TRIPLE NEGATIVE BREAST CANCER CELLS EXPOSED TO ARYL HYDROCARBON RECEPTOR LIGANDS HEXACHLOROBENZENE AND CHLORPYRIFOS ACTIVATE ENDOTHELIAL CELLS.

Breast cancer is currently one of the most prevalent cancers worldwide. The mechanisms by which pesticides can increase breast cancer risk are multiple and complex. We have previously observed that two aryl hydrocarbon receptor (AhR) agonists ‒pesticides hexachlorobenzene (HCB) and chlorpyrifos (CPF)‒ act on tumor progression, stimulating cell migration and invasion in vitro and tumor growth in animal models. Elevated levels of hypoxia inducible factor-1α (HIF-1α) are found in malignant breast tumors, and HIF-1α is known to induce proangiogenic factors such as vascular endothelial growth factor (VEGF), nitric oxide synthase-2 (NOS-2) and cyclooxygenase-2 (COX-2), which are fundamental in breast cancer progression. In this work, we studied HCB (0.005, 0.05, 0.5 and 5 µM) and CPF (0.05, 0.5, 5 and 50 µM) action on the expression of these proangiogenic factors in triple negative breast cancer cells MDA-MB-231, as well as the effect of their conditioned medium (CM) on endothelial cells. Exposure to pesticides increased HIF-1α and VEGF protein expression in an AhR-dependent manner. In addition, HCB and CPF boosted NOS-2 and COX-2 content and VEGF secretion in MDA-MB-231 cells. The treatment of endothelial cells with CM from tumor cells exposed to pesticides increased cell proliferation, migration, and tubule formation, enhancing both tubule length and branching points. Of note, these effects were VEGF-dependent, as they were blocked in the presence of a VEGF receptor-2 (VEGFR-2) inhibitor. In sum, our results highlight the harmful impact of HCB and CPF in modulating the interaction between breast cancer and endothelial cells and promoting angiogenesis.

Chronic exposure to low concentrations of chlorpyrifos affects normal cyclicity and histology of the uterus in female rats.

Chlorpyrifos (CPF), the most used insecticide in Argentina, can act as an endocrine disruptor at low doses. We previously demonstrated that chronic exposure to CPF induces hormonal imbalance in vivo. The aim of this work was to study the effects of low concentrations of CPF (0.01 and 1 mg/kg/day) on the reproductive system of virgin adult rats. In the ovary, we studied the effects of CPF on steroidogenesis by determining steroid hormone content by RIA and CYP11 and CYP19 enzyme expression by qRT-PCR. The estrous cycle was evaluated by microscopic observation of vaginal smear, as well as by changes in uterine histology. In endometrium, we determined the fractal dimension and expression of PCNA, ERα and PR by IHC. Our results showed that chronic exposure to CPF affects ovarian steroid synthesis, causing alterations in the normal cyclicity of animals. In addition, CPF induced proliferative changes in the uterus, suggesting that it could affect reproduction or act as a risk factor in the development of uterine proliferative pathologies.

Angiogenesis signaling in breast cancer models is induced by hexachlorobenzene and chlorpyrifos, pesticide ligands of the aryl hydrocarbon receptor.

Breast cancer incidence is increasing globally and pesticides exposure may impact risk of developing this disease. Hexachlorobenzene (HCB) and chlorpyrifos (CPF) act as endocrine disruptors, inducing proliferation in breast cancer cells. Vascular endothelial growth factor-A (VEGF-A), cyclooxygenase-2 (COX-2) and nitric oxide (NO) are associated with angiogenesis. Our aim was to evaluate HCB and CPF action, both weak aryl hydrocarbon receptor (AhR) ligands, on angiogenesis in breast cancer models. We used: (1) in vivo xenograft model with MCF-7 cells, (2) in vitro breast cancer model with MCF-7, and (3) in vitro neovasculogenesis model with endothelial cells exposed to conditioned medium from MCF-7. Results show that HCB (3 mg/kg) and CPF (0.1 mg/kg) stimulated vascular density in the in vivo model. HCB and CPF low doses enhanced VEGF-A and COX-2 expression, accompanied by increased levels of nitric oxide synthases (NOS), and NO release in MCF-7. HCB and CPF high doses intensified VEGF-A and COX-2 levels but rendered different effects on NOS, however, both pesticides reduced NO production. Moreover, our data indicate that HCB and CPF-induced VEGF-A expression is mediated by estrogen receptor and NO, while the increase in COX-2 is through AhR and NO pathways in MCF-7. In conclusion, we demonstrate that HCB and CPF environmental concentrations stimulate angiogenic switch in vivo. Besides, pesticides induce VEGF-A and COX-2 expression, as well as NO production in MCF-7, promoting tubulogenesis in endothelial cells. These findings show that pesticide exposure could stimulate angiogenesis, a process that has been demonstrated to contribute to breast cancer progression.

