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1
Brookes, Paul, Andrew Tompkins, Kimberly Morse, Shannon Hilchey, Suhail Salim, Denise Ray, Richard Phipps, and Steven H. Bernstein. "The Triterpenoids 2-cyano-3,12-dioxooleana-1,9-dien-28-oic Acid (CDDO) and Their Imidazole (CDDO-Im) and Dinitrile Derivatives (DI-CDDO) Elicit Apoptosis through a Novel Mitochondrial Pathway." Blood 106, no. 11 (November 16, 2005): 2426. http://dx.doi.org/10.1182/blood.v106.11.2426.2426.
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Abstract We have recently shown that B-cell non Hodgkin’s lymphoma express the transcription factor PPARγ and undergo apoptosis upon exposure to PPARγ ligands. The synthetic triterpenoid CDDO is a specific ligand for PPARγ, and CDDO and its derivatives, CDDO-Im and DI-CDDO, induce diffuse large cell lymphoma (DLCL) death (OCI Ly10 and OCI Ly19 cells), with a potency of DI-CDDO>CDDO-Im>CDDO, suggesting that such agents have therapeutic potential in lymphoma. The natural PPARγ ligand, 15d-PGJ2 (which also elicits DLCL death), has previously been shown to inhibit mitochondrial complex I, enhance mitochondrial reactive oxygen species (ROS) generation, and react with protein thiols. Given that CDDO is structurally similar to 15d-PGJ2 we hypothesized that CDDO-induced cell death may similarly be mediated via complex I inhibition, ROS generation, thiol oxidation, and opening of a large membrane pore complex in the mitochondrial membrane, termed the “permeability transition” (PT) pore. Studies on isolated rat liver mitochondria however showed that none of the CDDO-derivatives inhibited complex I activity or affected mitochondrial protein thiols. However, all three compounds did induce PT pore opening and mitochondrial swelling, with a concurrent loss of mitochondrial membrane potential, in a Ca2+ dependent manner (potency DI-CDDO>CDDO-Im>CDDO). This is consistent with a previously shown role for Ca2+ in CDDO-induced cell death. Interestingly, this mitochondrial swelling was not inhibited by the classical PT pore inhibitor cyclosporin A (CsA). This is supported by our findings that the induction of OCI-Ly19 cell death by CDDO was also not inhibited by CsA, or by another classical PT pore inhibitor, nortriptyline. These phenomena may be partially explained by invoking the “unregulated PT pore”. In addition to the classical PT pore, a non-CsA sensitive “unregulated PT pore” also exists, which is generated by the aggregation of misfolded mitochondrial membrane proteins that are induced by oxidants and thiol reactive agents. That exposure of mitochondria to CDDO results in the formation of “unregulated PT pores” is supported by our findings that the proteosome inhibitor PS341, potentiates CDDO-induced cell death, suggesting the involvement of a protein folding response. The temporal role of ROS in CDDO-induced cell death was also investigated, and it was found that the antioxidant N-acetyl-cysteine did not inhibit PT pore opening, but did inhibit cell death. This is consistent with our observation that ROS generation in isolated mitochondria was not immediately triggered by CDDO, but rather increased at delayed time points, placing it downstream of PT pore opening. This proposes the following novel model of a direct mitochondrial effect of CDDO and its derivatives: \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[CDDO{\rightarrow}\ mitochondrial\ protein\ misfolding\ {\rightarrow}\ unregulated\ PT\ pore\ formation\ {\rightarrow}\ ROS\ {\rightarrow}\ cell\ death\] \end{document} In summary: CDDO and its derivatives have direct effects on mitochondria, and represent novel therapeutic approaches for the treatment of patients with DLCL; and combinations of CDDO and its derivatives with proteosome inhibitors represent a rational combination to test in the context of clinical trials.
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Ross-Inta, Catherine, Chern-Yi Tsai, and Cecilia Giulivi. "The mitochondrial pool of free amino acids reflects the composition of mitochondrial DNA-encoded proteins: indication of a post- translational quality control for protein synthesis." Bioscience Reports 28, no. 5 (September 11, 2008): 239–49. http://dx.doi.org/10.1042/bsr20080090.
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Mitochondria can synthesize a limited number of proteins encoded by mtDNA (mitochondrial DNA) by using their own biosynthetic machinery, whereas most of the proteins in mitochondria are imported from the cytosol. It could be hypothesized that the mitochondrial pool of amino acids follows the frequency of amino acids in mtDNA-encoded proteins or, alternatively, that the profile is the result of the participation of amino acids in pathways other than protein synthesis (e.g. haem biosynthesis and aminotransferase reactions). These hypotheses were tested by evaluating the pool of free amino acids and derivatives in highly-coupled purified liver mitochondria obtained from rats fed on a nutritionally adequate diet for growth. Our results indicated that the pool mainly reflects the amino acid composition of mtDNA-encoded proteins, suggesting that there is a post-translational control of protein synthesis. This conclusion was supported by the following findings: (i) correlation between the concentration of free amino acids in the matrix and the frequency of abundance of amino acids in mtDNA-encoded proteins; (ii) the similar ratios of essential-to-non-essential amino acids in mtDNA-encoded proteins and the mitochondrial pool of amino acids; and (iii), lack of a correlation between codon usage or tRNA levels and amino-acid concentrations. Quantitative information on the mammalian mitochondrial content of amino acids, such as that presented in the present study, along with functional studies, will help us to better understand the pathogenesis of mitochondrial diseases or the biochemical implications in mitochondrial metabolism.
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Ryan, Lisa, Yvonne C. O'Callaghan, and Nora M. O'Brien. "The role of the mitochondria in apoptosis induced by 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide." British Journal of Nutrition 94, no. 4 (October 2005): 519–25. http://dx.doi.org/10.1079/bjn20051524.
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Oxysterols are oxygenated derivatives of cholesterol that may be formed endogenously or absorbed from the diet. Significant amounts of oxysterols have frequently been identified in foods of animal origin, in particular highly processed foods. To date, oxysterols have been shown to possess diverse biological activities; however, recent attention has focused on their potential role in the development of atherosclerosis. Oxysterols have been reported to induce apoptosis in cells of the arterial wall, a primary process in the development of atheroma. The aim of the present study was to identify the role of the mitochondria in the apoptotic pathways induced by the oxysterols 7β-hydroxycholesterol (7β-OH) and cholesterol-5β,6β-epoxide (β-epoxide) in U937 cells. To this end, we investigated the effects of these oxysterols on mitochondrial membrane potential, caspase-8 activity, the mitochondrial permeability transition pore and cytochrome c release. 7β-OH-induced apoptosis was associated with a loss in mitochondrial membrane potential after 2 h, accompanied by cytochrome c release from the mitochondria into the cytosol after 16 h. Pre-treatment with a range of inhibitors of the mitochondrial permeability transition pore protected against 7β-OH-induced cell death. In contrast, β-epoxide induced a slight increase in caspase-8 activity but had no effect on mitochondrial membrane potential or cytochrome c release. The present results confirm that 7β-OH-induced apoptosis occurs via the mitochondrial pathway and highlights differences in the apoptotic pathways induced by 7β-OH and β-epoxide in U937 cells.
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Reiter, Russel, Sergio Paredes, Ahmet Korkmaz, Mei-Jie Jou, and Dun-Xian Tan. "Melatonin combats molecular terrorism at the mitochondrial level." Interdisciplinary Toxicology 1, no. 2 (September 1, 2008): 137–49. http://dx.doi.org/10.2478/v10102-010-0030-2.
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Melatonin combats molecular terrorism at the mitochondrial levelThe intracellular environmental is a hostile one. Free radicals and related oxygen and nitrogen-based oxidizing agents persistently pulverize and damage molecules in the vicinity of where they are formed. The mitochondria especially are subjected to frequent and abundant oxidative abuse. The carnage that is left in the wake of these oxygen and nitrogen-related reactants is referred to as oxidative damage or oxidative stress. When mitochondrial electron transport complex inhibitors are used, e.g., rotenone, 1-methyl-1-phenyl-1,2,3,6-tetrahydropyridine, 3-nitropropionic acid or cyanide, pandemonium breaks loose within mitochondria as electron leakage leads to the generation of massive amounts of free radicals and related toxicants. The resulting oxidative stress initiates a series of events that leads to cellular apoptosis. To alleviate mitochondrial destruction and the associated cellular implosion, the cell has at its disposal a variety of free radical scavengers and antioxidants. Among these are melatonin and its metabolites. While melatonin stimulates several antioxidative enzymes it, as well as its metabolites (cyclic 3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine and N1-acetyl-5-methoxykynuramine), likewise effectively neutralize free radicals. The resulting cascade of reactions greatly magnifies melatonin's efficacy in reducing oxidative stress and apoptosis even in the presence of mitochondrial electron transport inhibitors. The actions of melatonin at the mitochondrial level are a consequence of melatonin and/or any of its metabolites. Thus, the molecular terrorism meted out by reactive oxygen and nitrogen species is held in check by melatonin and its derivatives.
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Inglut, Baglo, Liang, Cheema, Stabile, Woodworth, and Huang. "Systematic Evaluation of Light-Activatable Biohybrids for Anti-Glioma Photodynamic Therapy." Journal of Clinical Medicine 8, no. 9 (August 21, 2019): 1269. http://dx.doi.org/10.3390/jcm8091269.
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Photosensitizing biomolecules (PSBM) represent a new generation of light-absorbing compounds with improved optical and physicochemical properties for biomedical applications. Despite numerous advances in lipid-, polymer-, and protein-based PSBMs, their effective use requires a fundamental understanding of how macromolecular structure influences the physicochemical and biological properties of the photosensitizer. Here, we prepared and characterized three well-defined PSBMs based on a clinically used photosensitizer, benzoporphyrin derivative (BPD). The PSBMs include 16:0 lysophosphocholine-BPD (16:0 Lyso PC-BPD), distearoyl-phosphoethanolamine-polyethylene-glycol-BPD (DSPE-PEG-BPD), and anti-EGFR cetuximab-BPD (Cet-BPD). In two glioma cell lines, DSPE-PEG-BPD exhibited the highest singlet oxygen yield but was the least phototoxic due to low cellular uptake. The 16:0 Lyso PC-BPD was most efficient in promoting cellular uptake but redirected BPD’s subcellular localization from mitochondria to lysosomes. At 24 h after incubation, proteolyzed Cet-BPD was localized to mitochondria and effectively disrupted the mitochondrial membrane potential upon light activation. Our results revealed the variable trafficking and end effects of PSBMs, providing valuable insights into methods of PSBM evaluation, as well as strategies to select PSBMs based on subcellular targets and cytotoxic mechanisms. We demonstrated that biologically informed combinations of PSBMs to target lysosomes and mitochondria, concurrently, may lead to enhanced therapeutic effects against gliomas.
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Everard-Gigot, Valerie, Cory D. Dunn, Brigid M. Dolan, Susanne Brunner, Robert E. Jensen, and Rosemary A. Stuart. "Functional Analysis of Subunit e of the F1Fo-ATP Synthase of the Yeast Saccharomyces cerevisiae: Importance of the N-Terminal Membrane Anchor Region." Eukaryotic Cell 4, no. 2 (February 2005): 346–55. http://dx.doi.org/10.1128/ec.4.2.346-355.2005.
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ABSTRACT Mitochondrial F1Fo-ATP synthase complexes do not exist as physically independent entities but rather form dimeric and possibly oligomeric complexes in the inner mitochondrial membrane. Stable dimerization of two F1Fo-monomeric complexes involves the physical association of two membrane-embedded Fo-sectors. Previously, formation of the ATP synthase dimeric-oligomeric network was demonstrated to play a critical role in modulating the morphology of the mitochondrial inner membrane. In Saccharomyces cerevisiae, subunit e (Su e) of the Fo-sector plays a central role in supporting ATP synthase dimerization. The Su e protein is anchored to the inner membrane via a hydrophobic region located at its N-terminal end. The hydrophilic C-terminal region of Su e resides in the intermembrane space and contains a conserved coiled-coil motif. In the present study, we focused on characterizing the importance of these regions for the function of Su e. We created a number of C-terminal-truncated derivatives of the Su e protein and expressed them in the Su e null yeast mutant. Mitochondria were isolated from the resulting transformant strains, and a number of functions of Su e were analyzed. Our results indicate that the N-terminal hydrophobic region plays important roles in the Su e-dependent processes of mitochondrial DNA maintenance, modulation of mitochondrial morphology, and stabilization of the dimer-specific Fo subunits, subunits g and k. Furthermore, we show that the C-terminal coiled-coil region of Su e functions to stabilize the dimeric form of detergent-solubilized ATP synthase complexes. Finally, we propose a model to explain how Su e supports the assembly of the ATP synthase dimers-oligomers in the mitochondrial membrane.
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Williams, C. H., J. Lawson, and F. R. C. Backwell. "Oxidation of 3-amino-1-phenylprop-1-enes by monoamine oxidase and their use in a continuous assay of the enzyme." Biochemical Journal 256, no. 3 (December 15, 1988): 911–15. http://dx.doi.org/10.1042/bj2560911.
