RESUMEN
Cancers harboring dominant-negative p53 mutations are often aggressive and difficult to treat. Direct attempts to restore wild-type p53 function have produced little clinical benefit. We investigated whether targeting a p53-target gene could induce certain tumor-suppressor characteristics. We found that inhibition of stathmin, a microtubule regulator that can be transcriptionally repressed by wild-type p53, restored certain wild-type functions to cancer cells with mutant p53. Silencing of stathmin by small interfering RNA (siRNA) in mutant p53 cell lines lowered expression to that observed following activation of wild-type p53 by DNA damage in wild-type p53 cell lines. siRNA-induced repression of stathmin decreased cell proliferation, viability and clonogenicity in mutant p53 cell lines. Furthermore, knockdown of stathmin partially restored cell-cycle regulation and activation of apoptosis. Therefore, targeting stathmin, a gene product that is overexpressed in the presence of mutant p53, may represent a novel approach to treating cancers with aberrant p53 function.
Asunto(s)
Neoplasias de la Mama/metabolismo , Silenciador del Gen/fisiología , Inhibidores de Crecimiento/biosíntesis , Inhibidores de Crecimiento/genética , Mutación , Estatmina/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Femenino , Marcación de Gen/métodos , Inhibidores de Crecimiento/fisiología , Humanos , Estatmina/biosíntesis , Estatmina/genética , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Resistance to multiple, unrelated cancer chemotherapeutic drugs can be mediated by P-glycoprotein, the MDR1 gene product. Numerous substances, including chemotherapeutic drugs, heavy metals, growth factors, activated oncogenes, or changes in temperature increase MDR1 gene expression. Because several of these factors regulate cellular function through the activation of phospholipase C (PLC), we postulated that PLC-mediated signaling could be central to regulating the expression of MDR1. Transfection of NIH 3T3 cells with a pMJ30-PLC-gamma 1 expression vector increased the activity of the MDR1 promoter by 2- to 10-fold. PLC-mediated activation required a region between -106 and -99 of the MDR1 promoter. Treatment of cotransfected cells with platelet-derived growth factor further enhanced the activity of the MDR1 promoter. The stimulatory effect of PLC on the MDR1 promoter was increased by cotransfection with constitutively active v-raf and was blocked by the dominant-negative mutant, c-Raf-C4. The activity of mitogen-activated protein kinase (MAPK) was also increased in PLC-gamma 1-transfected cells. Furthermore, PD-98059 and U0126, two MAPK inhibitors, blocked PLC-gamma 1-induced expression of MDR1. The results of Northern blot analysis showed that activation of PLC by heat shock and growth factors increased expression of endogenous MDR1 mRNA in human renal carcinoma cells. These effects were blocked by inhibitors of the PLC-MAPK pathway. In summary, our results indicate for the first time that activation of PLC by a variety of cellular stimuli can regulate the expression of MDR1 and that the transcriptional modulation of MDR1 expression by PLC is mediated by the Raf-MAPK pathway.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Expresión Génica/fisiología , Isoenzimas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Fosfolipasas de Tipo C/metabolismo , Células 3T3 , Animales , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Isoenzimas/antagonistas & inhibidores , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fosfolipasa C gamma , Regiones Promotoras Genéticas/fisiología , Transfección , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
The search for a simple, sensitive test to reliably determine prognosis and predict response to therapy in patients with cancer is an important area of translational research. In this issue of Clinical Cancer Research, Hayes et al. (Clin. Cancer Res., 7: 601-604, 2001) report the results of an ancillary Cancer and Leukemia Group B protocol designed to determine whether the circulating extracellular domain of HER-2/neu (ECD-HER-2) was indicative of prognosis or predictive of response to therapy in women with metastatic breast cancer. Results were drawn from a sample of 242 patients of whom 89 had elevated values of the protein. These women had been enrolled in a variety of Cancer and Leukemia Group B protocols evaluating either the efficacy of dose in the use of megestrol acetate as second-line hormonal treatment or in patients enrolled into several chemotherapeutic protocols, many containing doxorubicin. They report that patients with pretreatment elevation of ECD-HER-2 had a worse prognosis than those who did not, but that there was no convincing correlation of elevated ECD-HER-2 with response to either endocrine or chemotherapy. Although the small number of patients and the retrospective study design allows one to draw only tentative conclusions, this report raises several important issues for the conduct of translational research and points to several new hypotheses for future testing.
