J. Neurochem. (2008) 104, 1599–1612. Mitochondrial alterations have been associated with the cytotoxic effect of 6‐hydroxydopamine (6‐OHDA), a widely used toxin to study Parkinson’s disease. In previous work, we have demonstrated that 6‐OHDA increases mitochondrial membrane permeability leading to cytochrome c release, but the precise mechanisms involved in this process remain unknown. Herein we studied the mechanism of increased mitochondrial permeability of SH‐SY5Y neuroblastoma cells in response to 6‐OHDA. Cytochrome c release induced by 6‐OHDA occurred, in both SH‐SY5Y cells and primary cultures, in the absence of mitochondrial swelling or a decrease in mitochondrial calcein fluorescence, suggesting little involvement of the mitochondrial permeability transition pore in this process. In contrast, 6‐OHDA‐induced cell death was associated with a significant translocation of the pro‐apoptotic Bax protein from the cytosol to mitochondria and with a significant induction of the BH3‐only protein PUMA. Experiments in mouse embryonic fibroblasts deficient in Bax or PUMA demonstrated a role for both proteins in 6‐OHDA‐induced apoptosis. Although 6‐OHDA elevated both total and nuclear p53 protein levels, activation of p53 was not essential for subsequent cell death. In contrast, we found that p38 mitogen‐activated protein kinase (MAPK) was activated early during 6‐OHDA‐induced apoptosis, and that treatment with the p38 MAPK inhibitor SKF86002 potently inhibited PUMA induction, green fluorescent protein‐Bax redistribution and apoptosis in response to 6‐OHDA. These data demonstrate a critical involvement of p38 MAPK, PUMA, and Bax in 6‐OHDA‐induced apoptosis.

Malonate, an inhibitor of mitochondrial complex II, is a widely used toxin to study neurodegeneration in Huntington's disease and ischemic stroke. We have shown previously that malonate increased reactive oxygen species (ROS) production in human SH-SY5Y neuroblastoma cells, leading to oxidative stress, cytochrome c release, and apoptotic cell death. Expression of a green fluorescent protein-Bax fusion protein in SH-SY5Y neuroblastoma cells demonstrated a Bax redistribution from the cytosol to mitochondria after 12 to 24 h of malonate treatment that coincided with mitochondrial potential collapse and chromatin condensation. Inhibition of Bax translocation using furosemide, as well as Bax gene deletion, afforded significant protection against malonate-induced apoptosis. Further experiments revealed that malonate induced a prominent increase in the level of activated p38 mitogen-activated protein (MAP) kinase and that treatment with the p38 MAP kinase inhibitor SKF86002 potently blocked malonate-induced Bax translocation and apoptosis. Treatment with vitamin E diminished ROS production, reduced the activation status of p38 MAP kinase, inhibited Bax translocation, and protected against malonate-induced apoptosis. Our data suggest that malonate-induced ROS production and subsequent p38 MAP kinase activation mediates the activation of the pro-apoptotic Bax protein to induce mitochondrial membrane permeabilization and neuronal apoptosis.

While the deregulation of iron homeostasis in breast epithelial cells is acknowledged, iron-related alterations in stromal inflammatory cells from the tumor microenvironment have not been explored. Immunohistochemistry for hepcidin, ferroportin 1 (FPN1), transferrin receptor 1 (TFR1) and ferritin (FT) was performed in primary breast tissues and axillary lymph nodes in order to dissect the iron-profiles of epithelial cells, lymphocytes and macrophages. Furthermore, breast carcinoma core biopsies frozen in optimum cutting temperature (OCT) compound were subjected to imaging flow cytometry to confirm FPN1 expression in the cell types previously evaluated and determine its cellular localization. We confirm previous results by showing that breast cancer epithelial cells present an ‘iron-utilization phenotype’ with an increased expression of hepcidin and TFR1, and decreased expression of FT. On the other hand, lymphocytes and macrophages infiltrating primary tumors and from metastized lymph nodes display an ‘iron-donor’ phenotype, with increased expression of FPN1 and FT, concomitant with an activation profile reflected by a higher expression of TFR1 and hepcidin. A higher percentage of breast carcinomas, compared to control mastectomy samples, present iron accumulation in stromal inflammatory cells, suggesting that these cells may constitute an effective tissue iron reservoir. Additionally, not only the deregulated expression of iron-related proteins in epithelial cells, but also on lymphocytes and macrophages, are associated with clinicopathological markers of breast cancer poor prognosis, such as negative hormone receptor status and tumor size. The present results reinforce the importance of analyzing the tumor microenvironment in breast cancer, extending the contribution of immune cells to local iron homeostasis in the tumor microenvironment context.