Mitochondria-mediated Apoptotic Cell Death Research Articles

Mixed-ligand Cu(II) hydrazone complexes designed to enhance anticancer activity

The ligand quantity, ligand type, and coordination geometry have important influences on the anticancer activity of metal-based complexes. On the basis of the structures of previously reported 1:1 Cu(II)/ligand complexes ([Cu(L1)Cl]·2H2O 1a, [Cu(L2)Cl]·H2O 2a, and [Cu(L2)NO3]·H2O 3a), we subsequently designed, developed, and characterized a series of corresponding 1:1:1 Cu(II)/ligand/co-ligand complexes ([Cu(L1)(Py)Cl]·H2O 1b, [Cu(L2)(Py)Cl] 2b, and [Cu(L2)(Py)NO3] 3b), where L1 = (E)-N'-(2-hydroxybenzylidene)acetohydrazide, L2 = (E)-N'-(2- hydroxybenzylidene)benzohydrazide, and Py = pyridine. All six Cu(II) complexes were assessed for their in vitro anticancer properties against a panel of human cancer cells, including cisplatin-resistant A549cisR cell lines. Interestingly, we observed that the 1:1:1 Cu/ligand/co-ligand mixed-ligand Cu(II) complexes exhibited higher anticancer activity than the corresponding 1:1 Cu(II)/ligand complexes. In particular, the 1:1:1 Cu(II)/ligand/co-ligand complex 3b displayed the greatest toxicity toward several cancer cells with better IC50 (1.12–3.77 μM) than cisplatin. Further mechanistic explorations showed that the 3b complex induced DNA damage, thus resulting in mitochondria-mediated apoptotic cell death. Furthermore, the 3b complex displayed pronounced cytostatic effects in the MCF-7 3D spheroid model.

\u03b2-Sitosterol targets Trx/Trx1 reductase to induce apoptosis in A549 cells via ROS mediated mitochondrial dysregulation and p53 activation

β-Sitosterol (BS), a major bioactive constituent present in plants and vegetables has shown potent anticancer effect against many human cancer cells, but the underlying mechanism remain elusive on NSCLC cancers. We found that BS significantly inhibited the growth of A549 cells without harming normal human lung and PBMC cells. Further, BS treatment triggered apoptosis via ROS mediated mitochondrial dysregulation as evidenced by caspase-3 & 9 activation, Annexin-V/PI positive cells, PARP inactivation, loss of MMP, Bcl-2-Bax ratio alteration and cytochrome c release. Moreover, generation of ROS species and subsequent DNA stand break were found upon BS treatment which was reversed by addition of ROS scavenger (NAC). Indeed BS treatment increased p53 expression and its phosphorylation at Ser15, while silencing the p53 expression by pifithrin-α, BS induced apoptosis was reduced in A549 cells. Furthermore, BS induced apoptosis was also observed in NCI-H460 cells (p53 wild) but not in the NCI-H23 cells (p53 mutant). Down-regulation of Trx/Trx1 reductase contributed to the BS induced ROS accumulation and mitochondrial mediated apoptotic cell death in A549 and NCI-H460 cells. Taken together, our findings provide evidence for the novel anti-cancer mechanism of BS which could be developed as a promising chemotherapeutic drug against NSCLC cancers.

PH and NIR-light-responsive magnetic iron oxide nanoparticles for mitochondria-mediated apoptotic cell death induced by chemo-photothermal therapy

