Mcl-1-mediated mitochondrial fission protects against stress but impairs cardiac adaptation to exercise.

Myeloid cell leukemia-1 (Mcl-1) is a structurally and functionally unique anti-apoptotic Bcl-2 protein. While elevated levels of Mcl-1 contribute to tumor cell survival and drug resistance, loss of Mcl-1 in cardiac myocytes leads to rapid mitochondrial dysfunction and heart failure development. Although Mcl-1 is an anti-apoptotic protein, previous studies indicate that its functions extend beyond regulating apoptosis. Mcl-1 is localized to both the mitochondrial outer membrane and matrix. Here, we have identified that Mcl-1 in the outer mitochondrial membrane mediates mitochondrial fission, which is independent of its anti-apoptotic function. We demonstrate that Mcl-1 interacts with Drp1 to promote mitochondrial fission in response to various challenges known to perturb mitochondria morphology. Induction of fission by Mcl-1 reduces nutrient deprivation-induced cell death and the protection is independent of its BH3 domain. Finally, cardiac-specific overexpression of Mcl-1OM, but not Mcl-1Matrix, contributes to a shift in the balance towards fission and leads to reduced exercise capacity, suggesting that a pre-existing fragmented mitochondrial network leads to decreased ability to adapt to an acute increase in workload and energy demand. Overall, these findings highlight the importance of Mcl-1 in maintaining mitochondrial health in cells.

Staying young at heart: autophagy and adaptation to cardiac aging.

Aging is a predominant risk factor for developing cardiovascular disease. Therefore, the cellular processes that contribute to aging are attractive targets for therapeutic interventions that can delay or prevent the development of age-related diseases. Our understanding of the underlying mechanisms that contribute to the decline in cell and tissue functions with age has greatly advanced over the past decade. Classical hallmarks of aging cells include increased levels of reactive oxygen species, DNA damage, accumulation of dysfunctional organelles, oxidized proteins and lipids. These all contribute to a progressive decline in the normal physiological function of the cell and to the onset of age-related conditions. A major cause of the aging process is progressive loss of cellular quality control. Autophagy is an important quality control pathway and is necessary to maintain cardiac homeostasis and to adapt to stress. A reduction in autophagy has been observed in a number of aging models and there is compelling evidence that enhanced autophagy delays aging and extends life span. Enhancing autophagy counteracts age-associated accumulation of protein aggregates and damaged organelles in cells. In this review, we discuss the functional role of autophagy in maintaining homeostasis in the heart, and how a decline is associated with accelerated cardiac aging. We also evaluate therapeutic approaches being researched in an effort to maintain a healthy young heart.

Novel sorafenib-based structural analogues: in-vitro anticancer evaluation of t-MTUCB and t-AUCMB.

In the current work, we carried out a mechanistic study on the cytotoxicity of two compounds, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-N-methyl-benzamide (t-AUCMB) and trans-N-methyl-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzamide (t-MTUCB), that are structurally similar to sorafenib. These compounds show strong cytotoxic responses in various cancer cell lines, despite significant differences in the induction of apoptotic events such as caspase activation and lactate dehydrogenase release in hepatoma cells. Both compounds induce autophagosome formation and LC3I cleavage, but there was little observable effect on mTORC1 or the downstream targets, S6K1 and 4E-binding protein. In addition, there was an increase in the activity of upstream signaling through the IRS1/PI3K/Akt-signaling pathway, suggesting that, unlike sorafenib, both compounds induce mammalian target of rapamycin (mTOR)-independent autophagy. The autophagy observed correlates with mitochondrial membrane depolarization, apoptosis-inducing factor release, and oxidative stress-induced glutathione depletion. However, there were no observable changes in the endoplasmic reticulum-stress markers such as binding immunoglobulin protein, inositol-requiring enzyme-α, phosphorylated eukaryotic initiation factor 2, and the lipid peroxidation marker, 4-hydroxynonenal, suggesting endoplasmic reticulum-independent oxidative stress. Finally, these compounds do not have the multikinase inhibitory activity of sorafenib, which may be reflected in their difference in the ability to halt cell cycle progression compared with sorafenib. Our findings indicate that both compounds have anticancer effects comparable with sorafenib in multiple cell lines, but they induce significant differences in apoptotic responses and appear to induce mTOR-independent autophagy. t-AUCMB and t-MTUCB represent novel chemical probes that are capable of inducing mTOR-independent autophagy and apoptosis to differing degrees, and may thus be potential tools for further understanding the link between these two cellular stress responses.

