ABSTRACT
Organophosphorus chlorpyrifos (CPF) is currently considered an endocrine disruptor (ED), as it can imitate hormone actions both in vitro and in vivo. We recently reported that CPF induces migration and invasion in 2D cultures and changes the expression of key molecular markers involved in epithelial mesenchymal transition in MCF-7 and MDA-MB-231 cell lines. In this study, we investigated whether CPF could behave as a predisposing factor for tumors to become more metastatic and aggressive using 3D culture models. In MCF-7 cells, 0.05 µM CPF induced an increase in the number and size of mammospheres via estrogen receptor alpha (ERα) and c-SRC. Furthermore, 0.05 µM CPF increased the area of spheroids generated from MCF-7 cells, induced invasion using both Matrigel® and type 1 collagen matrices, and increased cell migration capacity via ERα in this 3D model. In turn, 50 µM CPF increased cell migration capacity and invasion using type 1 collagen matrix. In monolayers, CPF increased the phosphorylation and membrane translocation of c-SRC at both concentrations assayed. CPF at 0.05 µM boosted p-AKT, p-GSK-3ß and p-P38. While p-AKT rose in a ERα-dependent way, p-GSK-3ß was dependent on ERα- and c-SRC, and p-P38 was only dependent on c-SRC. On the other hand, the increase in p-AKT and p-P38 induced by 50 µM CPF was dependent on the c-SRC pathway. We also observed that 0.05 µM CPF increased IGF-1R and IRS-1 expression and that 50 µM CPF induced IGF-1Rß phosphorylation. In the MDA-MB-231 cell line, 0.05 and 50 µM CPF increased p-c-SRC. Finally, p-AKT and p-GSK-3ß were also induced by CPF at 0.05 and 50 µM, and an increase in p-P38 was observed at 50 µM. Taken together, these data provide support for the notion that CPF may represent a risk factor for breast cancer development and progression.
Subject(s)
Breast Neoplasms , Chlorpyrifos , Endocrine Disruptors , Cell Line, Tumor , Cell Proliferation , Chlorpyrifos/toxicity , Endocrine Disruptors/toxicity , Female , Glycogen Synthase Kinase 3 beta , Humans , Phenotype , PhosphorylationABSTRACT
Chlorpyrifos (CPF) is one of the most frequently used pesticide in extensive agriculture around the world and can be incorporated by humans and animals with possible consequences on health. The effects of this pesticide on carcinogenesis are not clear and there is no consensus concerning the risks of this compound. In previous work, we demonstrated that CPF induces proliferation of breast cancer cells both in vivo and in vitro. In this work we investigate whether CPF promotes the epithelial-mesenchymal transition (EMT) in breast cancer cells. Herein, we demonstrate that 50 µM CFP induces invasion in MCF-7 and MDA-MB-231 cells. In addition, 0.05 and 50 µM CPF increases migration in both cell lines. In MCF-7 cells, 0.05 and 50 µM CPF increase the metalloprotease MMP2 expression and decrease E-Cadherin and ß-Catenin expression diminishing their membrane location. Furthermore, 50 µM CPF induces Vimentin expression and Slug nuclear translocation in MCF-7 cells. 0.05 and 50 µM CPF increase MMP2 gelatinolytic activity and expression, decrease ß-Catenin expression and increase Vimentin expression in MDA-MB-231 cells. Inhibition of the oncoprotein c-Src reverses all the effects induced by CPF in MDA-MB-231 but not in MCF-7 indicating that c-Src is a kinase with a crucial role in the cells which grow in an estrogen-independent way. In MCF-7 cells both c-Src and estrogen receptor alpha must be blocked to completly inhibit the CPF-mediated effects. Our results show for the first time that the exposure to subthreshold concentrations of CPF promotes the modulation of EMT-molecular markers and pathways. These results, together with the ubiquitous distribution of the pesticide CPF, make it of utmost importance to take measures to minimize the risk of exposure to this compound.
