Mitochondrial Pore Formation Research Articles

Abstract 5265: Targeting mtDNA dynamics enhances immunogenicity and sensitizes KRAS mutant cancers to PD-1 blockade

Abstract Mitochondrial DNA (mtDNA) can stimulate an innate immune response that potentiates the efficacy of cancer immunotherapy. However, mtDNA-targeting therapy has not been developed to enhance this innate immune system in specific cancer subtypes efficiently. Here we discover that mtDNA is enriched in KRAS mutant tumors where the DNA sensor machinery cGAS-STING signaling is primed for innate immune activation. Intriguingly, MEK inhibitor induces cytosolic mtDNA release to potently activate the cGAS-STING signaling, which in turn induces PRKN-mediated mitophagy in response to mitochondrial stress in KRAS mutant cancer cells. During the process of mitophagy, PRKN, as an E3 ubiquitin ligase, degrades BAX protein and attenuates mitochondrial pore formation, which is essential for mtDNA efflux upon MEK inhibition. Combined MEK and mitophagy inhibitors efficiently stimulate type I IFNs in both mtDNA- and STING-dependent manners, and this combination enhances the efficacy of PD-1 blockade in Kras mutant murine syngeneic tumors. In the clinic, PRKN is associated with a ‘cold tumor’ phenotype among KRAS mutant tumors. Together, targeting mtDNA dynamics by co-inhibition of MEK and mitophagy represents a promising strategy to convert ‘cold to hot tumor’ in mtDNA-enriched KRAS mutant cancers. Citation Format: Kosuke Tanaka, Yasuki Adachi, Miyuki Yoshiya, Takuya Owari, Tomoko Yamamori-Morita, Akihiro Ohashi, Susumu Kobayashi, Shohei Koyama, Hiroyoshi Nishikawa. Targeting mtDNA dynamics enhances immunogenicity and sensitizes KRAS mutant cancers to PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5265.

Molecular mechanisms of the effect of standardized placental hydrolysate peptides on mitochondria functioning

Human placenta hydrolysates (HPH), the study of which was initiated by the scientific school of Vladimir P. Filatov, are currently being investigated using modern proteomic technologies. HPH is a promising tool for maintaining the function of mitochondria and regenerating tissues and organs with a high content of mitochondria (liver, heart muscle, skeletal muscles, etc.). The molecular mechanisms of action of HPH are practically not studied. Identification of mitochondrial support mitochondrial function-supporting peptides in HPH (Laennec, produced by Japan Bioproducts). Data on the chemical structure of the peptides were collected through a mass spectrometric experiment. Then, to establish the amino acid sequences of the peptides, de novo peptide sequencing algorithms based on the mathematical theory of topological and metric analysis of chemographs were applied. Bioinformatic analysis of the peptide composition of HPH was carried out using the integral protein annotation method. The biological functions of 41 peptides in the composition of HPH have been identified and described. Among the target proteins, the activity of which is regulated by the identified peptides and significantly affects the function of mitochondria, are caspases (CASP1, CASP3, CASP4) and other proteins regulating apoptosis (BCL2, CANPL1, PPARA), MAP kinases (MAPK1, MAPK3, MAPK4, MAPK8, MAPK9 , MAPK10, MAPK14), AKT1/GSK3B/MTOR cascade kinases, and a number of other target proteins (ADGRG6 receptor, inhibitor of NF-êB kinase IKKE, pyruvate dehydrogenase 2/3/4, SIRT1 sirtuin deacetylase, ULK1 kinase). HPH peptides have been identified that promote inhibition of mitochondrial pore formation, apoptosis, and excessive mitochondrial autophagy under conditions of oxidative/toxic stress, chronic inflammation, and/or hyperinsulinemia.

Discovery of a Novel, First-in-Class Bfl-1 BH3 Mimetic with Pro-Apoptotic Activity

