Maresin conjugates in tissue regeneration biosynthesis enzymes in human macrophages.

Macrophages are central in coordinating immune responses, tissue repair, and regeneration, with different subtypes being associated with inflammation-initiating and proresolving actions. We recently identified a family of macrophage-derived proresolving and tissue regenerative molecules coined maresin conjugates in tissue regeneration (MCTR). Herein, using lipid mediator profiling we identified MCTR in human serum, lymph nodes, and plasma and investigated MCTR biosynthetic pathways in human macrophages. With human recombinant enzymes, primary cells, and enantiomerically pure compounds we found that the synthetic maresin epoxide intermediate 13S,14S-eMaR (13S,14S-epoxy- 4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid) was converted to MCTR1 (13R-glutathionyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) by LTC4S and GSTM4. Incubation of human macrophages with LTC4S inhibitors blocked LTC4 and increased resolvins and lipoxins. The conversion of MCTR1 to MCTR2 (13R-cysteinylglycinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) was catalyzed by γ-glutamyl transferase (GGT) in human macrophages. Biosynthesis of MCTR3 was mediated by dipeptidases that cleaved the cysteinyl-glycinyl bond of MCTR2 to give 13R-cysteinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid. Of note, both GSTM4 and GGT enzymes displayed higher affinity to 13S,14S-eMaR and MCTR1 compared with their classic substrates in the cysteinyl leukotriene metabolome. Together these results establish the MCTR biosynthetic pathway and provide mechanisms in tissue repair and regeneration.

Resolvin D3 and aspirin-triggered resolvin D3 are potent immunoresolvents.

Resolvins are a family of n-3 lipid mediators initially identified in resolving inflammatory exudates that temper inflammatory responses to promote catabasis. Here, temporal metabololipidomics with self-limited resolving exudates revealed that resolvin (Rv) D3 has a distinct time frame from other lipid mediators, appearing late in the resolution phase. Using synthetic materials prepared by stereocontrolled total organic synthesis and metabololipidomics, we established complete stereochemistry of RvD3 and its aspirin-triggered 17R-epimer (AT-RvD3). Both synthetic resolvins potently regulated neutrophils and mediators, reducing murine peritonitis and dermal inflammation. RvD3 and AT-RvD3 displayed leukocyte-directed actions, e.g., blocking human neutrophil transmigration and enhancing macrophage phagocytosis and efferocytosis. These results position RvD3 uniquely within the inflammation-resolution time frame to vantage and contribute to the beneficial actions of aspirin and essential n-3 fatty acids.

Preclinical development of novel anti-glioma drugs targeting the endoplasmic reticulum stress response.

The endoplasmic reticulum (ER) stress response represents a cellular "yin-yang" process, where low to moderate activity is cell protective and supports chemoresistance (yang), but where more severe conditions will aggravate these mechanisms to the point where they abandon their protective efforts and instead turn on a cell death program (yin). Because tumor cells frequently experience chronic stress conditions (due to hypoxia, hypoglycemia, acidification, etc.), the protective yang components of their ER stress response are continuously engaged and thus less able to neutralize additional insults taxing the ER stress response. This tumor-specific situation may provide therapeutic opportunities for pharmacologic intervention, where further aggravation of ER stress would lead to the activation of pro-apoptotic yin components and result in tumor cell death. This review will describe the yin-yang principle of ER stress, and will present pharmacologic agents and combination strategies aimed at exploiting the ER stress response for improved therapeutic outcomes, particularly in the setting of difficult to treat tumor types such as glioblastoma.

Discovery and preclinical evaluation of a novel class of cytotoxic propynoic acid carbamoyl methyl amides (PACMAs).

Herein, we discovered a series of propynoic acid carbamoyl methyl-amides (PACMAs) with potent cytotoxicity against a panel of cancer cell lines. These compounds interrupted cell cycle progression at low micromolar concentrations and induced early and late stage apoptosis. A representative compound suppressed tumor growth without apparent toxicity in an MDA-MB-435 mouse xenograft model. We used a Kinexus 628-antibody microarray and the Ingenuity Pathway Analysis (IPA) bioinformatics tools to better understand their mechanisms. The IPA analysis revealed the initiation of Nrf2-mediated oxidative stress through modulating the expression of SOD1 and STIP1 by compound 1. The involvement of the oxidative stress pathway was further validated by measuring the levels of the PACMA-induced mitochondrial superoxide species. To our knowledge, this is the first report on the discovery and biological evaluations of PACMAs as anticancer agents. Their broad-spectrum in vitro cytotoxicity, possibly through an oxidative stress-mediated pathway, and in vivo efficacy warrant further preclinical investigations.

Anti-inflammatory and pro-resolving properties of benzo-lipoxin A(4) analogs.

Lipoxins (LXs) are potent endogenous counter-regulatory lipid mediators that dampen acute inflammation and promote its resolution. Here, we present our investigation of a new class of thermally and metabolically stable benzo-LXA(4) analogs that are potently anti-inflammatory and easier to synthesize. Replacement of the tetraene unit of native LXA(4) with a benzo-fused ring system not only increases the thermal stability but also enables highly convergent and efficient syntheses of these analogs. In addition, they resist rapid catalysis and inactivation by eicosanoid oxidoreductase. Like native LXs, o-[9, 12]-benzo-omega6-epi-LXA(4), o-[9, 12]-benzo-deoxy-LXA(4), m-[9, 12]-benzo-omega6-epi-LXA(4) and [9, 14]-benzo-omega6-(R/S)-LXA(4) demonstrated potent time-dependent reduction, at nanogram dosages, of PMN infiltration and pro-inflammatory cytokine generation in vivo in murine peritonitis and were organ protective in hind limb ischemia-reperfusion injury of the lung. The o-[9, 12]-benzo-omega6-epi-LXA(4) and m-[9, 12]-benzo-omega6-epi-LXA(4) were most potent in nanogram doses; both decreased PMN infiltration by approximately 32%, while o-[9, 12]-benzo-deoxy-LXA(4) and [9, 15]-omega6-(R/S)-LXA(4) were less potent. The [9,12]-benzo-omega6-epi-LXA(4) also activated a lipoxin A(4) GPCR and increased macrophage phagocytic activity. Taken together, these findings demonstrate a new generation of LXA(4) stable analogs that are easy to synthesize and anti-inflammatory. These benzo-LXA(4) analogs are promising tools for new therapeutic approaches as well as assessing endogenous mechanisms in anti-inflammation and resolution.