Differential expression of the long and truncated Hv1 isoforms in breast-cancer cells.

Metabolic reprogramming of cancer cells results in a high production of acidic substances that must be extruded to maintain tumor-cell viability. The voltage-gated proton channel (Hv1) mediates highly selective effluxes of hydronium-ion (H+ ) that prevent deleterious cytoplasmic acidification. In the work described here, we demonstrated for the first time that the amino-terminal-truncated isoform of Hv1 is more highly expressed in tumorigenic breast-cancer-cell lines than in nontumorigenic breast cells. With respect to Hv1 function, we observed that pharmacologic inhibition of that channel, mediated by the specific blocker 5-chloro-2-guanidinobenzimidazole, produced a drop in intracellular pH and a decrease in cell viability, both in monolayer and in three-dimensional cultures, and adversely affected the cell-cycle in tumorigenic breast cells without altering the cycling of nontumorigenic cells. In conclusion, our results demonstrated that the Hv1 channel could be a potential tool both as a biomarker and as a therapeutic target in breast-cancer disease.

AhR ligands reactivate LINE-1 retrotransposon in triple-negative breast cancer cells MDA-MB-231 and non-tumorigenic mammary epithelial cells NMuMG.

Breast cancer is the most common cancer type in females worldwide. Environmental exposure to pesticides affecting hormonal homeostasis does not necessarily induce DNA mutations but may influence gene expression by disturbances in epigenetic regulation. Expression of long interspersed nuclear element-1 (LINE-1) has been associated with tumorigenesis in several cancers. In nearly all somatic cells, LINE-1 is silenced by DNA methylation in the 5́'UTR and reactivated during disease initiation and/or progression. Strong ligands of aryl hydrocarbon receptor (AhR) activate LINE-1 through the transforming growth factor-ß1 (TGF-ß1)/Smad pathway. Hexachlorobenzene (HCB) and chlorpyrifos (CPF), both weak AhR ligands, promote cell proliferation and migration in breast cancer cells, as well as tumor growth in rat models. In this context, our aim was to examine the effect of these pesticides on LINE-1 expression and ORF1p localization in the triple-negative breast cancer cell line MDA-MB-231 and the non-tumorigenic epithelial breast cell line NMuMG, and to evaluate the role of TGF-ß1 and AhR pathways. Results show that 0.5 µM CPF and 0.005 µM HCB increased LINE-1 mRNA expression through Smad and AhR signaling in MDA-MB-231. In addition, the methylation of the first sites in 5́'UTR of LINE-1 was reduced by pesticide exposure, although the farther sites remained unaffected. Pesticides modulated ORF1p localization in MDA-MB-231: 0.005 µM HCB and 50 µM CPF increased nuclear translocation, while both induced cytoplasmic retention at 0.5 and 5 µM. Moreover, both stimulated double-strand breaks, enhancing H2AX phosphorylation, coincidentally with ORF1p nuclear localization. In NMuMG similar results were observed, since they heighten LINE-1 mRNA levels. CPF effect was through AhR and TGF-ß1 signaling, whereas HCB action depends only of AhR. In addition, both pesticides increase ORF1p expression and nuclear localization. Our results provide experimental evidence that HCB and CPF exposure modify LINE-1 methylation levels and induce LINE-1 reactivation, suggesting that epigenetic mechanisms could contribute to pesticide-induced breast cancer progression.

Impact of endocrine disruptor hexachlorobenzene on the mammary gland and breast cancer: The story thus far.