Asunto(s)
Neoplasias de la Mama/sangre , Receptor ErbB-2/sangre , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Femenino , Humanos , Metástasis de la Neoplasia , Valor Predictivo de las Pruebas , Pronóstico , Análisis de SupervivenciaRESUMEN
The vesicular monoamine transporter 2 (VMAT2) has sequence homology with bacterial multidrug transporters which in turn share homology with mammalian P-glycoprotein (P-GP). Both VMAT2 and P-GP can detoxify cells. 1-Methyl-4-phenylpyridinium (MPP(+)), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a substrate for VMAT2 that has several structural features in common with P-GP substrates and inhibitors. The present studies investigated whether P-GP is responsible for the elimination of MPP(+) from the brain. Additionally, VMAT2 and P-GP are inhibited by many of the same compounds. Thus we also investigated whether VMAT2 inhibitors could block P-GP in vitro and vice versa whether P-GP inhibitors could block VMAT2 mediated transport of [3H]-DA into synaptic vesicles. In mice treated with MPTP and a P-GP inhibitor (quinidine, trans-flupentixol or cyclosporine A), the elimination of MPP(+) from the striatum was significantly delayed. However, in experiments using various cell lines expressing either mouse or human P-GP, MPP(+) did not reverse the P-GP mediated resistance to vincristine, suggesting that MPP(+) is a poor substrate for P-GP. Additional experiments were performed using mdr1a/b double knockout mice which lack functional P-GP encoded by these two genes. Data from mdr1a/b knockout mice treated with MPTP also suggest that MPP(+) is not extruded from the brain by P-GP. In other studies, we demonstrated that the VMAT2 inhibitors tetrabenazine and Ro 4-1284 inhibit P-GP and that the P-GP inhibitors trans-flupentixol and quinidine inhibit VMAT2. Thus, several new drugs can be added to the list of compounds that are able to inhibit both VMAT2 and P-GP, providing further evidence of the similarity between these two transporters.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacocinética , 1-Metil-4-fenilpiridinio/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Encéfalo/efectos de los fármacos , Interacciones Farmacológicas/fisiología , Glicoproteínas de Membrana/antagonistas & inhibidores , Proteínas de Transporte de Membrana , Neuropéptidos , Trastornos Parkinsonianos/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/metabolismo , 1-Metil-4-fenilpiridinio/análogos & derivados , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Antiarrítmicos/farmacología , Encéfalo/metabolismo , Encéfalo/fisiopatología , Bloqueadores de los Canales de Calcio/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Dopamina/metabolismo , Antagonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Inmunosupresores/farmacología , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Trastornos Parkinsonianos/fisiopatología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Células Tumorales Cultivadas , Proteínas de Transporte Vesicular de Aminas Biógenas , Proteínas de Transporte Vesicular de MonoaminasRESUMEN
Glioblastoma multiforme is the most treatment-resistant brain tumor. Elongation factor-2 (EF-2) kinase (calmodulin kinase III) is a unique protein kinase that is overexpressed in glioma cell lines and in human surgical specimens. Several mitogens activate this kinase and inhibitors block mitogen activation and produce cell death. Geldanamycin (GA) is a benzoquinone ansamycin antibiotic that disrupts Hsp90-protein interactions. Because EF-2 kinase is chaperoned by Hsp90, we investigated the effects of GA on the viability of glioma cells, the expression of EF-2 kinase protein, and the interaction between Hsp90 and EF-2 kinase. GA was a potent inhibitor of the clonogenicity of four glioma cells lines with IC(50)s ranging from 1 to 3 nM. 17-allylamino-17-demethoxygeldanamycin (17-AAG), a less toxic and less potent derivative of GA, inhibited the clonogenicity of glioma cells with IC(50) values of 13 nM in C6 cells and 35 nM in T98G cells. Treatment of cell lines for 24-48 h of GA or 17-AAG disrupted EF-2-kinase/Hsp90 interactions as measured by coimmunoprecipitation, resulting in a decreased amount of recoverable kinase in cell lysates. The ability of GA to inhibit the growth of glioma cells was abrogated by overexpressing EF-2 kinase. In addition, 17-AAG significantly inhibited the growth of a glioma xenograft in nude mice. These studies demonstrate for the first time the activity of GAs against human gliomas in vitro and in vivo and suggest that destruction of EF-2 kinase may be an important cytotoxic mechanism of this unique class of drug.