Recently, various therapeutic strategies in anticancer drug development are focused to reduce adverse side effects and to enhance the therapeutic efficacy. Mostly, the iron oxide (Fe3O4) nanoparticles have widely been utilized as an efficient drug delivery system towing to their unique properties such as excellent magnetic behavior, considerably low toxicity, easy surface modification and high drug-loading efficacy. In the present study, we synthesized a multifunctional, DMSA coated, water soluble Fe3O4 nanoparticles (Fe3O4@DMSA/DOX) for an effective pH and NIR-light triggered delivery of anticancer drug (DOX) in cancer therapy. The combination of photothermal therapy combined with chemotherapy results demonstrated that the synthesized Fe3O4@DMSA/DOX is an excellent candidate for pH- and NIR-light induced phothothermal agent for an effective delivery of anticancer drug (DOX) into the target sub-cellular level into the human breast cancer (MDA-MB-231) cells. Furthermore, the Fe3O4@DMSA/DOX nanoparticles induced an excellent temperature elevation upon NIR light irradiation and controlled DOX release in vitro. The Fe3O4@DMSA/DOX nanoparticles exhibited synergistic effect when combining chemotherapy with photothermal therapy and showed an excellent cell toxicity to MDA-MB-231 cells. In addition, the combined chemo-photothermal therapy of Fe3O4@DMSA/DOX nanoparticles promoted an effective cell death by mitochondrial disruption mediated by ROS generation. Thus, the synthesized Fe3O4@DMSA/DOX nanoparticles could be utilized as potential anticancer agents for breast cancer treatment.

Mitochondrial potassium channels in cell death

Mitochondria are intracellular organelles involved in several processes from bioenergetics to cell death. In the latest years, ion channels are arising as new possible targets in controlling several cellular functions. The discovery that several plasma membrane located ion channels have intracellular counterparts, has now implemented this consideration and the number of studies enforcing the understanding of their role in different metabolic pathways. In this review, we will discuss the recent updates in the field, focusing our attention on the involvement of potassium channels during mitochondrial mediated apoptotic cell death. Since mitochondria are one of the key organelles involved in this process, it is not surprising that potassium channels located in their inner membrane could be involved in modulating mitochondrial membrane potential, ROS production, and respiratory chain complexes functions. Eventually, these events lead to changes in the mitochondrial fitness that prelude to the cytochrome c release and apoptosis. In this scenario, both the inhibition and the activation of mitochondrial potassium channels could cause cell death, and their targeting could be a novel pharmacological way to treat different human diseases.

Ph-Induced Oligomerization of the Voltage Dependent Anion Channel

The Voltage-Dependent Anion Channel (VDAC) is the most abundant protein in the outer mitochondrial membrane facilitating the passage of ions and metabolites between the cytoplasm and the intermembrane space. In addition to its critical role in bioenergetics, VDAC is capable of modulating the organelle's permeability, implicating VDAC in metabolic stress and mitochondria-mediated apoptotic cell death. VDAC functions as a monomer but exhibits various oligomeric states, as shown by electron microscopy and atomic force microscopy. Oligomerization of VDAC can be linked to apoptosis and therefore is crucial for understanding mitochondrial regulation. Double Electron-Electron Resonance (DEER) on 4 singly spin-labeled mutants of mVDAC1 revealed a specific pH-induced dimerization. These inter-molecular distances were used to generate a model of a dimer interface that contains Glutamate 73. Mutation of Glutamate 73, to either Alanine or Glutamine, prevented pH induced oligomerization confirming the vital role of this residue. This dimer, induced by acidification, differs from any oligomeric form of VDAC previously described and likely plays a significant role in mitochondrial regulation and apoptosis in the response to cytosolic acidification during cellular stress.

Luteolin induces cholangiocarcinoma cell apoptosis through the mitochondrial-dependent pathway mediated by reactive oxygen species

To investigate the apoptosis-inducing effect and underlying mechanisms of luteolin in cholangiocarcinoma (CCA) cells. Cell viability was determined by sulphorhodamine B. Apoptosis was detected using acridine orange/ethidium bromide dye staining and annexin V/PI staining followed by flow cytometry. The effect of luteolin on the oxidative status of CCA cells was evaluated by measuring intracellular reactive oxygen species (ROS) levels using the dihydroethidium method and quantifying glutathione levels. The mitochondria transmembrane potential (ΔΨm) was examined through JC-1 staining. The protein levels were determined by Western blot. Caspase activity was determined using specific fluorogenic substrates. Luteolin decreased KKU-100 CCA cells' viability by induction of apoptosis. Luteolin treatment increased ROS production and decreased glutathione levels. These changes were associated with the decrease of Nrf2, γ-glutamylcysteine ligase and heme oxygenase-1 proteins. Moreover, luteolin induced mitochondrial depolarization, which was accompanied by the release of cytochrome c and a decrease of Bcl-2 and Bcl-XL proteins. Pretreatment with antioxidants, 4-hydroxy-TEMPO and N-acetyl-L-cysteine significantly prevented luteolin-induced CCA cell death and loss of ΔΨm. In addition, luteolin induced the activation of caspase-9 and caspase-3. Luteolin exerts its pro-apoptotic action partly through generating intracellular ROS that then contributes to the activation of mitochondria-mediated apoptotic cell death.