Subject(s)
Cell Movement/drug effects , Chlorpyrifos/toxicity , Endocrine Disruptors/toxicity , Epithelial-Mesenchymal Transition/drug effects , Pesticides/toxicity , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , CSK Tyrosine-Protein Kinase/genetics , Cell Line, Tumor , Cell Movement/genetics , Dose-Response Relationship, Drug , Epithelial-Mesenchymal Transition/genetics , Estrogen Receptor alpha/genetics , Female , Humans , MCF-7 Cells , Matrix Metalloproteinase 2/genetics , Signal TransductionABSTRACT
Hexachlorobenzene (HCB) is an organochlorine pollutant widely distributed in the environment around the entire world. Previous reports from our group and others have demonstrated that this compound is as an endocrine disruptor. We have also reported that HCB presents a co-carcinogenic effect in N-Nitroso-N-methyl-urea-induced mammary tumours in rats. In this work, we studied the effects of HCB on cell cycle progression and cell cycle regulating protein expression in the estrogen-sensitive breast cancer cell line, MCF-7. Here, we show that HCB alters cell cycle in a concentration-dependent way. The lowest assessed concentration (0.005µM) promotes the cell cycle progression, enhances cyclin D1 expression, and reduces the nuclear localization of p27 accompanied by an increased interaction between p27 and c-Src kinase. On the other hand, 5µM HCB delays the cell cycle progression and promotes the formation of the cyclin E-CDK2-p27 protein complex. Our results show that HCB stimulates cell proliferation through cell cycle modulation and c-Src involvement in MCF-7 cells. Here, we report for the first time that differential mechanisms of action of HCB on mammary cell cycle progression are triggered at different concentrations of this pollutant.
Subject(s)
Cell Cycle/drug effects , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Hexachlorobenzene/toxicity , Oncogene Proteins/metabolism , src-Family Kinases/metabolism , CSK Tyrosine-Protein Kinase , Cell Division/drug effects , Cell Proliferation/drug effects , Cyclin D1/genetics , Cyclin D1/metabolism , Cyclin E/genetics , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase Inhibitor p27/genetics , Humans , MCF-7 Cells , Oncogene Proteins/genetics , Phosphorylation , src-Family Kinases/geneticsSubject(s)
Histamine/metabolism , Intestinal Mucosa/pathology , Intestinal Mucosa/radiation effects , Animals , Cell Line, Tumor , Cell Survival/radiation effects , Humans , Immunohistochemistry , Intestinal Mucosa/metabolism , Intestine, Small/metabolism , Intestine, Small/pathology , Intestine, Small/radiation effects , Mice , Radiation, IonizingABSTRACT
OBJECTIVE: The aim of this work was to analyze the effect of estradiol (E(2)), medroxyprogesterone and the two selective estrogen receptor modulators (SERMs) (tamoxifen (Tam) and raloxifene (Ral)) on the estrogen receptor (ER) conformers profile performed by size exclusion HPLC in relation to hormone dependence of mammary tumors. MATERIALS AND METHODS: Two types of mammary tumors were studied: tumors transplanted in BALB/c mice that are medroxyprogesterone acetate (MPA)-dependent for growth, and tumors induced in Sprague-Dawley rats by intraperitoneal injection of N-nitroso-N-methylurea (NMU). Tumors from mice treated with MPA, E(2), Tam or Ral and NMU-treated rats were analyzed and compared to that of control. RESULTS: The tumor conformer profiles were as follows: control and MPA-treated mice showed only one peak (oligomeric form); E(2)-treated mice also showed only one peak (dimer); Tam-treated mice showed one peak corresponding to a possible proteolytic fragment, and Ral-treated mice showed two peaks (oligomeric and a possible proteolytic fragment). On the other hand, NMU-induced mammary tumors from rats showed three peaks (oligomeric, monomeric and proteolytic). CONCLUSION: Our findings may indicate that SERMs affect the aggregation state of ER and thereby its ability to modulate genomic transcription mechanisms related to growth rate.