Structure-Based Optimization of a Series of Covalent, Cell Active Bfl-1 Inhibitors.

Bfl-1, a member of the Bcl-2 family of proteins, plays a crucial role in apoptosis regulation and has been implicated in cancer cell survival and resistance to venetoclax therapy. Due to the unique cysteine residue in the BH3 binding site, the development of covalent inhibitors targeting Bfl-1 represents a promising strategy for cancer treatment. Herein, the optimization of a covalent cellular tool from a lead-like hit using structure based design is described. Informed by a reversible X-ray fragment screen, the strategy to establish interactions with a key glutamic acid residue (Glu78) and optimize binding in a cryptic pocket led to a 1000-fold improvement in biochemical potency without increasing reactivity of the warhead. Compound (R,R,S)-26 has a kinact/KI of 4600 M-1 s-1, shows <1 µM caspase activation in a cellular assay and cellular target engagement, and has good physicochemical properties and a promising in vivo profile.

5-Benzylglycinyl-amiloride kills proliferating and nonproliferating malignant glioma cells through caspase-independent necroptosis mediated by apoptosis-inducing factor.

5'-Βenzylglycinyl-amiloride (UCD38B) and glycinyl-amiloride (UCD74A) are cell-permeant and cell-impermeant derivatives of amiloride, respectively, and used here to identify the cellular mechanisms of action underlying their antiglioma effects. UCD38B comparably kills proliferating and nonproliferating gliomas cells when cell cycle progression is arrested either by cyclin D1 siRNA or by acidification. Cell impermeant UCD74A inhibits plasmalemmal urokinase plasminogen activator (uPA) and the type 1 sodium-proton exchanger with potencies analogous to UCD38B, but is cytostatic. In contrast, UCD38B targets intracellular uPA causing mistrafficking of uPA into perinuclear mitochondria, reducing the mitochondrial membrane potential, and followed by the release of apoptotic inducible factor (AIF). AIF nuclear translocation is followed by a caspase-independent necroptotic cell death. Reduction in AIF expression by siRNA reduces the antiglioma cytotoxic effects of UCD38B, while not activating the caspase pathway. Ultrastructural changes shortly following treatment with UCD38B demonstrate dilation of endoplasmic reticulum (ER) and mitochondrial swelling followed by nuclear condensation within hours consistent with a necroptotic cell death differing from apoptosis and from autophagy. These drug mechanism of action studies demonstrate that UCD38B induces a cell cycle-independent, caspase-independent necroptotic glioma cell death that is mediated by AIF and independent of poly (ADP-ribose) polymerase and H2AX activation.

Prognostic factors for recurrence in patients with papillary thyroid carcinoma.

BACKGROUND: The prognostic factors for tumor recurrence and mortality of patients diagnosed with Papillary Thyroid Carcinoma (PTC) with immediate surgery in Colombia has not been reported. OBJECTIVE: To retrospectively evaluate the risk factors for recurrence and survival at 10 years in patients with the diagnosis of PTC treated at Fundación Santa Fe deBogota (FSFB). METHODS: A total of 486 patients with thyroid surgery accompanied by medical follow-up were recruited. Demographic, clinical, and pathological variables were followed-up for a median period of 10 years. RESULTS: The most significant variables for recurrence were tumors with > 4 cm of size (hazard ratio [HR] = 8.1; 95% confidence interval [CI] = 1.7-55) and extrathyroidal spread (HR = 26.7; 95% CI = 3.1-228). CONCLUSION: PTC in our population has low rates of mortality (0.6%) and recurrence (9.6%), with an average time of recurrence of 3 years. Size of the lesion, positive surgical margins, extrathyroidal spread, and high postoperative serum thyroglobulin (Tg) level act as prognostic factors that determine the likelihood of recurrence. Unlike other studies, the influence of age and gender does not act as a prognostic factor.

