RESUMEN
Genistein, an estrogenic, soy-derived isoflavone, may play a protective role against hormone-related cancers. We have reported that a high concentration of genistein inhibits cell proliferation and induces apoptosis in human uterine smooth muscle cells, but not in leiomyoma (fibroid) cells. To better understand the differential cell death responses of normal and tumor cells to a high concentration of genistein, we treated uterine smooth muscle cells and uterine leiomyoma cells with 50 µg/ml of genistein for 72 h and 168 h, and assessed for mediators of apoptosis, cytotoxicity and autophagy. We found that leiomyoma cells had increased protection from apoptosis by expressing an increased ratio of Bcl-2: bak at 72 h and 168 h; however, in smooth muscle cells, the Bcl-2: bak ratio was decreased at 72 h, but significantly rebounded by 168 h. The apoptosis extrinsic factors, Fas ligand and Fas receptor, were highly expressed in uterine smooth muscle cells following genistein treatment at both time points as evidenced by confocal microscopy. This was not seen in the uterine leiomyoma cells; however, cytotoxicity as indicated by elevated lactate dehydrogenase levels was significantly enhanced at 168 h. Increased immunoexpression of an autophagy/autophagosome marker was also observed in the leiomyoma cells, although minimally present in smooth muscle cells at 72 h. Ultrastructurally, there was evidence of autophagic vacuoles in the leiomyoma cells; whereas, the normal smooth muscle cells showed nuclear fragmentation indicative of apoptosis. In summary, our data show differential cell death pathways induced by genistein in tumor and normal uterine smooth muscle cells, and suggest novel cell death pathways that can be targeted for preventive and intervention strategies for inhibiting fibroid tumor cell growth in vivo.
RESUMEN
Previously, we found that high doses of genistein show an inhibitory effect on uterine leiomyoma (UtLM) cell proliferation. In this study, using microarray analysis and Ingenuity Pathways Analysis™, we identified genes (up- or down-regulated, ≥ 1.5 fold, P ≤ 0.001), functions and signaling pathways that were altered following treatment with an inhibitory concentration of genistein (50 µg/ml) in UtLM cells. Downregulation of TGF-ß signaling pathway genes, activin A, activin B, Smad3, TGF-ß2 and genes related to cell cycle regulation, with the exception of the upregulation of the CDK inhibitor P15, were identified and validated by real- time RT-PCR studies. Western blot analysis further demonstrated decreased protein expression of activin A and Smad3 in genistein-treated UtLM cells. Moreover, we found that activin A stimulated the growth of UtLM cells, and the inhibitory effect of genistein was partially abrogated in the presence of activin A. Overexpression of activin A and Smad3 were found in tissue samples of leiomyoma compared to matched myometrium, supporting the contribution of activin A and Smad3 in promoting the growth of UtLM cells. Taken together, these results suggest that downregulation of activin A and Smad3, both members of the TGF-ß pathway, may offer a mechanistic explanation for the inhibitory effect of a high-dose of genistein on UtLM cells, and might be potential therapeutic targets for treatment of clinical cases of uterine leiomyomas.
