Chemical genetics analysis of an aniline mustard anticancer agent reveals complex I of the electron transport chain as a target

Supplementary information is available at the Journal of Biological Chemistry website.The antitumor agent 11β (CAS 865070-37-7), consisting of a DNA-damaging aniline mustard linked to an androgen receptor (AR) ligand, is known to form covalent DNA adducts and to induce apoptosis potently in AR-positive prostate cancer cells in vitro; it also strongly prevents growth of LNCaP xenografts in mice. The present study describes the unexpectedly strong activity of 11β against the AR-negative HeLa cells, both in cell culture and tumor xenografts, and uncovers a new mechanism of action that likely explains this activity. Cellular fractionation experiments indicated that mitochondria are the major intracellular sink for 11β; flow cytometry studies showed that 11β exposure rapidly induced oxidative stress, mitochondria being an important source of reactive oxygen species (ROS). Additionally, 11β inhibited oxygen consumption both in intact HeLa cells and in isolated mitochondria. Specifically, 11β blocked uncoupled oxygen consumption when mitochondria were incubated with complex I substrates, but it had no effect on oxygen consumption driven by substrates acting downstream of complex I in the mitochondrial electron transport chain. Moreover, 11β enhanced ROS generation in isolated mitochondria, suggesting that complex I inhibition is responsible for ROS production. At the cellular level, the presence of antioxidants (N-acetylcysteine or vitamin E) significantly reduced the toxicity of 11β, implicating ROS production as an important contributor to cytotoxicity. Collectively, our findings establish complex I inhibition and ROS generation as a new mechanism of action for 11β, which supplements conventional DNA adduct formation to promote cancer cell death.National Institutes of Health (U.S.) (Grant R01 CA077743)United States. Dept. of Defense (Prostate Cancer Research Program Award DAMD17-98-1-8520

Selective Adaptation in Networks of Heterogeneous Populations: Model, Simulation, and Experiment

Biological systems often change their responsiveness when subject to persistent stimulation, a phenomenon termed adaptation. In neural systems, this process is often selective, allowing the system to adapt to one stimulus while preserving its sensitivity to another. In some studies, it has been shown that adaptation to a frequent stimulus increases the system's sensitivity to rare stimuli. These phenomena were explained in previous work as a result of complex interactions between the various subpopulations of the network. A formal description and analysis of neuronal systems, however, is hindered by the network's heterogeneity and by the multitude of processes taking place at different time-scales. Viewing neural networks as populations of interacting elements, we develop a framework that facilitates a formal analysis of complex, structured, heterogeneous networks. The formulation developed is based on an analysis of the availability of activity dependent resources, and their effects on network responsiveness. This approach offers a simple mechanistic explanation for selective adaptation, and leads to several predictions that were corroborated in both computer simulations and in cultures of cortical neurons developing in vitro. The framework is sufficiently general to apply to different biological systems, and was demonstrated in two different cases

MTADV 5-MER peptide suppresses chronic inflammations as well as autoimmune pathologies and unveils a new potential target-Serum Amyloid A.

Despite the existence of potent anti-inflammatory biological drugs e.g., anti-TNF and anti IL-6 receptor antibodies, for treating chronic inflammatory and autoimmune diseases, these are costly and not specific. Cheaper oral available drugs remain an unmet need. Expression of the acute phase protein Serum Amyloid A (SAA) is dependent on release of pro-inflammatory cytokines IL-1, IL-6 and TNF-α during inflammation. Conversely, SAA induces pro-inflammatory cytokine secretion, including Th17, leading to a pathogenic vicious cycle and chronic inflammation. 5- MER peptide (5-MP) MTADV (methionine-threonine-alanine-aspartic acid-valine), also called Amilo-5MER, was originally derived from a sequence of a pro-inflammatory CD44 variant isolated from synovial fluid of a Rheumatoid Arthritis (RA) patient. This human peptide displays an efficient anti-inflammatory effects to ameliorate pathology and clinical symptoms in mouse models of RA, Inflammatory Bowel Disease (IBD) and Multiple Sclerosis (MS). Bioinformatics and qRT-PCR revealed that 5-MP, administrated to encephalomyelytic mice, up-regulates genes contributing to chronic inflammation resistance. Mass spectrometry of proteins that were pulled down from an RA synovial cell extract with biotinylated 5-MP, showed that it binds SAA. 5-MP disrupted SAA assembly, which is correlated with its pro-inflammatory activity. The peptide MTADV (but not scrambled TMVAD) significantly inhibited the release of pro-inflammatory cytokines IL-6 and IL-1β from SAA-activated human fibroblasts, THP-1 monocytes and peripheral blood mononuclear cells. 5-MP suppresses the pro-inflammatory IL-6 release from SAA-activated cells, but not from non-activated cells. 5-MP could not display therapeutic activity in rats, which are SAA deficient, but does inhibit inflammations in animal models of IBD and MS, both are SAA-dependent, as shown by others in SAA knockout mice. In conclusion, 5-MP suppresses chronic inflammation in animal models of RA, IBD and MS, which are SAA-dependent, but not in animal models, which are SAA-independent

Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli

Abstract Background Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-β1 and substrate stiffness affect fibroblast Fas expression are not well understood. Methods Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young’s moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-β1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. Results Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-β1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-β1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-β1. Conclusions Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.https://deepblue.lib.umich.edu/bitstream/2027.42/143539/1/12931_2018_Article_801.pd

Chemosensitization by phenothiazines in human lung cancer cells: impaired resolution of γH2AX and increased oxidative stress elicit apoptosis associated with lysosomal expansion and intense vacuolation