Background: Intrinsic apoptosis is regulated by the B Cell lymphoma 2 (BCL-2) protein family. Anti-apoptotic family members, such as BCL-2, MCL-1 and BFL-1, sequester their pro-apoptotic counterparts through a highly conserved BH3 binding groove. Disruption of this balance leads to mitochondrial pore formation and subsequent cell death. To evade apoptosis, tumors often upregulate the expression of one or more anti-apoptotic family members and as such they have become attractive targets for anti-cancer drug development. BFL-1 (‘Bcl-2 related gene expressed in fetal liver’, gene name BCL2A1) is a lesser-known anti-apoptotic family member, physiologically mainly expressed in the hematopoietic system. BCL2A1 overexpression has been reported in many types of B cell lymphoma. Diffuse Large B Cell Lymphoma (DLBCL) patient sample analysis showed that BCL2A1 expression levels are second only to MCL1 within the anti-apoptotic family. Expression of BFL-1 was shown to be upregulated in MYC+/BCL2+ double hit lymphoma cell lines treated with the BCL-2 inhibitor, Venetoclax, in vivo. Interestingly, BFL-1 positive lymphomas are less sensitive to inhibition of BCL-2 and MCL-1. Hence, targeting BFL-1 is a rational and attractive strategy in DLBCL to improve clinical outcome. Methods: Using hit identification screens followed by iterative structure-activity relationship (SAR) analyses and structure-based design, compound A, a first-in-class, selective BFL-1 BH3 mimetic, was identified. Compound A was evaluated using biochemical, cellular in vitro and in vivo tumor efficacy models. Results: Compound A is a highly potent and selective compound in biochemical assays. In BFL-1 sensitive lymphoma cell lines characterized by high BFL-1 protein expression, compound A was shown to displace BAK and BIM from BFL-1, leading to downstream cleavage of caspase 3 and cell death. Interestingly, these cellular models were resistant to Venetoclax. Compound A exhibited tumor growth inhibition in an engineered BFL-1 overexpressing, MCL-1 KO human multiple myeloma xenograft model. However, in the high BFL-1 expressing SU-DHL-2 DLBCL human xenograft model, compound A did not induce significant PD (cleaved caspase 3) and efficacy in vivo. Conclusion: First-in-class selective BFL-1 BH3 mimetics are shown to specifically inhibit cell growth of lymphoma cell lines in vitro and can induce efficacy in vivo in a BFL-1 overexpressing model. Further studies may be useful to determine whether compound A elicits anti-tumor efficacy in lymphoma alone or in combination with other anti-apoptotic agents.

CALCIUM-ACCUMULATING ABILITY OF RAT LIVER MITOCHONDRIA DURING HYPOTHERMIA OF VARIOUS DURATIONS

Calcium is one of the most important intracellular messengers that regulate physiological and biochemical processes in the cell. Mitochondria are able to deposit calcium ions and are involved in the regulation of the calcium signal. Hypothermic conditions in homoiothermic animals can lead to disruption of this important function of mitochondria and pathological consequences. The aim of this study is to study the effects of moderate (30°C) hypothermia of varying duration on the calcium-accumulating capacity of rat liver mitochondria. The experiments were carried out on male rats Wistar. Hypothermia was induced by external cooling of the animals in Plexiglas chambers with a jacket through which cold water circulated. Mitochondria were isolated from the liver of decapitated rats by differential centrifugation. The calcium-accumulating capacity of mitochondria was assessed by the kinetics of calcium-induced swelling of mitochondria and their calcium capacity. A study of the kinetics of calcium-induced swelling of mitochondria showed that during short-term moderate (30°C) hypothermia, the rate of swelling of mitochondria decreases, prolongation of hypothermia to 1 h contributes to a further decrease in the swelling rate, and to 3 h, its normalization. A positive correlation was found between the rate of calcium-induced swelling and the calcium capacity of mitochondria (r = 0.79). Thus, with prolonged exposure to the cold factor in rats, a number of compensatory-adaptive reactions are activated. The decrease in the rate of Ca2+ – induced swelling and calcium capacity of mitochondria at the initial stages of hypothermia may be associated with the formation of mitochondrial pores and is reversible.

Characterization of Mitochondrial Alterations in Aicardi-Goutières Patients Mutated in RNASEH2A and RNASEH2B Genes.

Aicardi-Goutières syndrome (AGS) is a rare encephalopathy characterized by neurological and immunological features. Mitochondrial dysfunctions may lead to mitochondrial DNA (mtDNA) release and consequent immune system activation. We investigated the role of mitochondria and mtDNA in AGS pathogenesis by studying patients mutated in RNASEH2B and RNASEH2A genes. Lymphoblastoid cell lines (LCLs) from RNASEH2A- and RNASEH2B-mutated patients and healthy control were used. Transmission Electron Microscopy (TEM) and flow cytometry were used to assess morphological alterations, reactive oxygen species (ROS) production and mitochondrial membrane potential variations. Seahorse Analyzer was used to investigate metabolic alterations, and mtDNA oxidation and VDAC1 oligomerization were assessed by immunofluorescence. Western blot and RT-qPCR were used to quantify mtTFA protein and mtDNA release. Morphological alterations of mitochondria were observed in both mutated LCLs, and loss of physiological membrane potential was mainly identified in RNASEH2A LCLs. ROS production and 8-oxoGuanine levels were increased in RNASEH2B LCLs. Additionally, the VDAC1 signal was increased, suggesting a mitochondrial pore formation possibly determining mtDNA release. Indeed, higher cytoplasmic mtDNA levels were found in RNASEH2B LCLs. Metabolic alterations confirmed mitochondrial damage in both LCLs. Data highlighted mitochondrial alterations in AGS patients' LCLs suggesting a pivotal role in AGS pathogenesis.