Breast cancer incidence is increasing globally and exposure to endocrine disruptors has gained importance as a potential risk factor. Hexachlorobenzene (HCB) was once used as a fungicide and, despite being banned, considerable amounts are still released into the environment. HCB acts as an endocrine disruptor in thyroid, uterus and mammary gland and was classified as possibly carcinogenic to human. This review provides a thorough analysis of results obtained in the last 15 years of research and evaluates data from assays in mammary gland and breast cancer in diverse animal models. We discuss the effects of environmentally relevant HCB concentrations on the normal mammary gland and different stages of carcinogenesis, and attempt to elucidate its mechanisms of action at molecular level. HCB weakly binds to the aryl hydrocarbon receptor (AhR), activating both membrane (c-Src) and nuclear pathways. Through c-Src stimulation, AhR signaling interacts with other membrane receptors including estrogen receptor-α, insulin-like growth factor-1 receptor, epidermal growth factor receptor and transforming growth factor beta 1 receptors. In this way, several pathways involved in mammary morphogenesis and breast cancer development are modified, inducing tumor progression. HCB thus stimulates epithelial cell proliferation, preneoplastic lesions and alterations in mammary gland development as well as neoplastic cell migration and invasion, metastasis and angiogenesis in breast cancer. In conclusion, our findings support the hypothesis that the presence and bioaccumulation of HCB in high-fat tissues and during highly sensitive time windows such as pregnancy, childhood and adolescence make exposure a risk factor for breast tumor development.

Exposure to environmental concentrations of hexachlorobenzene induces alterations associated with endometriosis progression in a rat model.

Hexachlorobenzene (HCB) is a dioxin-like compound widely distributed and is a weak ligand of the aryl hydrocarbon receptor (AhR). Endometriosis is a disease characterized by growth of endometrial tissue in ectopic sites. Our aim was to investigate the impact of HCB on the endocrine, invasion and inflammatory parameters in a rat endometriosis model surgically induced. Female rats were exposed to HCB (1, 10 and 100 mg/kg b.w.) during 30 days. Results showed that HCB increases endometriotic like-lesions (L) volume in a dose-dependent manner. In L, HCB10 increases microvessel density (immunohistochemistry) and the vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2) and AhR levels (Western Blot), while HCB1 enhances aromatase expression (Western Blot). In addition, in eutopic endometrium (EU), HCB10/HCB100 augments microvessel density, VEGF and MMP-9 expression, while HCB1/HCB10 increases tumor necrosis factor-α (TNF-α) content in peritoneal fluid (ELISA). Interestingly, both L and EU from HCB-treated rats exhibited higher estrogen receptor α (ERα) (immunohistochemistry) and metalloproteases (MMP)-2 and -9 levels (Western Blot), as well as lower progesterone receptor (PR) expression (immunohistochemistry) than in control rats. Environmentally relevant concentrations of HCB could contribute to abnormal changes associated with endometriosis progression and development.

El hexaclorobenceno como factor de riesgo en el cáncer de mama / Hexachlorobenzene as a risk factor in breast cancer

Hexachlorobenzene (HCB) is a widespread environmental pollutant and an endocrine disruptor. Chronic exposure of humans to HCB elicits porphyria, neurologic symptoms, immune disorders and thyroid dysfunctions. It is a dioxin-like compound and a weak ligand of the AhR (aryl hydrocarbon receptor), a transcription factor that modulates genes related to detoxification, proliferation, migration and invasion. This study was carried out to revise the results of HCB action on mammary gland and breast cancer, summarizing the main ideas of its mechanism of action. HCB increases tumor development and active c-Src/EGFR (epidermal growth factor receptor) signaling pathways, while reducing tyrosine537-ER-alpha (estrogen receptor-alpha) phosphorylation, and promoting a phenotype with enhanced malignancy and lung metastasis in different animal models. In a rat mammary gland, HCB promotes an estrogenic microenvironment by activation of ER-alpha and Insulin/IGFs (insulin growth factors) pathways. HCB induces cell proliferation, promoting cell cycle progression and enhancing cyclin D1 expression and c-Src/p27 interaction in (ER-alpha) MCF-7 human breast cancer cell line. In (ER-alpha)(-) MDA-MB-231 breast cancer cells, the pesticide enhances cell migration and invasion as well as metalloproteases and TGF-beta1 (transformig growth factor-beta1) expression. In conclusion our current study suggests that alterations in the estrogenic microenvironment may influence the biological behavior of mammary gland or breast tumors, leading to preneoplastic lesions or enhanced malignancy, respectively. Our findings suggest that HCB may be a risk factor for human breast cancer progression.