The Small GTPase Rab7 Regulates Release of Mitochondria in Extracellular Vesicles in Response to Lysosomal Dysfunction.

Mitochondrial quality control is critical for cardiac homeostasis as these organelles are responsible for generating most of the energy needed to sustain contraction. Dysfunctional mitochondria are normally degraded via intracellular degradation pathways that converge on the lysosome. Here, we identified an alternative mechanism to eliminate mitochondria when lysosomal function is compromised. We show that lysosomal inhibition leads to increased secretion of mitochondria in large extracellular vesicles (EVs). The EVs are produced in multivesicular bodies, and their release is independent of autophagy. Deletion of the small GTPase Rab7 in cells or adult mouse heart leads to increased secretion of EVs containing ubiquitinated cargos, including intact mitochondria. The secreted EVs are captured by macrophages without activating inflammation. Hearts from aged mice or Danon disease patients have increased levels of secreted EVs containing mitochondria indicating activation of vesicular release during cardiac pathophysiology. Overall, these findings establish that mitochondria are eliminated in large EVs through the endosomal pathway when lysosomal degradation is inhibited.

Mitochondria are secreted in extracellular vesicles when lysosomal function is impaired.

Mitochondrial quality control is critical for cardiac homeostasis as these organelles are responsible for generating most of the energy needed to sustain contraction. Dysfunctional mitochondria are normally degraded via intracellular degradation pathways that converge on the lysosome. Here, we identified an alternative mechanism to eliminate mitochondria when lysosomal function is compromised. We show that lysosomal inhibition leads to increased secretion of mitochondria in large extracellular vesicles (EVs). The EVs are produced in multivesicular bodies, and their release is independent of autophagy. Deletion of the small GTPase Rab7 in cells or adult mouse heart leads to increased secretion of EVs containing ubiquitinated cargos, including intact mitochondria. The secreted EVs are captured by macrophages without activating inflammation. Hearts from aged mice or Danon disease patients have increased levels of secreted EVs containing mitochondria indicating activation of vesicular release during cardiac pathophysiology. Overall, these findings establish that mitochondria are eliminated in large EVs through the endosomal pathway when lysosomal degradation is inhibited.

Cardiolipin Remodeling Defects Impair Mitochondrial Architecture and Function in a Murine Model of Barth Syndrome Cardiomyopathy.

BACKGROUND: Cardiomyopathy is a major clinical feature in Barth syndrome (BTHS), an X-linked mitochondrial lipid disorder caused by mutations in Tafazzin (TAZ), encoding a mitochondrial acyltransferase required for cardiolipin remodeling. Despite recent description of a mouse model of BTHS cardiomyopathy, an in-depth analysis of specific lipid abnormalities and mitochondrial form and function in an in vivo BTHS cardiomyopathy model is lacking. METHODS: We performed in-depth assessment of cardiac function, cardiolipin species profiles, and mitochondrial structure and function in our newly generated Taz cardiomyocyte-specific knockout mice and Cre-negative control mice (n≥3 per group). RESULTS: Taz cardiomyocyte-specific knockout mice recapitulate typical features of BTHS and mitochondrial cardiomyopathy. Fewer than 5% of cardiomyocyte-specific knockout mice exhibited lethality before 2 months of age, with significantly enlarged hearts. More than 80% of cardiomyocyte-specific knockout displayed ventricular dilation at 16 weeks of age and survived until 50 weeks of age. Full parameter analysis of cardiac cardiolipin profiles demonstrated lower total cardiolipin concentration, abnormal cardiolipin fatty acyl composition, and elevated monolysocardiolipin to cardiolipin ratios in Taz cardiomyocyte-specific knockout, relative to controls. Mitochondrial contact site and cristae organizing system and F1F0-ATP synthase complexes, required for cristae morphogenesis, were abnormal, resulting in onion-shaped mitochondria. Organization of high molecular weight respiratory chain supercomplexes was also impaired. In keeping with observed mitochondrial abnormalities, seahorse experiments demonstrated impaired mitochondrial respiration capacity. CONCLUSIONS: Our mouse model mirrors multiple physiological and biochemical aspects of BTHS cardiomyopathy. Our results give important insights into the underlying cause of BTHS cardiomyopathy and provide a framework for testing therapeutic approaches to BTHS cardiomyopathy, or other mitochondrial-related cardiomyopathies.