Asunto(s)
Activinas/genética , Anticarcinógenos/farmacología , Genisteína/farmacología , Leiomioma/metabolismo , Proteína smad3/genética , Factor de Crecimiento Transformador beta/genética , Neoplasias Uterinas/metabolismo , Activinas/metabolismo , Activinas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo , Femenino , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal/efectos de los fármacos , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
Uterine leiomyomas (fibroids) are benign tumors that are prevalent in women of reproductive age. Research suggests that activated receptor tyrosine kinases (RTKs) play an important role in the enhanced proliferation observed in fibroids. In this study, a phospho-RTK array technique was used to detect RTK activity in leiomyomas compared with myometrial tissue. We found that fifteen out of seventeen RTKs evaluated in this study were highly expressed (P < 0.02-0.03) in the leiomyomas, and included the IGF-I/IGF-IR, EGF/EGFR, FGF/FGF-R, HGF/HGF-R, and PDGF/PDGF-R gene families. Due to the higher protein levels of IGF-IR observed in leiomyomas by us in earlier studies, we decided to focus on the activation of the IGF-IR, its downstream effectors, and MAPKp44/42 to confirm our earlier findings; and validate the significance of the increased IGF-IR phosphorylation observed by RTK array analysis in this study. We used immunolocalization, western blot, or immunoprecipitation studies and confirmed that leiomyomas overexpressed IGF-IRbeta and phosphorylated IGF-IRbeta. Additionally, we showed that the downstream effectors, Shc, Grb2, and MAPKp44/42 (P < 0.02-0.001) were also overexpressed and involved in IGF-IR signaling in these tumors, while IRS-I, PI3K, and AKT were not. In vitro studies showed that IGF-I (100 ng/mL) increased the proliferation of uterine leiomyoma cells (UtLM) (P < 0.0001), and that phosphorylated IGF-IRbeta, Shc, and MAPKp44/42 were also overexpressed in IGF-I-treated UtLM cells (P < 0.05), similar to the tissue findings. A neutralizing antibody against the IGF-IRbeta blocked these effects. These data indicate that overexpression of RTKs and, in particular, activation of the IGF-IR signaling pathway through Shc/Grb2/MAPK are important in mediating uterine leiomyoma growth. These data may provide new anti-tumor targets for noninvasive treatment of fibroids.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Leiomioma/patología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Neoplasias Uterinas/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Inmunoprecipitación , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Leiomioma/genética , Leiomioma/metabolismo , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Hibridación de Ácido Nucleico/métodos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMEN
In order to find the biorational pesticides, eight novel 4beta-substituted phenoxyaniline derivatives of podophyllotoxin (4a-h) have been synthesized with significant stereoselectivity and improved yields by employing the BF(3).Et(2)O/NaI reagent system and evaluated for insecticidal activity against Pieris rapae as well as for their antifeedant effect against fifth instar larvae of P. rapae. The results showed that all these derivatives of PPT showed delayed insecticidal activity, which is different from the traditional neurotoxic insecticides. Among them, compounds possessing a 4beta-phenoxyaniline moiety substituted at para (CO(2)C(2)H(5), Cl, and OH) position exhibited greater insecticidal activity against P. rapae than podophyllotoxin. Also, the antifeedant activities showed that these compounds exhibited less potency than podophyllotoxin.
Asunto(s)
Conducta Alimentaria/efectos de los fármacos , Insecticidas , Juniperus/química , Lepidópteros/efectos de los fármacos , Plantas Medicinales/química , Podofilotoxina , Animales , Frutas/química , Insecticidas/síntesis química , Insecticidas/farmacología , Larva/efectos de los fármacos , Resonancia Magnética Nuclear Biomolecular , Podofilotoxina/análogos & derivados , Podofilotoxina/síntesis química , Podofilotoxina/farmacologíaRESUMEN
In the course of screening for naturally occurring insecticides from plants from the northwestern part of China, a petroleum ether extract of Juniperus sabina L was found to show insecticidal activity against fifth-instar larvae of Pieris rapae L. From the extract, an insecticidal compound was isolated by bioassay-guided fractionation. The compound was identified as deoxypodophyllotoxin (1) by comparison of its spectroscopic characteristics with literature data. In bioassays, 1 showed antifeedant activity against fifth-instar larvae of P rapae at 0.05-1.00 g litre(-1) and its AFC50 (concentration for 50% antifeedant activity) values at 12 and 48h were 0.170 and 0.060 g litre(-1), respectively. In that concentration range, all treated insects died within 48 h after treatment and compound 1 showed delayed insecticidal activity. At 0.015-0.100 g litre(-1), 1 showed insecticidal activity, with an LC50 of 0.020 g litre(-1). The related compound deoxypicropodophyllotoxin (2), however, showed lower antifeedant and insecticidal activities than 1 in bioassay. This indicated that the trans-lactone ring is an important moiety for enhancing activity in these compounds. Comparison of the insecticidal activities of 1 and another related compound, podophyllotoxin (3), suggested that varying the substituent at C-4 is an exciting possibility for synthesizing more potent analogues.