El hexaclorobenceno (HCB) es un contaminante ambiental ampliamente distribuido y un desorganizador endocrino. Su exposición crónica en seres humanos produce porfiria, síntomas neurológicos, trastornos inmunitarios y disfunciones tiroideas. Es un agonista débil del receptor de hidrocarburos aromáticos (AhR), un factor de transcripción que modula genes relacionados con el metabolismo de xenobióticos, la proliferación, la migración y la invasión. Nuestro objetivo es revisar los efectos del HCB en la glándula mamaria y el cáncer mamario, resumiendo los principales mecanismos de acción. El HCB aumenta el desarrollo tumoral y activa vías de señalización de c-Src/receptor del factor de crecimiento epidérmico (EGFR), mientras que disminuye la fosforilación de tirosina 537/receptor de estrógenos alfa (RE-alfa), promoviendo un fenotipo de mayor malignidad y metástasis pulmonar en diferentes modelos con animales. En la glándula mamaria de rata genera un microambiente estrogénico por activación del RE-alfa y las vías de insulina/factores de crecimiento similares a la insulina (IGF). En células de cáncer mamario humanas MCF-7 (RE-alfa) induce proliferación celular, promoviendo la progresión del ciclo, aumentando la ciclina D1 y la interacción p27/c-Src. En MDA-MB-231 (-RE-alfa) estimula la migración e invasión, así como la expresión de metaloproteasas y factor de crecimiento transformante beta 1 (TGF-beta 1). Estos estudios indican que las alteraciones en el microambiente estrogénico podrían influir el comportamiento biológico de la glándula mamaria y los tumores, lo que provoca lesiones preneoplásicas o aumento en la malignidad tumoral mamaria. Nuestros hallazgos sugieren que el HCB podría ser un factor de riesgo para la progresión del cáncer de mama humano.

Arsenite in drinking water produces glucose intolerance in pregnant rats and their female offspring.

Drinking water is the main source of arsenic exposure. Chronic exposure has been associated with metabolic disorders. Here we studied the effects of arsenic on glucose metabolism, in pregnant and post-partum of dams and their offspring. We administered 5 (A5) or 50 (A50) mg/L of sodium arsenite in drinking water to rats from gestational day 1 (GD1) until two months postpartum (2MPP), and to their offspring from weaning until 8 weeks old. Liver arsenic dose-dependently increased in arsenite-treated rats to levels similar to exposed population. Pregnant A50 rats gained less weight than controls and recovered normal weight at 2MPP. Arsenite-treated pregnant animals showed glucose intolerance on GD16-17, with impaired insulin secretion but normal insulin sensitivity; they showed dose-dependent increased pancreas insulin on GD18. All alterations reverted at 2MPP. Offspring from A50-treated mothers showed lower body weight at birth, 4 and 8 weeks of age, and glucose intolerance in adult females, probably due to insulin secretion and sensitivity alterations. Arsenic alters glucose homeostasis during pregnancy by altering beta-cell function, increasing risk of developing gestational diabetes. In pups, it induces low body weight from birth to 8 weeks of age, and glucose intolerance in females, demonstrating a sex specific response.

Hexachlorobenzene modulates the crosstalk between the aryl hydrocarbon receptor and transforming growth factor-ß1 signaling, enhancing human breast cancer cell migration and invasion.

Given the number of women affected by breast cancer, considerable interest has been raised in understanding the relationships between environmental chemicals and disease onset. Hexachlorobenzene (HCB) is a dioxin-like compound that is widely distributed in the environment and is a weak ligand of the aryl hydrocarbon receptor (AhR). We previously demonstrated that HCB acts as an endocrine disruptor capable of stimulating cell proliferation, migration, invasion, and metastasis in different breast cancer models. In addition, increasing evidence indicates that transforming growth factor-ß1 (TGF-ß1) can contribute to tumor maintenance and progression. In this context, this work investigated the effect of HCB (0.005, 0.05, 0.5, and 5µM) on TGF-ß1 signaling and AhR/TGF-ß1 crosstalk in the human breast cancer cell line MDA-MB-231 and analyzed whether TGF-ß1 pathways are involved in HCB-induced cell migration and invasion. RT-qPCR results indicated that HCB reduces AhR mRNA expression through TGF-ß1 signaling but enhances TGF-ß1 mRNA levels involving AhR signaling. Western blot analysis demonstrated that HCB could increase TGF-ß1 protein levels and activation, as well as Smad3, JNK, and p38 phosphorylation. In addition, low and high doses of HCB were determined to exert differential effects on AhR protein levels, localization, and activation, with a high dose (5µM) inducing AhR nuclear translocation and AhR-dependent CYP1A1 expression. These findings also revealed that c-Src and AhR are involved in HCB-mediated activation of Smad3. HCB enhances cell migration (scratch motility assay) and invasion (Transwell assay) through the Smad, JNK, and p38 pathways, while ERK1/2 is only involved in HCB-induced cell migration. These results demonstrate that HCB modulates the crosstalk between AhR and TGF-ß1 and consequently exacerbates a pro-migratory phenotype in MDA-MB-231 cells, which contributes to a high degree of malignancy. Taken together, our findings help to characterize the molecular mechanism underlying the effects of HCB on breast cancer progression.