ROS mediated crosstalk between endoplasmic reticulum and mitochondria by Phloxine B under environmental UV irradiation

Phloxine B (PhB) is a most commonly used dye in cosmetic products throughout the world. It shows an absorption in visible and ultraviolet radiations. PhB was photodegraded within 4h of UV exposure. It generates reactive oxygen species (ROS) photochemically and intracellularly. Photosensitized PhB caused dose dependent cell viability reduction of human keratinocyte cell line (HaCaT) which was measured through MTT (75.4%) and NRU (77.3%) assays. It also induces cell cycle arrest and DNA damage. Photosensitized PhB induces Ca2+ release from endoplasmic reticulum (ER). It causes the upregulation of ER stress marker genes ATF6 (1.79 fold) and CHOP (1.93 fold) at transcription levels. The similar response of ATF6 (3.6 fold) and CHOP (2.38 fold) proteins was recorded at translation levels. CHOP targeted the mitochondria and reduced the mitochondrial membrane potential analyzed through JC-1 staining. It further increases Bax/Bcl2 ratio (3.58 fold) and promotes the release of cytochrome c, finally leads to caspase-dependent apoptosis. Upregulation of APAF1 (1.79 fold) in PhB treated cells under UV B exposure supports the mitochondrial-mediated apoptotic cell death. The results support the involvement of ER and mitochondria in ROS mediated PhB phototoxicity. Therefore, the use of PhB in cosmetic products may be deleterious to users during sunlight exposure.

Nimbolide inhibits pancreatic cancer growth and metastasis through ROS-mediated apoptosis and inhibition of epithelial-to-mesenchymal transition.

The mortality and morbidity rates of pancreatic cancer are high because of its extremely invasive and metastatic nature. Its lack of symptoms, late diagnosis and chemo–resistance and the ineffective treatment modalities warrant the development of new chemo–therapeutic agents for pancreatic cancer. Agents from medicinal plants have demonstrated therapeutic benefits in various human cancers. Nimbolide, an active molecule isolated from Azadirachta indica, has been reported to exhibit several medicinal properties. This study assessed the anticancer properties of nimbolide against pancreatic cancer. Our data reveal that nimbolide induces excessive generation of reactive oxygen species (ROS), thereby regulating both apoptosis and autophagy in pancreatic cancer cells. Experiments with the autophagy inhibitors 3-methyladenine and chloroquine diphosphate salt and the apoptosis inhibitor z-VAD-fmk demonstrated that nimbolide-mediated ROS generation inhibited proliferation (through reduced PI3K/AKT/mTOR and ERK signaling) and metastasis (through decreased EMT, invasion, migration and colony forming abilities) via mitochondrial-mediated apoptotic cell death but not via autophagy. In vivo experiments also demonstrated that nimbolide was effective in inhibiting pancreatic cancer growth and metastasis. Overall, our data suggest that nimbolide can serve as a potential chemo–therapeutic agent for pancreatic cancer.