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Glioblastoma/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Quinonas/farmacología , Animales , Benzoquinonas , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasa del Factor 2 de Elongación , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Lactamas Macrocíclicas , Ratones , Ratones Desnudos , Ratas , Rifabutina/análogos & derivados , Rifabutina/farmacología , Células Tumorales CultivadasRESUMEN
Prostate cancer is a devastating disease that will be diagnosed in approximately 200,000 men in 2001. New methods for screening, prevention, and treatment are being developed. In addition, novel agents for the treatment of resistant prostate cancer are being developed in clinical trials. This review summarizes the recent efforts in diet, screening, novel systemic therapies, and alternative medicine for prostate cancer.
Asunto(s)
Medicamentos Herbarios Chinos , Neoplasias de la Próstata/terapia , Adulto , Anciano , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ensayos Clínicos como Asunto , Predicción , Genes bcl-2/genética , Genes p53/genética , Humanos , Inmunoterapia Activa , Interferones/administración & dosificación , Interferones/uso terapéutico , Masculino , Tamizaje Masivo , Persona de Mediana Edad , Mutación , Orquiectomía , Fitoterapia , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/radioterapia , Calidad de Vida , Investigación , Retinoides/administración & dosificación , Retinoides/uso terapéutico , Testosterona/sangre , Factores de TiempoRESUMEN
The expression of several drug-resistance genes, including MRP and p53, increases with advancing stage of human prostate cancer. Altered transcription could account for the genotypic alterations associated with prostate cancer progression, and it was recently reported that the promoter of MRP1 is activated in the presence of mutant p53. To determine whether there is a relationship between p53 status and the expression of MRP1, a human, temperature-sensitive p53 mutant (tsp Val(138)) was transfected into LNCaP human prostate cancer cells. In the transfected cell line (LVCaP), the wild-type p53 produced growth arrest at the G1/S interface of the cell cycle, inhibited colony formation, and induced p21(waf1/cip1). Temperature shifting to 38 degrees C (p53 mutant) produced a time-dependent increase in expression of MRP1. This change in MRP1 expression was also seen in isogenic cell lines in which p53 was inactivated by human papilloma virus (HPV)16E6 protein or by a dominant-negative mutant. Functional assays revealed a decrease in drug accumulation and drug sensitivity associated with mutant p53 and increased MRP1 expression. These results provide the first mechanistic link between expression of MRP1 and mutation of p53 in human prostate cancer and support recent clinical associations. Furthermore, these data suggest a mechanism tying accumulation of p53 mutations to the multidrug resistance phenotype seen in this disease.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Unión al ADN/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Neoplasias de la Próstata/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Carcinoma/genética , Ciclo Celular , Células Clonales , Neoplasias del Colon/genética , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Citometría de Flujo , Humanos , Leucotrieno C4/metabolismo , Masculino , Melanoma/genética , Proteína 3 Homóloga de MutS , Proteínas Recombinantes/metabolismo , Transfección , Proteína p53 Supresora de Tumor/genética , Vincristina/farmacologíaRESUMEN