COVID-19 spread, detection, and dynamics in Bogota, Colombia.

Latin America has been severely affected by the COVID-19 pandemic but estimations of rates of infections are very limited and lack the level of detail required to guide policy decisions. We implemented a COVID-19 sentinel surveillance study with 59,770 RT-PCR tests on mostly asymptomatic individuals and combine this data with administrative records on all detected cases to capture the spread and dynamics of the COVID-19 pandemic in Bogota from June 2020 to early March 2021. We describe various features of the pandemic that appear to be specific to a middle income countries. We find that, by March 2021, slightly more than half of the population in Bogota has been infected, despite only a small fraction of this population being detected. The initial buildup of immunity contributed to the containment of the pandemic in the first and second waves. We also show that the share of the population infected by March 2021 varies widely by occupation, socio-economic stratum, and location. This, in turn, has affected the dynamics of the spread with different groups being infected in the two waves.

Isolation of Rab5-positive endosomes reveals a new mitochondrial degradation pathway utilized by BNIP3 and Parkin.

Degradation of mitochondria is an important cellular quality control mechanism mediated by two distinct pathways: one involving Parkin-mediated ubiquitination and the other dependent on mitophagy receptors. It is known that mitochondria are degraded by the autophagy pathway; however, we recently reported that the small GTPase Rab5 and early endosomes also participate in Parkin-mediated mitochondrial clearance. Here, we have developed a protocol to isolate Rab5-positive vesicles from cells for proteomics analysis and provide additional data confirming that mitophagy regulators and mitochondrial proteins are present in these vesicles. We also demonstrate that the mitophagy receptor BNIP3 utilizes the Rab5-endosomal pathway to clear mitochondria in cells. These findings indicate that a redundancy exists in the downstream degradation pathways to ensure efficient mitochondrial clearance.

Nuclear Parkin Activates the ERRα Transcriptional Program and Drives Widespread Changes in Gene Expression Following Hypoxia.

Parkin is an E3 ubiquitin ligase well-known for facilitating clearance of damaged mitochondria by ubiquitinating proteins on the outer mitochondrial membrane. However, knowledge of Parkin's functions beyond mitophagy is still limited. Here, we demonstrate that Parkin has functions in the nucleus and that Parkinson's disease-associated Parkin mutants, ParkinR42P and ParkinG430D, are selectively excluded from the nucleus. Further, Parkin translocates to the nucleus in response to hypoxia which correlates with increased ubiquitination of nuclear proteins. The serine-threonine kinase PINK1 is responsible for recruiting Parkin to mitochondria, but translocation of Parkin to the nucleus occurs independently of PINK1. Transcriptomic analyses of HeLa cells overexpressing wild type or a nuclear-targeted Parkin revealed that during hypoxia, Parkin contributes to both increased and decreased transcription of genes involved in regulating multiple metabolic pathways. Furthermore, a proteomics screen comparing ubiquitinated proteins in hearts from Parkin-/- and Parkin transgenic mice identified the transcription factor estrogen-related receptor α (ERRα) as a potential Parkin target. Co-immunoprecipitation confirmed that nuclear-targeted Parkin interacts with and ubiquitinates ERRα. Further analysis uncovered that nuclear Parkin increases the transcriptional activity of ERRα. Overall, our study supports diverse roles for Parkin and demonstrates that nuclear Parkin regulates transcription of genes involved in multiple metabolic pathways.