Induction of autophagosomes, a SOS mechanism against organophosphate pesticide exposure in cultured human neuronal cells

Event Abstract Back to Event Induction of autophagosomes, a SOS mechanism against organophosphate pesticide exposure in cultured human neuronal cells Akriti Srivastava1*, Vivek Kumar1, Ankita Pandey1, Dipak Kumar1, Vinay K. Khanna1 and Aditya B. Pant1 1 CSIR-Indian Institute of Toxicology Research (CSIR-IITR), System Toxicology and Health Risk Assessment Group, India Developmental Neurotoxicity (DNT) of organophosphate (OP) pesticide – Monocrotophos (MCP) and its underlying mechanism has already been demonstrated by our research group. The present investigation elucidates the possible protective mechanism via induction of autophagy in MCP exposed human neuroblastoma cells. The cells were exposed to biologically safe concentration of MCP (1X10-4 M) for varying time points. We observed elevated ROS levels, MMP reduction and increased apoptosis rate in a time-dependent fashion in MCP treated SHSY5Y cells. Besides the end-points mentioned above the investigations were focused on expression of autophagy markers, i.e. LC3, p62, etc. which were upregulated in response to MCP exposure. MCP induced autophagy was also observed to be associated with decreased mTOR activity and enhanced AMP kinase activity. Pre-treatment with the autophagy inducer, rapamycin significantly enhanced the cell viability of MCP-exposed cells, which was partially due to alleviation of MCP-induced neuronal death via decrease in levels of oxidative stress and caspase-3. However, autophagy inhibition either pharmacologically (3-Methyladenine) or genetically (knockdown of ATG5 or ATG7), sensitized cells towards MCP-induced mitochondria-mediated apoptotic cell death. Hence, our data indicate that pharmacologically induced autophagy serves as a potent therapy against OP pesticide-triggered neurodegeneration. Keywords: Apoptosis, Neurotoxicity, in vitro, pesticide, Autophagosomes Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: O03: Postgraduate Travel Awardees Oral Session 3 Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Srivastava A, Kumar V, Pandey A, Kumar D, Khanna VK and Pant AB (2016). Induction of autophagosomes, a SOS mechanism against organophosphate pesticide exposure in cultured human neuronal cells. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00093 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Ms. Akriti Srivastava, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), System Toxicology and Health Risk Assessment Group, Lucknow, Uttar Pradesh, India, aks.srvstv@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Akriti Srivastava Vivek Kumar Ankita Pandey Dipak Kumar Vinay K Khanna Aditya B Pant Google Akriti Srivastava Vivek Kumar Ankita Pandey Dipak Kumar Vinay K Khanna Aditya B Pant Google Scholar Akriti Srivastava Vivek Kumar Ankita Pandey Dipak Kumar Vinay K Khanna Aditya B Pant PubMed Akriti Srivastava Vivek Kumar Ankita Pandey Dipak Kumar Vinay K Khanna Aditya B Pant Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy

The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼ 2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.

Cell Cycle Arrest and Induction of Apoptosis in Colon Adenocarcinoma Cells by a DNA Intercalative Quinoline Derivative, 4-Morpholinopyrimido [4′,5′:4,5] Selenolo (2,3-b) Quinoline

Circular dichroism, topological studies, molecular docking, absorbance, and fluorescence spectral titrations were employed to study the interaction of 4-morpholinopyrimido [4′,5′:4,5] selenolo (2,3-b) quinoline (MPSQ) with DNA. The association constants of MPSQ–DNA interactions were of the order of 104 M−1. Melting temperature, topological, and docking studies confirmed that the mode of interaction was by intercalation with preference to d(GpC)–d(CpG) site of DNA. Cytotoxicity studies showed the MPSQ-induced dose-dependent inhibitory effect on the proliferation of different cancer cells. Colon adenocarcinoma (COLO 205) cells are more sensitive among the cell lines tested, with an IC50 value of 15 μM. Flow cytometry revealed that MPSQ affects the cell cycle progression by arresting at G2M phase. Further, Annexin V staining, mitochondrial membrane potential assay, and caspase-3 activity assay confirmed that MPSQ leads to mitochondria-mediated apoptotic cell death in COLO 205 cells.