The prognosis of patients with tumors expressing P-glycoprotein (P-gp), the MDR1 gene product, is generally poor. It is assumed that this is due to decreased tumor responsiveness that results from decreased drug accumulation. We observed that treatment of animals bearing MDR1-transfected leukemic cells with P-gp substrates (i.e., drugs that are transported by P-gp) significantly worsened host survival compared to treatment with vehicle or non-P-gp substrates. This effect was seen with cancer chemotherapeutic agents (paclitaxel and vincristine) and with the MDR modulator, trans-flupenthixol. To determine the mechanism(s) underlying this observation, we studied alterations in pharmacokinetics, pharmacodynamics, and metastasis. We found that the drug-induced acceleration of disease was associated with increased metastases. P-gp(+) cells treated with P-gp substrates demonstrated several pro-metastatic features, including membrane ruffling and invasion through a hepatocyte monolayer. These results suggest that the treatment of MDR tumors with P-gp substrates may produce changes in malignant behavior that could adversely affect therapeutic outcomes.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Antineoplásicos , Leucemia P388/patología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Progresión de la Enfermedad , Femenino , Flupentixol/farmacología , Humanos , Concentración de Iones de Hidrógeno , Leucemia P388/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/secundario , Mecloretamina/farmacología , Ratones , Invasividad Neoplásica , Trasplante de Neoplasias , Paclitaxel/farmacocinética , Paclitaxel/farmacología , Tasa de Supervivencia , Células Tumorales Cultivadas , Vincristina/farmacocinética , Vincristina/farmacologíaRESUMEN
PURPOSE: Recent studies demonstrate that retinoids decrease expression of the anti-apoptotic protein bcl-2, enhance the effect of chemotherapy, and act synergistically with interferon alfa (IFNalpha) to inhibit tumor cell growth in vitro. A phase I trial of 13-cis-retinoic acid (CRA), IFNalpha, and paclitaxel (TAX) was conducted to determine the toxicity and recommended phase II dose of this combination. Pharmacodynamic studies were performed to determine whether CRA and IFNalpha could modulate bcl-2 expression in vitro and in patients. PATIENTS AND METHODS: Twenty-two patients with prostate cancer or other advanced malignancies were treated with CRA/IFNalpha and escalating doses of TAX. The effect of CRA/IFNalpha on TAX pharmacokinetics was analyzed in both patients and human liver microsomes. The effect of CRA/IFNalpha on bcl-2 expression was assessed in vitro and in peripheral-blood mononuclear cells (PBMCs) by immunoblotting. RESULTS: CRA 1 mg/kg on days 1 to 4, IFNalpha 6 MU/m(2) subcutaneously on days 1 to 4, and TAX 175 mg/m(2) on day 3 was well tolerated. Pharmacokinetic studies demonstrated that CRA/IFNalpha caused a 33% decrease in TAX clearance and a 23% decrease in the area under the concentration-time curve values of the TAX metabolite 6-alfa-hydroxytaxol (6-HT). CRA alone reduced conversion of TAX to 6-HT by 41% in human liver microsomes. CRA/IFNalpha decreased bcl-2 expression in vitro and in PBMCs. CONCLUSION: CRA/IFNalpha and TAX is a well-tolerated regimen. CRA/IFNalpha increases TAX area under the concentration-time curve through an inhibitory effect of CRA on the metabolism of TAX to 6-HT. CRA/IFNalpha can modulate bcl-2 expression in vitro and demonstrates similar biologic activity in patients. Further studies will determine the activity of CRA/IFNalpha/TAX and validate the assessment of bcl-2 in PBMCs as a marker of tumor response.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas In Vitro , Interferón-alfa/administración & dosificación , Interferón-alfa/farmacología , Isotretinoína/administración & dosificación , Isotretinoína/farmacología , Leucocitos Mononucleares , Hígado/efectos de los fármacos , Masculino , Microsomas/efectos de los fármacos , Persona de Mediana Edad , Paclitaxel/administración & dosificación , Paclitaxel/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Taxanes and Vinca alkaloids are among the most active classes of drugs in the treatment of cancer. Yet, fewer than 50% of previously untreated patients respond, and clinicians have few ways of predicting who will benefit from treatment and who will not. Mutations in p53 occur in more than half of human malignancies and may alter the sensitivity to a variety of anticancer therapies. We have shown that the transcriptional status of p53 determines the sensitivity to antimicrotubule drugs and that this is mediated through the regulation of microtubule-associated protein 4 (MAP4). Expression of MAP4 is transcriptionally repressed by wild-type p53. Increased