the proto-oncogene EGFR tightly interacts with ERBB2 on the protein level, the hypothesis that high-level EGFR expression might contribute to resistance against ERBB2-directed therapies was experimentally validated. SKBR3 and HCC1954 cells were chosen as model systems of EGFR-high/ERBB2-amplified breast cancer and exposed to trastuzumab, pertuzumab and erlotinib, respectively, and in combination. Drug impact was quantified in cell viability assays and on the proteomic level using reverse-phase protein arrays. Phosphoprotein dynamics revealed a significant downregulation of AKT signaling after exposure to trastuzumab, pertuzumab or a coapplication of both antibodies in SKBR3 cells but no concomitant impact on ERK1/2, RB or RPS6 phosphorylation. On the other hand, signaling was fully downregulated in SKBR3 cells after coinhibition of EGFR and ERBB2. Inhibitory effects in HCC1954 cells were driven by erlotinib alone, and a significant upregulation of RPS6 and RB phosphorylation was observed after coincubation with pertuzumab and trastuzumab. In summary, proteomic data suggest that high-level expression of EGFR in ERBB2-amplified breast cancer cells attenuates the effect of anti-ERBB2-directed antibodies. In conclusion, EGFR expression may serve as diagnostic and predictive biomarker to advance personalized treatment concepts of patients with ERBB2-amplified breast cancer.
RESUMEN
PURPOSE: The monoclonal antibody pertuzumab represents the first HER2 dimerization inhibitor with unknown activity in colon cancer treatment. We examined the antitumor activity of pertuzumab as a single agent or in combination with erlotinib or irinotecan in human colon cancer cells in vitro and in vivo. METHODS: Colon cancer cell lines were tested for HER1/HER2 expression by western blot analysis. The effect of pertuzumab on cell cycle distribution was analyzed by FACS. Nude mice bearing xenograft tumors were treated with pertuzumab alone, or in combination either with irinotecan or with erlotinib. Tumor volume was measured repeatedly. Tumor histology was analyzed for necrosis. RESULTS: Six of nine cell lines showed high expression of HER1/HER2. Pertuzumab inhibited cell cycle progression in various cell lines. Pertuzumab showed minor antitumor activity in xenograft tumors, but significantly inhibited tumor growth when combined with erlotinib (P < 0.001). Combination of pertuzumab with irinotecan had no additional effect on growth of additional tumors. Pertuzumab treated DLD-1 xenograft tumors did not show enhanced necrosis, which, however, was found in HCT116 derived xenografts. CONCLUSIONS: Pertuzumab has some antitumor activity on human colon cancer cells in vitro and in vivo, in particular when combined with erlotinib. In vivo, pertuzumab combination treatment was not superior to irinotecan monotherapy. These data warrant further investigation of simultaneous HER1/EGFR TKI inhibition and HER1/HER2 dimerization inhibition for colorectal cancer therapy.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Multimerización de Proteína/efectos de los fármacos , Receptor ErbB-2/metabolismo , Animales , Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Western Blotting , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Dimerización , Quimioterapia Combinada , Factor de Crecimiento Epidérmico/farmacología , Clorhidrato de Erlotinib , Femenino , Humanos , Técnicas In Vitro , Irinotecán , Ratones , Ratones Desnudos , Quinazolinas/administración & dosificación , Receptor ErbB-2/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The human epidermal growth factor receptors HER1/EGFR and HER2 offer potential targets for treating non-small cell lung cancer (NSCLC). The antitumor efficacy of erlotinib (Tarceva, F. Hoffmann-La Roche, Ltd., Basel, Switzerland), a HER1/EGFR tyrosine-kinase inhibitor, was investigated in relation to HER1/EGFR and HER2 expression in five NSCLC xenograft models. MATERIALS AND METHODS: Tumor-bearing mice were randomized to daily oral erlotinib, 50 mg/kg, or vehicle (controls) for 20-50 days. The antitumor efficacy of erlotinib was measured through tumor volume, serum tumor markers and tumor biomarkers. Tumor HER1/EGFR and HER2 expression were analyzed immunohistochemically. RESULTS: Erlotinib reduced tumor volume in three NSCLC models. It also reduced serum tumor marker levels and the extent of inhibition correlated with tumor growth inhibition. HER1/EGFR and HER2 expression differed between the five tumor models, suggesting that expression level does not predict response to treatment. CONCLUSION: Erlotinib showed differing antitumor activity in five NSCLC models, suggesting that its antitumor effect is independent of HER1/EGFR and HER2 overexpression.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Modelos Animales de Enfermedad , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinas/uso terapéutico , Receptor ErbB-2/metabolismo , Animales , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