Subject(s)
Mammary Neoplasms, Experimental/metabolism , Raloxifene Hydrochloride/pharmacology , Receptors, Estrogen/metabolism , Selective Estrogen Receptor Modulators/pharmacology , Tamoxifen/pharmacology , Animals , Chromatography, High Pressure Liquid , Female , Mammary Neoplasms, Experimental/chemically induced , Mammary Neoplasms, Experimental/pathology , Methylnitrosourea , Mice , Mice, Inbred BALB C , Neoplasms, Hormone-Dependent/metabolism , Protein Conformation , Rats , Rats, Sprague-Dawley , Receptors, Estrogen/chemistrySubject(s)
Cell Division/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Histamine/pharmacology , Receptors, Histamine H2/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Humans , Phosphatidylinositols/metabolism , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
La insulina, miembro de la familia de factores de crecimiento que incluyen al factor de crecimiento tipo insulina I (IGF-I) y II (IGF-II), presenta efectos mitogénicos sobre células epiteliales mamarias normales y malignas (Goodwin y col., 2002). Se postula que altos niveles de insulina permiten identificar mujeres con una mala evolución de su cáncer de mama, en quienes deberán aplicarse estrategias terapéuticas más efectivas. Se estudiaron 32 pacientes con cáncer de mama, de las cuales 18 presentaron carcinoma ductal invasor, incluidos 3 multifocales (56 por ciento), 6 carcinoma lobulillar infiltrante (19 por ciento), 3 carcinoma papilar (10 por ciento) y el resto otros tipos (15 por ciento). Dos pacientes (7 por ciento) presentan diabetes mellitus no-insulino dependiente. Los niveles de insulina plasmática en ayunas determinados por RIA (Insulin-CT kit) resultaron en: 18 pacientes (56 por ciento) con niveles normales (5,5 a 19,9 AUI/ml), el resto (44 por ciento) con insulinemias superiores al normal. La insulinemia plasmática en ayunas en voluntarias sanas resultó ser de 13,9ñ4,3 AUI/ml (n=10)...(AU)
Subject(s)
Humans , Adult , Female , Middle Aged , Aged , Breast Neoplasms/diagnosis , Biomarkers, Tumor , Carcinoma, Intraductal, Noninfiltrating , Carcinoma, Lobular , Carcinoma, Papillary , Insulin/blood , Insulin Antagonists/therapeutic use , Breast Neoplasms/physiopathology , Breast Neoplasms/pathology , Prognosis , Insulin/diagnosis , Receptors, Estrogen , Lymphatic Metastasis , Receptors, Progesterone , Receptors, Somatomedin , Survival RateABSTRACT
La insulina, miembro de la familia de factores de crecimiento que incluyen al factor de crecimiento tipo insulina I (IGF-I) y II (IGF-II), presenta efectos mitogénicos sobre células epiteliales mamarias normales y malignas (Goodwin y col., 2002). Se postula que altos niveles de insulina permiten identificar mujeres con una mala evolución de su cáncer de mama, en quienes deberán aplicarse estrategias terapéuticas más efectivas. Se estudiaron 32 pacientes con cáncer de mama, de las cuales 18 presentaron carcinoma ductal invasor, incluidos 3 multifocales (56 por ciento), 6 carcinoma lobulillar infiltrante (19 por ciento), 3 carcinoma papilar (10 por ciento) y el resto otros tipos (15 por ciento). Dos pacientes (7 por ciento) presentan diabetes mellitus no-insulino dependiente. Los niveles de insulina plasmática en ayunas determinados por RIA (Insulin-CT kit) resultaron en: 18 pacientes (56 por ciento) con niveles normales (5,5 a 19,9 µUI/ml), el resto (44 por ciento) con insulinemias superiores al normal. La insulinemia plasmática en ayunas en voluntarias sanas resultó ser de 13,9ñ4,3 µUI/ml (n=10)...
