Mariel Núñez

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.

The role of histamine in human mammary carcinogenesis: H3 and H4 receptors as potential therapeutic targets for breast cancer treatment.

There is increasing evidence that describes a histamine role in normal and cancer cell proliferation. To better understand the importance of histamine in breast cancer development, the expression of histamine H3 (H3R) and H4 (H4R) receptors and their association with proliferating cell nuclear antigen (PCNA), histidine decarboxylase (HDC) and histamine content were explored in mammary biopsies. Additionally, we investigated whether H3R and H4R were implicated in the biological responses triggered by histamine in MDA-MB-231 breast cancer cells. The expression levels of H3R, H4R, PCNA, HDC and histamine content were determined by immunohistochemistry in 40 benign and malignant lesions. MDA-MB-231 cells proliferation (clonogenic assay and BrdU incorporation) and cell cycle distribution (flow cytometry) were evaluated upon treatment with histamine, H3R and H4R agonists and antagonists. Apoptosis was determined by Annexin staining and TUNEL assay. Cell migration was assessed by transwell system. Results indicate that H3R was detected in 67% (10/15) of benign lesions and in almost all carcinomas (24/25), being the level of its expression significantly higher in carcinomas (P=0.0016). The non-tumoral breast tissue surrounding carcinomas revealed a lower H3R expression compared to the tumor cells. Only 13% (2/15) of the benign lesions expressed H4R compared to 44% (11/25) of the carcinomas. Interestingly, H3R expression was correlated in carcinomas with the expression of HDC and PCNA (P

Histamine-Mediated Signaling Processes in Human Malignant Mammary Cells

Histamine is a biogenic amine responsible for multiple biological actions including regulation of physiological functions of mammary gland. It has been postulated that histamine plays a critical role in proliferation of normal and cancer cells. To investigate the biological responses that histamine exerts in malignant cells derived from human mammary gland, we evaluated in MDA-MB-231 line the expression of histamine receptors, histamine intracellular content, the capacity of histamine to influence proliferation, cell cycle progression, differentiation and apoptosis. We also studied histamine involvement in cellular response to ionizing radiation. HBL-100 cells were used as control of non-tumorigenic breast cells. Proliferation and surviving fraction were assessed by clonogenic assay. Cell cycle progression and lipid accumulation were determined by flow cytometry while apoptosis was studied by Annexin V and DNA fragmentation assays. Both cell lines expressed the four histamine receptors subtypes as evaluated by western blot and RT-PCR analyses, and present endogenous histamine. Histamine regulated proliferation of cancer cells in a dose-dependent way and 10 μM histamine reduced significantly proliferation to 23% inducing cell cycle arrest in G2/M phase, differentiation by 26% and a significant increase in the number of apoptotic cells (p

Mechanisms underlying the radioprotective effect of histamine on small intestine.

Purpose: To examine the protective effects of histamine on intestinal damage produced by gamma-radiation. Materials and methods: 56 mice were divided into 4 groups. Histamine and Histamine-10 Gy groups received a daily subcutaneous histamine injection (0.1 mg/kg) starting 20 hours before irradiation and continued until the end of the experimental period; the untreated group received saline. Histamine-10 Gy and untreated-10 Gy groups were irradiated with a single dose on whole-body using Cesium-137 source (7 Gy/min) and were sacrificed 3 days after irradiation. Small intestine was removed, fixed and stained with hematoxylin and eosin. The number of intestinal crypts per circumference, and other histological characteristics of intestinal cells were evaluated. We further determined by immunohistochemistry the expression of proliferating cell nuclear antigen (PCNA), Bax, Bcl-2 (pro- and anti-apoptotic protein, respectively), antioxidant enzymes (Superoxide dismutase (SOD), Catalase and Glutathione peroxidase), histamine content and apoptosis by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) assay. Cells in the S phase of the cell cycle were identified by immunohistochemical detection of 5-bromo-2′-deoxyuridine (BrdU) incorporation. Results: Histamine treatment reduced mucosal atrophy, edema and preserved villi, crypts and nuclear and cytoplasmic characteristics of small intestine after radiation exposure. Additionally, histamine treatment increased PCNA expression and the BrdU-positive cell number, histamine content, decreased the number of apoptotic cells and significantly increased Catalase and copper-zinc-containing SOD of irradiated mice. Conclusions: Histamine prevents radiation-induced toxicity by increasing proliferation of damaged intestinal mucosa and suppressing apoptosis that was associated with an increase in SOD and Catalase levels. This effect might be of clinical value in patients undergoing radiotherapy.

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.

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.

Histamine regulates the MAPK pathway via the H(2) receptor in PANC-1 human cells.

The role of histamine (HA) in normal and neoplastic growth has been extensively investigated. We have previously demonstrated that the human pancreatic carcinoma cell line PANC-1 overexpresses H1 and H2 receptors (H1R, H2R). Stimulation of the H2R by high HA concentrations or by specific agonists increases intracellular cAMP levels and inhibits cell growth without inducing apoptosis but producing a partial activation of cell differentiation. On the other hand, cells synthesize and release HA and low concentrations of HA increase cell proliferation suggesting that the H1R could be also involved in the regulation of cell growth [1, 2]. It has been reported that HA modulates the Mitogen Activated Protein Kinase (MAPK) pathway via H1R in normal and tumoral cells [3]. In this work we evaluated the possible regulation of expression of MAPK activity through H2R.

Histamine inhibits proliferation of a pancreatic carcinoma cell line without inducing apoptosis significantly

Previously we demonstrated that human pancreatic carcinoma cell line PANC-1 overexpresses histamine (Hi) H1 and H2 receptors. Both H1 and H2 agonists stimulate phosphoinositide turnover while only H2 agonists increase intracellular cAMP production [1]. Also these cells can synthesize and release Hi and the treatment with Hi, H2 agonists or forskolin produces a significant inhibition on cell proliferation [1]. In this work we used cAMP analogues to confirm the association between inhibition of cell growth and cAMP levels. We also evaluated induction of apoptosis and expression of differentiation markers to investigate the mechanism by which Hi inhibits cell proliferation.

Histamine modulates cellular events involved in tumour invasiveness in pancreatic carcinoma cells.

No Abstract..