BNIP3L/NIX and FUNDC1-mediated mitophagy is required for mitochondrial network remodeling during cardiac progenitor cell differentiation.

Cell-based therapies represent a very promising strategy to repair and regenerate the injured heart to prevent progression to heart failure. To date, these therapies have had limited success due to a lack of survival and retention of the infused cells. Therefore, it is important to increase our understanding of the biology of these cells and utilize this information to enhance their survival and function in the injured heart. Mitochondria are critical for progenitor cell function and survival. Here, we demonstrate the importance of mitochondrial autophagy, or mitophagy, in the differentiation process in adult cardiac progenitor cells (CPCs). We found that mitophagy was rapidly induced upon initiation of differentiation in CPCs. We also found that mitophagy was mediated by mitophagy receptors, rather than the PINK1-PRKN/PARKIN pathway. Mitophagy mediated by BNIP3L/NIX and FUNDC1 was not involved in regulating progenitor cell fate determination, mitochondrial biogenesis, or reprogramming. Instead, mitophagy facilitated the CPCs to undergo proper mitochondrial network reorganization during differentiation. Abrogating BNIP3L- and FUNDC1-mediated mitophagy during differentiation led to sustained mitochondrial fission and formation of donut-shaped impaired mitochondria. It also resulted in increased susceptibility to cell death and failure to survive the infarcted heart. Finally, aging is associated with accumulation of mitochondrial DNA (mtDNA) damage in cells and we found that acquiring mtDNA mutations selectively disrupted the differentiation-activated mitophagy program in CPCs. These findings demonstrate the importance of BNIP3L- and FUNDC1-mediated mitophagy as a critical regulator of mitochondrial network formation during differentiation, as well as the consequences of accumulating mtDNA mutations. Abbreviations: Baf: bafilomycin A1; BCL2L13: BCL2 like 13; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CPCs: cardiac progenitor cells; DM: differentiation media; DNM1L: dynamin 1 like; EPCs: endothelial progenitor cells; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FUNDC1: FUN14 domain containing 1; HSCs: hematopoietic stem cells; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MFN1/2: mitofusin 1/2; MSCs: mesenchymal stem cells; mtDNA: mitochondrial DNA; OXPHOS: oxidative phosphorylation; PPARGC1A: PPARG coactivator 1 alpha; PHB2: prohibitin 2; POLG: DNA polymerase gamma, catalytic subunit; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester.

A Rab5 endosomal pathway mediates Parkin-dependent mitochondrial clearance.

Damaged mitochondria pose a lethal threat to cells that necessitates their prompt removal. The currently recognized mechanism for disposal of mitochondria is autophagy, where damaged organelles are marked for disposal via ubiquitylation by Parkin. Here we report a novel pathway for mitochondrial elimination, in which these organelles undergo Parkin-dependent sequestration into Rab5-positive early endosomes via the ESCRT machinery. Following maturation, these endosomes deliver mitochondria to lysosomes for degradation. Although this endosomal pathway is activated by stressors that also activate mitochondrial autophagy, endosomal-mediated mitochondrial clearance is initiated before autophagy. The autophagy protein Beclin1 regulates activation of Rab5 and endosomal-mediated degradation of mitochondria, suggesting cross-talk between these two pathways. Abrogation of Rab5 function and the endosomal pathway results in the accumulation of stressed mitochondria and increases susceptibility to cell death in embryonic fibroblasts and cardiac myocytes. These data reveal a new mechanism for mitochondrial quality control mediated by Rab5 and early endosomes.