Atmospheric-pressure plasma jet induces DNA double-strand breaks that require a Rad51-mediated homologous recombination for repair in Saccharomyces cerevisiae

Non-thermal plasma generated under atmospheric pressure produces a mixture of chemically reactive molecules and has been developed for a number of biomedical applications. Recently, plasma jet has been proposed as novel cancer therapies based on the observation that free radicals generated by plasma jet induce mitochondria-mediated apoptotic cell death. We show here that air plasma jet induces DNA double-strand breaks (DSBs) in yeast chromosomes leading to genomic instability and loss of viability, which are alleviated by Rad51, the yeast homolog of Escherichiacoli RecA recombinase, through DNA damage repair by a homologous recombination (HR) process. Hypersensitivity of rad51 mutant to air plasma was not restored by antioxidant treatment unlike sod1 mutant that was highly sensitive to reactive oxygen species (ROS) challenge, suggesting that plasma jet induces DSB-mediated cell death independent of ROS generation. These results may provide a new insight into the mechanism of air plasma jet-induced cell death.

Pathway of Programmed Cell Death and Oxidative Stress Induced by β-Hydroxybutyrate in Dairy Cow Abomasum Smooth Muscle Cells and in Mouse Gastric Smooth Muscle

The administration of exogenous β-hydroxybutyrate (β-HB), as well as fasting and caloric restriction, is a condition associated with β-HB abundance and decreased appetite in animals. Increased β-HB and decreased appetite exist simultaneously in some diseases, such as bovine left displaced abomasums (LDA) and human chronic gastritis. However, the effects of β-HB on stomach injuries have not been explored. To elucidate the possible effects of exogenous β-HB on the stomach, mice were injected intraperitoneally with β-HB, and bovine abomasum smooth muscle cells (BSMCs) were treated with different concentrations of β-HB. We found that β-HB induced BSMCs endoplasmic reticulum- and mitochondria-mediated apoptotic cell death. β-HB promoted Bax expression and caspase-12, -9, and -3 activation while blocking Bcl-2 expression. β-HB also promoted AIF, EndoG release and p53 expression. β-HB acted on key molecules in the apoptotic cell death pathway and increased p38 and c-June NH2-terminal kinase phosphorylation while inhibiting ERK phosphorylation and PCNA expression. β-HB upregulated P27 and P21 mRNA levels while downregulating cyclin and CDK mRNA levels, arresting the cell cycle. These results suggest that BSMCs treated with β-HB can induce oxidative stress, which can be prevented by intracellular calcium chelators BAPTA/AM but not antioxidant NAC. Additionally, these results suggest that β-HB causes ROS generation through a Ca2+-dependent mechanism and that intracellular Ca2+ levels play a critical role in β-HB -induced apoptotic cell death. The impact of β-HB on programmed cell death and oxidative stress in vivo was confirmed in murine experiments. For the first time, we show oxidative stress effects of β-HB on smooth muscle. We propose that β-HB is a possible cause of some stomach diseases, including bovine LDA.

Nanoparticle-directed sub-cellular localization of doxorubicin and the sensitization breast cancer cells by circumventing GST-Mediated drug resistance

Resistance to single or multiple chemotherapeutic drugs is a major complication in clinical oncology and is one of the most common treatment limitations in patients with reoccurring cancers. Nanoparticle (NP)-based drug delivery systems (DDS's) have been shown to overcome drug resistance in cancer cells mainly by avoiding the activation of efflux pumps in these cells. We demonstrate in this work that polyester-based hyperbranched dendritic-linear (HBDL)-based NPs carrying doxorubicin (Dox) can effectively overcome microsomal glutathione transferase 1 (MGST1)-mediated drug resistance in breast cancer cells. Our DDS was much more effective at considerably lower intracellular Dox concentrations (IC50 6.3 μm vs. 36.3 μm) and achieved significantly greater reductions in viability and induced higher degrees of apoptosis (31% vs. 14%) compared to the free drug in the resistant cells. Dox-loaded HBDL NPs were found to translocate across the membranes of resistant cells via active endocytic pathways and to be transported to lysosomes, mitochondria, and the endoplasmic reticulum. A significantly lower amount of Dox accumulated in these cytoplasmic compartments in resistant cells treated with free Dox. Moreover, we found that Dox-HBDL significantly decreased the expression of MGST1 and enhanced mitochondria-mediated apoptotic cell death compared to free Dox. Dox-HBDL also markedly activated the JNK pathway that contributes to the apoptosis of drug-resistant cells. These results suggest that HBDL NPs can modulate subcellular drug distribution by specific endocytic and trafficking pathways and that this results in drug delivery that alters enzyme levels and cellular signaling pathways and, most importantly, increases the induction of apoptosis. Our findings suggest that by exploiting the cell transport machinery we can optimize the polymeric vehicles for controlled drug release to overcome drug resistance combat drug resistance with much higher efficacy.