FK866 is a novel anticancer agent that was previously shown to interfere with NAD superset+ biosynthesis by inhibition of nicotinamide phosphoribosyltransferase and to initiate apoptosis in cancer cells. As NAD superset+ is involved in cellular DNA repair processes, the present in vitro study on THP-1 and K562 leukemia cells was conducted to investigate the cytotoxicity of FK866 combination treatment with various cytotoxic agents: the antimetabolite Ara-C, the DNA-intercalating agent daunorubicin and the alkylating compounds 1-methyl-3-nitro-1-nitrosoguanidinium (MNNG) and melphalan. Cell viability after drug exposure was assessed by propidium iodide (PI) staining. Non-cytotoxic concentrations of FK866 (10 superset-9 M or less), applied simultaneously or 24 hours before adding cytotoxic agents, caused a depletion in the intracellular NAD superset+ and--to a lesser extent-- NADH levels in THP-1 cells. After 48 and 72 hours treatment with daunorubicin and Ara-C, respectively, increased cell death was observed in THP-1 cells that were pretreated with FK866, as compared to cells exposed to antineoplastic drugs alone. However, this effect was transient, and there was no difference in cell survival after 72 hours incubation with daunorubicin or 96 hours with Ara-C. - Non-toxic concentrations of FK866 added 8, 16, or 24 hours before starting treatment with the PARP-activating agent MNNG synergistically decreased intracellular NAD superset+ contents, and increased MNNG-induced cytotoxicity both in THP-1 and K562 cells for at least 72 hours. This effect was less pronounced when FK866 was used in combination with another alkylating agent, melphalan. The PARP inhibitor 3-aminobenzamide delayed MNNG-induced cytotoxicity by 24 hours both in cells that were pretreated with FK866 and in non-pretreated cells. 48 hours later, the protective effect of 3-aminobenzamide could no longer be observed, but FK866-pretreated cells retained increased sensitivity to MNNG. - In conclusion, the chemosensitizing effect of FK866 on cell death induced by antineoplastic drugs was particularly obvious in combination with substances like MNNG that cause NAD superset+ depletion per se. It was less pronounced and only transiently measurable in combination with daunorubicin, Ara-C, and melphalan, respectively. These results may indicate different levels of DNA damage implicated in the action of the cytotoxic agents used.
Asunto(s)
Acrilamidas/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Monocitos/efectos de los fármacos , NAD/antagonistas & inhibidores , Piperidinas/farmacología , Antibióticos Antineoplásicos/farmacología , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/metabolismo , Antineoplásicos Alquilantes/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citarabina/farmacología , Daunorrubicina/farmacología , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Melfalán/farmacología , Metilnitronitrosoguanidina/farmacología , Monocitos/citología , NAD/metabolismoRESUMEN
Polyadenosylation of nuclear enzymes is well known to regulate the cellular repair capacity after DNA damage. PARP mediates the transfer of poly-ADP-ribose moieties on itself and other nuclear proteins by the breakdown of NAD+. The present study investigated how modulation of PARP activity interferes with cell death induced by two different alkylating agents used in cancer chemotherapy. 1-methyl-3-nitro-1-nitrosoguanidinium (MNNG) decreased cellular reduction capacity (WST-1 assay) in HL60 and CCRF-CEM cells, accompanied by increased activity of PARP and depletion of intracellular NAD+ and ATP. Pretreatment with the PARP inhibitors 3-AB or 4-AN resulted in transient cell protection, which was associated with a switch from necrosis to apoptosis in CCRF-CEM cells and enhanced apoptosis in HL60 cells. Both PARP inhibitors delayed the drop in WST-1 reduction and retained NAD+ and ATP levels required for apoptosis. Furthermore, 3-AB or 4-AN prevented progressive DNA degradation in MNNG-treated CCRF-CEM cells. In contrast to MNNG, we did not observe early activation of PARP, decrease in WST-1 reduction, or wasteful consumption of NAD+ and ATP after treatment with melphalan. However, preincubation with 3-AB or 4-AN resulted in decreased HL60 cell membrane blebbing and reduced formation of apoptotic bodies. In conclusion, the cell death preventing effects of PARP inhibitors are mediated by their ability to maintain cellular energy metabolism, to inhibit the activation of endonucleolytic DNA degradation and to prevent cell blebbing. Surprisingly, these protective effects of PARP inhibitors on different cell functions seem to be independent of each other and are rather determined by the respective cytotoxic mechanisms implicated by different drugs. Our results support the hypothesis, that PARP activation and/or cleavage plays a regulatory role in the induction of apoptosis.