Spontaneous inflammatory cytokine gene expression in normal human peripheral blood mononuclear cells.

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Measurement of cytokine levels has yielded useful information on the pathological process of different diseases such as AIDS, endotoxic shock, sepsis, asthma, and cancer. It may also be of use in the monitoring of disease progression and/or inflammation. To determine spontaneous cytokine gene expression in whole blood and PBMCs, whole blood was obtained from healthy volunteers and total mRNA was isolated from PBMCs. The kinetics of response were determined by sequential testing of cytokine gene expression by RT-PCR analysis. Our results demonstrated that isolated and incubated PBMCs expressed TNF-alpha and high levels of IL-1beta, IL-6, IL-8, and IL-10. In contrast, WB only expressed the mRNA cytokines of TNF-alpha and IL-8 (p < 0.05). These results suggest that spontaneous myriad mRNA cytokine expression can be avoided with the use of WB incubation and the rapid collection of PBMCs. Furthermore, this method should be employed in all cases where the levels of cytokine gene expression can be evaluated.

Hexamethylene amiloride engages a novel reactive oxygen species- and lysosome-dependent programmed necrotic mechanism to selectively target breast cancer cells.

Anticancer chemotherapeutics often rely on induction of apoptosis in rapidly dividing cells. While these treatment strategies are generally effective in debulking the primary tumor, post-therapeutic recurrence and metastasis are pervasive concerns with potentially devastating consequences. We demonstrate that the amiloride derivative 5-(N,N-hexamethylene) amiloride (HMA) harbors cytotoxic properties particularly attractive for a novel class of therapeutic agent. HMA is potently and specifically cytotoxic toward breast cancer cells, with remarkable selectivity for transformed cells relative to non-transformed or primary cells. Nonetheless, HMA is similarly cytotoxic to breast cancer cells irrespective of their molecular profile, proliferative status, or species of origin, suggesting that it engages a cell death mechanism common to all breast tumor subtypes. We observed that HMA induces a novel form of caspase- and autophagy-independent programmed necrosis relying on the orchestration of mitochondrial and lysosomal pro-death mechanisms, where its cytotoxicity was attenuated with ROS-scavengers or lysosomal cathepsin inhibition. Overall, our findings suggest HMA may efficiently target the heterogeneous populations of cancer cells known to reside within a single breast tumor by induction of a ROS- and lysosome-mediated form of programmed necrosis.

Rapid Discovery of Functional Small Molecule Ligands against Proteomic Targets through Library-Against-Library Screening.

Identifying "druggable" targets and their corresponding therapeutic agents are two fundamental challenges in drug discovery research. The one-bead-one-compound (OBOC) combinatorial library method has been developed to discover peptides or small molecules that bind to a specific target protein or elicit a specific cellular response. The phage display cDNA expression proteome library method has been employed to identify target proteins that interact with specific compounds. Here, we combined these two high-throughput approaches, efficiently interrogated approximately 10(13) possible molecular interactions, and identified 91 small molecule compound beads that interacted strongly with the phage library. Of 19 compounds resynthesized, 4 were cytotoxic against cancer cells; one of these compounds was found to interact with EIF5B and inhibit protein translation. As more binding pairs are confirmed and evaluated, the "library-against-library" screening approach and the resulting small molecule-protein domain interaction database may serve as a valuable tool for basic research and drug development.

Bovine dialyzable leukocyte extract modulates the nitric oxide and pro-inflammatory cytokine production in lipopolysaccharide-stimulated murine peritoneal macrophages in vitro.