Abstract 2133: In pursuit of MCL-1 inhibitors with improved therapeutic window for the treatment of hematological malignancies: Discovery of JNJ-4355

Abstract Appropriate control of cell death is a fundamental biological process which is frequently dysregulated during tumor development and therapeutic resistance. Apoptosis is a form of regulated cell death initiated by either the extracellular environment (extrinsic) or following internal cellular damage (intrinsic). It is controlled by the BCL-2 family which includes anti-apoptotic regulators like BCL-2, BCL-XL and MCL-1 that bind and sequester various pro-apoptotic BH3-only proteins (BIM, BAD, BID, NOXA, PUMA, etc.), and the pro-apoptotic effectors (BAK, BAX, etc.) responsible for mitochondrial pore formation and MOMP (mitochondrial outer membrane permeabilization). MOMP results in intermembrane space protein release, leading to caspase activation in an irreversible path to programmed cell death. Of the anti-apoptotic regulators, MCL-1 is one of the most frequently and highly amplified genes in human cancers such as myeloid leukemia making it a compelling therapeutic target. Since BCL-2 proteins interact through protein-protein interactions, they have long been elusive targets. The success of selective BCL-2 protein inhibitor Venetoclax in the treatment of various hematological cancers, however spurred interest in MCL-1 as an oncology target. Using structure-based drug design, major breakthroughs were made in the development of MCL-1 inhibitors, with several candidates entering clinical studies in the past five years. JNJ-4355, a highly potent 1,4-indolyl macrocycle (MCL-1 Ki = 18 pM, Cell (MOLP8) AC50 = 8.7 nM) was optimized to address shortcomings from first generation MCL-1 inhibitors: it has improved physicochemical properties (CHI LogD7.4 = 2.35, EPSA = 151 Å2), resulting in greatly improved equilibrium solubility (3.14 mM in buffer pH 7) and reduced protein binding (99.93%). JNJ-4355 showed promising in vitro potency data in cancer cell lines and AML patient-derived samples (cell killing AC50 0.29-75 nM in 25/27 evaluable samples). In vivo MCL-1:BAK complex disruption was confirmed in a mouse MOLM13 (AML) xenograft. Efficacy was demonstrated in a mouse MOLP8 (multiple myeloma) xenograft resulting in complete tumor regression after a single IV dose of JNJ-4355. Citation Format: Frederik J. Rombouts, Lento William, Ingrid Velter, Ann Vos, Aldo Peschiulli, Reuillon Tristan, Maria Dominguez Blanco, Matthieu Jouffroy, Lisa McQueen, Helena Steyvers, Mariette Bekkers, Cristina Altrocchi, Beth Pietrak, Seong Joo Koo, Lawrence Szewczuk, David Walker, Kathryn Packman, Ruud Bueters, Petra Vinken, Amy Johnson, Ricardo Attar, Ulrike Philippar. In pursuit of MCL-1 inhibitors with improved therapeutic window for the treatment of hematological malignancies: Discovery of JNJ-4355 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2133.

Gasdermin D promotes hyperinflammation by triggering necroptosis in the presence of mitochondrial stress

Abstract Human genetic variants associated with mitochondrial dysfunction have been linked to chronic inflammatory diseases as well as susceptibility to infection. However, the mechanistic impact that these variants have on the immune system is poorly understood. We have discovered that macrophages harboring the common Parkinson’s disease associated variant, Lrrk2G2019S are more prone to cell death in response to both Mycobacterium tuberculosis (Mtb) infection and AIM2 inflammasome activation. Unexpectedly, the enhanced cell death in Lrrk2G2019S macrophages is driven by increased susceptibility to gasdermin D (GSDMD)-mediated mitochondrial pore formation, which releases accumulated mitochondrial ROS and pushes Lrrk2G2019S cells to undergo RIPK3-mediated necroptosis. Consistent with elevated necroptotic cell death, infection of Lrrk2G2019S mice with Mtb elicits dramatic hyperinflammation and exacerbated pathogenesis via enhanced neutrophil infiltration. Remarkably, expression of hLRRK2G2019S in Drosophila melanogaster recapitulates a similar phenotype, suggesting that Lrrk2G2019S plays an evolutionarily conserved role in regulating innate immunity. Our findings demonstrate that altered mitochondrial function can reprogram canonical innate immune and cell death pathways to elicit distinct immune outcomes, providing mechanistic insights into why mutations in LRRK2 are associated with susceptibility to chronic inflammatory and infectious diseases. Supported by grants from the Parkinson's Foundation (PF-FBS-1932), NIH (R01 AI125512), Michael J. Fox Foundation (grant 12185), and the Texas A&M Clinical Science and Translational Research (CSTR) Pilot Grant Program.

