Reprogramming human A375 amelanotic melanoma cells by catalase overexpression: Upregulation of antioxidant genes correlates with regression of melanoma malignancy and with malignant progression when downregulated

Reactive oxygen species (ROS) are implicated in tumor transformation. The antioxidant system (AOS) protects cells from ROS damage. However, it is also hijacked by cancers cells to proliferate within the tumor. Thus, identifying proteins altered by redox imbalance in cancer cells is an attractive prognostic and therapeutic tool. Gene expression microarrays in A375 melanoma cells with different ROS levels after overexpressing catalase were performed. Dissimilar phenotypes by differential compensation to hydrogen peroxide scavenging were generated. The melanotic A375-A7 (A7) upregulated TYRP1, CNTN1 and UCHL1 promoting melanogenesis. The metastatic A375-G10 (G10) downregulated MTSS1 and TIAM1, proteins absent in metastasis. Moreover, differential coexpression of AOS genes (EPHX2, GSTM3, MGST1, MSRA, TXNRD3, MGST3 and GSR) was found in A7 and G10. Their increase in A7 improved its AOS ability and therefore, oxidative stress response, resembling less aggressive tumor cells. Meanwhile, their decrease in G10 revealed a disruption in the AOS and therefore, enhanced its metastatic capacity.These gene signatures, not only bring new insights into the physiopathology of melanoma, but also could be relevant in clinical prognostic to classify between non aggressive and metastatic melanomas.Fil: Bracalente, Candelaria. Comisión Nacional de Energía Atómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ibañez, Irene Laura. Comisión Nacional de Energía Atómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Berenstein, Ariel José. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Notcovich, Cintia. Comisión Nacional de Energía Atómica; ArgentinaFil: Cerda, María B.. Comisión Nacional de Energía Atómica. Gerencia Química. CAC; ArgentinaFil: Klamt, Fabio. Universidade Federal do Rio Grande do Sul; BrasilFil: Chernomoretz, Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Durán, Hebe. Comisión Nacional de Energía Atómica; Argentin

Diagnóstico y seguimiento de cáncer de piel no-melanoma utilizando 99mTc-MIBI : estudios en un modelo animal

El objetivo consistió en evaluar la utilidad del 99mTc-MIBI como marcador para diagnóstico y seguimiento de la progresión tumoral del NMSC en un modelo de carcinogénesis completa en ratones. Los animales en estudio fueron inyectados con 99mTc-MIBI a diferentes tiempos y eutanasiados. Se disecaron muestras de tumor y piel sana para evaluar la captación del radiofármaco y realizar el diagnóstico histológico. En animales con 22 semanas de progresión tumoral se observó una diferencia significativa en la captación del 99mTc-MIBI entre piel sana y NMSC. El protocolo que mejor se adapta al uso del 99mTc-MIBI como marcador para el diagnóstico y seguimiento de la progresión tumoral en ratones portadores de NMSC inducidos es la administración i.v de 1 mCi de 99mTc-MIBI con adquisición de datos a los 30 minutos post inyección. Se observó que a medida que los tumores progresan, la captación de 99mTc-MIBI disminuye respecto a la piel normal.The aim of the work was to evaluate the usefulness of 99mTc-MIBI as a tracer for the tumor diagnosis and progression of NMSC in a chemically induced model in mice. After administration of 99mTc-MIBI animals were sacrificed at different times. Samples of tumor and healthy skin were dissected in order to perform histological analysis and to evaluate 99mTc-MIBI uptake. Animals under 22 weeks of tumor evolution showed a statistically difference in 99mTc-MIBI uptake between healthy skin and NMSC. Our results showed that the better protocol for the study of the tumor diagnosis and progression of NMSC in mice is the administration of 1 mCi of 99mTc-MIBI and acquisition of images 30 minutes post injection. Results showed that, as tumor progresses, the uptake of 99mTc-MIBI is significantly lower than healthy skin.Fil: Salgueiro, María Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Tesan, Fiorella Carla. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Palmieri, Mónica Alejandra. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; ArgentinaFil: Durán, Hebe. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Comisión Nacional de Energía Atómica; ArgentinaFil: Medina, Vanina Araceli. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Leonardi, Natalia. Laboratorios BACON; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Goldman, Cinthia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Boccio, Jose Ruben. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Zubillaga, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Análisis de la promoción y progresión tumoral inducidas por peróxido de benzoílo en el modelo de carcinogénesis en múltiples estadios en piel de ratón

Fil:Durán, Hebe Alicia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Propiedades antioxidantes y anticancerígenas del lúpulo

El objetivo de este artículo es describir las propiedades antioxidantes y anticancerígenas del lúpulo. Con este fin, se detallan las fuentes celulares de especies oxidativas y las defensas antioxidantes, en particular, polifenoles y flavonoides. Con respecto a la inducción de cáncer, se describen nociones básicas sobre los mecanismos de carcinogénesis y la participación de especies oxidativas en dicho proceso. Los extractos de la flor del lúpulo contienen los ácidos amargos, humulonas y lupulonas, y flavonoides prenilados. La cerveza representa uno de los productos de la dieta más importantes en cuanto al contenido de prenilflavonoides. Con respecto a los ácidos amargos, en particular, la humulona, se ha demostrado su capacidad antioxidante y otros efectos biológicos relacionados con su actividad anticancerígena: inhibición de la proliferación de células tumorales, inducción de apoptosis, inhibición de la promoción tumoral, actividad antinflamatoria y antiangiogénica. Con respecto a los prenilflavonoides presentes en el lúpulo, se ha demostrado que poseen capacidad antioxidante, actividad antiproliferativa de células tumorales, inhibición de la activación metabólica de procarcinógenos e inducción de enzimas detoxificadoras de ciertos carcinógenos. Estos antecedentes sumados a futuras investigaciones permitirán evaluar el posible papel de los antioxidantes del lúpulo incorporados en el organismo a través del consumo de cerveza en la quimioprevención y la posible aplicación de compuestos del lúpulo en terapias antioxidantes

Phosphorylation and subcellular localization of p27Kip1 regulated by hydrogen peroxide modulation in cancer cells.

