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.

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.

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.

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.

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.

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.

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.

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.

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.

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 is a tumor co-carcinogen and induces alterations in insulin-growth factors signaling pathway in the rat mammary gland.

Hexachlorobenzene (HCB) is a widespread environmental pollutant. Controversy still exists about the breast carcinogenic properties of organochlorines in humans. The ligands, receptors, and related signaling proteins of the insulin growth factor family are involved in the regulation of breast-cancer cell growth. The aims of this study were to determine: (1) whether HCB is co-carcinogenic in a medium term assay of N-nitroso N-methylurea (NMU)-induced mammary tumors in rats; (2) the effect of HCB on insulin receptor (IR), insulin-like growth factor-I receptor (IGF-IR) and insulin receptor substrate-1 (IRS-1) levels and on IRS-1 phosphorylation; (3) microsomal and cytosolic Protein Tyrosine Kinase (PTK) activities in mammary glands and NMU-induced tumors. Sprague Dawley rats were injected with 50 mg/kg body weight of NMU at 50, 80, and 110 days old. HCB (100 mg/kg body weight) was administered three times a week from 65 to 110 days of age. Rats were separated in four groups: control, NMU, HCB, and NMU-HCB. HCB alone did not induce tumor development. Parameters of tumor development were increased in NMU-HCB compared to NMU rats. A higher cellular undifferentiation was observed in NMU-HCB tumors. IR, IGF-IR, and IRS-1 levels were higher in HCB than in controls. Conversely IGF-IR levels decreased in NMU-HCB vs. NMU group. The IRS-1 phosphorylation increased in HCB rats; however, it decreased in NMU-HCB vs. NMU. HCB decreased microsomal PTK activity in tumors. This study showed for the first time that HCB is a co-carcinogenic agent in NMU-induced mammary tumors in rats. Our results suggest that the IR and/or IGF-IR signaling pathway may be involved in the mechanism of action of HCB.

Effect of rosiglitazone on N-nitroso-N-methylurea-induced mammary tumors in rat.

The objective of this study was to evaluate the in vivo antitumor action of rosiglitazone (Rosi) alone or in combination with tamoxifen (Tam) on experimental mammary tumors induced by N-nitroso-N-methylurea (NMU) in Sprague-Dawley rats. Animals bearing mammary tumors were treated with 0.06 mg/kg/day or 0.12 mg/kg/day of Rosi orally, 1 mg/kg/day of Tam s.c., or with the combined treatment (Rosi+Tam). After 25 days of treatment, the following responses were observed: 45% of tumors were responsive to 0.06 mg/kg/day of Rosi treatment, while 55% of tumors under Tam treatment responded. The results of the combined Rosi+Tam treatment indicated that 75% of tumors were responsive. Similar results were obtained with 0.12 mg/kg/day of Rosi. Apoptosis, necrosis and glandular hypersecretion were observed in Rosi-treated tumors. In all cases, the combined Rosi+Tam treatment potentiated the antitumor effect of Tam alone. No side-effects were observed after treatment at any assayed dose.

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.

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.

Enhancement of BRCA1 gene expression by the peroxisome proliferator-activated receptor gamma in the MCF-7 breast cancer cell line.

BRCA1 has been linked to the genetic susceptibility of a majority of familial breast and ovarian cancers. Several lines of evidence indicate that BRCA1 is a tumor suppressor and its expression is downregulated in sporadic breast and ovarian cancer cases. Therefore, the identification of genes involved in the regulation of BRCA1 gene expression might lead to new insights into the pathogenesis and treatment of these tumors. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily that has well-established roles in the regulation of adipocyte development and glucose homeostasis. More recently, it has been shown that ligands of PPARgamma have a potent antitumorigenic activity in breast cancer cells. In the present study we have found that two distinct ligands of PPARgamma; 15-deoxy-delta-(12,14)-prostaglandin J2 (15dPG-J2) and rosiglitazone, increase the levels of BRCA1 protein in human MCF-7 breast cancer cells. Immunofluorescence microscopy analysis showed that, after treatment with 15dPG-J2, the BRCA1 protein is mainly localized in the nucleus. Functional analysis by transient transfection of different 5'-flanking region fragments, as well as gel mobility shift assays and mutagenic analysis, suggests that the effects of 15dPG-J2 and rosiglitazone are mediated through a functional DR1 located between the nucleotides -241 and -229, which is a canonical PPARgamma type response element. Our data suggest that PPARgamma is a crucial gene regulating BRCA1 gene expression and might therefore be important for the BRCA1 regulatory pathway involved in the pathogenesis of sporadic breast and ovarian cancer.