expression of MAP4, which occurs when p53 is transcriptionally inactive, increases microtubule polymerization, paclitaxel binding, and sensitivity to paclitaxel, a drug that stabilizes polymerized microtubules. In contrast, overexpression of MAP4 decreases microtubule binding and sensitivity to Vinca alkaloids, which promotes microtubule depolymerization. To determine whether induction of endogenous wild-type p53 by DNA-damaging agents alters the expression of MAP4 and changes the sensitivity to antimicrotubule drugs, we assayed cell lines with wild-type or mutant p53 for the expression of MAP4 and drug sensitivity before and after DNA damage. UV irradiation, bleomycin, and doxorubicin increased wild-type p53 expression and decreased MAP4 expression. These changes were associated with decreased sensitivity to paclitaxel and increased sensitivity to vinblastine. These changes in drug sensitivity were no longer observed when p53 and MAP4 returned to baseline levels. Changes in drug sensitivity following DNA-damaging agents were associated with decreased binding of paclitaxel and increased binding of Vinca alkaloids. In contrast, DNA damage did not alter the sensitivity to non-microtubule-active drugs, such as 1-beta-D-arabinofuranosylcytosine and doxorubicin. Changes in drug sensitivity following DNA-damaging drugs were not observed in cells with mutant p53. These studies demonstrate that induction of wild-type p53 by DNA-damaging agents can affect the sensitivity to antimicrotubule drugs through the regulation of MAP4 expression and may have implications for the design of clinical anticancer therapies.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Daño del ADN , ADN de Neoplasias/efectos de los fármacos , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas de Neoplasias/biosíntesis , Paclitaxel/farmacología , Taxoides , Proteína p53 Supresora de Tumor/fisiología , Alcaloides de la Vinca/farmacología , Animales , Biopolímeros , Bleomicina/farmacología , Citarabina/farmacología , ADN de Neoplasias/efectos de la radiación , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Genes p53 , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/efectos de los fármacos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Ratas , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Rayos Ultravioleta , Vinblastina/farmacologíaRESUMEN
Calmodulin-dependent protein kinase III (CaM kinase III, elongation factor-2 kinase) is a unique member of the Ca2+/CaM-dependent protein kinase family. Activation of CaM kinase III leads to the selective phosphorylation of elongation factor 2 (eEF-2) and transient inhibition of protein synthesis. Recent cloning and sequencing of CaM kinase III revealed that this enzyme represents a new superfamily of protein kinases. The activity of CaM kinase III is selectively activated in proliferating cells; inhibition of the kinase blocked cells in G0/G1-S and decreased viability. To determine the significance of CaM kinase III in breast cancer, we measured the activity of the kinase in human breast cancer cell lines as well as in fresh surgical specimens. The specific activity of CaM kinase III in human breast cancer cell lines was equal to or greater than that seen in a variety of cell lines with similar rates of proliferation. The specific activity of CaM kinase III was markedly increased in human breast tumour specimens compared with that of normal adjacent breast tissue. The activity of this enzyme was regulated by breast cancer mitogens. In serum-deprived MDA-MB-231 cells, the combination of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulated cell proliferation and activated CaM kinase III to activities observed in the presence of 10% serum. Inhibition of enzyme activity blocked cell proliferation induced by growth factors. In MCF-7 cells separated by fluorescence-activated cell sorting. CaM kinase III was increased in S-phase over that of other phases of the cell cycle. In summary, the activity of Ca2+/CaM-dependent protein kinase III is controlled by breast cancer mitogens and appears to be constitutively activated in human breast cancer. These results suggest that CaM kinase III may contribute an important link between growth factor/receptor interactions, protein synthesis and the induction of cellular proliferation in human breast cancer.
