Claudia Cocca

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.

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.

Thiols and polyamines in the potentiation of malathion toxicity in larval stages of the toad Bufo arenarum.

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.

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 induces cell proliferation and IGF-I signaling pathway in an estrogen receptor α-dependent manner in MCF-7 breast cancer cell line

Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the biosphere. ERalpha regulates the expression of genes involved in growth and development, and plays an important role in breast cancer. The present study focuses attention on the effect of HCB (0.005, 0.05, 0.5, 5 microM) on cell proliferation in estrogen receptor alpha (ERalpha)(+) MCF-7, and ERalpha(-) MDA-MB-231 breast cancer cell lines. We also studied the insulin growth factor-I (IGF-I) signaling pathway in MCF-7 cells. HCB (0.005 and 0.05 microM) stimulated cell proliferation in MCF-7, but not in MDA-MB-231 cells. The pesticide increased apoptosis in MCF-7, at HCB (0.5 and 5 microM). At these doses, HCB induced the expression of the aryl hydrocarbon receptor (AhR)-regulated gene cytochrome P4501A1. MCF-7 cells exposed to HCB (0.005 and 0.05 microM) overexpressed IGF-IR and insulin receptor (IR). IRS-1-phosphotyrosine content was increased in a dose dependent manner. ICI 182,780 prevented HCB-induced cell proliferation and IGF-I signaling in MCF-7 cells incubated in phenol-red free media. In addition, HCB (0.005 microM) increased c-Src activation, Tyr537-ERalpha phosphorylation and ERalpha down-regulation. Taken together, our data indicate that HCB stimulation of cell proliferation and IGF-I signaling is ERalpha dependent in MCF-7 cells.

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.

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.

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.

An Experimental Model of Diabetes and Cancer in Rats

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.

Suppression of mammary gland tumorigenesis in diabetic rats

The aim of this study was to compare mammary gland tumorigenesis in diabetic and non-diabetic rats. Streptozotocin and N-nitroso-N-methylurea were used to induce diabetes and mammary tumors, respectively. A suppression of mammary carcinogenesis in diabetic rats was shown by a longer latency period, a lower number of tumors per animal and a smaller final tumor volume. An 84% of the lesions developed in diabetic animals were benign tumors. Eighty day-old diabetic rats had significantly lower plasma levels of total-IGF-I and insulin versus non-diabetic rats. We postulate that the decrease in the total IGF-I and insulin levels during the promotion phase of carcinogenesis in this model plays an important role in retarding the tumor development in diabetic animals and in favoring the development of benign mammary lesions.