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3-Amino-1-phenylprop-1-ene (cinnamylamine) and some derivatives were examined as substrates for monoamine oxidases A and B in mitochondria. All of the amines examined were readily oxidized by monoamine oxidase B but much less readily by monoamine oxidase A. E-Cinnamylamine was found to have Km 0.025 mM and Vmax. 3.9 nmol/min per mg of mitochondrial protein. Corresponding values with monoamine oxidase A were 0.026 mM and 0.85 nmol/min per mg respectively. Despite their different stereochemistry, E- and Z-N-methylcinnamylamines were almost equally effective as substrates for monoamine oxidase B. The characteristic u.v. absorbance and high absorption coefficient of cinnamaldehyde, the product produced by enzymic oxidation of cinnamylamine, is utilized in a sensitive continuous spectrophotometric assay for both enzymes in the rat and for the assay of a purified monoamine oxidase B from bovine liver.
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Horvath, Orsolya, Katalin Ordog, Kitti Bruszt, Laszlo Deres, Ferenc Gallyas, Balazs Sumegi, Kalman Toth, and Robert Halmosi. "BGP-15 Protects against Heart Failure by Enhanced Mitochondrial Biogenesis and Decreased Fibrotic Remodelling in Spontaneously Hypertensive Rats." Oxidative Medicine and Cellular Longevity 2021 (January 30, 2021): 1–13. http://dx.doi.org/10.1155/2021/1250858.
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Heart failure (HF) is a complex clinical syndrome with poor clinical outcomes despite the growing number of therapeutic approaches. It is characterized by interstitial fibrosis, cardiomyocyte hypertrophy, activation of various intracellular signalling pathways, and damage of the mitochondrial network. Mitochondria are responsible for supplying the energy demand of cardiomyocytes; therefore, the damage of the mitochondrial network causes cellular dysfunction and finally leads to cell death. BGP-15, a hydroxylamine derivative, is an insulin-sensitizer molecule and has a wide range of cytoprotective effects in animal as well as in human studies. Our recent work was aimed at examining the effects of BGP-15 in a chronic hypertension-induced heart failure model. 15-month-old male SHRs were used in our experiment. The SHR-Baseline group represented the starting point ( n = 7 ). Animals received BGP-15 (SHR-B, n = 7 ) or placebo (SHR-C, n = 7 ) for 18 weeks. WKY rats were used as age-matched normotensive controls ( n = 7 ). The heart function was monitored by echocardiography. Histological preparations were made from cardiac tissue. The levels of signalling proteins were determined by Western blot. At the end of the study, systolic and diastolic cardiac function was preserved in the BGP-treated animals. BGP-15 decreased the interstitial collagen deposition via decreasing the activity of TGFβ/Smad signalling factors and prevented the cardiomyocyte hypertrophy in hypertensive animals. BGP-15 enhanced the prosurvival signalling pathways (Akt/Gsk3β). The treatment increased the activity of MKP1 and decreased the activity of p38 and JNK signalling routes. The mitochondrial mass of cardiomyocytes was also increased in BGP-15-treated SHR animals due to the activation of mitochondrial biogenesis. The mitigation of remodelling processes and the preserved systolic cardiac function in hypertension-induced heart failure can be a result—at least partly—of the enhanced mitochondrial biogenesis caused by BGP-15.
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Sato-Kaneko, Fumi, Shiyin Yao, Fitzgerald S. Lao, Jason Nan, Jonathan Shpigelman, Annette Cheng, Tetsuya Saito, et al. "Mitochondria-dependent synthetic small-molecule vaccine adjuvants for influenza virus infection." Proceedings of the National Academy of Sciences 118, no. 23 (June 2, 2021): e2025718118. http://dx.doi.org/10.1073/pnas.2025718118.
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Vaccine adjuvants enhance and prolong pathogen-specific protective immune responses. Recent reports indicate that host factors—such as aging, pregnancy, and genetic polymorphisms—influence efficacies of vaccines adjuvanted with Toll-like receptor (TLR) or known pattern-recognition receptor (PRR) agonists. Although PRR independent adjuvants (e.g., oil-in-water emulsion and saponin) are emerging, these adjuvants induce some local and systemic reactogenicity. Hence, new TLR and PRR-independent adjuvants that provide greater potency alone or in combination without compromising safety are highly desired. Previous cell-based high-throughput screenings yielded a small molecule 81 [N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide], which enhanced lipopolysaccharide-induced NF-κB and type I interferon signaling in reporter assays. Here compound 81 activated innate immunity in primary human peripheral blood mononuclear cells and murine bone marrow-derived dendritic cells (BMDCs). The innate immune activation by 81 was independent of TLRs and other PRRs and was significantly reduced in mitochondrial antiviral-signaling protein (MAVS)-deficient BMDCs. Compound 81 activities were mediated by mitochondrial dysfunction as mitophagy inducers and a mitochondria specific antioxidant significantly inhibited cytokine induction by 81. Both compound 81 and a derivative obtained via structure–activity relationship studies, 2F52 [N-benzyl-N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] modestly increased mitochondrial reactive oxygen species and induced the aggregation of MAVS. Neither 81 nor 2F52 injected as adjuvants caused local or systemic toxicity in mice at effective concentrations for vaccination. Furthermore, vaccination with inactivated influenza virus adjuvanted with 2F52 demonstrated protective effects in a murine lethal virus challenge study. As an unconventional and safe adjuvant that does not require known PRRs, compound 2F52 could be a useful addition to vaccines.
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Filho, Ricardo Imbroisi, Daniel T. G. Gonzaga, Thainá M. Demaria, João G. B. Leandro, Dora C. S. Costa, Vitor F. Ferreira, Mauro Sola-Penna, Fernando de C. da Silva, and Patricia Zancan. "A Novel Triazole Derivative Drug Presenting In Vitro and In Vivo Anticancer Properties." Current Topics in Medicinal Chemistry 18, no. 17 (November 26, 2018): 1483–93. http://dx.doi.org/10.2174/1568026618666180917100640.
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Background: Cancer is a major cause of death worldwide, despite many different drugs available to treat the disease. This high mortality rate is largely due to the complexity of the disease, which results from several genetic and epigenetic changes. Therefore, researchers are constantly searching for novel drugs that can target different and multiple aspects of cancer. Experimental: After a screening, we selected one novel molecule, out of ninety-four triazole derivatives, that strongly affects the viability and proliferation of the human breast cancer cell line MCF-7, with minimal effects on non-cancer cells. The drug, named DAN94, induced a dose-dependent decrease in MCF-7 cells viability, with an IC50 of 3.2 ± 0.2 µM. Additionally, DAN94 interfered with mitochondria metabolism promoting reactive oxygen species production, triggering apoptosis and arresting the cancer cells on G1/G0 phase of cell cycle, inhibiting cell proliferation. These effects are not observed when the drug was tested in the non-cancer cell line MCF10A. Using a mouse model with xenograft tumor implants, the drug preventing tumor growth presented no toxicity for the animal and without altering biochemical markers of hepatic function. Results and Conclusion: The novel drug DAN94 is selective for cancer cells, targeting the mitochondrial metabolism, which culminates in the cancer cell death. In the end, DAN94 has been shown to be a promising drug for controlling breast cancer with minimal undesirable effects.
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Cerqueira, Fernanda M., Noga Kozer, Anton Petcherski, Boris M. Baranovski, Dane Wolf, Essam A. Assali, Yaelle Roth, et al. "MitoTimer-based high-content screen identifies two chemically-related benzothiophene derivatives that enhance basal mitophagy." Biochemical Journal 477, no. 2 (January 31, 2020): 461–75. http://dx.doi.org/10.1042/bcj20190616.
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Mitochondrial turnover is required for proper cellular function. Both mitochondrial biogenesis and mitophagy are impaired in several degenerative and age-related diseases. The search for mitophagy activators recently emerged as a new therapeutical approach; however, there is a lack in suitable tools to follow mitochondrial turnover in a high-throughput manner. We demonstrate that the fluorescent protein, MitoTimer, is a reliable and robust probe to follow mitochondrial turnover. The screening of 15 000 small molecules led us to two chemically-related benzothiophenes that stimulate basal mitophagy in the beta-cell line, INS1. Enhancing basal mitophagy was associated with improved mitochondrial function, higher Complex I activity and Complex II and III expressions in INS1 cells, as well as better insulin secretion performance in mouse islets. The possibility of further enhancing mitophagy in the absence of mitochondrial stressors points to the existence of a ‘basal mitophagy spare capacity'. To this end, we found two small molecules that can be used as models to better understand the physiological regulation of mitophagy.
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Wei, Shu-jun, Min Shi, Jun-hua He, M. Sharkey, and Xue-xin Chen. "The complete mitochondrial genome of Diadegma semiclausum (Hymenoptera: Ichneumonidae) indicates extensive independent evolutionary events." Genome 52, no. 4 (April 2009): 308–19. http://dx.doi.org/10.1139/g09-008.
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Few complete mitochondrial genomes representing limited families in the order Hymenoptera have been sequenced. Here, we sequenced the complete mitochondrial genome of Diadegma semiclausum (Hymenoptera: Ichneumonidae). This genome is 18 728 bp long, the second largest hexapod mitochondrial genome sequenced in its entirety and that with the highest A+T content at 87.4%. Four tRNAs are rearranged compared with the ancestral arrangement. Gene rearrangement mechanisms are different among all three rearranged regions. Six tRNAs have a large variable loop, which is not found in other metazoan mitochondrial genomes. trnS(AGY) uses the abnormal anticodon TCT but trnK uses the normal CTT. The A+T-rich region is very long (2161 bp). An extremely A+T-rich (99.1%) 1515 bp tandem repeat region with three types of repeat elements is located between cox1 and cox2, and the most likely ancestral element originated from the 3′ end of cox1. Independent tandem duplications followed by mutation–insertion–deletion is the best model to explain the formation of this region. These results indicate that independent evolutionary events occurred extensively, such as gene rearrangement events, gene rearrangement mechanisms, derivation of tRNA variable loops, and tandem repeat region evolutionary processes, all of which likely contribute to the diversified features of hymenopteran mitochondrial genomes.
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Busenbender, Tobias, Sergey Dyshlovoy, Moritz Kaune, Lukas Boeckelmann, Tobias Lange, Udo Schumacher, Dmitry N. Pelageev, et al. "In vitro and in vivo investigations of novel 1,4-napthoquinone sulphomethylene carbohydrate conjugates in prostate cancer." Journal of Clinical Oncology 39, no. 6_suppl (February 20, 2021): 104. http://dx.doi.org/10.1200/jco.2021.39.6_suppl.104.
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104 Background: The Warburg effect describes the ability of cancer cells to consume larger amounts of glucose in comparison to normal tissue, due to the overexpression of insulin-independent glucose transporters (e.g. GLUT1). This effect can be used to enhance the selectivity and reduce side effects of cytotoxic anticancer molecules by its conjugation to sugar residues, thus, generating cytotoxic agents showing higher selectivity to cancer cells. In continuation of our research on anticancer natural 1,4-naphthoquinones we have investigated a large series of novel semi-synthetic molecules containing 1,4-naphthoquinones element conjugated with glucose molecule via -S-CH2- bond. Methods: We performed screening examinations for 35 novel synthetic molecules in human prostate cancer in vitro. The selected most active compounds were tested in several human prostate cancer cell lines harboring different levels of drug resistance, as well as in non-malignant cells to specify their selectivity. Compounds with the highest cytotoxicity and selectivity were further investigated. The mode of action was assessed including effects on apoptosis induction, oxidative stress, mitochondria, AR-signaling as well as glucose uptake and ER stress were assessed. In vivo dose finding and efficacy analyses were performed. Results: We identified two promising derivatives, showing IC50s at low micro- and nanomolar concentrations. Glucose depletion from the culture media led to increased cytotoxicity and cotreatment with a GLUT1-inhibitor showed an antagonistic effect, suggesting a concurrent uptake and therefore a Warburg effect targeting. The selected compounds exhibited most pronounced cytotoxic activity in DU145 cells as well as 22Rv1 cells. Non-malignant cells were generally less affected. The mode of action involves a loss of mitochondrial membrane potential, a release of cytochrome c and AIF into the cytosol and an upregulation of caspase-9, caspase-3 and cleaved PARP, as well as downregulation of Bcl-2 and Survivin, indicating that mitochondria are a major target, leading to the activation of the intrinsic apoptotic pathway. Early events in treated cells are ROS production and calcium release into the cytosol, a marker of ER-stress. Furthermore, downregulation of the AR and its signaling was observed on mRNA- and protein-level. In vivo experiments revealed antitumor activity in a 22Rv1-xenograft mouse model without severe side effects. Conclusions: In conclusion, we were able to identify two glucose-conjugated 1,4-naphthoquinones exhibiting potent in vitro and in vivoactivity and selectivity in human prostate cancer cells due to the Warburg effect targeting. Cytotoxic activity was exerted via initial ROS production and ER stress leading to mitochondrial damage and the induction of the intrinsic apoptotic pathway.