Asunto(s)
Neoplasias de la Mama/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Animales , Quinasa del Factor 2 de Elongación , Activación Enzimática , Humanos , Células Tumorales CultivadasRESUMEN
BACKGROUND: Herbal mixtures are popular alternatives to demonstrated therapies. PC-SPES, a commercially available combination of eight herbs, is used as a nonestrogenic treatment for cancer of the prostate. Since other herbal medicines have estrogenic effects in vitro, we tested the estrogenic activity of PC-SPES in yeast and mice and in men with prostate cancer. METHODS: We measured the estrogenic activity of PC-SPES with transcriptional-activation assays in yeast and a biologic assay in mice. We assessed the clinical activity of PC-SPES in eight patients with hormone-sensitive prostate cancer by measuring serum prostate-specific antigen and testosterone concentrations during and after treatment. RESULTS: In complementary yeast assays, a 1:200 dilution of an ethanol extract of PC-SPES had estrogenic activity similar to that of 1 nM estradiol, and in ovariectomized CD-1 mice, the herbal mixture increased uterine weights substantially. In six of six men with prostate cancer, PC-SPES decreased serum testosterone concentrations (P<0.05), and in eight of eight patients it decreased serum concentrations of prostate-specific antigen. All eight patients had breast tenderness and loss of libido, and one had venous thrombosis. High-performance liquid chromatography, gas chromatography, and mass spectrometry showed that PC-SPES contains estrogenic organic compounds that are distinct from diethylstilbestrol, estrone, and estradiol. CONCLUSIONS: PC-SPES has potent estrogenic activity. The use of this unregulated mixture of herbs may confound the results of standard or experimental therapies and may produce clinically significant adverse effects.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Medicamentos Herbarios Chinos/farmacología , Extractos Vegetales/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Receptores de Estrógenos/efectos de los fármacos , Animales , Antineoplásicos Fitogénicos/efectos adversos , Antineoplásicos Fitogénicos/química , Mama/efectos de los fármacos , Medicamentos Herbarios Chinos/efectos adversos , Medicamentos Herbarios Chinos/química , Femenino , Humanos , Libido/efectos de los fármacos , Masculino , Ratones , Extractos Vegetales/efectos adversos , Extractos Vegetales/química , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/fisiopatología , Saccharomyces cerevisiae/efectos de los fármacos , Testosterona/sangre , Tromboflebitis/inducido químicamente , Útero/efectos de los fármacos , Levaduras/efectos de los fármacosRESUMEN
Prostate cancer progresses from a localized disease to a widely disseminated malignancy. Each step along this progression pathway involves multiple genetic alterations that impart a survival advantage to the tumor cell over its normal counterparts and may confer resistance to therapy. Because metastatic prostate cancer is one of the most therapy-resistant human neoplasms, we studied the expression of certain molecular determinants of drug resistance in the context of tumor progression. Paraffin-embedded formalin-fixed resected prostates were chosen based on Gleason grade and surgical stage. Immunohistochemistry was used to detect the expression of multidrug resistance protein (MRP), topoisomerase II alpha, p53, glutathione S-transferase pi, Bcl-2, and P-glycoprotein in these specimens. We found that all of the proteins were expressed in resected prostate except for P-glycoprotein. The expression of MRP, topoisomerase II alpha, p53, and Bcl-2 increased with the Gleason grade. In addition, the expression of MRP, topoisomerase II alpha, and p53 increased with the surgical stage. In contrast, the glutathione S-transferase pi and Bcl-2 expression decreased with the increasing surgical stage. Stage was the strongest indicator of protein expression. These results suggest that drug resistance gene products are expressed in prostate cancer at the time of surgical resection. Thus, although the emergence of the "pan-resistance" phenotype in prostate cancer may partly be a function of the selection pressure exerted by therapeutic interventions, certain determinants of chemoresistance may be caused by genetic changes accompanying tumorigenesis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/análisis , Resistencia a Múltiples Medicamentos , Neoplasias de la Próstata/patología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/biosíntesis , ADN-Topoisomerasas de Tipo II/análisis , ADN-Topoisomerasas de Tipo II/biosíntesis , Progresión de la Enfermedad , Glutatión Transferasa/análisis , Glutatión Transferasa/biosíntesis , Humanos , Inmunohistoquímica , Isoenzimas/análisis , Isoenzimas/biosíntesis , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Estadificación de Neoplasias , Oportunidad Relativa , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/cirugía , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
