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.

Extracellular acidosis stimulates breast cancer cell motility through aryl hydrocarbon receptor and c-src kinase activation.

A reduction in extracellular pH (pHe) is a characteristic of most malignant tumors. The aryl hydrocarbon receptor (AhR) is a transcription factor localized in a cytosolic complex with c-Src, which allows it to trigger nongenomic effects through c-Src. Considering that the slightly acidic tumor microenvironment promotes breast cancer progression in a similar way to the AhR/c-Src axis, our aim was to evaluate whether this pathway could be activated by low pHe. We examined the effect of pHe 6.5 on AhR/c-Src axis using two breast cancer cell lines (MDA-MB-231 and LM3) and mammary epithelial cells (NMuMG) and found that acidosis increased c-Src phosphorylation only in tumor cells. Moreover, the presence of AhR inhibitors prevented c-Src activation. Low pHe reduced intracellular pH (pHi), while amiloride treatment, which is known to reduce pHi, induced c-Src phosphorylation through AhR. Analyses were conducted on cell migration and metalloproteases (MMP)-2 and -9 activities, with results showing an acidosis-induced increase in MDA-MB-231 and LM3 cell migration and MMP-9 activity, but no changes in NMuMG cells. Moreover, all these effects were blocked by AhR and c-Src inhibitors. In conclusion, acidosis stimulates the AhR/c-Src axis only in breast cancer cells, increasing cell migration and MMP-9 activity. Although the AhR activation mechanism still remains elusive, a reduction in pHi may be thought to be involved. These findings suggest a critical role for the AhR/c-Src axis in breast tumor progression stimulated by an acidic microenvironment.

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.

Hexachlorobenzene induces cell proliferation, and aryl hydrocarbon receptor expression (AhR) in rat liver preneoplastic foci, and in the human hepatoma cell line HepG2. AhR is a mediator of ERK1/2 signaling, and cell cycle regulation in HCB-treated HepG2 cells.

Hexachlorobenzene (HCB) is a widespread environmental pollutant, and a liver tumor promoter in rodents. Depending on the particular cell lines studied, exposure to these compounds may lead to cell proliferation, terminal differentiation, or apoptosis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in drug and xenobiotic metabolism. AhR can also modulate a variety of cellular and physiological processes that can affect cell proliferation and cell fate determination. The mechanisms by which AhR ligands, both exogenous and endogenous, affect these processes involve multiple interactions between AhR and other signaling pathways. In the present study, we examined the effect of HCB on cell proliferation and AhR expression, using an initiation-promotion hepatocarcinogenesis protocol in rat liver and in the human-derived hepatoma cell line, HepG2. Female Wistar rats were initiated with a single dose of 100 mg/kg of diethylnitrosamine (DEN) at the start of the experiment. Two weeks later, daily dosing of 100 mg/kg HCB was maintained for 10 weeks. Partial hepatectomy was performed 3 weeks after initiation. The number and area of glutathione S-transferase-P (GST-P)-positive foci, in the rat liver were used as biomarkers of liver precancerous lesions. Immunohistochemical staining showed an increase in proliferating cell nuclear antigen (PCNA)-positive cells, along with enhanced AhR protein expression in hepatocytes within GST-P-positive foci of (DEN HCB) group, when compared to DEN. In a similar manner, Western blot analysis demonstrated that HCB induced PCNA and AhR protein expression in HepG2 cells. Flow cytometry assay indicated that the cells were accumulated at S and G2/M phases of the cell cycle. HCB increased cyclin D1 protein levels and ERK1/2 phosphorylation in a dose-dependent manner. Treatment of cells with a selective MEK1 inhibitor, prevented HCB-stimulatory effect on PCNA and cyclinD1, indicating that these effects are mediated by ERK1/2. Pretreatment with an AhR antagonist, prevented HCB-induced PCNA protein levels, ERK1/2 phosphorylation and alterations in cell cycle distribution. These results demonstrate that HCB-induced HepG2 proliferation and cell cycle progression depend on ERK1/2 phosphorylation which is mediated by the AhR. Our results provide a clue to the molecular events involved in the mechanism of action of HCB-induced hepatocarcinogenesis.

Hexachlorobenzene induces TGF-ß1 expression, which is a regulator of p27 and cyclin D1 modifications.

Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the environment. In this study we have demonstrated that HCB induced loss of cell viability and alterations in cell cycle regulation in FRTL-5 rat thyroid cells. Analysis of cell cycle distribution by flow cytometric analysis demonstrated that HCB induced cell cycle arrest at G2/M and at G0/G1 phase, inhibiting cell cycle progression at the G1/S phase, after 24 h and 72 h of treatment. HCB-treatment resulted in an increase in transforming growth factor-beta (TGF-ß1) mRNA levels, a negative regulator of cell growth in thyroid epithelial cells. Time-dependent studies showed that both cytosolic and nuclear p27 protein levels were increased by 5 µM HCB. After 24 h of treatment, total p27 in whole cells lysate was increased. Dose-dependent studies, demonstrated that HCB (0.005, 0.05, 0.5 and 5 µM) increased p27, both in the cytosol and nucleus. HCB (5 µM) induced a concomitant decrease in nuclear cyclin D1 protein levels, in a time-dependent manner. We have also demonstrated that TGF-ß1 Smad signaling is involved in HCB-induced alterations of p27 and cyclin D1 protein levels. On the other hand, ERK1/2 activation is not involved in the alteration of cell cycle regulatory proteins.