Effect of glibenclamide on N-nitroso-N-methylurea-induced mammary tumors in diabetic and nondiabetic rats.

The objective of this study was to evaluate the antitumor effect of glibenclamide (Gli) alone or in combination with tamoxifen (Tam) on experimental mammary tumors induced by N-nitroso-N-methylurea (NMU) in nondiabetic and diabetic rats. For experimental diabetes induction, Sprague-Dawley rats were injected with streptozotocin (STZ) on the second day of life. For experimental mammary tumor induction, nondiabetic and diabetic rats were injected IP with NMU at 50, 80, and 110 days of life. Nondiabetic and diabetic rats bearing mammary tumors were treated with 0.06 mg/day of Gli orally, Tam 1 mg/kg/day SC, or with the combined treatment (Gli + Tam). After 20 days of treatment, different responses were observed. In nondiabetic rats, 64% of tumors were responsive to Gli treatment (they regressed or remained stable), whereas 57% of tumors under treatment with Tam exhibited a response. Results of the combined Gli + Tam treatment indicated that all tumors were responsive: 58% regressed and 42% remained stable. Diabetic rats receiving Gli treatment did not show response to this treatment, while 65% of the tumors of Tam-treated diabetic rats showed regression. Histopathologic observation indicated an important intratumor secretion in all tumors of Gli-, Tam-, or Gli + Tam-treated rats. No secondary toxic effect was observed after treatment at any assayed doses. In conclusion, the present data demonstrate the in vivo antitumor action of Gli treatment on the experimental mammary tumors employed, indicating that Gli exerted a direct effect on tumor cells in nondiabetic rats. The combined Gli + Tam treatment potentiated the antitumor effect of each drug alone. Future research will examine the molecular aspects of these findings.

Insulin-like growth factor-I receptor expression and localization in benign and malignant rat mammary tumors.

The aim of this study was to investigate the expression and localization of the insulin growth factor type 1 receptor (IGF-IR) in malignant and benign mammary tumors induced in rats by N-nitroso-N-methylurea (NMU) and its correlation with histopathology and hormone dependence. Also, protein tyrosine kinase activities (PTKs) were analyzed in order to study the activation of the intracellular cascade. The results showed that IGF-IR is present in NMU tumors (analyzed by binding assay and Western blot), that a variable content is expressed in tumors that continued growing post-ovariectomy (OVX) of rats, and that it is undetectable in tumors that regressed post-OVX. IGF-IR was principally localized (by immunohistochemistry) in the epithelial cells of malignant tumors and in the fibrous cells of benign ones. Also, a significantly lower expression of both cytosolic and microsomal PTKs were found in benign tumors. Our results suggest a different expression and role of IGF-IR in benign and malignant tumors.

Mitochondrial apoptotic pathways.

Apoptosis or programmed cell death (PCD) is a physiological process characteristic of pluricellular organisms leading to self-destruction of the cell. It is therefore involved in development, homeostasis and host defense. However, a significant difference has been shown between mammalian cell apoptosis and non-mammalian cell apoptosis: mitochondria are implicated only in the former. Execution of PCD includes the release of several proapoptotic proteins from the intermembrane space of mitochondria. They could exert their actions through a caspase dependent as well as a caspase independent way. On the other hand, regulation of PCD is mainly given by the Bcl-2 family members, which are in turn essentially regulated by activation of death receptors and/or DNA damage. Nowadays, execution of apoptosis is better known than its regulation. Nevertheless, we are still far of a complete understanding of the apoptotic process.

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.

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.