Mutations in p53 change the sensitivity to cancer chemotherapeutic drugs. Whereas many drugs, including the vinca alkaloids, often become less effective when p53 is transcriptionally inactivated, several, most notably paclitaxel, may become more effective. In studying the underlying mechanism(s), we found that increased MAP4 expression, which occurs with transcriptionally silent p53, is associated with increased sensitivity to paclitaxel and decreased sensitivity to vinca alkaloids. Using murine fibroblasts transfected with MAP4, we directly demonstrated that the changes in drug sensitivity were associated with parallel alterations in drug-induced apoptosis and cell-cycle arrest. Immunofluorescent staining of the microtubule network revealed that cells with increased MAP4 expression displayed an increase in polymerized microtubules and an increased binding of fluorsceinated paclitaxel. Since MAP4 stabilizes polymerized microtubules, overexpression of this gene provides a plausible mechanism to explain the altered sensitivity to microtubule-active drugs in the presence of mutant p53.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Microtúbulos/biosíntesis , Mutación/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Células 3T3 , Animales , Antineoplásicos/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular , Riñón , Ratones , Ratones Endogámicos BALB C , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Paclitaxel/metabolismo , Paclitaxel/farmacología , Polímeros/metabolismo , Ratas , Receptores de Droga/análisis , Proteína p53 Supresora de Tumor/efectos de los fármacos , Vinblastina/farmacología , Vincristina/farmacologíaRESUMEN
Many multidrug-resistant (MDR) cell lines overexpress the epidermal growth factor receptor (EGFR) as well as P-glycoprotein (P-gp). However, the role of the increased EGFR in P-gp-mediated drug resistance remains unclear. Since recent studies suggest that activation of phospholipase C (PLC) could increase the phosphorylation of P-gp, and activation of the EGFR would also activate PLC, we investigated whether the effect of epidermal growth factor (EGF) on the phosphorylation of P-gp was mediated through PLC. Treatment of the human MDR breast cancer cell line, MCF-7/AdrR, with EGF increased the phosphorylation of P-gp by 20-50%. The increased phosphorylation of P-gp was accompanied by stimulation of PLC activity, as measured by the production of inositol, 1,4,5-trisphosphate and diacylglycerol, products of phosphatidylinositol-4,5-bisphosphate hydrolysis. Treatment of MDR cells with EGF also had detectable effects on P-gp function. For example, following incubation of MCF-7/AdrR cells with ECF, we observed a consistent decrease in total vinblastine (VBL) accumulation. Kinetic analysis revealed this change to be due to an increase in membrane efflux. The latter was measured by the initial uptake velocity, which was inhibited by EGF. VBL uptake measured at 0-320 sec was inhibited by 20-40%, which was associated with a similar increase in VBL efflux. EGF had no effect on drug accumulation, uptake, or efflux in sensitive MCF-7 cells. These data indicate that EGF can modulate the phosphorylation and function of P-gp, and suggest that this effect may be initiated by the activation of PLC.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Factor de Crecimiento Epidérmico/farmacología , Fosfolipasas de Tipo C/metabolismo , Resistencia a Múltiples Medicamentos , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/biosíntesis , Receptores ErbB/efectos de los fármacos , Humanos , Fosfatos de Inositol/análisis , Fosforilación , Células Tumorales CultivadasRESUMEN
The several hundred members of the eukaryotic protein kinase superfamily characterized to date share a similar catalytic domain structure, consisting of 12 conserved subdomains. Here we report the existence and wide occurrence in eukaryotes of a protein kinase with a completely different structure. We cloned and sequenced the human, mouse, rat, and Caenorhabditis elegans eukaryotic elongation factor-2 kinase (eEF-2 kinase) and found that with the exception of the ATP-binding site, they do not contain any sequence motifs characteristic of the eukaryotic protein kinase superfamily. Comparison of different eEF-2 kinase sequences reveals a highly conserved region of approximately 200 amino acids which was found to be homologous to the catalytic domain of the recently described myosin heavy chain kinase A (MHCK A) from Dictyostelium. This suggests that eEF-2 kinase and MHCK A are members of a new class of protein kinases with a novel catalytic domain structure.