Subject(s)
Humans , Adult , Female , Middle Aged , Breast Neoplasms , Carcinoma, Intraductal, Noninfiltrating , Carcinoma, Lobular , Carcinoma, Papillary , Insulin , Insulin Antagonists , Biomarkers, Tumor , Breast Neoplasms , Insulin , Lymphatic Metastasis , Prognosis , Receptors, Estrogen , Receptors, Progesterone , Receptors, Somatomedin , Survival RateABSTRACT
Treatment with exogenous spermidine enhanced acute malathion toxicity during larval development of the toad Bufo arenarum Hensel. The polyamine was rapidly incorporated in the larvae with a subsequent metabolization to putrescine and spermine, which were excreted to the media. Endogenous polyamine levels were not changed by either spermidine or malathion treatments. However, 0.5-mM spermidine modified malathion uptake and bioavailability increasing the concentration of the xenobiotic in the larvae. The amount of reduced thiols was decreased by both compounds, but the depletion was insufficient to induce cytotoxicity. The oxidative degradation of polyamines competes for the pool of reduced glutathione used in the conjugation of malathion in the larvae, thus leading to the reported potentiation of toxicity. Our results suggest that exposure to thiols-depleting agents may induce alteration of organophosphate degradation in amphibian larvae.
Subject(s)
Bufo arenarum/growth & development , Malathion/toxicity , Pesticide Synergists/pharmacology , Spermidine/pharmacology , Sulfhydryl Compounds/metabolism , Animals , Biological Availability , Biotransformation/drug effects , Dose-Response Relationship, Drug , Drug Synergism , Glutathione Disulfide/drug effects , Larva/drug effects , Lipid Peroxidation/drug effects , Malathion/pharmacokinetics , Oxidation-Reduction , Pesticide Synergists/pharmacokinetics , Spermidine/pharmacokineticsABSTRACT
Las terapias oncológicas conllevan generalmente efectos secundarios indeseados por lo que el mejor conocimiento de los mecanismos regulatorios del desarrollo y crecimiento tumoral puede abrir el camino a enfoques terapeúticos más adecuados. El objetivo de éste trabajo fue profundizar el estudio de la implicancia de factores que regulan el crecimiento del cáncer mamario empleando un modelo experimental químicamente inducido en rata, el que presenta similitudes con el cáncer mamario humano principalmente en lo que respecta a la regulación hormonal de su crecimiento. El tumor mamario fue inducido químicamente en ratas normales y diabéticas. Se analizó la expresión de receptores a factor de crecimiento insulínico tipo I (RIGF-I), el que forma parte de un sistema formado por factores de crecimiento, sus receptores y proteínas transportadas; éste sistema se encuentra alterado en pacientes con diabetes mellitus no dependiente de insulina. También se analizó la expresión de las proteínas c-FOS y PCNA (antígeno nuclear de proliferación celular), ambas relacionadas con la proliferación celular. Los resultados experimentales mostraron significativas diferencias en los tumores mamarios desarrollados: los de las ratas diabéticas presentaron mayor período de latencia (p<0,001), menor número de tumores desarrollados por rata (p<0,02) y una velocidad de crecimiento menor (p<0,05) con respecto a los tumores desarrollados en ratas normales. Asimismo, mostraron un patrón histológico de marcada benignidad, en contraste con los adenocarcinomas malignos ductales desarrollados en los animales normales. La expresión de las proteínas c-FOS y PCNA detectada por métodos inmunohistoquímicos fue significativamente menor en los tumores de las ratas diabéticas que en ratas normales. En cuanto a la expresión de RIGF-I, los resultados indicaron que la misma estaría regulada por las hormonas esteroides en animales diabéticos y normales. El trabajo permitió analizar experimentalmente la interrelación entre factores de crecimiento insulínicos y hormonas esteroides en el desarrollo y crecimiento tumoral mamario, particularmente cuando están presentes la patología mamaria y la diabetes (AU)
Subject(s)
Animals , Rats , Mammary Neoplasms, Experimental/physiopathology , Receptor, IGF Type 1 , Proliferating Cell Nuclear Antigen , Mammary Neoplasms, Experimental/pathology , Receptor, IGF Type 1/drug effects , Diabetes Mellitus , Diabetes Mellitus, Experimental , Genes, fos , Methylurea Compounds , Estrogen Antagonists , Immunohistochemistry , TamoxifenABSTRACT