Pesticide chlorpyrifos acts as an endocrine disruptor in adult rats causing changes in mammary gland and hormonal balance.

Endocrine disruptors (EDs) are compounds that interfere with hormone regulation and influence mammary carcinogenesis. We have previously demonstrated that the pesticide chlorpyrifos (CPF) acts as an ED in vitro, since it induces human breast cancer cells proliferation through estrogen receptor alpha (ERα) pathway. In this work, we studied the effects of CPF at environmental doses (0.01 and 1mg/kg/day) on mammary gland, steroid hormone receptors expression and serum steroid hormone levels. It was carried out using female Sprague-Dawley 40-days-old rats exposed to the pesticide during 100 days. We observed a proliferating ductal network with a higher number of ducts and alveolar structures. We also found an increased number of benign breast diseases, such as hyperplasia and adenosis. CPF enhanced progesterone receptor (PgR) along with the proliferating cell nuclear antigen (PCNA) in epithelial ductal cells. On the other hand, the pesticide reduced the expression of co-repressors of estrogen receptor activity REA and SMRT and it decreased serum estradiol (E2), progesterone (Pg) and luteinizing hormone (LH) levels. Finally, we found a persistent decrease in LH levels among ovariectomized rats exposed to CPF. Therefore, CPF alters the endocrine balance acting as an ED in vivo. These findings warn about the harmful effects that CPF exerts on mammary gland, suggesting that this compound may act as a risk factor for breast cancer.

El organofosforado clorpirifos como disruptor estrogénico y factor de riesgo para el cáncer de mama / The organophosphorus chlorpyrifos as endocrine disruptor and breast cancer factor risk

El clorpirifos (CPF) es un insecticida de amplio espectro que se utiliza en Argentina y en otros países de Latinoamérica. Se emplea para el control de plagas en la producción de frutas, hortalizas, cereales y plantas ornamentales. El principal mecanismo de acción descripto para este insecticida es la inhibición de la acetilcolinesterasa. Sin embargo, reportes más recientes sugieren múltiples efectos del plaguicida independientes de la inhibición de esa enzima. El objetivo de este trabajo es transmitir a la comunidad los resultados de nuestras investigaciones obtenidos utilizando diferentes dosis de CPF en distintos modelos experimentales, tanto in vitro como in vivo. En relación a esto, hemos evidenciado una acción del CPF sobre el sistema redox celular que conduce al incremento de especies reactivas del oxígeno y consecuentemente a la activación de diferentes vías de señalización. Además, hemos determinado que el insecticida CPF puede comportarse como un disruptor endócrino modulando la acción de los estrógenos y alterando la normal estructura del tejido mamario. Nuestros resultados alertan sobre el impacto que este compuesto podría tener sobre la salud, sugiriendo la necesidad de revisar su uso dado que manifiesta acciones a dosis encontradas en el ambiente.

Chlorpyrifos (CPF) is a broad spectrum insecticide used in Argentina and other Latin American countries. It is commonly used for pest control in the production of fruits, vegetables, cereals and ornamental plants. The main mechanism of action described for this insecticide is the inhibition of acetylcholinesterase activity. However, more recent reports suggest multiple effects for this pesticide in an independent way from the inhibition of this enzyme. The objective of this work is to convey to the community the results of our investigations obtained using different doses of CPF in various experimental models, both in vitro and in vivo. In this connection, we have shown a CPF action on the cellular redox system which leads to increased reactive oxygen species and the consequent activation of different signaling pathways. In addition, we have determined that the insecticide CPF acts as an endocrine disruptor modulating the action of estrogen and altering the normal structure of breast tissue. Our findings warn about the impact that this compound might have on health, suggesting the need to review its use since adverse actions were found at environmentally relevant doses.