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Matthias, Jessica, Thines Kanagasundaram, Klaus Kopka, and Carsten S. Kramer. "Synthesis of a dihalogenated pyridinyl silicon rhodamine for mitochondrial imaging by a halogen dance rearrangement." Beilstein Journal of Organic Chemistry 15 (October 1, 2019): 2333–43. http://dx.doi.org/10.3762/bjoc.15.226.
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Background: Since their first synthesis, silicon xanthenes and the subsequently developed silicon rhodamines (SiR) gained a lot of attention as attractive fluorescence dyes offering a broad field of application. We aimed for the synthesis of a fluorinable pyridinyl silicon rhodamine for the use in multimodal (PET/OI) medical imaging of mitochondria in cancerous cells. Results: A dihalogenated fluorinatable pyridinyl rhodamine could be successfully synthesized with the high yield of 85% by application of a halogen dance (HD) rearrangement. The near-infrared dye shows a quantum yield of 0.34, comparable to other organelle targeting SiR derivatives and absorbs at 665 nm (εmax = 34 000 M−1cm−1) and emits at 681 nm (τ = 1.9 ns). Using colocalization experiments with MitoTracker® Green FM, we could prove the intrinsic targeting ability to mitochondria in two human cell lines (Pearson coefficient >0.8). The dye is suitable for live cell STED nanoscopy imaging and shows a nontoxic profile which makes it an appropriate candidate for medical imaging. Conclusions: We present a biocompatible, nontoxic, small molecule near-infrared dye with the option of subsequent radiolabelling and excellent optical properties for medical and bioimaging. As a compound with intrinsic mitochondria targeting ability, the radiolabelled analogue can be applied in multimodal (PET/OI) imaging of mitochondria for diagnostic and therapeutic use in, e.g., cancer patients.
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Adetutu, Adisa Rahmat, Sulaimon Lateef Adegboyega, Okeke Ebele Geraldine, Ariyo OC, and Abdulkareem Fatimah Biade. "Mitoubiquinol Mesylate Attenuated Diethyl Nitrosamine–Induced Hepatocellular Carcinoma Through Modulation of Mitochondrial Antioxidant Defense Systems." JCO Global Oncology 6, Supplement_1 (July 2020): 9. http://dx.doi.org/10.1200/go.20.20000.
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PURPOSE Hepatocellular carcinoma (HCC) is a highly malignant cancer, with a high recurrence rate and a poor prognosis. Diethyl nitrosamine (DEN) cirrhosis HCC–induced model has revealed an association of cancer progression with oxidative stress and mitochondrial dysfunction. This study investigated the effects of mitoubiquinol mesylate (MitoQ), a mitochondrial targeted antioxidant derivative from ubiquinone on DEN-induced oxidative damage in HCC Wistar rats. METHODS Fifty male Wistar rats were randomly divided into 5 groups, 10 rats per group. Groups A, B, and C received distilled water 10 mL/kg DEN, and MitoQ orally for 16 weeks, respectively. Animals in group D were pretreated with MitoQ for 1 week followed by coadministration of MitoQ and DEN (protective effect), whereas group E received DEN for 8 weeks, then coadministration of DEN and MitoQ (therapeutic effect) until the end of the study. Survival index, tumor incidence, liver function indices, hematologic profile, mitochondrial respiratory enzymes, and antioxidant defense status were assessed. RESULTS Data obtained show that rats in groups D and E had 80% survival and decreased tumor incidence (40% and 60%, respectively) compared with group B. Similarly, MitoQ significantly ( P < .05) decreased the activities of liver function enzymes, while hemoglobin concentration, red blood cell count, and lymphocytes levels were significantly elevated compared with the DEN-only group. Furthermore, MitoQ significantly ( P < .05) protected the liver from DEN-induced oxidative damage; however, there was no significant difference ( P > .05) between activities of mitochondrial F1F0-ATPase and succinate dehydrogenase of groups A, B, D, and E, respectively, although these enzyme activities were significantly ( P < .05) elevated in group C. Macroscopic and microscopic features indicated a reversal of DEN-induced cellular degeneration in hepatocytes. CONCLUSION These data suggest that MitoQ treatment for 16 weeks attenuated DEN-induced oxidative stress indices via modulation of mitochondrial antioxidant defense systems and could alleviate the burden of HCC as a chemotherapeutic agent.
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Belov, George A., Lyudmila I. Romanova, Elena A. Tolskaya, Marina S. Kolesnikova, Yuri A. Lazebnik, and Vadim I. Agol. "The Major Apoptotic Pathway Activated and Suppressed by Poliovirus." Journal of Virology 77, no. 1 (January 1, 2003): 45–56. http://dx.doi.org/10.1128/jvi.77.1.45-56.2003.
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ABSTRACT Cells respond to poliovirus infection by switching on the apoptotic program, implementation of which is usually suppressed by viral antiapoptotic functions. We show here that poliovirus infection of HeLa cells or derivatives of MCF-7 cells was accompanied by the efflux of cytochrome c from mitochondria. This efflux occurred during both abortive infection (e.g., interrupted by guanidine-HCl and ending with apoptosis) and productive infection (leading to cytopathic effect). The former type of infection, but not the latter, was accompanied by truncation of the proapoptotic protein Bid. The virus-triggered cytochrome c efflux was suppressed by overexpression of Bcl-2. Both abortive and productive infections also resulted in a decreased level of procaspase-9, as revealed by Western blotting. In the former case, this decrease was accompanied by the accumulation of a protein with the electrophoretic mobility of active caspase-9. In contrast, in the productively infected cells, the latter protein was absent but caspase-9-related polypeptides with altered mobility could be detected. Both caspase-9 and caspase-3 were shown to be essential for the development of such hallmarks of virus-induced apoptosis as chromatin condensation, DNA degradation, and nuclear fragmentation. These and some other results suggest the following scenario. Poliovirus infection activates the apoptotic pathway, involving mitochondrial damage, cytochrome c efflux, and consecutive activation of caspase-9 and caspase-3. The apoptotic signal appears to be amplified by a loop which includes secondary processing of Bid. The implementation of the apoptotic program in productively infected cells may be suppressed, however, by the viral antiapoptotic functions, which act at a step(s) downstream of the cytochrome c efflux. The suppression appears to be caused, at least in part, by aberrant processing and degradation of procaspase-9.
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Hertz, R., and J. Bar–Tana. "Prevention of peroxisomal proliferation by carnitine palmitoyltransferase inhibitors in cultured rat hepatocytes and in vivo." Biochemical Journal 245, no. 2 (July 15, 1987): 387–92. http://dx.doi.org/10.1042/bj2450387.
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1. The induction of peroxisomal beta-oxidation activities by bezafibrate in cultured rat hepatocytes and in the rat in vivo was prevented by inhibitors of carnitine acyltransferase, e.g. 2-bromopalmitate, 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate or 2-tetradecylglycidic acid. 2. The prevention of peroxisomal proliferation by carnitine palmitoyltransferase inhibitors could not be accounted for by inhibition of mitochondrial beta-oxidation, since 2-bromo-octanoate, acting as an inhibitor of beta-oxidation, did not prevent the induction of peroxisomal activities in cultured rat hepatocytes. 3. The putative role of the acylcarnitine derivative of bezafibrate was analysed by studying the formation of bezafibroylcarnitine with bezafibroyl-CoA as substrate. However, no bezafibroylcarnitine formation was demonstrated in the presence of rat liver preparations capable of catalysing transfer to carnitine of medium- or long-chain fatty acids. 4. The prevention of peroxisomal proliferation by carnitine acyltransferase inhibitors may help in dissecting the causal relationship between the multiple effects mediated by peroxisomal proliferators.
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Parikh, V. S., H. Conrad-Webb, R. Docherty, and R. A. Butow. "Interaction between the yeast mitochondrial and nuclear genomes influences the abundance of novel transcripts derived from the spacer region of the nuclear ribosomal DNA repeat." Molecular and Cellular Biology 9, no. 5 (May 1989): 1897–907. http://dx.doi.org/10.1128/mcb.9.5.1897.
Full textAbstract:
We have identified stable transcripts from the so-called nontranscribed spacer region (NTS) of the nuclear ribosomal DNA repeat in certain respiration-deficient strains of Saccharomyces cerevisiae. These RNAs, which are transcribed from the same strand as is the 37S rRNA precursor, are 500 to 800 nucleotides long and extend from the 5' end of the 5S rRNA gene to three major termination sites about 1,780, 1,830, and 1,870 nucleotides from the 3' end of the 26S rRNA gene. A survey of various wild-type and respiration-deficient strains showed that NTS transcript abundance depended on the mitochondrial genotype and a single codominant nuclear locus. In strains with that nuclear determinant, NTS transcripts were barely detected in [rho+] cells, were slightly more abundant in various mit- derivatives, and were most abundant in petites. However, in one petite that was hypersuppressive and contained a putative origin of replication (ori5) within its 757-base-pair mitochondrial genome, NTS transcripts were no more abundant than in [rho+] cells. The property of low NTS transcript abundance in the hypersuppressive petite was unstable, and spontaneous segregants that contained NTS transcripts as abundant as in the other petites examined could be obtained. Thus, respiration deficiency per se is not the major factor contributing to the accumulation of these unusual RNAs. Unlike RNA polymerase I transcripts, the abundant NTS RNAs were glucose repressible, fractionated as poly(A)+ RNAs, and were sensitive to inhibition by 10 micrograms of alpha-amanitin per ml, a concentration that had no effect on rRNA synthesis. Abundant NTS RNAs are therefore most likely derived by polymerase II transcription.
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Parikh, V. S., H. Conrad-Webb, R. Docherty, and R. A. Butow. "Interaction between the yeast mitochondrial and nuclear genomes influences the abundance of novel transcripts derived from the spacer region of the nuclear ribosomal DNA repeat." Molecular and Cellular Biology 9, no. 5 (May 1989): 1897–907. http://dx.doi.org/10.1128/mcb.9.5.1897-1907.1989.
Full textAbstract:
We have identified stable transcripts from the so-called nontranscribed spacer region (NTS) of the nuclear ribosomal DNA repeat in certain respiration-deficient strains of Saccharomyces cerevisiae. These RNAs, which are transcribed from the same strand as is the 37S rRNA precursor, are 500 to 800 nucleotides long and extend from the 5' end of the 5S rRNA gene to three major termination sites about 1,780, 1,830, and 1,870 nucleotides from the 3' end of the 26S rRNA gene. A survey of various wild-type and respiration-deficient strains showed that NTS transcript abundance depended on the mitochondrial genotype and a single codominant nuclear locus. In strains with that nuclear determinant, NTS transcripts were barely detected in [rho+] cells, were slightly more abundant in various mit- derivatives, and were most abundant in petites. However, in one petite that was hypersuppressive and contained a putative origin of replication (ori5) within its 757-base-pair mitochondrial genome, NTS transcripts were no more abundant than in [rho+] cells. The property of low NTS transcript abundance in the hypersuppressive petite was unstable, and spontaneous segregants that contained NTS transcripts as abundant as in the other petites examined could be obtained. Thus, respiration deficiency per se is not the major factor contributing to the accumulation of these unusual RNAs. Unlike RNA polymerase I transcripts, the abundant NTS RNAs were glucose repressible, fractionated as poly(A)+ RNAs, and were sensitive to inhibition by 10 micrograms of alpha-amanitin per ml, a concentration that had no effect on rRNA synthesis. Abundant NTS RNAs are therefore most likely derived by polymerase II transcription.
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White, F. C., M. D. McKirnan, E. A. Breisch, B. D. Guth, Y. M. Liu, and C. M. Bloor. "Adaptation of the left ventricle to exercise-induced hypertrophy." Journal of Applied Physiology 62, no. 3 (March 1, 1987): 1097–110. http://dx.doi.org/10.1152/jappl.1987.62.3.1097.
Full textAbstract:
Cardiac functional and structural adaptations to exercise-induced hypertrophy were studied in 68 pigs. Pigs were exercise trained on a treadmill for 10 wk. Sequential measurements were made of cardiac dimensions, [left ventricular end-diastolic diameter (EDD), changes in diameter (delta D%), wall thickness (WTh), wall thickening (WTh%), left ventricular pressure (LVP), time derivative of pressure (dP/dt), stroke volume, total body O2 consumption (VO2), blood gases, and systemic hemodynamics] at rest and during moderate and severe exercise. Postmortem studies included morphometric measurements of capillary density, arteriolar density, mitochondria, and myofibrils. All of the exercise-trained pigs showed significant increases in aerobic capacity. Maximum O2 consumption (VO2 max) increased by 37.5% in group 1 (moderate exercise training) and 34% in group 3 (heavy exercise training). Cardiac hypertrophy ranged from less than 15% in a group (n = 8) subjected to moderate exercise training to greater than 30% in a group (n = 11) subjected to heavy exercise training. Before training, exercise was characterized by a decreasing EDD during progressive exercise; this was reversed after exercise training. Stroke volume and end-diastolic volumes during exercise showed a highly significant increase after exercise training and hypertrophy. Morphometric measurements showed that mitochondria and cell membranes increased with increasing myocyte growth in all exercise groups, but there was only a partially compensated adaptation of capillary proliferation. Arteriolar number and length increased in all exercise groups. Intrinsic contractility as measured by delta D%, WTh%, or left ventricular dP/dt did not increase with exercise training and in some instances decreased. Therefore, left ventricular adaptation to strenuous exercise in the pig heart is primarily one of changes in left ventricular dimensions and a compensated hypertrophy. Exercise-induced increases in EDD and stroke volume can be accounted for by decreases in peripheral resistance and increased cardiac dimensions.