Asunto(s)
1-Naftilamina/análogos & derivados , Alquilantes/antagonistas & inhibidores , Alquilantes/farmacología , Inhibidores Enzimáticos/farmacología , Leucemia/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , 1-Naftilamina/farmacología , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Benzamidas/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Tamaño de la Célula/efectos de los fármacos , Células HL-60 , Humanos , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Melfalán/farmacología , Metilnitronitrosoguanidina/farmacología , NAD/metabolismo , Naftalimidas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Quinolonas/farmacologíaRESUMEN
OBJECTIVES: Antineoplastic activity of tiazofurin (Tz) and selenazofurin (Se) depends on their conversion to substances which are analogs of NAD. NAD performs pleiotropic and essential cellular functions, both as a cofactor in oxidation-reduction reactions and as a substrate for poly- and mono-ADP-ribosylation reactions. The therapeutic potential of modulating intracellular NAD levels and activity of NAD-dependent enzymes by concomitant administration of conventional anticancer agents merits further research. Our aim was to investigate the cytotoxic effects of Tz and Se in hematopoietic cells and to test their ability to potentiate the effects of DNA strand-disrupting agents. MATERIAL: THP-1, a cell line, derived from human acute monoblastic leukemia, was used. CLL lymphocytes were obtained from 8 patients with CLL. METHODS: The WST-l test was used to detect the function of NAD(P)-dependent dehydrogenases after exposure of THP-1 cells to Tz or Se. Cytotoxicity of Tz, Se, MNNG and chlorambucil was assessed using the membrane permeability assay (PI test). RESULTS: THP-1 cells were sensitive to cytotoxic effects of Tz and Se, with IC50 values of 2.5 x 10(-5) M for Tz and 2 x 10(-6) M for Se, as determined with the WST-1 test; 10 microM Se induced cell membrane disruption in more than 20% of THP-1 cells 48 hours after commencement of treatment, whereas the same concentration of Tz failed to increase membrane permeability. Pretreatment of THP-1 cells with 0.5 - 1.5 microM Se had no effect on the time course of cell death, induced by treatment with the DNA-damaging agent 1-methyl-3-nitro-1 - nitrosoguanidinium (MNNG) for 36 hours. However, when incubation of THP-1 cells with MNNG was prolonged (72 hours) without changing the incubation medium, pretreatment with Se had the following effects: the relative number of cells that died spontaneously decreased, and the cytotoxicity of MNNG was diminished. This effect was also demonstrated ex vivo in 6 of 8 cases of CLL, treated with MNNG and chlorambucil. CONCLUSIONS: Contrary to other investigations, we here demonstrate that preincubation with Se may partially protect cells from cell death induced by the alkylating agents MNNG and chlorambucil in the THP-1 cell line and in CLL lymphocytes presumably by affecting spontaneous cell death.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Compuestos de Organoselenio/farmacología , Compuestos de Organoselenio/uso terapéutico , Ribavirina/análogos & derivados , Ribonucleósidos/farmacología , Ribonucleósidos/uso terapéutico , Antineoplásicos/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Clorambucilo/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Monocítica Aguda/tratamiento farmacológico , Metilnitronitrosoguanidina/farmacología , Compuestos de Organoselenio/metabolismo , Ribavirina/metabolismo , Ribavirina/farmacología , Ribavirina/uso terapéutico , Ribonucleósidos/metabolismoRESUMEN