Hexachlorobenzene promotes angiogenesis in vivo, in a breast cancer model and neovasculogenesis in vitro, in the human microvascular endothelial cell line HMEC-1.

Exposure to environmental pollutants may alter proangiogenic ability and promotes tumor growth. Hexachlorobenzene (HCB) is an organochlorine pesticide found in maternal milk and in lipid foods, and a weak ligand of the aryl hydrocarbon receptor (AhR). HCB induces migration and invasion in human breast cancer cells, as well as tumor growth and metastasis in vivo. In this study, we examined HCB action on angiogenesis in mammary carcinogenesis. HCB stimulates angiogenesis and increases vascular endothelial growth factor (VEGF) expression in a xenograft model with the human breast cancer cell line MDA-MB-231. Human microvascular endothelial cells HMEC-1 exposed to HCB (0.005, 0.05, 0.5 and 5µM) showed an increase in cyclooxygenase-2 (COX-2) and VEGF protein expression involving AhR. In addition, we found that HCB enhances VEGF-Receptor 2 (VEGFR2) expression, and activates its downstream pathways p38 and ERK1/2. HCB induces cell migration and neovasculogenesis in a dose-dependent manner. Cells pretreatment with AhR, COX-2 and VEGFR2 selective inhibitors, suppressed these effects. In conclusion, our results show that HCB promotes angiogenesis in vivo and in vitro. HCB-induced cell migration and tubulogenesis are mediated by AhR, COX-2 and VEGFR2 in HMEC-1. These findings may help to understand the association among HCB exposure, angiogenesis and mammary carcinogenesis.

Chlorpyrifos inhibits cell proliferation through ERK1/2 phosphorylation in breast cancer cell lines.

It is well known the participation of oxidative stress in the induction and development of different pathologies including cancer, diabetes, neurodegeneration and respiratory disorders among others. It has been reported that oxidative stress may be induced by pesticides and it could be the cause of health alteration mediated by pollutants exposure. Large number of registered products containing chlorpyrifos (CPF) is used to control pest worldwide. We have previously reported that 50 µM CPF induces ROS generation and produces cell cycle arrest followed by cell death. The present investigation was designed to identify the pathway involved in CPF-inhibited cell proliferation in MCF-7 and MDA-MB-231 breast cancer cell lines. In addition, we determined if CPF-induced oxidative stress is related to alterations in antioxidant defense system. Finally we studied the molecular mechanisms underlying in the cell proliferation inhibition produced by the pesticide. In this study we demonstrate that CPF (50 µM) induces redox imbalance altering the antioxidant defense system in breast cancer cells. Furthermore, we found that the main mechanism involved in the inhibition of cell proliferation induced by CPF is an increment of p-ERK1/2 levels mediated by H2O2 in breast cancer cells. As PD98059 could not abolish the increment of ROS induced by CPF, we concluded that ERK1/2 phosphorylation is subsequent to ROS production induced by CPF but not the inverse.

Action of hexachlorobenzene on tumor growth and metastasis in different experimental models.

Hexachlorobenzene (HCB) is a widespread organochlorine pesticide, considered a possible human carcinogen. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR). We have found that HCB activates c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways and cell migration, in an AhR-dependent manner in MDA-MB-231 breast cancer cells. The aim of this study was to investigate in vitro the effect of HCB (0.005, 0.05, 0.5, 5µM) on cell invasion and metalloproteases (MMPs) 2 and 9 activation in MDA-MB-231 cells. Furthermore, we examined in vivo the effect of HCB (0.3, 3, 30mg/kg b.w.) on tumor growth, MMP2 and MMP9 expression, and metastasis using MDA-MB-231 xenografts and two syngeneic mouse breast cancer models (spontaneous metastasis using C4-HI and lung experimental metastasis using LM3). Our results show that HCB (5µM) enhances MMP2 expression, as well as cell invasion, through AhR, c-Src/HER1 pathway and MMPs. Moreover, HCB increases MMP9 expression, secretion and activity through a HER1 and AhR-dependent mechanism, in MDA-MB-231 cells. HCB (0.3 and 3mg/kg b.w.) enhances subcutaneous tumor growth in MDA-MB-231 and C4-HI in vivo models. In vivo, using MDA-MB-231 model, the pesticide (0.3, 3 and 30mg/kg b.w.) activated c-Src, HER1, STAT5b, and ERK1/2 signaling pathways and increased MMP2 and MMP9 protein levels. Furthermore, we observed that HCB stimulated lung metastasis regardless the tumor hormone-receptor status. Our findings suggest that HCB may be a risk factor for human breast cancer progression.