Reactive oxygen species and extracellular signal-regulated kinase 1/2 mediate hexachlorobenzene-induced cell death in FRTL-5 rat thyroid cells.

Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the environment. We have previously shown that chronic HCB exposure triggers apoptosis in rat thyroid follicular cells. This study was carried out to investigate the molecular mechanism by which the pesticide causes apoptosis in FRTL-5 rat thyroid cells exposed to HCB (0.005, 0.05, 0.5, and 5µM) for 2, 6, 8, 24, and 48h. HCB treatment lowered cell viability and induced apoptotic cell death in a dose- and time-dependent manner, as demonstrated by morphological nuclear changes and the increase of DNA fragmentation. The pesticide increased activation of caspases-3, -8, and full-length caspase-10 processing. HCB induced mitochondrial membrane depolarization, release of cytochrome c and apoptosis-inducing factor (AIF), from the mitochondria to the cytosol, and AIF nuclear translocation. Cell death was accompanied by an increase in reactive oxygen species (ROS) generation. Blocking of ROS production, with a radical scavenger (Trolox), resulted in inhibition of AIF nuclear translocation and returned cells survival to control levels, demonstrating that ROS are critical mediators of HCB-induced apoptosis. The pesticide increased ERK1/2, JNK, and p38 phosphorylation in a time- and dose-dependent manner. However, when FRTL-5 cells were treated with specific MAPK inhibitors, only blockade of MEK1/2 with PD98059 prevented cell loss of viability, as well as caspase-3 activation. In addition, we demonstrated that HCB-induced production of ROS has a critical role in ERK1/2 activation. These results demonstrate for the first time that HCB induces apoptosis in FRTL-5 cells, by ROS-mediated ERK1/2 activation, through caspase-dependent and -independent pathways.

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.

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.

Hexachlorobenzene induces deregulation of cellular growth in rat liver.

Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the biosphere. The aim of the present study was to investigate the effect of HCB on the homeostasis of liver cell growth, analyzing parameters of cell proliferation and apoptosis, in HCB (0.1, 1, 10 and 100 mg/kg body weight)-treated rats, during 4 weeks. Cell proliferation and ERK1/2 phosphorylation, associated with survival mechanisms, were increased at HCB 100 mg/kg. The pesticide increased the number of apoptotic cells, and the activation of caspase-3, -9 and -8, in a dose-dependent manner, suggesting that HCB-induced apoptosis is mediated by caspases. Increased Fas and FasL protein levels indicate that the death receptor pathway is also involved. This process is associated with decreased Bid, and increased cytosolic cytochrome c protein levels. Transforming growth factor-beta1 (TGF-ß1) intervenes in apoptotic and/or proliferative processes in hepatocytes. TGF-ß1 cDNA and protein levels are dose-dependently increased, suggesting that this cytokine might be involved in HCB-induced dysregulation of cell proliferation and apoptosis. In conclusion, this study reports for the first time that HCB induces loss of the homeostatic balance between cell growth and cell death in rat liver. Induced apoptosis occurs by mechanisms involving signals emanating from death receptors, and the mitochondrial pathway.

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.

Hexachlorobenzene triggers apoptosis in rat thyroid follicular cells.

Hexachlorobenzene (HCB) is a widespread environmental pollutant. Chronic exposure of humans to HCB produces a number of effects, such as triggering of porphyria, increased synthesis of liver microsomal enzymes, neurological symptoms, immunological disorders and thyroid dysfunctions. In rats, HCB induced hepatic porphyria, neurotoxic effects, and toxic effects on the reproductive system, thyroid function, and immune system. HCB is also known to cause tumors of the liver, thyroid and mammary gland in laboratory animals. The aim of this study was to investigate parameters of thyroid growth regulation, mainly cell proliferation and apoptosis in thyroid tissue from HCB (0.1, 1, 10, 100, and 500 mg/kg body weight)-treated female Wistar rats. The current study demonstrates that only the exposure to the highest HCB dose for 30 days, has adverse effects on thyroid endpoints examined related to thyroid gland morphology, and 3,3'5,5'-tetraiodothyronine (T(4), thyroxine) serum levels, without changes in thyroid-stimulating hormone concentrations or in thyroid gland weight. Morphological changes, included flattened epithelium and increased colloid size compared with control tissue. Transforming growth factor (TGF-beta1) mRNA levels, evaluated by RT-PCR, revealed a significant upregulation after exposure to HCB (1, 10, 100 mg/kg body weight). Cell proliferation evaluated by 5'-Br deoxiuridine incorporation into DNA, was not altered at any dose. HCB (1, 10, 100 mg/kg body weight) induces apoptosis, evaluated by in situ end labeling of fragmented DNA, terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick-end labeling, in rat thyroid glands. This process is associated with dose-dependent increases in cytochrome c release from the mitochondria and procaspase-9 processing to its active product. Caspase-8 was not activated. These studies indicate that doses of HCB that do not disrupt thyroid economy induce TGF-beta1 expression and apoptosis in the thyroid gland, involving the mitochondrial pathway.