Las terapias oncológicas conllevan generalmente efectos secundarios indeseados por lo que el mejor conocimiento de los mecanismos regulatorios del desarrollo y crecimiento tumoral puede abrir el camino a enfoques terapeúticos más adecuados. El objetivo de éste trabajo fue profundizar el estudio de la implicancia de factores que regulan el crecimiento del cáncer mamario empleando un modelo experimental químicamente inducido en rata, el que presenta similitudes con el cáncer mamario humano principalmente en lo que respecta a la regulación hormonal de su crecimiento. El tumor mamario fue inducido químicamente en ratas normales y diabéticas. Se analizó la expresión de receptores a factor de crecimiento insulínico tipo I (RIGF-I), el que forma parte de un sistema formado por factores de crecimiento, sus receptores y proteínas transportadas; éste sistema se encuentra alterado en pacientes con diabetes mellitus no dependiente de insulina. También se analizó la expresión de las proteínas c-FOS y PCNA (antígeno nuclear de proliferación celular), ambas relacionadas con la proliferación celular. Los resultados experimentales mostraron significativas diferencias en los tumores mamarios desarrollados: los de las ratas diabéticas presentaron mayor período de latencia (p<0,001), menor número de tumores desarrollados por rata (p<0,02) y una velocidad de crecimiento menor (p<0,05) con respecto a los tumores desarrollados en ratas normales. Asimismo, mostraron un patrón histológico de marcada benignidad, en contraste con los adenocarcinomas malignos ductales desarrollados en los animales normales. La expresión de las proteínas c-FOS y PCNA detectada por métodos inmunohistoquímicos fue significativamente menor en los tumores de las ratas diabéticas que en ratas normales. En cuanto a la expresión de RIGF-I, los resultados indicaron que la misma estaría regulada por las hormonas esteroides en animales diabéticos y normales. El trabajo permitió analizar experimentalmente la interrelación entre factores de crecimiento insulínicos y hormonas esteroides en el desarrollo y crecimiento tumoral mamario, particularmente cuando están presentes la patología mamaria y la diabetes
Subject(s)
Animals , Rats , Proliferating Cell Nuclear Antigen , Mammary Neoplasms, Experimental , Receptor, IGF Type 1 , Estrogen Antagonists , Diabetes Mellitus , Diabetes Mellitus, Experimental , Genes, fos , Immunohistochemistry , Mammary Neoplasms, Experimental , Methylurea Compounds , Receptor, IGF Type 1 , TamoxifenSubject(s)
Adenocarcinoma/pathology , Histamine/physiology , Pancreatic Neoplasms/pathology , Cell Division/drug effects , Histamine/pharmacology , Histamine Agonists/pharmacology , Histamine H1 Antagonists/pharmacology , Histamine H2 Antagonists/pharmacology , Humans , Receptors, Histamine H1/analysis , Receptors, Histamine H1/physiology , Receptors, Histamine H2/analysis , Receptors, Histamine H2/physiology , Tumor Cells, CulturedABSTRACT
The aim of this study was to develop an experimental model for the study of cancer associated with diabetes. For diabetes induction, Sprague-Dawley rats were given streptozotocin (STZ, 90 mg/kg body weight (BW), by intraperitoneal injection on the second day of life. For mammary tumour induction, rats were injected with 50 mg/kg BW of N-nitroso-N-methylurea (NMU) at 50, 80 and 110 days old. The neoplastic process and the effect of tamoxifen treatment was examined in non-diabetic and diabetic rats. The latency period, NMU-induced tumour incidence and the number of tumours per rat in diabetic rats versus controls were 117 +/- 7 days versus 79 +/- 9 days (P < 0.001); 93% versus 95% (NS); and 5.2 +/- 1.6 versus 2.7 +/- 0.5 (P < 0.02). A more benign histological pattern for tumours in diabetic animals was observed. Mammary tumours in diabetic rats grew more slowly than in controls. Tamoxifen (1 mg/kg/day) treated diabetic rats showed tumour regression in 67% of NMU-induced mammary tumours versus 53% in controls (NS). Our results show that tumour progression seems to be affected by diabetes in this experimental model. We suggest this is the result of changes to insulin-like growth factors and their receptors, which occur in diabetics, and our future research will examine this hypothesis.
