S100A8 and S100A9, highly expressed by neutrophils, activated macrophages, and microvascular endothelial cells, are secreted during inflammatory processes. Our earlier studies showed S100A8 to be an avid scavenger of oxidants, and, together with its dependence on IL-10 for expression in macrophages, we postulated that this protein has a protective role. S-nitrosylation is an important posttranslational modification that regulates NO transport, cell signaling, and homeostasis. Relatively few proteins are targets of S-nitrosylation. To date, no inflammation-associated proteins with NO-shuttling capacity have been identified. We used HPLC and mass spectrometry to show that S100A8 and S100A9 were readily S-nitrosylated by NO donors. S-nitrosylated S100A8 (S100A8-SNO) was the preferred nitrosylated product. No S-nitrosylation occurred when the single Cys residue in S100A8 was mutated to Ala. S100A8-SNO in human neutrophils treated with NO donors was confirmed by the biotin switch assay. The stable adduct transnitrosylated hemoglobin, indicating a role in NO transport. S100A8-SNO suppressed mast cell activation by compound 48/80; intravital microscopy was used to demonstrate suppression of leukocyte adhesion and extravasation triggered by compound 48/80 in the rat mesenteric microcirculation. Although S100A8 is induced in macrophages by LPS or IFN-γ, the combination, which activates inducible NO synthase, did not induce S100A8. Thus, the antimicrobial functions of NO generated under these circumstances would not be compromised by S100A8. Our results suggest that S100A8-SNO may regulate leukocyte-endothelial cell interactions in the microcirculation, and suppression of mast cell-mediated inflammation represents an additional anti-inflammatory property for S100A8.

We have shown previously that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stressinduced apoptosis, but resistance can be reversed through activation of caspase-4 by inhibition of the MEK/ERK pathway. We report in this study that apoptosis was induced by the ER stress inducer thapsigargin or tunicamycin via a caspase-8-mediated pathway in the melanoma cell line

Purpose: Lactate dehydrogenase (LDH) levels in blood of patients with melanoma have proven to be an accurate predictor of prognosis and response to some treatments. Exclusion of patients with high LDH levels from many trials of new treatments has created a need for treatments aimed at patients with high LDH levels. This article reviews the metabolic basis for the association of LDH with prognosis and the treatment initiatives that may be successful in this patient group. Experimental Design: Review of current literature on the topic. The growth rates and sites of spread of metastatic melanoma vary widely between patients. In some instances, the disease shows indolent growth rates and limited spread to skin, lymph nodes, or lungs. In other patients, the disease has rapid growth rates and early spread to multiple sites, including internal organs. Several studies have identified lactate dehydrogenase (LDH) levels as the most reliable marker of the more rapidly growing highly metastatic forms of the disease (1, 2). LDH levels also identify melanoma that are resistant to certain forms of treatment (3) and is widely used by sponsors to exclude patients with unfavorable prognosis from clinical trials of new agents. Data from the Genasense study (3) suggest that approximately one third of patients are excluded from trials if a cutoff of >1.2 upper limit of normal is taken as an entry criteria.LDH is coded for by two genes, LDH-A (M-muscle type) and LDH-B (H-heart type), which code for two polypeptide chains that form five isoenzymes depending on the combinations of the two chains. LDH-5 is composed of four M subunits, whereas LDH-1 is composed of four H subunits (4). LDH-5 is the most efficient isotype in catalyzing conversion of pyruvate to lactate (5). LDH-5 expression is readily detected in histologic sections of melanoma and was shown to be a strong correlate of prognosis in primary melanoma. It was not an independent predictor and correlated with the other prognostic determinants of thickness and mitotic rate. Increased levels of LDH within the cell are believed to be due largely to upregulation by the transcription factor hypoxiainducible factor 1α (HIF-1α). The latter is normally degraded by the E3 ligase von Hippel-Lindau protein under normal oxygen levels but not under hypoxic conditions, leading to posttranslational increases in HIF-1α (6). Hypoxia also activates the endoplasmic reticulum (ER) stress response, which indirectly upregulates mRNA for HIF-1α (7) through the transcription factor XBP-1 and Akt activation (8); however, this needs to be confirmed. Hypoglycemia and other factors resulting from the demands of the malignant process also contribute to ER stress and the resultant unfolded protein response (9-11).One of the consequences of ER stress is a change in metabolism of the cell due to activation of HIF-1α and activation of the Akt pathway. These changes are believed to result in reduced mitochondrial-dependent oxidation of glucose by cancer cells referred to as the Warburg effect (12). As ...

Phenoxodiol is a chemically modified analogue of the plant hormone isoflavone with antitumour activities. In the present study, we have examined its ability to induce apoptosis in human melanoma cells and the mechanisms involved. Apoptosis was observed in Phenoxodiol-treated cells by using annexin V/propidium iodide staining and determining mitochondrial membrane potential. To determine which caspase pathways were involved in Phenoxodiol-induced apoptosis, studies were performed using specific caspase inhibitors. Western studies were performed to ascertain which proteins of the apoptosis cascade were affected to cause Phenoxodiol-induced apoptosis. We found that induction of apoptosis by Phenoxodiol was maximal at 48 h with a range of apoptosis of 12+/-4 to 48+/-5% in different melanoma lines. This apoptosis was mainly dependent on activation of caspase-3 and caspase-9. Apoptosis was associated with induction of changes in mitochondrial membrane potential and was inhibited by over-expression of Bcl-2. Variation in sensitivity to Phenoxodiol appeared related to events upstream of the mitochondria and the degree of conformational change in Bax. The p53-regulated BH3-only proteins (Bad, PUMA and Noxa) were increased in the sensitive, but not in the resistant lines, whereas Bim was increased in all the lines tested. Bim appeared, however, to be partially involved because reduction of Bim by RNA interference resulted in decreased levels of apoptosis. Together, these studies suggest that Phenoxodiol induces apoptosis of melanoma cells by induction of p53-dependent BH3 proteins (Bad, PUMA and Noxa) and the p53-independent Bim protein, resulting in activation of Bax and its downstream events.