P-glycoprotein(P-gp)- related resistance is one of the major obstacles in treating leukemia patients. Therefore, it is of clinical interest to find new potential modulators and compare their P-gp-modulating efficacy. The present analysis investigated the influence of P-gp modulators, such as verapamil, tamoxifen, droloxifene E, droloxifene Z, SDZ PSC 833 (PSC 833) and dexniguldipine in a leukemic T-cell line (CCRF-CEM) and its P-gp-resistant counterparts (CCRF-CEM/ACT400 and CCRF-CEM/VCR1000). P-gp expression was assessed with an immunocytological technique using the monoclonal antibody 4E3.16. It was characterized as the percentage of P-gp positive cells and also expressed as a D value by using the Kolmogorov Smirnov statistic. The efficacy of P-gp modulators was determined with the rhodamine-123 accumulation test and the MTT test. An in vitro modulator concentration between 0.1 microM and 3 microM was determined, where no genuine antiproliferative effect was apparent. The modulators PSC 833 and dexniguldipine were the significant (pAsunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores
, Ciclosporinas/farmacología
, Dihidropiridinas/farmacología
, Resistencia a Múltiples Medicamentos
, Resistencia a Antineoplásicos
, Leucemia de Células T/patología
, Proteínas de Neoplasias/antagonistas & inhibidores
, Tamoxifeno/análogos & derivados
, Tamoxifeno/farmacología
, Verapamilo/farmacología
, Transportadoras de Casetes de Unión a ATP/análisis
, Anticuerpos Monoclonales/inmunología
, División Celular/efectos de los fármacos
, Ensayos de Selección de Medicamentos Antitumorales
, Humanos
, Proteínas Asociadas a Resistencia a Múltiples Medicamentos
, Células Tumorales Cultivadas/efectos de los fármacos
, Partículas Ribonucleoproteicas en Bóveda
RESUMEN
In the human gastric carcinoma cell line EPG85-257P (parent) induction of resistance to daunorubicin (DAU) was achieved by selection with stepwise increased concentrations of the drug. The new variant was named EPG85-257DAU and was shown to overexpress the mdr1 gene product 170 kDa P-glycoprotein (P-Gp) as demonstrated by immunocytochemistry and mdr1-specific RT-PCR. To investigate the intracellular pathway of DAU the subcellular distribution of this autofluorescent drug was studied in the resistant cells and compared to its chemosensitive counterpart EPG85-257P. When sensitive cells were exposed to DAU the drug rapidly accumulated in the nucleus until cell death. No redistribution of DAU to the cytoplasm was observed. In resistant cells exposed to the drug DAU also accumulated in the nucleus but to a lesser extent than in parent cells. Following exposure, nuclear fluorescence was observed to decrease over a time period of up to 48 h. Six hours after DAU exposure formation of fluorescent vesicle formation started in the perinuclear region and increased continuously. After 48 h nuclear fluorescence was no longer detectable and DAU was located exclusively in vesicles. During this period the vesicles moved from the region of origin to the cell periphery. A pulse chase experiment showed, that vesicles may contain DAU derived from the nucleus. Treatment of EPG85-257DAU cells with DAU in conjunction with the chemosensitizer cyclosporin A (CsA) increased nuclear fluorescence without impairing vesicle formation. Disruption of microtubules by nocodazole led to an accumulation of vesicles in the perinuclear region indicating that microtubules are involved in vesicular transport. Treatment of EPG85-257DAU cells with the actin disruptor cytochalasin B led to accumulation of vesicles in the cell periphery indicating that actin may be involved in exocytosis. Uptake and efflux of DAU and rhodamin (RH) were determined in sensitive and resistant cells using a fluorescence activated cell sorter. Uptake of both compounds was distinctly lower in resistant than in sensitive cells. When resistant cells preloaded for 2 h with RH subsequently were incubated in drug free medium the substance was rapidly released indicating transmembrane transport by P-Gp. In contrast, despite expression of P-Gp in resistant cells no considerable release of DAU was observed for up to 2 h under the same experimental protocol. This indicates that in resistant cells intracellular DAU at least in part may be inaccessible for P-Gp and that vesicular drug transport appears to contribute to DAU resistance by removing intracellular DAU via exocytosis.