Lipopolysaccharides (LPS) released from Gram-negative bacteria after infection initiate an exagerated response that leads to a cascade of pathophysiological events termed sepsis. Monocytes or macrophages produce many of the mediators found in septic patients. Targeting of these mediators, especially tumor necrosis factor (TNF)-alpha and nitric oxide (NO), has been pursued as a mean of reducing mortality in sepsis. Bovine dialyzable leukocyte extract (bDLE) is a dialysate of a heterogeneous mixture of low-molecular-weight substances released from disintegrated leukocytes of the blood or tissue lymphoid. In this study, to determine whether bDLE modulates NO and pro-inflammatory cytokine production, murine peritoneal macrophages were treated with bDLE (0.05 or 0.5 U/mL) before LPS (20 mg/mL) stimulation, and also LPS-stimulated murine peritoneal macrophages were treated with bDLE (0.05 or 0.5 U/mL) at 0, 4, 8, 12, and 24 hours. The bDLE significantly decreased NO production, and also decreased TNF-alpha and interleukin (IL)-6 but increased IL-10 production in LPS-stimulated murine peritoneal macrophages. Our results demonstrate that bDLE plays an important role in modulating TNF-alpha, IL-6, and NO production through IL-10, and this may offer therapeutic potential in clinical endotoxic shock.

Bovine dialyzable leukocyte extract protects against LPS-induced, murine endotoxic shock.

The pathophysiology of endotoxic shock is characterized by the activation of multiple pro-inflammatory genes and their products which initiate the inflammatory process. Endotoxic shock is a serious condition with high mortality. Bovine dialyzable leukocyte extract (bDLE) is a dialyzate of a heterogeneous mixture of low molecular weight substances released from disintegrated leukocytes of the blood or lymphoid tissue obtained from homogenized bovine spleen. bDLE is clinically effective for a broad spectrum of diseases. To determine whether bDLE improves survival and modulates the expression of pro-inflammatory cytokine genes in LPS-induced, murine endotoxic shock, Balb/C mice were treated with bDLE (1 U) after pretreatment with LPS (17 mg/kg). The bDLE improved survival (90%), suppressed IL-10 and IL-6, and decreased IL-1beta, TNF-alpha, and IL-12p40 mRNA expression; and decreased the production of IL-10 (P<0.01), TNF-alpha (P<0.01), and IL-6 (P<0.01) in LPS-induced, murine endotoxic shock. Our results demonstrate that bDLE leads to improved survival in LPS-induced endotoxic shock in mice, modulating the pro-inflammatory cytokine gene expression, suggesting that bDLE is an effective therapeutic agent for inflammatory illnesses associated with an unbalanced expression of pro-inflammatory cytokine genes such as in endotoxic shock, rheumatic arthritis and other diseases.

A cell-permeant amiloride derivative induces caspase-independent, AIF-mediated programmed necrotic death of breast cancer cells.

Amiloride is a potassium-sparing diuretic that has been used as an anti-kaliuretic for the chronic management of hypertension and heart failure. Several studies have identified a potential anti-cancer role for amiloride, however the mechanisms underlying its anti-tumor effects remain to be fully delineated. Our group previously demonstrated that amiloride triggers caspase-independent cytotoxic cell death in human glioblastoma cell lines but not in primary astrocytes. To delineate the cellular mechanisms underlying amiloride's anti-cancer cytotoxicity, cell permeant and cell impermeant derivatives of amiloride were synthesized that exhibit markedly different potencies in cancer cell death assays. Here we compare the cytotoxicities of 5-benzylglycinyl amiloride (UCD38B) and its free acid 5-glycinyl amiloride (UCD74A) toward human breast cancer cells. UCD74A exhibits poor cell permeability and has very little cytotoxic activity, while UCD38B is cell permeant and induces the caspase-independent death of proliferating and non-proliferating breast cancer cells. UCD38B treatment of human breast cancer cells promotes autophagy reflected in LC3 conversion, and induces the dramatic swelling of the endoplasmic reticulum, however these events do not appear to be the cause of cell death. Surprisingly, UCD38B but not UCD74A induces efficient AIF translocation from the mitochondria to the nucleus, and AIF function is necessary for the efficient induction of cancer cell death. Our observations indicate that UCD38B induces programmed necrosis through AIF translocation, and suggest that its cytosolic accessibility may facilitate drug action.