Chemotherapy resistance poses severe limitations on the efficacy of anti-cancer medications. Recently, the notion of using novel combinations of ‘old' drugs for new indications has garnered significant interest. The potential of using phenothiazines as chemosensitizers has been suggested earlier but so far our understanding of their molecular targets remains scant. The current study was designed to better define phenothiazine-sensitive cellular processes in relation to chemosensitivity. We found that phenothiazines shared the ability to delay γH2AX resolution in DNA-damaged human lung cancer cells. Accordingly, cells co-treated with chemotherapy and phenothiazines underwent protracted cell-cycle arrest followed by checkpoint escape that led to abnormal mitoses, secondary arrest and/or a form of apoptosis associated with increased endogenous oxidative stress and intense vacuolation. We provide evidence implicating lysosomal dysfunction as a key component of cell death in phenothiazine co-treated cells, which also exhibited more typical hallmarks of apoptosis including the activation of both caspase-dependent and -independent pathways. Finally, we demonstrated that vacuolation in phenothiazine co-treated cells could be reduced by ROS scavengers or the vacuolar ATPase inhibitor bafilomycin, leading to increased cell viability. Our data highlight the potential benefit of using phenothiazines as chemosensitizers in tumors that acquire molecular alterations rendering them insensitive to caspase-mediated apoptosis

Protective effect of geranylgeranylacetone, an inducer of heat shock protein 70, against drug-induced lung injury/fibrosis in an animal model

<p>Abstract</p> <p>Background</p> <p>To determine whether oral administration of geranylgeranylacetone (GGA), a nontoxic anti-ulcer drug that is an inducer of heat shock protein (HSP) 70, protects against drug-induced lung injury/fibrosis <it>in vivo</it>.</p> <p>Methods</p> <p>We used a bleomycin (BLM)-induced lung fibrosis model in which mice were treated with oral 600 mg/kg of GGA before and after BLM administration. Inflammation and fibrosis were evaluated by histological scoring, hydroxyproline content in the lung and inflammatory cell count, and quantification by ELISA of macrophage inflammatory protein-2 (MIP-2) in bronchoalveolar lavage fluid. Apoptosis was evaluated by the TUNEL method. The induction of HSP70 in the lung was examined with western blot analysis and its localization was determined by immunohistochemistry.</p> <p>Results</p> <p>We confirmed the presence of inflammation and fibrosis in the BLM-induced lung injury model and induction of HSP70 by oral administration of GGA. GGA prevented apoptosis of cellular constituents of lung tissue, such as epithelial cells, most likely related to the <it>de novo </it>induction of HSP70 in the lungs. GGA-treated mice also showed less fibrosis of the lungs, associated with the findings of suppression of both production of MIP-2 and inflammatory cell accumulation in the injured lung, compared with vehicle-treated mice.</p> <p>Conclusion</p> <p>GGA had a protective effect on drug-induced lung injury/fibrosis. Disease-modifying antirheumatic drugs such as methotrexate, which are indispensable for the treatment of rheumatoid arthritis, often cause interstitial lung diseases, an adverse event that currently cannot be prevented. Clinical use of GGA for drug-induced pulmonary fibrosis might be considered in the future.</p

Role of Versican, Hyaluronan and CD44 in Ovarian Cancer Metastasis

There is increasing evidence to suggest that extracellular matrix (ECM) components play an active role in tumor progression and are an important determinant for the growth and progression of solid tumors. Tumor cells interfere with the normal programming of ECM biosynthesis and can extensively modify the structure and composition of the matrix. In ovarian cancer alterations in the extracellular environment are critical for tumor initiation and progression and intra-peritoneal dissemination. ECM molecules including versican and hyaluronan (HA) which interacts with the HA receptor, CD44, have been shown to play critical roles in ovarian cancer metastasis. This review focuses on versican, HA, and CD44 and their potential as therapeutic targets for ovarian cancer

Bistable Percepts in the Brain: fMRI Contrasts Monocular Pattern Rivalry and Binocular Rivalry

The neural correlates of binocular rivalry have been actively debated in recent years, and are of considerable interest as they may shed light on mechanisms of conscious awareness. In a related phenomenon, monocular rivalry, a composite image is shown to both eyes. The subject experiences perceptual alternations in which the two stimulus components alternate in clarity or salience. The experience is similar to perceptual alternations in binocular rivalry, although the reduction in visibility of the suppressed component is greater for binocular rivalry, especially at higher stimulus contrasts. We used fMRI at 3T to image activity in visual cortex while subjects perceived either monocular or binocular rivalry, or a matched non-rivalrous control condition. The stimulus patterns were left/right oblique gratings with the luminance contrast set at 9%, 18% or 36%. Compared to a blank screen, both binocular and monocular rivalry showed a U-shaped function of activation as a function of stimulus contrast, i.e. higher activity for most areas at 9% and 36%. The sites of cortical activation for monocular rivalry included occipital pole (V1, V2, V3), ventral temporal, and superior parietal cortex. The additional areas for binocular rivalry included lateral occipital regions, as well as inferior parietal cortex close to the temporoparietal junction (TPJ). In particular, higher-tier areas MT+ and V3A were more active for binocular than monocular rivalry for all contrasts. In comparison, activation in V2 and V3 was reduced for binocular compared to monocular rivalry at the higher contrasts that evoked stronger binocular perceptual suppression, indicating that the effects of suppression are not limited to interocular suppression in V1