Subject(s)
Anticarcinogenic Agents/therapeutic use , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Type 2/complications , Mammary Neoplasms, Experimental/etiology , Tamoxifen/therapeutic use , Animals , Anti-Bacterial Agents , Carcinogens/toxicity , Cell Division , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/pathology , Female , Insulin/blood , Mammary Neoplasms, Experimental/blood , Mammary Neoplasms, Experimental/pathology , Methylnitrosourea/toxicity , Rats , StreptozocinABSTRACT
In this work we analyze the hypothesis that tumors induced by i.p. N-nitroso-N-methylurea injection express EGF-like peptides and EGF receptors which could be involved in the response to hormone manipulation. EGF receptors (EGFR) were determined in the purified membrane fraction of tumors from control and ovariectomized (OVX) animals and no significant differences were found in either maximal binding capacities (Q) or dissociation constants (Kd) between them. Neither did we observe differences between tumors that regressed (HR) or continued growing (HU) after ovariectomy. In order to test the ability of EGFR to trigger a biological response we measured the production of second messengers inositol triphosphates (IP3) and cAMP levels; we found that EGF increases IP3 production in a dose-dependent way, while cAMP levels were not affected. In addition, EGF was able to induce in vitro cell proliferation in a concentration-dependent manner when tested in primary cultures of tumor cells by the clonogenic soft agar technique. EGF/TGF-alpha activity was determined by a radioreceptor assay in tumor cytosols from control and OVX rats. Results showed a trend to lower values in tumors from OVX rats, but no differences between HR and HU tumors. A positive correlation was found between EGF/TGF-alpha activity and progesterone receptor maximal binding capacity. When we tested the action of estradiol and EGF added together to primary cultures of tumor cells we found an additive effect on cell proliferation. The study of steady state mRNA levels showed that E2 increases PgR and c-myc mRNA levels in HR but not in HU tumors. In conclusion, the autocrine loop EGFR-EGF/TGF-alpha present in all tumors is hormonally regulated, possibly by Pg, but is not related to the tumor response to ovariectomy.
Subject(s)
Epidermal Growth Factor/analysis , ErbB Receptors/analysis , Mammary Neoplasms, Animal/metabolism , Animals , Carcinogens , Cyclic AMP/analysis , Epidermal Growth Factor/pharmacology , Female , Mammary Neoplasms, Animal/chemically induced , Mammary Neoplasms, Animal/pathology , Methylnitrosourea , Ovariectomy , Phosphatidylinositols/metabolism , Proto-Oncogene Proteins c-myc/analysis , Rats , Rats, Sprague-Dawley , Receptors, Estrogen/analysis , Receptors, Progesterone/analysis , Transforming Growth Factor alpha/analysisABSTRACT
Mammary adenocarcinomas induces in female Sprague-Dawley rats by three intraperitoneal injections of N-nitroso-N-methylurea were studied in order to characterize their estrogen (ER), progesterone (PgR), prolactin (PRLR) and epidermal growth factor (EGFR) receptors. All samples evaluated showed the presence of ER and PgR in the cytosol fraction and PRLR amd EGFR in the membrane fraction. Q (fmol/mg) and K(d) (nM) values were as follows: ER, 56 +/- 11 and 0.5 +/- 0.1; PgR, 109 +/- 25 and 2.2 +/- 0.5 and PRLR, 335 +/- 75 and 0.5 +/- 0.2, respectively. In all tumors studied, two specific sites were found for EGFR, one with Q(1) = 22 +/- 9 and K(d1) = 0.6 +/- 0.3, and the other with Q(2) = 125 +/- 33 and K(d2) = 2.1 +/- 0.5. Receptor content was found to be independent of tumor histopathological variety. Displacement index (DI) with estradiol and tamoxifen of [I(3)H]E2-ER binding showed great heterogeneity, with values ranging from 0.01 to1.54. No correlation between ER content and DI values was found. Antiestrogenic binding sites were not found in the microsomal fraction of ten mammary tumors examined. Proliferation of this experimental mammary tumor may be regulated by a complex interaction of steroid and polypeptide hormones, as well as growth factors.