Glibenclamide inhibits cell growth by inducing G0/G1 arrest in the human breast cancer cell line MDA-MB-231.

BACKGROUND: Glibenclamide (Gli) binds to the sulphonylurea receptor (SUR) that is a regulatory subunit of ATP-sensitive potassium channels (KATP channels). Binding of Gli to SUR produces the closure of KATP channels and the inhibition of their activity. This drug is widely used for treatment of type 2-diabetes and it has been signaled as antiproliferative in several tumor cell lines. In previous experiments we demonstrated the antitumoral effect of Gli in mammary tumors induced in rats. The aim of the present work was to investigate the effect of Gli on MDA-MB-231 breast cancer cell proliferation and to examine the possible pathways involved in this action. RESULTS: The mRNA expression of the different subunits that compose the KATP channels was evaluated in MDA-MB-231 cells by reverse transcriptase-polymerase chain reaction. Results showed the expression of mRNA for both pore-forming isoforms Kir6.1 and Kir6.2 and for the regulatory isoform SUR2B in this cell line. Gli inhibited cell proliferation assessed by a clonogenic method in a dose dependent manner, with an increment in the population doubling time. The KATP channel opener minoxidil increased clonogenic proliferation, effect that was counteracted by Gli. When cell cycle analysis was performed by flow cytometry, Gli induced a significant cell-cycle arrest in G0/G1 phase, together with an up-regulation of p27 levels and a diminution in cyclin E expression, both evaluated by immunoblot. However, neither differentiation evaluated by neutral lipid accumulation nor apoptosis assessed by different methodologies were detected. The cytostatic, non toxic effect on cell proliferation was confirmed by removal of the drug.Combination treatment of Gli with tamoxifen or doxorubicin showed an increment in the antiproliferative effect only for doxorubicin. CONCLUSIONS: Our data clearly demonstrated a cytostatic effect of Gli in MDA-MB-231 cells that may be mediated through KATP channels, associated to the inhibition of the G1-S phase progression. In addition, an interesting observation about the effect of the combination of Gli with doxorubicin leads to future research for a potential novel role for Gli as an adjuvant in breast cancer treatment.

PANC-1 cells proliferative response to ionizing radiation is related to GSK-3ß phosphorylation.

Radiotherapy may be used to treat pancreatic cancer and relieve pain. We have previously reported that histamine modulates pancreatic adenocarcinoma PANC-1 cell proliferation. This work was aimed to evaluate whether histamine improves radiosensitivity of PANC-1 cells in relation to phosphorylation/inhibition of glycogen synthase kinase-3ß (GSK-3ß). Immediately after γ irradiation, intracellular hydrogen peroxide was markedly decreased together with a rapid increase in catalase activity. Although histamine diminished catalase activity in nonirradiated cells, it only partially hindered the increase observed in irradiated cells and could not modify radiosensitivity. In control cells, a high expression of total and a very low expression of phosphorylated/inactive GSK-3ß were found. An increment in reactive oxygen species levels produced an augmentation in GSK-3ß phosphorylation and suppressed cell proliferation. In both control and histamine-treated irradiated cells, the rise in catalase activity lowered reactive oxygen species levels and only a small increase in phosphorylated GSK-3ß was detected. Alternatively, 3-aminotriazole, an irreversible inhibitor of catalase, reduced the survival fraction in irradiated control cells along with an increment in phosphorylated GSK-3ß. These results suggest that upon irradiation, early catalase activation may be responsible for keeping GSK-3ß active conceding cells a survival advantage toward cytotoxic effects of ionizing radiation.

Differential mechanisms of action are involved in chlorpyrifos effects in estrogen-dependent or -independent breast cancer cells exposed to low or high concentrations of the pesticide.

It has reported that many environmental compounds may display estrogenic actions and these findings led to researchers to associate breast cancer risk with the use of some pesticides. The aim of this work was to investigate the effect of chlorpyrifos (CPF) on cell proliferation and the ERα-dependence of this action employing MCF-7 and MDA-MB-231 breast cancer cell lines. We have also analyzed CPF action on the cell cycle distribution and the cyclins that are implicated in G1-S and intra-S checkpoints. Finally, the action on cell death and ROS production were studied. We demonstrated the ability of CPF 0.05µM to induce cell proliferation through ERα in hormone-dependent breast cancer cells. In contrast, CPF 50µM induces intra-S arrest modifying checkpoints proteins, through a mechanism that may involve changes in redox balance in MCF-7. In MDA-MB-231, we have found that CPF 50µM produces an arrest in G2/M phase which could be related to the capacity of the pesticide for binding to tubulin sites altering microtubules polymerization. Altogether, our results provide new evidences on the action of the pesticide CPF as an environmental breast cancer risk factor due to the effects that causes on the mechanisms that modulate breast cell proliferation.