Combination treatment with 2-methoxyestradiol overcomes bortezomib resistance of multiple myeloma cells

Bortezomib is a proteasome inhibitor used for the treatment of relapsed/refractory multiple myeloma (MM). However, intrinsic and acquired resistance to bortezomib has already been observed in MM patients. In a previous report, we demonstrated that changes in the expression of mitochondrial genes lead to changes in mitochondrial activity and bortezomib susceptibility or resistance, and their combined effects contribute to the differential sensitivity or resistance of MM cells to bortezomib. Here we report that the combination treatment of bortezomib and 2-methoxyestradiol (2ME), a natural estrogen metabolite, induces mitochondria-mediated apoptotic cell death of bortezomib-resistant MM KMS20 cells via mitochondrial reactive oxygen species (ROS) overproduction. Bortezomib plus 2ME treatment induces a higher level of cell death compared with treatment with bortezomib alone and increases mitochondrial ROS and Ca2+ levels in KMS20 cells. Pretreatment with the antioxidant N-acetyl-L-cysteine scavenges mitochondrial ROS and decreases cell death after treatment with bortezomib plus 2ME in KMS20 cells. Moreover, we observed that treatment with bortezomib plus 2ME maintains the activation of c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase kinase kinase 4/7 (MKK4/7). Collectively, combination treatment with bortezomib and 2ME induces cell death via JNK-MKK4/7 activation by overproduction of mitochondrial ROS. Therefore, combination therapy with specific mitochondrial-targeting drugs may prove useful to the development of novel strategies for the treatment of bortezomib-resistant MM patients.

Protective effects of Peroxiredoxin 6 overexpression on amyloid β-induced apoptosis in PC12 cells

Oxidative stress triggered by amyloid beta (Aβ) accumulation contributes substantially to the pathogenesis of Alzheimer's disease (AD). In the present study, we examined the involvement of the antioxidant activity of peroxiredoxin 6 (Prdx 6) in protecting against Aβ25–35-induced neurotoxicity in rat PC12 cells. Treatment of PC12 cells with Aβ25-35 resulted in a dose- and time-dependent cytotoxicity that was associated with increased accumulation of intracellular reactive oxygen species (ROS) and mitochondria-mediated apoptotic cell death, including activation of Caspase 3 and 9, inactivation of poly ADP-ribosyl polymerse (PARP), and dysregulation of Bcl-2 and Bax. This apoptotic signaling machinery was markedly attenuated in PC12 cells that overexpress wild-type Prdx 6, but not in cells that overexpress the C47S catalytic mutant of Prdx 6. This indicates that the peroxidase activity of Prdx 6 protects PC12 cells from Aβ25-35-induced neurotoxicity. The neuroprotective role of the antioxidant Prdx 6 suggests its therapeutic and/or prophylactic potential to slow the progression of AD and limit the extent of neuronal cell death caused by AD.