Green tea polyphenols block the anticancer effects of bortezomib and other boronic acid-based proteasome inhibitors.

The anticancer potency of green tea and its individual components is being intensely investigated, and some cancer patients already self-medicate with this "miracle herb" in hopes of augmenting the anticancer outcome of their chemotherapy. Bortezomib (BZM) is a proteasome inhibitor in clinical use for multiple myeloma. Here, we investigated whether the combination of these compounds would yield increased antitumor efficacy in multiple myeloma and glioblastoma cell lines in vitro and in vivo. Unexpectedly, we discovered that various green tea constituents, in particular (-)-epigallocatechin gallate (EGCG) and other polyphenols with 1,2-benzenediol moieties, effectively prevented tumor cell death induced by BZM in vitro and in vivo. This pronounced antagonistic function of EGCG was evident only with boronic acid-based proteasome inhibitors (BZM, MG-262, PS-IX), but not with several non-boronic acid proteasome inhibitors (MG-132, PS-I, nelfinavir). EGCG directly reacted with BZM and blocked its proteasome inhibitory function; as a consequence, BZM could not trigger endoplasmic reticulum stress or caspase-7 activation, and did not induce tumor cell death. Taken together, our results indicate that green tea polyphenols may have the potential to negate the therapeutic efficacy of BZM and suggest that consumption of green tea products may be contraindicated during cancer therapy with BZM.

Rapid appearance of resolvin precursors in inflammatory exudates: novel mechanisms in resolution.

Resolution of inflammation is essential. Although supplementation of omega-3 fatty acids is widely used, their availability at sites of inflammation is not known. To this end, a multidisciplinary approach was taken to determine the relationship of circulating omega-3 to inflammatory exudates and the generation of resolution signals. In this study, we monitored resolvin precursors in evolving exudates, which initially paralleled increases in edema and infiltrating neutrophils. We also prepared novel microfluidic chambers to capture neutrophils from a drop of blood within minutes that permitted single-cell monitoring. In these, docosahexaenoic acid-derived resolvin D1 rapidly stopped neutrophil migration, whereas precursor docosahexaenoic acid did not. In second organ injury via ischemia-reperfusion, resolvin metabolically stable analogues were potent organ protectors reducing neutrophils. Together, these results indicate that circulating omega-3 fatty acids rapidly appear in inflammatory sites that require conversion to resolvins that control excessive neutrophil infiltration, protect organs, and foster resolution.

Celecoxib analogs that lack COX-2 inhibitory function: preclinical development of novel anticancer drugs.

Celecoxib is an NSAID that was developed as a selective inhibitor of COX-2 and approved by the FDA for the treatment of various forms of arthritis and the management of acute or chronic pain. In addition, it was more recently approved as an oral adjunct to prevent colon cancer development in patients with familial adenomatous polyposis and is presently being investigated for its chemotherapeutic potential in the therapy of advanced cancers. However, in laboratory studies it was discovered that celecoxib was able to suppress tumor growth in the absence of any apparent involvement of COX-2, and additional pharmacologic activities associated with this drug were found. Intriguingly, the two pharmacologic effects, inhibition of COX-2 and suppression of tumor growth, were found to reside in different structural aspects of the celecoxib molecule and, therefore, could be separated. This dualism enabled the synthesis of close structural analogs of celecoxib that exhibited increased antitumor potency in the absence of COX-2 inhibition. In theory, such compounds should be superior to celecoxib for antitumor purposes because they might reduce gastrointestinal and cardiovascular risks and the life-threatening side effects that appear during the long-term use of selective COX-2 inhibitors. In this review, the authors present the status of preclinical development of anticancer analogs of celecoxib that are COX-2 inactive, with an emphasis on 2,5-dimethyl-celecoxib (DMC) and OSU-03012.

Stereochemical assignment, antiinflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1.

The essential fatty acid eicosapentaenoic acid (EPA) present in fish oils displays beneficial effects in a range of human disorders associated with inflammation including cardiovascular disease. Resolvin E1 (RvE1), a new bioactive oxygenated product of EPA, was identified in human plasma and prepared by total organic synthesis. Results of bioaction and physical matching studies indicate that the complete structure of RvE1 is 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-EPA. At nanomolar levels, RvE1 dramatically reduced dermal inflammation, peritonitis, dendritic cell (DC) migration, and interleukin (IL) 12 production. We screened receptors and identified one, denoted earlier as ChemR23, that mediates RvE1 signal to attenuate nuclear factor-kappaB. Specific binding of RvE1 to this receptor was confirmed using synthetic [(3)H]-labeled RvE1. Treatment of DCs with small interference RNA specific for ChemR23 sharply reduced RvE1 regulation of IL-12. These results demonstrate novel counterregulatory responses in inflammation initiated via RvE1 receptor activation that provide the first evidence for EPA-derived potent endogenous agonists of antiinflammation.