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Wang, Ying, Shan He, Yongnian Liu, Robert Hooper, Hongshuang Yu, Yuanyuan Tian, Bui Tien, et al. "Artesunate Inhibits Graft-Versus-Host Disease in Mice Via a Mechanism of Inducing Mitochondrial Calcium Overloading in Activated T Cells." Blood 134, Supplement_1 (November 13, 2019): 4432. http://dx.doi.org/10.1182/blood-2019-131115.
Full textAbstract:
Despite pharmacological prophylaxis using calcineurin (CN) inhibitors (i.e., cyclosporin A and Tacrolimus), graft-versus-host disease (GVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Upon antigen stimulation, activated T cell receptor (TCR) signaling triggers rapid Ca2+ influx. This induces both CN-mediated NFAT activation, and increase of mitochondrial Ca2+ content, a major driver of metabolic activity. However, mitochondrial Ca2+ overload triggers opening of the mitochondrial permeability transition pore and cell death. We hypothesize that pharmacologically increasing mitochondrial Ca2+ load may decrease T cell survival capability, thereby reducing the GVH reaction. If this can be accomplished, it may lead to new strategies for inhibition of GVHD, which is conceptually different from the use of CN inhibitors. To test this hypothesis, we employed a high throughput drug screening system with the proliferation and cytotoxicity of TCR-activated human T cells as the readout, and screened the NPL-800 library (https://www.timtec.net) composed of 800 pure natural compounds. With a stringent criterion in consideration of dose-dependent effect, 26 compounds stood out for reducing the count of activated human T cells with a reduction rate of at least 30% at both 1.0uM and 10.0uM. Positive hits included inhibitors of DNA synthesis, the Na-K-ATPase and mitochondrial metabolism. We were particularly interested in artesunate (ART), which is a derivative of artemisinin that has been used for treating malaria in patients. While artemisinin acts by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in P. falciparum malaria, which causes passive endoplasmic reticulum (ER) Ca2+ depletion and the subsequent cytosolic Ca2+ influx, ART did not inhibit SERCA in T cells. Ex vivo culture assays showed that ART dose-dependently reduced the survival of TCR-activated murine T cells. This inhibitory effect of ART was abrogated by inhibiting Ca2+ influx using BTP2, a potent inhibitor of store-operated Ca2+ channels. Furthermore, treatment of murine CD8 T cells with ART induced significant increases in mitochondrial Ca2+ loading upon TCR activation. These data suggest that inhibition of T cell survival by ART was dependent on TCR activation-induced Ca2+ influx and associated with enhanced mitochondrial Ca2+ uptake. We examined the impact of ART on GVHD in Balb/c mice receiving C57BL/6 (B6) mouse T cell-depleted bone marrow (TCD-BM) and CD4+ T cells. Intraperitoneal injection of ART (10 mg/kg, every other day) from day 1 to day 28 after transplantation reduced clinical signs of GVHD in these recipients and significantly improved their overall survival. Similar inhibition effects of ART on GVHD were observed in miHA-mismatched B6 anti-Balb/b and haplo-identical B6 anti-BDF1 mouse models of GVHD. Further investigations showed that in vivo administration of ART caused significant decreases in the number of host-reactive donor T cells in the spleen and liver of Balb/c mice 7 days after transfer of B6 TCD-BM plus CD4+ T cells. ART treatment did not affect the capacity of donor T cells to produce effector cytokines (e.g., IFN-g and TNF-α) in individual cells. Importantly, in vivo administration of ART preserved anti-leukemia activity of donor T cells and did not impair the reconstitution of hematopoiesis and lymphocytes. Collectively, our findings indicate that pharmacologically increasing mitochondrial Ca2+ loading may have significant implications in the development of novel strategies to prevent GVHD and other T cell-mediated inflammatory disorders in a broad context. Since ART therapy has been clinically approved, this work could be immediately translated into patients. Disclosures No relevant conflicts of interest to declare.
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Xiao, Junjie, Dandan Liang, Hong Zhang, Ying Liu, Fajie Li, and Yi-Han Chen. "4′-Chlorodiazepam, a translocator protein (18 kDa) antagonist, improves cardiac functional recovery during postischemia reperfusion in rats." Experimental Biology and Medicine 235, no. 4 (April 2010): 478–86. http://dx.doi.org/10.1258/ebm.2009.009291.
Full textAbstract:
Inhibition of translocator protein (18 kDa) (TSPO) can effectively prevent reperfusion-induced arrhythmias and improve postischemic contractile performance. Mitochondrial permeability transition pore (mPTP) opening, mediated mainly through oxidative stress during ischemia/reperfusion (I/R), is a key event in reperfusion injury. 4′-Chlorodiazepam is a widely used TSPO antagonist. However, whether 4′-chlorodiazepam can improve cardiac functional recovery during postischemia reperfusion by affecting oxidative enzymes, reducing reactive oxygen species (ROS) and thereby inhibiting mPTP opening is still unknown. Cardiac function including heart rate, coronary flow rate, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximal time derivatives of pressure (±d P/d t max) and the severity of ventricular arrhythmias were analyzed in isolated rat hearts during I/R. mPTP opening, ROS and oxidative enzyme activities were measured with fluorometric or spectrophotometric techniques. 4′-Chlorodiazepam did not affect heart rate and coronary flow rate, but abolished the increase in LVEDP, accelerated the recovery of LVDP and ±d P/d t max, and reduced the severity of ventricular arrhythmias. The mPTP opening probability was reduced by 4′-chlorodiazepam, accompanied by a reduction in ROS level. In addition, the activities of mitochondrial electron transport chain complex I and complex III were increased, while those of xanthine oxidase and NADPH oxidase were reduced. Therefore, 4′-chlorodiazepam may improve cardiac functional recovery during reperfusion, potentially by affecting the activities of oxidative enzymes, reducing ROS and thereby inhibiting mPTP opening. The present study presents evidence that 4′-chlorodiazepam could be a novel adjunct to reperfusion.
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Ke, YJ, XD Qin, YC Zhang, H. Li, R. Li, JL Yuan, X. Yang, and SM Ding. "In vitro study on cytotoxicity and intracellular formaldehyde concentration changes after exposure to formaldehyde and its derivatives." Human & Experimental Toxicology 33, no. 8 (November 12, 2013): 822–30. http://dx.doi.org/10.1177/0960327113510538.
Full textAbstract:
HeLa cells were exposed to formaldehyde and its metabolic derivatives, methanol, formic acid, and acetaldehyde, to investigate that the toxicity of formaldehyde is not caused by the chemical group. After 1 h of treatment with formaldehyde, mitochondrial assays showed that low concentrations (e.g. 10 μmol/L) of formaldehyde promoted growth of the HeLa cells, while higher concentrations (e.g. ≥62.5 μmol/L) inhibited cell growth; while all four chemicals at a concentration of 125 μmol/L affected cell growth, formaldehyde affected the largest. Reactive oxygen species concentration increased with the concentration of the exposure chemical. The endogenous formaldehyde content increased the most in the formaldehyde group, but in other three groups, it did not increase as the exposure concentration increased. Expression of dehydrogenase (formaldehyde dehydrogenase (FDH)) in the formaldehyde (10.40) and methanol (10.60) groups increased significantly compared with the control (1), while it was similar to the control in formic acid (0.90) and acetaldehyde (1.10) groups. Our results suggest that formaldehyde could affect cell activity and even enter cells. Exposure to formaldehyde changes the endogenous formaldehyde concentration in cells within 24 h, and this induces expression of FDH for formaldehyde degradation to maintain the formaldehyde balance. The toxicity of formaldehyde is not caused by the carbon atoms in the aldehyde, hydroxyl, or carboxyl groups. Formaldehyde is hypothesized to be an important signaling molecule in the regulation of cell growth and maintenance of the endogenous formaldehyde level.
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Mitsiades, Nicholas, Ciaran J. McMullan, Vassiliki Poulaki, Joseph Negri, David C. Geer, Valsala Haridas, Nikhil C. Munshi, Jordan Gutterman, Kenneth C. Anderson, and Constantine S. Mitsiades. "Avicins: A Novel Class of Anti-Myeloma Agents." Blood 104, no. 11 (November 16, 2004): 3405. http://dx.doi.org/10.1182/blood.v104.11.3405.3405.
Full textAbstract:
Abstract Multiple myeloma (MM) remains an incurable neoplasia, despite recent development of several novel therapies. As part of our efforts to identify new compounds with anti-MM activity, we evaluated the class of avicins, which are triterpenoid saponins that have been shown to induce apoptosis of neoplastic cells by affecting mitochondrial function independently of membrane-bound death receptors. Because we have previously shown that mitochondria constitute key regulators of MM cell responsiveness to diverse anti-tumor agents, (e.g. the proteasome inhibitor bortezomib), we evaluated the in vitro anti-MM effects of this class of compounds. Our vitro drug-sensitivity studies showed that Avicin D and Avicin G, the main members of this class of compounds, are active against a broad panel of MM cell lines and primary tumor cells, including tumor cells resistant to conventional (e.g. dexamethasone, alkylating agents, anthracyclines) or novel (e.g. thalidomide, immunomodulatory thalidomide derivatives, proteasome inhibitor PS-341[bortezomib], Apo2L/TRAIL) anti-MM agents. Using MTT survival assays, we confirmed that the IC50 values for both Avicins were highly concordant and were less than 250 nM for the overwhelming majority of MM cell lines tested. Importantly, this potent in vitro anti-MM activity was triggered by concentrations of Avicins which had minimal, if any, effect on the viability of normal hematopoietic cells or bone marrow stromal cells. Furthermore these IC50 values were comparable with the in vitro activity of this agent among the most Avicin-sensitive tumor models that have been previously tested. This potent anti-MM effect was not inhibited by transfection of MM cells with construct for constitutively active Akt. Although cytokine- or cell adhesion-mediated interactions of the local bone marrow (BM) microenvironment (e.g. BM stromal cells) protects MM cells from conventional therapies (e.g. dexamethasone or cytotoxic chemotherapy), avicins were able to overcome this protective effect in co-culture models of MM cells with BM stromal cells and sensitized MM cells to cytotoxic chemotherapy-induced cell death. Using hierarchical clustering analyses and relevance network algorithms, we compared the patterns of MM cell sensitivity to Avicin D vs Avicin G vs. other anti-cancer drugs and found that the pattern of dose-response relationships of the 2 main members of this class of compounds are very similar to each other, but clearly distinct from the patterns of sensitivity or resistance to other drugs, either conventional or investigational. This further supports the notion that the anti-MM properties of Avicins are mediated by molecular mechanisms distinct from those of currently available anti-MM drugs, and also suggests that Avicins may have anti-tumor activity even against subgroups of MM which may be resistant to other novel therapies that are currently in clinical development. These results have provided the framework for ongoing in vivo studies of anti-tumor activity of these agents, to evaluate the feasibility of future clinical trials of Avicins to improve patient outcome in MM.
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Nasr, Soad, Mai Rady, Aya Sebak, Iman Gomaa, Walid Fayad, Menna El Gaafary, Mahmoud Abdel-Kader, Tatiana Syrovets, and Thomas Simmet. "A Naturally Derived Carrier for Photodynamic Treatment of Squamous Cell Carcinoma: In Vitro and In Vivo Models." Pharmaceutics 12, no. 6 (May 29, 2020): 494. http://dx.doi.org/10.3390/pharmaceutics12060494.
Full textAbstract:
Photodynamic therapy (PDT) is a non-invasive treatment strategy that includes the combination of three components—a photosensitizer, a light source, and tissue oxygen. PDT can be used for the treatment of skin diseases such as squamous cell carcinoma. The photosensitizer used in this study is the naturally derived chlorophyll derivative chlorin e6 (Ce6), which was encapsulated in ultradeformable ethosomes. Singlet oxygen production by Ce6 upon laser light irradiation was not significantly affected by encapsulation into ethosomes. PDT of squamous cell carcinoma cells treated with Ce6 ethosomes triggered increased mitochondrial superoxide levels and increased caspase 3/7 activity, resulting in concentration- and light-dose-dependent cytotoxicity. Ce6 ethosomes showed good penetration into 3D squamous cell carcinoma spheroids, which upon laser light irradiation exhibited reduced size, proliferation, and viability. The PDT effect of Ce6 ethosomes was specific and showed higher cytotoxicity against squamous cell carcinoma spheroids compared to normal skin fibroblast spheroids. In addition, PDT treatment of squamous cell carcinoma xenografts grown on chorioallantoic membranes of chick eggs (CAM) exhibited reduced expression of Ki-67 proliferation marker and increased terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining, indicating reduced proliferation and activation of apoptosis, respectively. The results demonstrate that Ce6-loaded ethosomes represent a convenient formulation for photodynamic treatment of squamous cell carcinoma.