Impact of Oxidized Phospholipids on Membrane Organization

Lipids have often been seen as basic structural membrane subunits with proteins doing the actual work. This view has changed in recent years where it has been shown that lipids are also directly involved in numerous physiological processes and are often required for specific membrane protein functions. However, how a membrane and its function becomes modified under intracellular oxidative conditions, which e.g. trigger mitochondria-mediated apoptotic cell death, is still not really known. Oxidative stress can generate oxidized phospholipids (OxPls), which have a great impact on mitochondrial membrane integrity. Therefore, we studied the impact of OxPls on DMPC based bilayers membranes by differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy. Incorporation of OxPls with functional groups (carboxyl or aldehyde) at their truncated sn-2-chain ends generated information about the effect which OxPl species exert on the basic structural and physico-chemical properties of DMPC bilayers. DSC experiments revealed significant changes in the thermotropic phase behavior of these vesicles in the presence of OxPls as a function of their concentration. In addition, solid state 31P NMR provided molecular information of the behavior of the DMPC headgroups when OxPls were present. In addition changes could also be monitored during temperature induced phase transitions, where OxPls induced a very complex phase behavior. Between 293 K (onset of Lα-phase) and 298 K two overlapping NMR subspectra occured which indicated the co-existence of two liquid-crystalline lamellar phases. Most likely one phase reflected an OxPls poor domain and the other an OxPl-rich domain. In summary, the presence of OxPls seems to alter the mitochondrial membrane organization, which has serious implication for the role of this membrane and its Bcl-2 proteins involved in mitochondrial apoptosis.

(+)-Medioresinol leads to intracellular ROS accumulation and mitochondria-mediated apoptotic cell death in Candida albicans

The phytochemical (+)-Medioresinol, a furofuran type lignan identification and isolation on the stem bark of Sambucus williamsii, which is a folk medicinal plant used in traditional medicine. (+)-Medioresinol is known to possess a lesishmanicidal activity and cardiovascular disease risk reduction but its antifungal effects have not yet been identified. In this study, to confirm (+)-Medioresinol's antifungal properties and mode of action, we observed morphological and physiological change in Candida albicans. In cells exposed to (+)-Medioresinol, arrested the cell cycle and intracellular reactive oxygen species (ROS) which is a major cause of apoptosis were increased. The increase of ROS induced oxidative stress and the mitochondria dysfunction which causes release of pro-apoptotic factors. We investigated a series of characteristic cellular changes of apoptosis by using various apoptosis detection methods. We report here for the first time that (+)-Medioresinol has effects on mitochondria and induced the accumulation of ROS in C.albicans cells. We demonstrated that one of the important features of apoptosis, mitochondrial membrane depolarization is caused by ROS. Substantially, we investigated the release of cytochrome c, which is one of the factors of metacaspase activity. We also show that the effects of (+)-Medioresinol are mediated at an early stage in apoptosis acting on the plasma membrane phosphatidylserine externalization. In addition, (+)-Medioresinol induced apoptotic morphological changes, showing the reduced cell size (low FSC) and enhanced intracellular density (high SSC). In late stage of confirmation of diagnostic markers in yeast apoptosis include the effects of nucleus morphological change, DNA fragmentation and condensation by influence of oxidative stress. These apoptotic phenomena represent that oxidative stress and mitochondria dysfunctions by inducing the phytochemical (+)-Medioresinol must be an important factors of the apoptotic process in C.albicans. These results support the elucidation of the underlying antifungal mechanisms of (+)-Medioresinol.

Abstract 1131: Blocking ATP delivery to hexokinase II in glioblastoma is a promising therapeutic strategy