Asunto(s)
Daunorrubicina/análisis , Daunorrubicina/metabolismo , Resistencia a Múltiples Medicamentos/fisiología , Neoplasias Gástricas/química , Neoplasias Gástricas/ultraestructura , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Ciclosporina/farmacología , Citocalasina B/farmacología , Citometría de Flujo , Humanos , Nocodazol/farmacología , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Neoplasias Gástricas/patología , Células Tumorales CultivadasRESUMEN
The new antiestrogen Droloxifene has a 10-60-fold higher binding affinity to the estrogen receptor (ER) compared to the related compound Tamoxifen. A similar relationship was found in growth inhibition studies which showed that Droloxifene inhibited the different ER positive human breast cancer cells more effectively than Tamoxifen, predominantly in drug concentrations which are found in humans during therapy. As another consequence of the high stability of the complex formed by Droloxifene binding to the ER, intermittent exposures with clinically relevant concentrations of Droloxifene brought about effective growth inhibition of human ER positive tumor cells even after short-term application. Droloxifene was found, like Tamoxifen, to block human breast cancer cells in G1-phase of the cell cycle. Moreover, cell-cycle data confirmed the superior growth-inhibiting potency of Droloxifene compared to Tamoxifen. Droloxifene was also found to effectively induce expression of the negative growth factor TGF-beta, to inhibit IGF-I stimulated cell growth and to prevent estrogen-stimulated proto-oncogene c-myc expression. Unlike Tamoxifen, Droloxifene is a potent inhibitor of protein biosynthesis in ER-positive breast cancer cells at physiologically relevant concentrations. Lower estrogenic and higher antiestrogenic effects on immature rat uterus indicate a higher therapeutic index for Droloxifene compared to Tamoxifen. In vivo, Droloxifene displayed increased growth inhibition of different tumors of animal (R3230AC and 13762) and human origin (T61). Furthermore, it was found that the two structurally similar drugs differ in their toxicologic characteristics in the following important respects: Droloxifene is devoid of any in vivo or in vitro carcinogenic or mutagenic effects, whereas Tamoxifen causes liver tumors in rats, induces DNA adduct formation in rats and hamsters and shows transforming activity in SHE-cells (Syrian hamster embryo fibroblasts). Considerably less toxicity and a lower level of intrinsic estrogenicity was observed even after maximum long-term exposure of different animal species to Droloxifene, in comparison with Tamoxifen. Therefore, it can be assumed that Droloxifene may represent an important step forward in the treatment of mammary carcinomas in women through its better tolerability and increased efficacy compared with Tamoxifen. For long-term adjuvant or preventive treatment of breast cancer, Droloxifene may well be the safer choice.
Asunto(s)
Antineoplásicos/uso terapéutico , Antagonistas de Estrógenos/uso terapéutico , Tamoxifeno/análogos & derivados , Animales , Neoplasias de la Mama/tratamiento farmacológico , Ciclo Celular , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Proto-Oncogenes Mas , Ratas , Ratas Endogámicas , Receptores de Estrógenos/metabolismo , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Recently, it was reported that the anti-estrogen tamoxifen not only inhibits estradiol-stimulated growth of MCF-7 cells but also significantly reduces the proliferation rate of cells stimulated by growth factors. We have confirmed this finding and also shown that the new anti-estrogen droloxifene inhibits the proliferation of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I)-stimulated MCF-7 cells. The growth-factor-induced proliferation was inhibited in a dose-dependent manner by the anti-estrogens in the complete absence of estrogen and FCS. Of the anti-estrogens, droloxifene was considerably more potent than tamoxifen. Because the expression of the proto-oncogenes c-fos and c-myc has been considered a key event in development of the mitogenic response, we examined the effects of anti-estrogens on c-myc and c-fos gene expression. We included in these investigations the steroidal anti-estrogen ICI 164,384 because this compound has no or very little estrogenic activity. The studies revealed that all 3 anti-estrogens transiently induced c-myc mRNA expression. However, the anti-estrogens inhibited estradiol-induced c-myc mRNA expression, although with different potencies. Pre-incubation of MCF-7 cells with droloxifene and tamoxifen resulted in elevated levels of growth-factor-induced c-myc mRNA expression. In contrast, the anti-estrogens did not induce c-fos mRNA or affect the expression of c-fos mRNA induced by growth factors. In conclusion, non-steroidal anti-estrogens inhibit growth-factor-stimulated proliferation of MCF-7 cells without inhibiting growth-factor-induced c-myc or c-fos mRNA expression.
Asunto(s)
Neoplasias de la Mama/patología , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Antagonistas de Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Neoplasias de la Mama/genética , División Celular/efectos de los fármacos , Estradiol/farmacología , Genes fos/efectos de los fármacos , Genes myc/efectos de los fármacos , Humanos , ARN Mensajero/efectos de los fármacos , ARN Neoplásico/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Virus-infected cells were analyzed using multiparameter flow cytometry. Two virus-cell systems were investigated: HSV-1-infected VF cells and influenza C virus JHB/1/66-infected MDCK cells. Analysis included the measurement of the appearance of virus specific antigens. On individual cells, with polyclonal antibodies, antigens were first detected at 12 h p.i., and the numbers of labeled cells were followed up to 96 h p.i. The efficacy of four antiviral agents was tested with this system. The results were in good agreement with those of plaque reduction tests and indicated that this new method may be extremely useful for the correlation of viral and cellular events with antiviral activity. Finally, it was demonstrated that infected cells in both systems have a considerably greater volume than non-infected cells.