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Ismail, Ismail, Zhuoyue Chen, Xiuru Ji, Lu Sun, Long Yi, and Zhen Xi. "A Fast-Response Red Shifted Fluorescent Probe for Detection of H2S in Living Cells." Molecules 25, no. 3 (January 21, 2020): 437. http://dx.doi.org/10.3390/molecules25030437.
Full textAbstract:
Near-infrared (NIR) fluorescent probes are attractive tools for bioimaging applications because of their low auto-fluorescence interference, minimal damage to living samples, and deep tissue penetration. H2S is a gaseous signaling molecule that is involved in redox homeostasis and numerous biological processes in vivo. To this end, we have developed a new red shifted fluorescent probe 1 to detect physiological H2S in live cells. The probe 1 is based on a rhodamine derivative as the red shifted fluorophore and the thiolysis of 7-nitro 1,2,3-benzoxadiazole (NBD) amine as the H2S receptor. The probe 1 displays fast fluorescent enhancement at 660 nm (about 10-fold turn-ons, k2 = 29.8 M−1s−1) after reacting with H2S in buffer (pH 7.4), and the fluorescence quantum yield of the activated red shifted product can reach 0.29. The probe 1 also exhibits high selectivity and sensitivity towards H2S. Moreover, 1 is cell-membrane-permeable and mitochondria-targeting, and can be used for imaging of endogenous H2S in living cells. We believe that this red shifted fluorescent probe can be a useful tool for studies of H2S biology.
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Machado, João Franco, João D. G. Correia, and Tânia S. Morais. "Emerging Molecular Receptors for the Specific-Target Delivery of Ruthenium and Gold Complexes into Cancer Cells." Molecules 26, no. 11 (May 25, 2021): 3153. http://dx.doi.org/10.3390/molecules26113153.
Full textAbstract:
Cisplatin and derivatives are highly effective in the treatment of a wide range of cancer types; however, these metallodrugs display low selectivity, leading to severe side effects. Additionally, their administration often results in the development of chemoresistance, which ultimately results in therapeutic failure. This scenario triggered the study of other transition metals with innovative pharmacological profiles as alternatives to platinum, ruthenium- (e.g., KP1339 and NAMI-A) and gold-based (e.g., Auranofin) complexes being among the most advanced in terms of clinical evaluation. Concerning the importance of improving the in vivo selectivity of metal complexes and the current relevance of ruthenium and gold metals, this review article aims to survey the main research efforts made in the past few years toward the design and biological evaluation of target-specific ruthenium and gold complexes. Herein, we give an overview of the inorganic and organometallic molecules conjugated to different biomolecules for targeting membrane proteins, namely cell adhesion molecules, G-protein coupled receptors, and growth factor receptors. Complexes that recognize the progesterone receptors or other targets involved in metabolic pathways such as glucose transporters are discussed as well. Finally, we describe some complexes aimed at recognizing cell organelles or compartments, mitochondria being the most explored. The few complexes addressing targeted gene therapy are also presented and discussed.
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Kolwicz, Stephen C., Suneet Purohit, and Rong Tian. "Cardiac Metabolism and its Interactions With Contraction, Growth, and Survival of Cardiomyocytes." Circulation Research 113, no. 5 (August 16, 2013): 603–16. http://dx.doi.org/10.1161/circresaha.113.302095.
Full textAbstract:
The network for cardiac fuel metabolism contains intricate sets of interacting pathways that result in both ATP-producing and non–ATP-producing end points for each class of energy substrates. The most salient feature of the network is the metabolic flexibility demonstrated in response to various stimuli, including developmental changes and nutritional status. The heart is also capable of remodeling the metabolic pathways in chronic pathophysiological conditions, which results in modulations of myocardial energetics and contractile function. In a quest to understand the complexity of the cardiac metabolic network, pharmacological and genetic tools have been engaged to manipulate cardiac metabolism in a variety of research models. In concert, a host of therapeutic interventions have been tested clinically to target substrate preference, insulin sensitivity, and mitochondrial function. In addition, the contribution of cellular metabolism to growth, survival, and other signaling pathways through the production of metabolic intermediates has been increasingly noted. In this review, we provide an overview of the cardiac metabolic network and highlight alterations observed in cardiac pathologies as well as strategies used as metabolic therapies in heart failure. Lastly, the ability of metabolic derivatives to intersect growth and survival are also discussed.
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Kawai, Keisuke, Fuzhong Qin, Junya Shite, Weike Mao, Shuji Fukuoka, and Chang-seng Liang. "Importance of antioxidant and antiapoptotic effects of β-receptor blockers in heart failure therapy." American Journal of Physiology-Heart and Circulatory Physiology 287, no. 3 (September 2004): H1003—H1012. http://dx.doi.org/10.1152/ajpheart.00797.2003.
Full textAbstract:
The present study was carried out to determine whether beneficial effects of carvedilol in congestive heart failure (CHF) are mediated via its β-adrenergic blocking, antioxidant, and/or α-adrenergic blocking action. Rabbits with heart failure induced by rapid cardiac pacing were randomized to receive subcutaneous carvedilol, metoprolol, propranolol plus doxazosin, or placebo pellets for 8 wk and compared with sham-operated rabbits without pacing. We found rapid cardiac pacing produced clinical heart failure, left ventricular dilation, and decline of left ventricular fractional shortening. This was associated with an increase in left ventricular end-diastolic pressure, decrease in left ventricular first derivative of left ventricular pressure, and myocyte hypertrophy. Tissue oxidative stress measured by GSH/GSSG was increased in the heart with increased oxidation product of mitochondrial DNA, 8-oxo-7,8-dihydro-2′-deoxyguanosine, increase of Bax, decrease of Bcl-2, and increase of apoptotic myocytes as measured by anti-single-stranded DNA monoclonal antibody. Administration of carvedilol and metoprolol, which had no effect in sham animals, attenuated cardiac ventricular remodeling, cardiac hypertrophy, oxidative stress, and myocyte apoptosis in CHF. In contrast, propranolol plus doxazosin, which has less antioxidant effects, produced smaller effects on left ventricular function and myocyte apoptosis. In all animals, GSH/GSSG correlated significantly with changes of left ventricular end-diastolic dimension ( r = −0.678, P < 0.0001), fractional shortening ( r = 0.706, P < 0.0001), and apoptotic myocytes ( r = −0.473, P = 0.0001). Thus our findings suggest antioxidant and antiapoptotic actions of carvedilol and metoprolol are important determinants of clinical beneficial effects of β-receptors in the treatment of CHF.
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Lountos, George T., Xue Zhi Zhao, Evgeny Kiselev, Joseph E. Tropea, Danielle Needle, Yves Pommier, Terrence R. Burke, and David S. Waugh. "Identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl-DNA phosphodiesterase I (TDP1) phosphoryl recognition by crystallographic fragment cocktail screening." Nucleic Acids Research 47, no. 19 (June 14, 2019): 10134–50. http://dx.doi.org/10.1093/nar/gkz515.
Full textAbstract:
Abstract Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3′ end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.
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Khusnutdinova, Elmira, Anastasiya Petrova, Zulfia Zileeva, Ulyana Kuzmina, Liana Zainullina, Yulia Vakhitova, Denis Babkov, and Oxana Kazakova. "Novel A-Ring Chalcone Derivatives of Oleanolic and Ursolic Amides with Anti-Proliferative Effect Mediated through ROS-Triggered Apoptosis." International Journal of Molecular Sciences 22, no. 18 (September 10, 2021): 9796. http://dx.doi.org/10.3390/ijms22189796.
Full textAbstract:
A series of A-ring modified oleanolic and ursolic acid derivatives including C28 amides (3-oxo-C2-nicotinoylidene/furfurylidene, 3β-hydroxy-C2-nicotinoylidene, 3β-nicotinoyloxy-, 2-cyano-3,4-seco-4(23)-ene, indolo-, lactame and azepane) were synthesized and screened for their cytotoxic activity against the NCI-60 cancer cell line panel. The results of the first assay of thirty-two tested compounds showed that eleven derivatives exhibited cytotoxicity against cancer cells, and six of them were selected for complete dose–response studies. A systematic study of local SARs has been carried out by comparative analysis of potency distributions and similarity relationships among the synthesized compounds using network-like similarity graphs. Among the oleanane type triterpenoids, C2-[4-pyridinylidene]-oleanonic C28-morpholinyl amide exhibited sub-micromolar potencies against 15 different tumor cell lines and revealed particular selectivity for non-small cell lung cancer (HOP-92) with a GI50 value of 0.0347 μM. On the other hand, superior results were observed for C2-[3-pyridinylidene]-ursonic N-methyl-piperazinyl amide 29, which exhibited a broad-spectrum inhibition activity with GI50 < 1 μM against 33 tumor cell lines and <2 μM against all 60 cell lines. This compound has been further evaluated for cell cycle analysis to decipher the mechanism of action. The data indicate that compound 29 could exhibit both cytostatic and cytotoxic activity, depending on the cell line evaluated. The cytostatic activity appears to be determined by induction of the cell cycle arrest at the S (MCF-7, SH-SY5Y cells) or G0/G1 phases (A549 cells), whereas cytotoxicity of the compound against normal cells is nonspecific and arises from apoptosis without significant alterations in cell cycle distribution (HEK293 cells). Our results suggest that the antiproliferative effect of compound 29 is mediated through ROS-triggered apoptosis that involves mitochondrial membrane potential depolarization and caspase activation.
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Kodr, David, Jarmila Stanková, Michaela Rumlová, Petr Džubák, Jiří Řehulka, Tomáš Zimmermann, Ivana Křížová, et al. "Betulinic Acid Decorated with Polar Groups and Blue Emitting BODIPY Dye: Synthesis, Cytotoxicity, Cell-Cycle Analysis and Anti-HIV Profiling." Biomedicines 9, no. 9 (August 28, 2021): 1104. http://dx.doi.org/10.3390/biomedicines9091104.
Full textAbstract:
Betulinic acid (BA) is a potent triterpene, which has shown promising potential in cancer and HIV-1 treatment. Here, we report a synthesis and biological evaluation of 17 new compounds, including BODIPY labelled analogues derived from BA. The analogues terminated by amino moiety showed increased cytotoxicity (e.g., BA had on CCRF-CEM IC50 > 50 μM, amine 3 IC50 0.21 and amine 14 IC50 0.29). The cell-cycle arrest was evaluated and did not show general features for all the tested compounds. A fluorescence microscopy study of six derivatives revealed that only 4 and 6 were detected in living cells. These compounds were colocalized with the endoplasmic reticulum and mitochondria, indicating possible targets in these organelles. The study of anti-HIV-1 activity showed that 8, 10, 16, 17 and 18 have had IC50i > 10 μM. Only completely processed p24 CA was identified in the viruses formed in the presence of compounds 4 and 12. In the cases of 2, 8, 9, 10, 16, 17 and 18, we identified not fully processed p24 CA and p25 CA-SP1 protein. This observation suggests a similar mechanism of inhibition as described for bevirimat.
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Giovannoni, G., S. J. R. Heales, J. M. Land, and E. J. Thompson. "The potential role of nitric oxide in multiple sclerosis." Multiple Sclerosis Journal 4, no. 3 (June 1998): 212–16. http://dx.doi.org/10.1177/135245859800400323.
Full textAbstract:
Nitric oxide (.NO) and its reactive derivative peroxynitrite (ONOO7) have been implicated in the pathogenesis of multiple sclerosis (MS). They are cytotoxic to oligodendrocytes and neurones in culture by inhibiting the mitochondrial respiratory chain (complexes II/III and IV) and inhibiting certain key intracellular enzymes. Recently .NO has been implicated as a possible aetiological factor in reversible conduction block in demyelinated axons. Inducible nitric oxide synthase (iNOS) is upregulated in the central nervous system of animals with experimental allergic encephalomyelitis (EAE) and in patients with MS. In some EAE models inhibiting iNOS activity decreases disease severity whilst in other models disease activity is exacerbated. Raised levels of nitrate and nitrite, stable end-products of .NO/ONOO7, are found in the cerebrospinal fluid, serum and urine of patients with MS. CSF levels of nitrate and nitrite correlate with blood-brain-barrier dysfunction, which suggests that .NO may play a role in inflammatory blood-brain-barrier dysfunction. In a longitudinal study on 24 patients with relapsing remitting and secondary progressive MS, raised serum nitrate and nitrite levels correlated with a relapsing course and infrequent relapses. However, no correlation was found between raised serum levels of nitrate and nitrite and MRI activity, disease progression, or the development of cerebral atrophy. In autoimmune mediated CNS demyelinating disease .NO may be a double-edged sword, mediating tissue damage on the one hand and on the other hand modulating complex immunological functions which may be protective.