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Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Caenorhabditis elegans/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , Dictyostelium/enzimología , Quinasa del Factor 2 de Elongación , Humanos , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Proteínas Protozoarias , Conejos , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reticulocitos/enzimología , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Calmodulin-dependent protein kinases phosphorylate certain substrates that have been implicated in regulating cellular proliferation. For example, upon mitogenic stimulation, there is a rapid activation of calmodulin-dependent protein kinase III (CaM kinase III), which leads to the phosphorylation of elongation factor 2. Recently, our laboratory demonstrated that the activity of CaM kinase III is increased in glioma cells following exposure to mitogens and is diminished or absent in nonproliferating glial tissue. Rottlerin, a 5,7-dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzy l)-8-cinnamoyl-1,2-chromene isolated from the pericarps of Mallotus phillippinensis, has been shown to be an effective CaM kinase III inhibitor. Therefore, we evaluated the effects of rottlerin on the growth and viability of glioblastoma cell lines. Rottlerin decreased growth and induced cytotoxicity in rat (C6) and two human gliomas (T98G and U138MG) at concentrations that inhibited the activity of CaM kinase III in vitro and in vivo. Far less demonstrable effects were observed on other Ca2++/CaM-sensitive kinases. Incubation of glial cells with rottlerin produced a block at the G1-S interface and the appearance of a population of cells with a <2N complement of DNA. In addition, rottlerin induced changes in cellular morphology such as cell shrinkage, accumulation of cytoplasmic vacuoles, and packaging of cellular components within membranes. These data suggest that CaM kinase III may be an important link between the activation of CaM-dependent signaling, proliferation, and viability in malignant cells, and that inhibition of CaM kinase III may represent an interesting pharmacological target in malignant gliomas.
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Acetofenonas/farmacología , Benzopiranos/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Glioblastoma/enzimología , Animales , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glioblastoma/ultraestructura , Humanos , Microscopía Electrónica , Ratas , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Calmodulin (CaM) is involved in cellular processes that are vital to cell proliferation and viability. Elevated CaM content is seen in transformed cells. Anti-CaM compounds alone are cytotoxic to tumor cells and are synergistic with certain cancer chemotherapeutic agents. However, all known CaM antagonists are nonselective, complicating interpretation of these studies. To more rigorously analyze the relationship between CaM protein expression and the behavior of cancer cells, tumor-derived cell lines were engineered such that CaM concentration could be manipulated by overexpressing CaM RNA. A full-length rat CaM I cDNA was inserted into the mammalian expression vector pMTCB6+ so that either CaM mRNA (sense) or antisense RNA was expressed under the control of an inducible metallothionein promoter. Constructs were introduced into C6 glioblastoma cells by liposome-mediated transfection and colonies were selected in G418. Significantly fewer clones were recovered from transfections with antisense vectors compared to CaM sense RNA or control (empty) vector alone. This difference was attributed to the cytotoxic effects of antisense CaM RNA as opposed to differences in transfection efficiencies. CaM expression was analyzed at the RNA level by Northern blotting and CaM protein concentrations were quantitated by immunofluorescence. Clones were identified in which CaM protein could be increased or decreased following exposure to zinc ions. Changes in CaM mRNA preceded changes in CaM protein by several hours. Overexpression of CaM had no significant effects on the growth of C6 cells. However, reductions in CaM lead to decreased growth rates of C6 cells and lowered cell viability.