Asunto(s)
Antivirales , Evaluación Preclínica de Medicamentos/métodos , Citometría de Flujo , Animales , Antígenos Virales/biosíntesis , Supervivencia Celular , Células Cultivadas , Estudios de Evaluación como Asunto , Fluoresceína-5-Isotiocianato , Fluoresceínas , Técnica del Anticuerpo Fluorescente , Humanos , Gammainfluenzavirus/efectos de los fármacos , Gammainfluenzavirus/inmunología , Simplexvirus/efectos de los fármacos , Simplexvirus/inmunología , Coloración y Etiquetado , Tiocianatos , Factores de Tiempo , Ensayo de Placa ViralRESUMEN
Using the fluorescent membrane potential probe, 3,3'-dihexyl-oxacarbocyanine (DiOC6(3], we found a 4-fold higher uptake in Adriamycin (ADM)-sensitive versus -resistant Friend leukemia cells (FLC). When sensitive cells were treated in the presence of high potassium (120 mM K+), there was a greater than 80% reduction of DiOC6(3) uptake. Using carbonylcyanide 4-trifluoromethoxy-phenylhydrazone (FCCP), a specific inhibitor of mitochondrial membrane potential, DiOC6(3) accumulation was reduced by less than 30% in these cells. Both results support the conclusion that a greater uptake of DiOC6(3) in ADM-sensitive than in -resistant cells indicates an increased plasma transmembrane potential. Since electronegative plasma membrane potentials are a driving force for the transport of lipophilic positively-charged compounds, differences in membrane potentials between sensitive and multiple drug resistant (MDR) tumor cells could have an important influence on drug accumulation and cytotoxicity. The drugs which our ADM-resistant FLC display multiple drug resistance to are positively charged. In MDR FLC, the calcium channel antagonist, verapamil, has been shown to block the efflux of Rhodamine 123 (Rho 123) and other positively-charged compounds. Since DiOC6(3) is also positively-charged, we used verapamil to investigate its effects on drug uptake. In MDR FLC, verapamil increased DiOC6(3) accumulation by 1.9-fold, whereas in sensitive cells it was increased 1.5-fold. In contrast, verapamil increased the levels of Rho 123 in resistant cells 7.8-fold but lowered them in sensitive cells 1.5-fold. The minimal loss of DiOC6(3) from both sensitive and MDR cells and the above results can best be interpreted as indicating that DiOC6(3) is not transported by the efflux "pump" system but that verapamil induces a plasma membrane potential increase in sensitive and resistant cells that DiOC6(3) is sensitive to. On the other hand, since Rho 123 did appear to be actively effluxed from these resistant cells, the enhancement of this compound by verapamil was more likely due to inhibition of the MDR "pump." How, or whether, plasma membrane potentials and the MDR efflux "pump" are related remains to be investigated. In the resistant cells, verapamil also induced an increase (13-fold) in the accumulation of the electrically neutral fluorescent probe for calcium, INDO-1/AM. However, verapamil had no effect on the efflux of this compound, which was equivalent in both resistant and sensitive cells. Thus, a new effect of verapamil on drug accumulation in MDR cells is identified here.
Asunto(s)
Membrana Celular/fisiología , Doxorrubicina/farmacología , Resistencia a Medicamentos , Potenciales de la Membrana , Animales , Carbocianinas/metabolismo , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Citometría de Flujo , Indoles/metabolismo , Ratones , Potasio/farmacología , Células Tumorales Cultivadas , Verapamilo/farmacologíaRESUMEN
The raw extracts of a series of microorganisms were screened for the presence of acetyl-coenzyme A: arylamine N-acetyltransferase (AAAT) using a radioactive assay with 3H-acetyl-coenzyme A and aniline as substrates. Enzyme activities were primarily detected in the soluble fractions of Bacillus and Nocardia species, and in some further soil organisms. Only strains of Bacillus cereus were able to acetylate 4-nitroaniline and 3,5-dimethyl-4-nitroaniline. The fermentation conditions for the production of the enzyme were optimized. The AAAT from one strain of Bacillus cereus was purified 24-fold and characterized.