Endocrine disruptor chlorpyrifos promotes migration, invasion, and stemness phenotype in 3D cultures of breast cancer cells and induces a wide range of pathways involved in cancer progression.

Organophosphorus chlorpyrifos (CPF) is currently considered an endocrine disruptor (ED), as it can imitate hormone actions both in vitro and in vivo. We recently reported that CPF induces migration and invasion in 2D cultures and changes the expression of key molecular markers involved in epithelial mesenchymal transition in MCF-7 and MDA-MB-231 cell lines. In this study, we investigated whether CPF could behave as a predisposing factor for tumors to become more metastatic and aggressive using 3D culture models. In MCF-7 cells, 0.05 µM CPF induced an increase in the number and size of mammospheres via estrogen receptor alpha (ERα) and c-SRC. Furthermore, 0.05 µM CPF increased the area of spheroids generated from MCF-7 cells, induced invasion using both Matrigel® and type 1 collagen matrices, and increased cell migration capacity via ERα in this 3D model. In turn, 50 µM CPF increased cell migration capacity and invasion using type 1 collagen matrix. In monolayers, CPF increased the phosphorylation and membrane translocation of c-SRC at both concentrations assayed. CPF at 0.05 µM boosted p-AKT, p-GSK-3ß and p-P38. While p-AKT rose in a ERα-dependent way, p-GSK-3ß was dependent on ERα- and c-SRC, and p-P38 was only dependent on c-SRC. On the other hand, the increase in p-AKT and p-P38 induced by 50 µM CPF was dependent on the c-SRC pathway. We also observed that 0.05 µM CPF increased IGF-1R and IRS-1 expression and that 50 µM CPF induced IGF-1Rß phosphorylation. In the MDA-MB-231 cell line, 0.05 and 50 µM CPF increased p-c-SRC. Finally, p-AKT and p-GSK-3ß were also induced by CPF at 0.05 and 50 µM, and an increase in p-P38 was observed at 50 µM. Taken together, these data provide support for the notion that CPF may represent a risk factor for breast cancer development and progression.

Chlorpyrifos subthreshold exposure induces epithelial-mesenchymal transition in breast cancer cells.

Chlorpyrifos (CPF) is one of the most frequently used pesticide in extensive agriculture around the world and can be incorporated by humans and animals with possible consequences on health. The effects of this pesticide on carcinogenesis are not clear and there is no consensus concerning the risks of this compound. In previous work, we demonstrated that CPF induces proliferation of breast cancer cells both in vivo and in vitro. In this work we investigate whether CPF promotes the epithelial-mesenchymal transition (EMT) in breast cancer cells. Herein, we demonstrate that 50 µM CFP induces invasion in MCF-7 and MDA-MB-231 cells. In addition, 0.05 and 50 µM CPF increases migration in both cell lines. In MCF-7 cells, 0.05 and 50 µM CPF increase the metalloprotease MMP2 expression and decrease E-Cadherin and ß-Catenin expression diminishing their membrane location. Furthermore, 50 µM CPF induces Vimentin expression and Slug nuclear translocation in MCF-7 cells. 0.05 and 50 µM CPF increase MMP2 gelatinolytic activity and expression, decrease ß-Catenin expression and increase Vimentin expression in MDA-MB-231 cells. Inhibition of the oncoprotein c-Src reverses all the effects induced by CPF in MDA-MB-231 but not in MCF-7 indicating that c-Src is a kinase with a crucial role in the cells which grow in an estrogen-independent way. In MCF-7 cells both c-Src and estrogen receptor alpha must be blocked to completly inhibit the CPF-mediated effects. Our results show for the first time that the exposure to subthreshold concentrations of CPF promotes the modulation of EMT-molecular markers and pathways. These results, together with the ubiquitous distribution of the pesticide CPF, make it of utmost importance to take measures to minimize the risk of exposure to this compound.

Characterization of dendritic cells in testicular draining lymph nodes in a rat model of experimental autoimmune orchitis.

The maturation state of dendritic cells (DC) is regarded as a control point for the induction of peripheral tolerance or autoimmunity. Experimental autoimmune orchitis (EAO) serves as a model to investigate inflammatory-based testicular impairment, which ranks as a significant cause of male infertility. This work aimed to determine whether DC enrichment occurs organotypically in testicular draining lymph nodes (TLN) compared with LN draining the site of immunization (ILN) and thus contributes to the pathogenesis of autoimmune orchitis. In this regard, we quantified and characterized the DC from TLN and ILN in rats with EAO. Flow cytometric analysis showed a significant increase in the percentage of DC (OX62+) only in TLN from EAO rats compared with normal (N) and adjuvant control (C) groups. The number of DC from ILN and TLN expressing CD80, CD86 and major histocompatibility complex (MHC) II was comparable among N, C and experimental (E) groups at 30 and 50 days after the first immunization. However, TLN DC from EAO rats (50 days) showed an increase in mean fluorescence intensity for MHC II compared with N, C and E groups (30 days). The mRNA expression level of IL-10 and IL-12p35 was significantly upregulated in enriched DC fraction from TLN in EAO rats with no significant changes observed in ILN DC. The expression of IL-23p19 mRNA remained unchanged. Functional data, using proliferation assays showed that EAO-DC from TLN, but not from ILN, significantly enhanced the proliferation of naïve T cells compared with C-DC. In summary, our data suggest that the DC in TLN from orchitis rats are mature, present antigens to T cells and stimulate an autoimmune response against testicular antigens, thus causing immunological infertility.