The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H(2)O(2)) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H(2)O(2) removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H(2)O(2) (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H(2)O(2) scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1

Intracellular ROS levels in melanoma cells and melanocytes determined by DCFH-DA assay.

<p>(A–D) Intracellular ROS levels decreased in melanoma cells either when treated with catalase or when overexpressing it. (A) Representative histograms of DCF fluorescence of melanoma cells treated with 500 (blue line) and 1000 (orange line) U/ml catalase or left untreated (green line) for 24 h. Control cells not exposed to DCFH-DA (black line) and control cells treated with catalase just before DCFH-DA incubation (red line). (B) DCF mean fluorescence (arbitrary units) vs. catalase (CAT) dose. Data are expressed as mean ± SD. **p<0.01 vs. untreated cells (0 U/ml catalase). (C) Representative histograms of DCF fluorescence of melanoma cells overexpressing catalase (A375-CAT-E9) and its controls (A375-pcDNA3 and untreated A375 cells). Control cells not exposed to DCFH-DA (black line), control cells treated with catalase just before DCFH-DA incubation (red line) and cells incubated with DCFH-DA (green line). (D) DCF mean fluorescence (arbitrary units) of A375-CAT-E9, A375-pcDNA3 and A375 control cells. Data are expressed as mean ± SD. **p<0.01 vs. A375 control. (E-F) Melanoma cells (A375) exhibited higher levels of intracellular ROS than their non-tumor counterpart (PIG-1 melanocytes). (E) Representative histograms of DCF fluorescence of PIG-1 and A375 cells: control cells not exposed to DCFH-DA (black lines), control cells treated with catalase just before DCFH-DA incubation (red line) and cells incubated with DCFH-DA (green line). (F) DCF mean fluorescence (arbitrary units) of PIG-1 melanocytes and A375 melanoma cells. Data are expressed as mean ± SD. **p<0.01 vs. PIG-1.</p

Adding 0.1 µM H₂O₂ to FBS starved cells regulates p27Kip1 phosphorylation and localization, favoring proliferation.

<p>Melanoma (A375) cells grown in complete medium with 10% FBS were arrested by FBS starvation (0% FBS) for a period of 24 h and then cells were incubated with different concentrations of H₂O₂ (0.01–10 µM) or to 10% FBS. (A) Intracellular ROS levels measured by DCFH-DA assay. (B) Cell proliferation rate evaluated by the MTT assay. (C) Representative images of p27Kip1 immunocytofluorescence showing the subcellular localization of the protein. DAPI: staining of nuclear DNA; p27Kip1: FITC staining of p27Kip1 protein. (D) Percentage of positive (□) cytoplasms and positive (■) nuclei for p27Kip1 relative to the total number of counted cells. (E) The expression of p27Kip1, p27pS10 and p27pT198 analyzed by western blot. (F) Relative densitometric values of () p27Kip1 levels, (□) p27pS10 and (■) p27pT198. Actin densitometric values were used to standardize for protein loading. Results are referred to control incubated with 10% FBS. (A, B, D and F) Data are expressed as mean ± SD. *p<0.05, **p<0.01 and ***p<0.001 vs. cells incubated with 10% FBS; ^#p<0.05, ^##p<0.01 and ^###p<0.001 vs. FBS-starved cells not-exposed to H₂O₂.</p

Nuclear localization of p27Kip1 in response to H₂O₂ scavenging and intrinsic low levels of H₂O₂.

<p>(A-F) Subcellular localization of p27Kip1 evaluated by immunocytofluorescence. (A–B) Melanoma cells treated with 500 and 1000 U/ml catalase (CAT) for periods of 6 or 24 h or left untreated. FBS starved cells were used as control of G1 arrest. (C–D) Catalase overexpression model (A375-CAT-E9 cells) vs. controls (A375-pcDNA3 and A375 control cells). (E-F) Non-tumor (PIG-1) vs. tumor (A375) cells. (A, C and E) Representative images of p27Kip1 immunocytofluorescence showing the subcellular localization of the protein. DAPI: staining of nuclear DNA; p27Kip1: FITC staining of p27Kip1 protein. (B, D and F) Percentage of positive (□) cytoplasms and positive (■) nuclei for p27Kip1 relative to the total number of counted cells. Data are expressed as mean ± SD. (B) **p<0.01 vs. control. (D) *p<0.05 and **p<0.01 vs. A375 control. (F) *p<0.05 and **p<0.01 vs. non-tumor cells. (G–H) Increased expression of nuclear p27Kip1 in A375 cells after 1000 U/ml catalase (CAT) treatment as compared with control A375 cells (treated with 1000 U/ml heat-inactivated catalase, IN-CAT) for 24 h, detected by western blot of nuclear and cytosolic extracts (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044502#s4" target="_blank">Methods</a>). (G) Representative immunoblot images are shown. C: Cytoplasmic extracts; N: Nuclear extracts. Actin and Ku-70 densitometric values were used to standardize for cytoplasmic and nuclear protein loading, respectively. (H) Relative densitometric values of (□) cytoplasmic and (■) nuclear p27Kip1 levels. Results are referred to control cells. Data are expressed as mean ± SD. *p<0.05 vs. control.</p