Abstract Glioblastoma has a 5-year survival of less than 5% and is responsible for more years of life lost than any other malignancy, making it a challenging therapeutic problem. Healthy cells mainly rely on oxidative phosphorylation to catabolize glucose, while glioblastoma cells use aerobic glycolysis. The first step in glycolysis, conversion of glucose to glucose-6-phosphate using ATP, is catalyzed by hexokinase and glioblastoma tumors employ an isoform (HKII) that is bound to mitochondria via interaction with the outer-membrane voltage dependent anion channel. The prevailing theory is that this localization affords HKII preferential access to the mitochondrial ATP via inner-membrane adenine nucleotide translocase (ANT) to rapidly phosphorylate and trap glucose, thus initiating glycolysis across the cell cytosol. We have tested this hypothesis using a novel water-soluble organoarsenical, PENAO, to inactivate ANT (1). Treatment of glioblastoma cell lines and primary glioma initiating cells with PENAO inhibited acid (lactate) production, a measure of cancer cell glycolysis, at a half-maximal inhibitory concentration (IC50) of 1.5 µM, which supports the hypothesis. Proper functioning of ANT is also important for mitochondrial integrity and low micromolar concentrations of PENAO increased the cytosolic levels of superoxide, disrupted the mitochondrial trans-membrane potential and inhibited O2 utilization in glioblastoma cells. Blocking ANT with PENAO resulted in inhibition of glioblastoma cell migration at IC50 value of 1.4 µM, proliferation arrest at IC50 values of 0.3-4.5 µM and mitochondrial-mediated apoptotic cell death at PENAO concentrations of 0.3-5 µM. These effects were observed under both normoxic and hypoxic (1% O2) conditions. PENAO has up to 440- and 171-fold greater anti-proliferative activity than temozolomide or carboplatin, typical drugs used to treat glioblastoma, in glioblastoma cell lines and primary glioma initiating cells. PENAO was found to cross the intact blood-brain-barrier in mice and administration of 1 mg/kg/day PENAO to ten mice bearing subcutaneous glioblastoma tumors resulted in eight partial and two complete tumor responses. There were no signs or symptoms of treatment toxicity. These findings indicate that blocking ATP delivery to hexokinase II in glioblastoma by inhibiting mitochondrial ANT is a promising therapeutic strategy. The efficacy of PENAO in in vitro and in vivo models of glioblastoma supports its clinical development as a treatment option for this cancer. 1. Dilda PJ, Decollogne S, Weerakoon L, Norris MD, Haber M, Allen JD, Hogg PJ (2009) Optimization of the antitumor efficacy of a synthetic mitochondrial toxin by increasing the residence time in the cytosol. J Med Chem 52, 6209-6216. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1131. doi:1538-7445.AM2012-1131

Methamphetamine-induced neurotoxicity linked to ubiquitin-proteasome system dysfunction and autophagy-related changes that can be modulated by protein kinase C delta in dopaminergic neuronal cells

A compromised protein degradation machinery has been implicated in methamphetamine (MA)-induced neurodegeneration. However, the signaling mechanisms that induce autophagy and ubiquitin-proteasome system (UPS) dysfunction are not well understood. The present study investigates the contributions of protein kinase C delta (PKCδ)-mediated signaling events in MA-induced autophagy, UPS dysfunction, and cell death. Using an in vitro mesencephalic dopaminergic cell culture model, we demonstrate that MA-induced early induction of autophagy is associated with reduction in proteasomal function and concomitant dissipation of mitochondrial membrane potential (MMP), followed by significantly increased PKCδ activation, caspase-3 activation, accumulation of ubiquitin-positive aggregates and microtubule-associated light chain-3 (LC3-II) levels. Interestingly, siRNA-mediated knockdown of PKCδ or overexpression of cleavage-resistant mutant of PKCδ dramatically reduced MA-induced autophagy, proteasomal function, and associated accumulation of ubiquitinated protein aggregates, which closely paralleled cell survival. Importantly, when autophagy was inhibited either pharmacologically (3-MA) or genetically (siRNA-mediated silencing of LC3), the dopaminergic cells became sensitized to MA-induced apoptosis through caspase-3 activation. Conversely, overexpression of LC3 partially protected against MA-induced apoptotic cell death, suggesting a neuroprotective role for autophagy in MA-induced neurotoxicity. Notably, rat striatal tissue isolated from MA-treated rats also exhibited elevated LC3-II, ubiquitinated protein levels, and PKCδ cleavage. Taken together, our data demonstrate that MA-induced autophagy serves as an adaptive strategy for inhibiting mitochondria-mediated apoptotic cell death and degradation of aggregated proteins. Our results also suggest that the sustained activation of PKCδ leads to UPS dysfunction, resulting in the activation of caspase-3-mediated apoptotic cell death in the nigrostriatal dopaminergic system.