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Stitt, A. W., and I. Fairweather. "Spermatogenesis and the fine structure of the mature spermatozoon of the liver fluke, Fasciola hepatica (Trematoda: Digenea)." Parasitology 101, no. 3 (December 1990): 395–407. http://dx.doi.org/10.1017/s0031182000060595.
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SUMMARYSpermatogenesis and the fine structure of the mature spermatozoon of Fasciola hepatica have been studied by transmission electron microscopy. The primary spermatogonia display a typical gonial morphology and occupy the periphery of the testis. They undergo 3 mitotic divisions to give rise to 8 primary spermatocytes forming a rosette of cells connected to a central cytophore. The primary spermatocytes undergo 2 meiotic divisions, resulting in 32 spermatids that develop into spermatozoa. Intranuclear synaptonemal complexes in primary spermatocytes confirm the first meiotic division. The onset of spermiogenesis is marked by the formation of the zone of differentiation which contains 2 basal bodies and a further centriole derivative, the central body. The zone extends away from the spermatid cell to form the median process; into this migrates the differentiated and elongate nucleus. Simultaneously, 2 axonemes develop from the basal bodies. During development, they rotate through 90° to extend parallel to the median process. The migration of the nucleus to the distal end of the median process coincides with the fusion of the axonemes to the latter to form a monopartite spermatozoon. The mature spermatozoon possesses 2 axonemes of the 9 + ‘1’ pattern typical of parasitic platyhelminths, 2 elongate mitochondria and a variable array of peripheral microtubules. The nuclear region of the spermatozoon is immotile. The value of sperm ultrastructure as a taxonomic tool in platyhelminth phylogeny is discussed.
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Moldogazieva, Nurbubu T., Innokenty M. Mokhosoev, Tatiana I. Mel’nikova, Yuri B. Porozov, and Alexander A. Terentiev. "Oxidative Stress and Advanced Lipoxidation and Glycation End Products (ALEs and AGEs) in Aging and Age-Related Diseases." Oxidative Medicine and Cellular Longevity 2019 (August 14, 2019): 1–14. http://dx.doi.org/10.1155/2019/3085756.
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Oxidative stress is a consequence of the use of oxygen in aerobic respiration by living organisms and is denoted as a persistent condition of an imbalance between the generation of reactive oxygen species (ROS) and the ability of the endogenous antioxidant system (AOS) to detoxify them. The oxidative stress theory has been confirmed in many animal studies, which demonstrated that the maintenance of cellular homeostasis and biomolecular stability and integrity is crucial for cellular longevity and successful aging. Mitochondrial dysfunction, impaired protein homeostasis (proteostasis) network, alteration in the activities of transcription factors such as Nrf2 and NF-κB, and disturbances in the protein quality control machinery that includes molecular chaperones, ubiquitin-proteasome system (UPS), and autophagy/lysosome pathway have been observed during aging and age-related chronic diseases. The accumulation of ROS under oxidative stress conditions results in the induction of lipid peroxidation and glycoxidation reactions, which leads to the elevated endogenous production of reactive aldehydes and their derivatives such as glyoxal, methylglyoxal (MG), malonic dialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) giving rise to advanced lipoxidation and glycation end products (ALEs and AGEs, respectively). Both ALEs and AGEs play key roles in cellular response to oxidative stress stimuli through the regulation of a variety of cell signaling pathways. However, elevated ALE and AGE production leads to protein cross-linking and aggregation resulting in an alteration in cell signaling and functioning which causes cell damage and death. This is implicated in aging and various age-related chronic pathologies such as inflammation, neurodegenerative diseases, atherosclerosis, and vascular complications of diabetes mellitus. In the present review, we discuss experimental data evidencing the impairment in cellular functions caused by AGE/ALE accumulation under oxidative stress conditions. We focused on the implications of ALEs/AGEs in aging and age-related diseases to demonstrate that the identification of cellular dysfunctions involved in disease initiation and progression can serve as a basis for the discovery of relevant therapeutic agents.
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Conserva, Geanne, Natalia Girola, Carlos Figueiredo, Ricardo Azevedo, Sasha Mousdell, Patricia Sartorelli, Marisi Soares, Guilherme Antar, and João Lago. "Terpenoids from Leaves of Guarea macrophylla Display In Vitro Cytotoxic Activity and Induce Apoptosis In Melanoma Cells." Planta Medica 83, no. 16 (April 10, 2017): 1289–96. http://dx.doi.org/10.1055/s-0043-107241.
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Abstract Guarea macrophylla is a Brazilian plant species that has been used in folk medicine to treat a range of diseases. Our ongoing work focuses on the discovery of new bioactive natural products derived from Brazilian flora. The current study describes the identification of cytotoxic compounds from the EtOH extract of leaves from G. macrophylla using bioactivity-guided fractionation. This approach resulted in the isolation and characterization of four compounds: cycloart-23E-ene-3β,25-diol (1), (23S*,24S*)-dihydroxycicloart-25-en-3-one (2), isopimara-7,15-diene-2α,3β-diol (3), and isopimara-7,15-dien-3β-ol (4), in which 2 and 3 are identified as new derivatives. In vitro assays were conducted to evaluate the cytotoxic activity of compounds 1–4 against a panel of cancer cell lines and to determine the possible mechanism(s) related to the activity of the compounds on B16F10Nex2 cells. The most active compound 1 induced cytotoxic effects on tumor cells, with IC50 values of 18.3, 52.1, and 58.9 µM against HL-60, HeLa, and B16F10-Nex2 tumor cells, respectively. Furthermore, it was observed in melanoma cells that compound 1 induced several specific apoptotic hallmarks, such as morphological changes in the cell shape structure, nuclear DNA condensation, specific chromatin fragmentation, and disruption in the mitochondrial membrane potential, which are related to the intrinsic apoptotic pathway.
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Qureshi, Irfan A., and Mark F. Mehler. "An evolving view of epigenetic complexity in the brain." Philosophical Transactions of the Royal Society B: Biological Sciences 369, no. 1652 (September 26, 2014): 20130506. http://dx.doi.org/10.1098/rstb.2013.0506.
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Recent scientific advances have revolutionized our understanding of classical epigenetic mechanisms and the broader landscape of molecular interactions and cellular functions that are inextricably linked to these processes. Our current view of epigenetics includes an increasing appreciation for the dynamic nature of DNA methylation, active mechanisms for DNA demethylation, differential functions of 5-methylcytosine and its oxidized derivatives, the intricate regulatory logic of histone post-translational modifications, the incorporation of histone variants into chromatin, nucleosome occupancy and dynamics, and direct links between cellular signalling pathways and the actions of chromatin ‘reader’, ‘writer’ and ‘eraser’ molecules. We also have an increasing awareness of the seemingly ubiquitous roles played by diverse classes of selectively expressed non-coding RNAs in transcriptional, post-transcriptional, post-translational and local and higher order chromatin modulatory processes. These perspectives are still evolving with novel insights continuing to emerge rapidly (e.g. those related to epigenetic regulation of mobile genetic elements, epigenetic mechanisms in mitochondria, roles in nuclear architecture and ‘RNA epigenetics’). The precise functions of these epigenetic factors/phenomena are largely unknown. However, it is unequivocal that they serve as key mediators of brain complexity and flexibility, including neural development and aging, cellular differentiation, homeostasis, stress responses, and synaptic and neural network connectivity and plasticity.
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Yang, Guangwei, Elijah Paintsil, Ginger E. Dutschman, Susan P. Grill, Chuan-Jen Wang, Jimin Wang, Hiromichi Tanaka, Takayuki Hamasaki, Masanori Baba, and Yung-Chi Cheng. "Impact of Novel Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutations P119S and T165A on 4′-Ethynylthymidine Analog Resistance Profile." Antimicrobial Agents and Chemotherapy 53, no. 11 (August 24, 2009): 4640–46. http://dx.doi.org/10.1128/aac.00686-09.
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ABSTRACT 2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T), a derivative of stavudine (d4T), has potent activity against human immunodeficiency virus and is much less inhibitory to mitochondrial DNA synthesis and cell growth than its progenitor, d4T. 4′-Ed4T triphosphate was a better reverse transcriptase (RT) inhibitor than d4T triphosphate, due to the additional binding of the 4′-ethynyl group at a presumed hydrophobic pocket in the RT active site. Previous in vitro selection for 4′-Ed4T-resistant viral strains revealed M184V and P119S/T165A/M184V mutations on days 26 and 81, respectively; M184V and P119S/T165A/M184V conferred 3- and 130-fold resistance to 4′-Ed4T, respectively. We investigated the relative contributions of these mutations, engineered into the strain NL4-3 background, to drug resistance, RT activity, and viral growth. Viral variants with single RT mutations (P119S or T165A) did not show resistance to 4′-Ed4T; however, M184V and P119S/T165A/M184V conferred three- and fivefold resistance, respectively, compared with that of the wild-type virus. The P119S/M184V and T165A/M184V variants showed about fourfold resistance to 4′-Ed4T. The differences in the growth kinetics of the variants were not more than threefold. The purified RT of mutants with the P119S/M184V and T165A/M184V mutations were inhibited by 4′-Ed4TTP with 8- to 13-fold less efficiency than wild-type RT. M184V may be the primary resistance-associated mutation of 4′-Ed4T, and P119S and T165A are secondary mutations. On the basis of our findings and the results of structural modeling, a virus with a high degree of resistance to 4′-Ed4T (e.g., more than 50-fold resistance) will be difficult to develop. The previously observed 130-fold resistance of the virus with P119S/T165A/M184V to 4′-Ed4T may be partly due to mutations both in the RT sequence and outside the RT sequence.
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Zhang, Yin Hua. "Nitric oxide signalling and neuronal nitric oxide synthase in the heart under stress." F1000Research 6 (May 23, 2017): 742. http://dx.doi.org/10.12688/f1000research.10128.1.
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Nitric oxide (NO) is an imperative regulator of the cardiovascular system and is a critical mechanism in preventing the pathogenesis and progression of the diseased heart. The scenario of bioavailable NO in the myocardium is complex: 1) NO is derived from both endogenous NO synthases (endothelial, neuronal, and/or inducible NOSs [eNOS, nNOS, and/or iNOS]) and exogenous sources (entero-salivary NO pathway) and the amount of NO from exogenous sources varies significantly; 2) NOSs are located at discrete compartments of cardiac myocytes and are regulated by distinctive mechanisms under stress; 3) NO regulates diverse target proteins through different modes of post-transcriptional modification (soluble guanylate cyclase [sGC]/cyclic guanosine monophosphate [cGMP]/protein kinase G [PKG]-dependent phosphorylation, S-nitrosylation, and transnitrosylation); 4) the downstream effectors of NO are multidimensional and vary from ion channels in the plasma membrane to signalling proteins and enzymes in the mitochondria, cytosol, nucleus, and myofilament; 5) NOS produces several radicals in addition to NO (e.g. superoxide, hydrogen peroxide, peroxynitrite, and different NO-related derivatives) and triggers redox-dependent responses. However, nNOS inhibits cardiac oxidases to reduce the sources of oxidative stress in diseased hearts. Recent consensus indicates the importance of nNOS protein in cardiac protection under pathological stress. In addition, a dietary regime with high nitrate intake from fruit and vegetables together with unsaturated fatty acids is strongly associated with reduced cardiovascular events. Collectively, NO-dependent mechanisms in healthy and diseased hearts are better understood and shed light on the therapeutic prospects for NO and NOSs in clinical applications for fatal human heart diseases.
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Kondo, Mitsuhiro, Tomoko Oya-Ito, Takeshi Kumagai, Toshihiko Osawa, and Koji Uchida. "Cyclopentenone Prostaglandins as Potential Inducers of Intracellular Oxidative Stress." Journal of Biological Chemistry 276, no. 15 (January 12, 2001): 12076–83. http://dx.doi.org/10.1074/jbc.m009630200.