Alterations in c-Src/HER1 and estrogen receptor α signaling pathways in mammary gland and tumors of hexachlorobenzene-treated rats.

Hexachlorobenzene (HCB) is an organochlorine pesticide that acts as an endocrine disruptor in humans and rodents. The development of breast cancer strongly depends on endocrine conditions modulated by environmental factors. We have demonstrated that HCB is a tumor co-carcinogen in rats and an inducer of proliferation in MCF-7 cells, in an estrogen receptor α (ERα)-dependent manner, and of migration in MDA-MB-231 breast cancer cell line. In the present study, we examined HCB effect on c-Src/human epidermal growth factor receptor (HER1) and ERα signaling pathways in mammary glands and in N-nitroso-N-methylurea (NMU)-induced mammary tumors in rats. Furthermore, we evaluated histopathological changes and serum hormone levels. Rats were separated into four groups: control, HCB (100 mg/kg b.w.), NMU (50 mg/kg b.w.) and NMU-HCB. Our data show that HCB increases c-Src and HER1 activation, c-Src/HER1 association, and Y699-STAT5b and ERK1/2 phosphorylation in mammary glands. HCB also enhances Y537-ERα phosphorylation and ERα/c-Src physical interaction. In tumors, HCB also induces c-Src and HER1 activation, c-Src/HER1 association, as well as T308-Akt and Y699-STAT5b phosphorylation. In addition, the pesticide increases ERα protein content and decreases p-Y537-ERα levels and ERα/c-Src association in tumors. HCB increases serum 17-beta estradiol and prolactin contents and decreases progesterone, FSH and LH levels in rats without tumors, while the opposite effect was observed in rats with tumors. Taken together, our results indicate that HCB induces an estrogenic effect in mammary gland, increasing c-Src/HER1 and ERα signaling pathways. HCB stimulates c-Src/HER1 pathway, but decreases ERα activity in tumors, appearing to shift them towards a higher malignancy phenotype.

Activation of c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways and cell migration by hexachlorobenzene in MDA-MB-231 human breast cancer cell line.

Hexachlorobenzene (HCB) is a widespread environmental pollutant. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR) protein. HCB is a tumor cocarcinogen in rat mammary gland and an inducer of cell proliferation and c-Src kinase activity in MCF-7 breast cancer cells. This study was carried out to investigate HCB action on c-Src and the human epidermal growth factor receptor (HER1) activities and their downstream signaling pathways, Akt, extracellular-signal-regulated kinase (ERK1/2), and signal transducers and activators of transcription (STAT) 5b, as well as on cell migration in a human breast cancer cell line, MDA-MB-231. We also investigated whether the AhR is involved in HCB-induced effects. We have demonstrated that HCB (0.05µM) produces an early increase of Y416-c-Src, Y845-HER1, Y699-STAT5b, and ERK1/2 phosphorylation. Moreover, our results have shown that the pesticide (15 min) activates these pathways in a dose-dependent manner (0.005, 0.05, 0.5, and 5µM). In contrast, HCB does not alter T308-Akt activation. Pretreatment with a specific inhibitor for c-Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine [PP2]) prevents Y845-HER1 and Y699-STAT5b phosphorylation. AG1478, a specific HER1 inhibitor, abrogates HCB-induced STAT5b and ERK1/2 activation, whereas 4,7-orthophenanthroline and α-naphthoflavone, two AhR antagonists, prevent HCB-induced STAT5b and ERK1/2 phosphorylation. HCB enhances cell migration evaluated by scratch motility and transwell assays. Pretreatment with PP2, AG1478, and 4,7-orthophenanthroline suppresses HCB-induced cell migration. These results demonstrate that HCB stimulates c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways in MDA-MB-231. c-Src, HER1, and AhR are involved in HCB-induced increase in cell migration. The present study makes a significant contribution to the molecular mechanism of action of HCB in mammary carcinogenesis.