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In the present study, we find that cyclopentenone prostaglandins (PGs) of the J2series, naturally occurring derivatives of PGD2, are potential inducers of intracellular oxidative stress that mediates cell degeneration. Based on an extensive screening of diverse chemical agents on induction of intracellular production of reactive oxygen species (ROS), we found that the cyclopentenone PGs, such as PGA2, PGJ2, Δ12-PGJ2, and 15-deoxy-Δ12,14-PGJ2, showed the most potent pro-oxidant effect on SH-SY5Y human neuroblastoma cells. As the intracellular events that mediate the PG cytotoxicity, we observed (i) the cellular redox alteration represented by depletion of antioxidant defenses, such as glutathione and glutathione peroxidase; (ii) a transient decrease in the mitochondrial membrane potential (Δψ); (iii) the production of protein-bound lipid peroxidation products, such as acrolein and 4-hydroxy-2-nonenal; and (iv) the accumulation of ubiquitinated proteins. These events correlated well with the reduction in cell viability. In addition, the thiol compound,N-acetylcysteine, could significantly inhibit the PG-induced ROS production, thereby preventing cytotoxicity, suggesting that the redox alteration is closely related to the pro-oxidant effect of cyclopentenone PGs. More strikingly, the lipid peroxidation end products, acrolein and 4-hydroxy-2-nonenal, detected in the PG-treated cells potently induced the ROS production, which was accompanied by the accumulation of ubiquitinated proteins and cell death, suggesting that the membrane lipid peroxidation products may represent one of the causative factors that potentiate the cytotoxic effect of cyclopentenone PGs by accelerating intracellular oxidative stress. These data suggest that the intracellular oxidative stress, represented by ROS production/lipid peroxidation and redox alteration, may underlie the well documented biological effects, such as antiproliferative and antitumor activities, of cyclopentenone PGs.
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Hong, Ming, Jinke Li, Siying Li, and Mohammed M.Almutairi. "Acetylshikonin Sensitizes Hepatocellular Carcinoma Cells to Apoptosis through ROS-Mediated Caspase Activation." Cells 8, no. 11 (November 19, 2019): 1466. http://dx.doi.org/10.3390/cells8111466.
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The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown strong and explicit cancer cell-selectivity, which results in little toxicity toward normal tissues, and has been recognized as a potential, relatively safe anticancer agent. However, several cancers are resistant to the apoptosis induced by TRAIL. A recent study found that shikonin b (alkannin, 5,8-dihydroxy-2-[(1S)-1-hydroxy-4-methylpent-3-en-1-yl]naphthalene-1,4-dione) might induce apoptosis in TRAIL-resistant cholangiocarcinoma cells through reactive oxygen species (ROS)-mediated caspases activation. However, the strong cytotoxic activity has limited its potential as an anticancer drug. Thus, the current study intends to discover novel shikonin derivatives which can sensitize the liver cancer cell to TRAIL-induced apoptosis while exhibiting little toxicity toward the normal hepatic cell. The trypan blue exclusion assay, western blot assay, 4′,6-diamidino-2-phenylindole (DAPI) staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as well as the ‘comet’ assay, were used to study the underlying mechanisms of cell death and to search for any mechanisms of an enhancement of TRAIL-mediated apoptosis in the presence of ASH. Herein, we demonstrated that non-cytotoxic doses of acetylshikonin (ASH), one of the shikonin derivatives, in combination with TRAIL, could promote apoptosis in HepG2 cells. Further studies showed that application of ASH in a non-cytotoxic dose (2.5 μM) could increase intracellular ROS production and induce DNA damage, which might trigger a cell intrinsic apoptosis pathway in the TRAIL-resistant HepG2 cell. Combination treatment with a non-cytotoxic dose of ASH and TRAIL activated caspase and increased the cleavage of PARP-1 in the HepG2 cell. However, when intracellular ROS production was suppressed by N-acetyl-l-cysteine (NAC), the synergistic effects of ASH and TRAIL on hepatocellular carcinoma (HCC) cell apoptosis was abolished. Furthermore, NAC could alleviate p53 and the p53 upregulated modulator of apoptosis (PUMA) expression induced by TRAIL and ASH. Small (or short) interfering RNA (siRNA) targeting PUMA or p53 significantly reversed ASH-mediated sensitization to TRAIL-induced apoptosis. In addition, Bax gene deficiency also abolished ASH-induced TRAIL sensitization. An orthotopical HCC implantation mice model further confirmed that co-treated ASH overcomes TRAIL resistance in HCC cells without exhibiting potent toxicity in vivo. In conclusion, the above data suggested that ROS could induce DNA damage and activating p53/PUMA/Bax signaling, and thus, this resulted in the permeabilization of mitochondrial outer membrane and activating caspases as well as sensitizing the HCC cell to apoptosis induced by TRAIL and ASH treatment.
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Speer, Hollie, and Andrew J. McKune. "Aging under Pressure: The Roles of Reactive Oxygen and Nitrogen Species (RONS) Production and Aging Skeletal Muscle in Endothelial Function and Hypertension—From Biological Processes to Potential Interventions." Antioxidants 10, no. 8 (August 4, 2021): 1247. http://dx.doi.org/10.3390/antiox10081247.
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The proportion of adults living with hypertension increases significantly with advancing age. It is therefore important to consider how health and vitality can be maintained by the aging population until end of life. A primary risk factor for the progression of cardiovascular diseases (CVD) is hypertension, so exploring the factors and processes central to this burden of disease is essential for healthy aging. A loss of skeletal muscle quantity and quality is characteristic in normal aging, with a reduction of vasodilatory capacity caused by endothelial dysfunction, and subsequent increase in peripheral resistance and risk for hypertension. Reactive Oxygen and Nitrogen Species (RONS) encompass the reactive derivatives of NO and superoxide, which are continuously generated in contracting skeletal muscle and are essential mediators for cellular metabolism. They act together as intra and intercellular messengers, gene expression regulators, and induce programmed cell death. In excessive amounts RONS can inflict damage to endothelial and skeletal muscle cells, alter signaling pathways or prematurely promote stress responses and potentially speed up the aging process. The age-related increase in RONS by skeletal muscle and endothelial mitochondria leads to impaired production of NO, resulting in vascular changes and endothelial dysfunction. Changes in vascular morphology is an early occurrence in the etiology of CVDs and, while this is also a normal characteristic of aging, whether it is a cause or a consequence of aging in hypertension remains unclear. This review serves to focus on the roles and mechanisms of biological processes central to hypertension and CVD, with a specific focus on the effects of aging muscle and RONS production, as well as the influence of established and more novel interventions to mediate the increasing risk for hypertension and CVD and improve health outcomes as we age.
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de Castro, Raquel V. G., Naira C. G. Pieri, Paulo Fantinato Neto, Bianca M. Grizendi, Renata G. S. Dória, Flavio V. Meirelles, Lawrence C. Smith, Joaquim M. Garcia, and Fabiana F. Bressan. "In Vitro Induction of Pluripotency from Equine Fibroblasts in 20% or 5% Oxygen." Stem Cells International 2020 (November 26, 2020): 1–16. http://dx.doi.org/10.1155/2020/8814989.
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The cellular reprogramming into pluripotency is influenced by external and internal cellular factors, such as in vitro culture conditions (e.g., environmental oxygen concentration), and the aging process. Herein, we aimed to generate and maintain equine iPSCs (eiPSCs) derived from fibroblasts of a horse older than 20 years and to evaluate the effect of different levels of oxygen tension (atmospheric 20% O2, 5% O2, or 20% to 5% O2) on these cells. Fibroblasts were reprogrammed, and putative eiPSCs were positive for positive alkaline phosphatase detection; they were positive for pluripotency-related genes OCT4, REX1, and NANOG; immunofluorescence-positive staining was presented for OCT4 and NANOG (all groups), SOX2 (groups 5% O2 and 20% to 5% O2), and TRA-1-60, TRA-1-81, and SSEA-1 (only in 20% O2); they formed embryoid bodies; and there is spontaneous differentiation in mesoderm, endoderm, and ectoderm embryonic germ layers. In addition to the differences in immunofluorescence analysis results, the eiPSC colonies generated at 20% O2 presented a more compact morphology with a well-defined border than cells cultured in 5% O2 and 20% to 5% O2. Significant differences were also observed in the expression of genes related to glucose metabolism, mitochondrial fission, and hypoxia (GAPDH, GLUT3, MFN1, HIF1α, and HIF2α), after reprogramming. Our results show that the derivation of eiPSCs was not impaired by aging. Additionally, this study is the first to compare high and low oxygen cultures of eiPSCs, showing the generation of pluripotent cells with different profiles. Under the tested conditions, the lower oxygen tension did not favor the pluripotency of eiPSCs. This study shows that the impact of oxygen atmosphere has to be considered when culturing eiPSCs, as this condition influences the pluripotency characteristics.
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Amaral de Mascena Costa, Luciana, Ashlyn C. Harmon, Alvaro Aguiar Coelho Teixeira, Filipe Cássio Silva de Lima, Silvany de Sousa Araújo, Fabio Del Piero, Helivaldo Diógenes da Silva Souza, et al. "Cytotoxic Activity of the Mesoionic Compound MIH 2.4Bl in Breast Cancer Cell Lines." Breast Cancer: Basic and Clinical Research 14 (January 2020): 117822342091333. http://dx.doi.org/10.1177/1178223420913330.
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In this work, we report the synthesis of a new 1,3-thiazolium-5-thiolate derivative of a mesoionic compound (MIH 2.4Bl) and the characterization of its selective cytotoxicity on a panel of breast cancer cells lines. The cytotoxic effect of MIH 2.4Bl on breast cancer cell lines was determined by XTT and crystal violet assays, flow cytometry analysis, electron microscopy characterization, and terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) apoptosis assays. As determined using XTT cell growth and survival assays, MIH 2.4Bl exhibited growth inhibition activity on most breast cancer cell lines tested, compared with normal human mammary epithelial cells. Three breast cancer cell lines (MCF-7, T-47D, and ZR-75-1) showed a more potent sensitivity index to growth inhibition by MIH 2.4Bl than the other breast cancer cell lines. Interestingly, these 3 cell lines were derived from tumors of Luminal A origin and have ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2) positive expression. Additional analysis of cytotoxicity mediated by MIH 2.4Bl was performed using the MCF-7 cell line. MCF-7 cells displayed both time- and dose-dependent decreases in cell growth and survival, with a maximum cytotoxic effect observed at 72 and 96 hours. The MCF-7 cells were also characterized for cell cycle changes upon treatment with MIH 2.4Bl. Using flow cytometry analysis of cell cycle distribution, a treatment-dependent effect was observed; treatment of cells with MIH 2.4Bl increased the G2/M population to 34.2% compared with 0.1% in untreated (control) cells. Ultrastructural analysis of MFC-7 cells treated with MIH 2.4Bl at 2 different concentrations (37.5 and 75 μM) was performed by transmission electron microscopy. Cells treated with 37.5 μM MIH 2.4Bl showed morphologic changes beginning at 6 hours after treatment, while cells treated with 75 μM showed changes beginning at 3 hours after treatment. These changes were characterized by an alteration of nuclear morphology and mitochondrial degeneration consistent with apoptotic cell death. Results of a TUNEL assay performed on cells treated for 96 hours with MIH 2.4Bl supported the observation of apoptosis. Together, these results suggest that MIH 2.4Bl is a promising candidate for treating breast cancer and support further in vitro and in vivo investigation.
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Leiba, Merav, Jana Jakubikova, Steffen Klippel, Constantine S. Mitsiades, Teru Hideshima, Yu-Tzu Tai, Paul Richardson, et al. "Halofuginone, a Novel Antimyeloma Agent, Upregulates C-Jun, JNK and P-53 Protein in Vitro, and Inhibits Tumor Growth and Improves Survival in in Vivo Multiple Myeloma(MM) Animal Models." Blood 112, no. 11 (November 16, 2008): 2754. http://dx.doi.org/10.1182/blood.v112.11.2754.2754.
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Abstract Halofuginone, a synthetic derivative of quinazolinone alkaloid, previously has been shown to have anti-cancer effects in various solid and hematological malignancies. Halofuginone inhibits mainly collagen type I synthesis, and extracellular matrix formation, via the inhibition of TGFβ signaling, matrix metalloproteinase 2(MMP2), and angiogenesis. Last year, we first reported, that Halofuginone in a low doses (IC50 of 50—100 nM) induces cytotoxicity in multiple MM cell lines, including cells resistant to conventional (e.g., dexamethasone, alkylating agents, and anthracyclines) or novel (e.g. thalidomide and bortezomib) anti-MM agents and overcomes the survival and growth advantages conferred by interleukin-6, insulin-like growth factor-1 and by bone marrow stroma cells. Halofuginone induced apoptosis in a caspase 3, 8, and 9 dependent mechanisms, reduced mitochondrial membrane potential, and down regulated MCL1 protein. We now assessed the cytotoxic effect of Halofuginone in primary MM patient cells in vitro and, its effect on tumor growth and survival in in vivo models. We found that Halofuginone also induces growth inhibition and cell death in primary MM cells (n=4, IC50: 100–200nM). Importantly, Halofuginone demonstrated additive or synergistic effects with some of the established anti-MM agents such as Melphalan, Dexamethasone, and Lenalidomide. In addition, Halofuginone inhibits IL6 production in the supernatant of a co-culture of MM.1S cells with HS-5 stromal cell line. Mechanistically, Halofuginone induces MM cell death, which involves the up-regulation of c-jun NH2-terminal kinase signaling (JNK), c-Jun, as well as the p-53 proapoptotic protein. Additionally, the in vivo anti-MM activity of Halofuginone was evaluated in 2 separate in vivo models, a xenograft model in SCID mice (subcutaneous injection of MM1S cells), and a model of diffuse MM lesions in SCID-beige mice (generated by i.v. injections of OPM-2 cells). In both models, mice were first sublethally irradiated (200 rads), injected s.c or i.v., respectively, with 1×106 MM cells and then randomly assigned to receive, either treatment with 0.75mg/kg halofuginone (IP or by oral gavage, respectively; n=10) or vehicle only (n=10) on a cyclical schedule of 5 days-on/2 days-off. In both models, Halofuginone inhibited MM tumor growth and improved survival, 70% vs 40% at 140 days (NS) in the treated vs control group respectively (figure). Clinical evidence of adverse events (weight loss, vomiting) were not observed. Halofuginone may, thus represents a promising novel orally bioavailable anti-MM agent that needs further evaluation for possible clinical trials in MM. Diffuse MM lesions in SCID-beige mice. Animals were sub lethally irradiated (200rads), injected i.v. with 1×106 OPM2 cells and then randomly assigned to receive, either halofuginone treatment (by oral gavage; 0.75mg/kg (n=10) or vehicle only (n=10) on a cyclical schedule of 5 days-on/2 days-off. Figure Figure
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Guzman, Monica L., Shama Nasim, Marlene Balys, Cheryl A. Corbett, Peter A. Crooks, and Craig T. Jordan. "Chemical Derivatives of the Anti-Leukemia Stem Cell Compound 4-Benzyl-2-Methyl-1,2,4-Thiadiazolidine-3,5-Dione (TDZD-8) with Improved Activity." Blood 114, no. 22 (November 20, 2009): 3764. http://dx.doi.org/10.1182/blood.v114.22.3764.3764.
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Abstract Abstract 3764 Poster Board III-700 We have recently described the novel and unique anti-leukemic properties of 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8). Indeed, TDZD-8 was shown to eradicate leukemia at the bulk, stem, and progenitor level with rapid kinetics (typically < 2 h) with minimal toxicity to normal hematopoietic cells. Moreover, the cytotoxic activity of this drug is observed only in hematologic malignancies. The precise mechanism of TDZD-8 is not fully appreciated, but the compound has been shown to inhibit NFkappaB, GSK3beta, protein kinase C, FMS-like tyrosine kinase (Flt3), AKT and KDR. In addition, TDZD-8 rapidly depletes free thiols and appears to disrupt membrane integrity. Interestingly, TDZD-8 results in rapid mitochondrial swelling, followed by vacuole formation and accumulation. Despite possessing potent and specific anti-leukemia activity, the clinical utility of TDZD-8 is limited by the need for high concentrations (20 microM) and poor solubility. Therefore, to improve the pharmacological properties of TDZD-8, we initiated efforts to generate derivatives with greater anti-leukemia activity at lower concentrations and with greater water-solubility. To this end, an extensive structure-activity relationship study was carried out to examine the effects on anti-leukemic activity resulting from introduction of substituents at the C-2 and C-4- positions of the thiadiazolidine ring of TDZD-8, and of replacement of the TDZD ring with isosteric scaffolds. In total, we have screened more than 60 new analogs. Preliminary screens were performed using two different AML cell lines, evaluating the LD50 for each of the analogs relative to the parental compound. We found that introducing substitutuents into the main TDZD ring resulted in loss of anti-leukemic activity of the compounds. Moreover, substitution in the benzyl/phenyl ring does not affect anti-leukemia activity of the TDZD analogs. Importantly, we found that N-2 halogenoethyl analogs, exhibit exceptional activity against leukemic cells. Of the halogenoethyl analogs evaluated, the iodoethyl analog TD-361 was the most active compound with an LD50 of 0.49 microM in MV-411 cells. Compounds exhibiting increased anti-leukemia activity were subjected to further testing in phenotypically-defined AML stem/progenitor cells. All analogs demonstrated efficacy in primary AML cells at lower concentrations than TDZD-8. Moreover, these analogs still maintained the rapid kinetics observed with TDZD-8. Finally, we performed colony assays to determine the effect of new analogs on progenitor cells from normal and leukemic cells. These studies demonstrated that the more active TDZD analogs retained the selective ability of TDZD-8 to abate AML progenitor cells without harming normal hematopoietic cells. In summary, we have identified the critical chemical moieties for the observed activity of TDZD-8, and have also discovered analogs with improved anti-leukemia activity. Going forward, the most active derivatives are being optimized for water-solubility and will then be evaluated using primary human AML specimens engrafted into immune deficient mice. Based on evidence to date, we propose that the TDZD family of compounds may represent a new class of drugs for the treatment of leukemia and related hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.
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Reich, Ieva L., Hans J. Reich, Nancy Kneer, and Henry Lardy. "Ergosteroids V: preparation and biological activity of various D-ring derivatives in the 7-oxo-dehydroepiandrosterone series1 1Abbreviations: DHEA, dehydroepiandrosterone or 3β-hydroxyandrost-5-ene-17-one; DEAD, diethylazodicarboxylate; MCPBA, m-chloroperbenzoic acid; DMAP, dimethylaminopyridine; LDA, lithium diisopropylamide; THF, tetrahydrofuran; TMSCl, trimethylchlorosilane; GPDH, liver mitochondrial glycerophosphate dehydrogenase; ME, cytosolic malic enzyme." Steroids 67, no. 3-4 (March 2002): 221–33. http://dx.doi.org/10.1016/s0039-128x(01)00155-6.
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Sharmeen, Sumaiya, Marko Skrtic, Mahadeo Sukhai, Joyce Sun, Rose Hurren, Marcela Gronda, Xiaoming Wang, et al. "Activation of Chloride Channels with the Anti-Parasitic Agent Ivermectin Induces Membrane Hyperpolarization and Cell Death in Leukemia Cells." Blood 114, no. 22 (November 20, 2009): 410. http://dx.doi.org/10.1182/blood.v114.22.410.410.
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Abstract Abstract 410 FDA-approved drugs with previously unrecognized anti-cancer activity could be rapidly repurposed for this new indication. To identify such compounds, we compiled a library of known drugs with high maximal tolerated doses and well-known toxicity profiles. We screened this library in a dose-response manner in 4 leukemia cell lines to identify cytotoxic compounds as measured by the MTS assay. From these screens, we identified the anti-parasitic agent ivermectin (IVM) that induced cell death at low micromolar concentrations in all four leukemia lines tested. IVM is a derivative of avermecin B1 and licensed for the treatment of strongyloidiasis and onchocerciasis parasitic infections, but also effective against other worm infestations (e.g., ascariasis, trichuriasis and enterobiasis). To evaluate the effects of IVM as a potential anti-cancer agent, leukemia and myeloma (n = 9) cell lines were treated with increasing concentrations of IVM. 72 hours after incubation, cell viability was determined by the MTS assay. IVM decreased the viability of 3/5 leukemia cell lines with an LD50 < 5uM and all other tested malignant cell lines with an LD50 < 10uM. Cell death was confirmed by Trypan blue staining and Annexin V staining. In clonogenic growth assays, 6uM IVM reduced clonogenic growth by ≥ 40% in 3/6 primary AML samples, but <15% in 3/3 samples of normal hematopoietic mononuclear cells. Given the effects of IVM in cell lines, we evaluated the drug in mouse models of leukemia. Here, K562, OCI-AML2, and MDAY-D2 leukemia cells were implanted subcutaneously into the flanks of sublethally irradiated NOD/SCID mice. One week after implantation, when the tumors were palpable, mice were treated with IVM at 3mg/kg/day by oral gavage or buffer control. Compared to control, IVM decreased tumor volume and weight in all 3 xenograft models by up to 72% without observable toxicity. Of note, a dose of 3mg/kg in mice translates to a dose of 0.24 mg/kg in humans based on scaling of body weight and surface area. This dose is readily achievable in human as patients routinely receive 0.2mg/kg for the treatment of parasitic disease and overdoses of up to 6g were not toxic. As an anti-parasitic, IVM binds and activates chloride ion channels, so we tested the effects of IVM on chloride flux in leukemia cells. OCI AML2 cells were treated with increasing concentrations of IVM and changes in intracellular chloride were measured using the fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium and flow cytometry. IVM increased intracellular chloride ion concentrations within 30 minutes of treatment. Chloride influx was accompanied by plasma membrane hyperpolarization within 1 hr of treatment, but no change in mitochondrial membrane potential was noted up to 24 hours after treatment. Plasma membrane hyperpolarization was dependent on chloride influx, as treatment with IVM in chloride-free media did not induce membrane hyperpolarization. Alternations in intracellular chloride and membrane hyperpolarization can lead to increased reactive oxygen species (ROS) generation. Therefore, we measured changes in ROS after treatment with IVM. IVM (6uM) increased ROS generation in OCI-AML2 cells up to 2 ± 0.2 fold within 4 hours of treatment. Increased ROS generation appeared functionally important for IVM-induced cell death as pre-treatment with the antioxidant N-acetyl-L-cysteine (NAC) inhibited IVM-induced cell death. Further supporting a mechanism of cell death related to increased ROS, IVM treatment increased expression of STAT1, IFIT3, OAS1, and TRIM22, members of the STAT1 signaling pathway that are known to be upregulated upon ROS generation. Cytarabine and daunorubicin are used in the treatment of AML and increase ROS production through mechanisms related to DNA damage. Therefore, we evaluated the combination of IVM and cytarabine and daunorubicin. By isobologram analysis, IVM synergized with cyatarabine (CI=0.51, 0.58, 0.65 at ED25, ED50, ED75, respectively) and daunorubicin (CI=0.48, 0.51, 0.54 at ED25, ED50, ED75, respectively). Thus, in summary, IVM activates chloride channels in leukemia cells leading to membrane hyperpolarization and increased ROS generation. In addition, it demonstrated preclinical activity in this disease at pharmacologically achievable concentrations. Therefore, IVM could be rapidly repurposed for the treatment of leukemia and highlights a potential new therapeutic strategy for this disease. Disclosures: Off Label Use: Ivermectin is an antiparasitic agent, licensed for the treatment of strongyloidiasis and onchocerciasis parasitic infections, but also effective against other worm infestations (e.g., ascariasis, trichuriasis and enterobiasis).
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Panickar, Kiran, and Harry Dawson. "Acute effects of all-trans-retinoic acid in ischemic injury." Translational Neuroscience 3, no. 2 (January 1, 2012). http://dx.doi.org/10.2478/s13380-012-0015-z.
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AbstractAll-trans-retinoic acid (ATRA) is a vitamin A derivative that is important in neuronal patterning, survival, and neurite outgrowth. Neuroprotective effects of ATRA in ischemia have been demonstrated but its effects on glial swelling are not known. We investigated the relatively acute effects of ATRA on cell swelling in ischemic injury and on key features hypothesized to contribute to cell swelling including increased reactive oxygen species/reactive nitrogen species (ROS/RNS), depolarization of the inner mitochondrial membrane potential (ΔΨm), and increased intracellular calcium ([Ca2+]i). C6 glial cultures were subjected to 5 hr oxygen-glucose deprivation (OGD). ATRA was added to separate groups after the end of OGD. OGD increased cell volume by 43%, determined at 90 min after the end of OGD, but this increase was significantly attenuated by ATRA. OGD induced an increase in ROS/RNS production in the whole cell and mitochondria, as assessed by the fluorescent dyes CM-H2DCFDA and MitoTracker CM-H2-XROS at the end of OGD. The increase in mitochondrial ROS, but not cellular ROS, was significantly attenuated by ATRA. OGD also induced a 67% decline in mitochondrial ΔΨm but this decline was significantly attenuated by ATRA. OGD-induced increase in [Ca2+]i was also significantly attenuated by ATRA. Taken together with our previous results where calcium channel blockers reduced cell swelling, the effects of ATRA in attenuating swelling are possibly mediated through its effects in regulating [Ca2+]i. Considering the paucity of agents in attenuating brain edema in ischemia, ATRA has the potential to reduce brain edema and associated neural damage in ischemic injury.
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Nass, Norbert, Alexandra Kukat, Peter Seibel, Hans-Jürgen Brömme, Reinhard Schinzel, Rolf-Edgar Silber, and Andreas Simm. "Advanced glycation end product accumulation in rho0 cells without a functional respiratory chain." Biological Chemistry 390, no. 9 (September 1, 2009). http://dx.doi.org/10.1515/bc.2009.083.
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Abstract Advanced glycation end products (AGEs) accumulate during ageing with reactive oxygen species from the mitochondrial respiratory chain discussed as a driving force. To determine the role of mitochondrial activity for AGE formation, a rho0 derivative of the 143B.TK- osteosarcoma cell line lacking the respiratory chain, was analysed. These cells exhibit decreased superoxide formation but unchanged mitochondrial SOD expression as well as unchanged antioxidative free sulfhydryl (SH) levels. Whereas total protein content shows no differences in AGE levels, cell fractionation and Western blotting demonstrates some changes in the AGE pattern. Thus, the absence of functional respiration has only a negligible impact on AGE accumulation.