Bile Acid-Induced Ca2+ Efflux from Liver Mitochondria as a Factor Preventing the Formation of Mitochondrial Pores

The effect of bile acids as inducers of Ca2+ efflux from the matrix was studied on isolated rat liver mitochondria. Mitochondria in the presence of cyclosporin A (CsA) were energized by succinate, then loaded with Ca2+ and after the addition of the calcium uniporter inhibitor ruthenium red were de-energized by malonate. It was shown that under these conditions hydrophobic bile acids lithocholic and chenodeoxycholic at concentrations of 10 and 30 μM respectively and hydrophilic bile acids ursodeoxycholic and cholic at a concentration of 400 μM induce Ca2+ efflux from the mitochondrial matrix. It is noted that the efflux of these ions is not associated with damage of the inner mitochondrial membrane by bile acids, since it is accompanied by the generation of Δψ, i.e., the formation of the diffusion potential. It is assumed that along with induction of calcium efflux from the matrix, bile acids are also capable of transporting hydrogen and potassium ions in the opposite direction, i.e., perform H+/Ca2+ and K+/Ca2+ exchange. It was found that ruthenium red added to Ca2+-loaded energized mitochondria prevents the return of these ions to the matrix and weakens the effect of chenodeoxycholic acid as an inducer of the CsA-sensitive mitochondrial pore and the effect of ursodeoxycholic acid as an inducer of CsA-insensitive permeability of the inner mitochondrial membrane. We conclude that in the conditions of the calcium uniporter activity decrease, Ca2+ efflux from the matrix induced by bile acids can be considered as one of the mechanisms reducing their effectiveness as inducers of the Ca2+-dependent CsA-sensitive pore and CsA-insensitive permeability transition in mitochondria.

Modulation of Immune Response by Organophosphate Pesticides: Mammals as Potential Model

Organophosphates (OPs) are most widely used pesticides and primarily induce toxicity by inhibition of acetylcholinesterase (AChE) in the nerve terminals of central and peripheral nervous system, leading to a variety of short-term and chronic effects in the non-target animals. In addition to acetylcholinesterase, OPs are known to potent inhibitors of serine hydrolases which are vital component of the immune system and therefore influence the immune functions. OPs induce several immunomodulatory effects in vertebrates by altering neutrophil function, macrophage production, antibody production, immunosuppression, reduced interleukin production and T cell proliferation. Immunotoxicity due to OP exposure is mediated through perturbation of the cholinergic response of lymphocytes, altering signal transduction, mutilating granule exocytosis pathway and impairing FasL/Fas pathway of natural killer cell and other immune related cells. Apoptosis of lymphocytes or immune related cells is promoted through mitochondrial pore formation and DNA fragmentation. In this review an attempt has been made to document the immunomodulatory effects of organophosphate pesticides using mammals as potential model with an additional information on the probable mechanism of immunotoxicity induced by OPs.

Phytochemicals prevent mitochondrial membrane permeabilization and protect SH-SY5Y cells against apoptosis induced by PK11195, a ligand for outer membrane translocator protein.

Epidemiological studies present the beneficial effects of dietary habits on prevention of aging-associated decline of brain function. Phytochemicals, the second metabolites of food, protect neuronal cells from cell death in cellular models of neurodegenerative disorders, and the neuroprotective activity has been ascribed to the anti-oxidant and anti-inflammatory functions. In this paper, the cellular mechanism of neuroprotection by phytochemicals was investigated, using the cellular model of mitochondrial apoptosis induced by PK11195, a ligand of outer membrane translocator protein, in SH-SY5Y cells. PK11195 induced mitochondrial membrane permeabilization with rapid transit production of superoxide (superoxide flashes) and calcium release from mitochondria, and activated apoptosis signal pathway. Study on the structure-activity relationship of astaxanthin, ferulic acid derivatives, and sesame lignans revealed that these phytochemicals inhibited mitochondrial membrane permeabilization and protected cells from apoptosis. Ferulic acid derivatives and sesame lignans inhibited or enhanced the mitochondrial pore formation and cell death by PK11195 according to their amphiphilic properties, not directly depending on the antioxidant activity. Regulation of pore formation at mitochondrial membrane is discussed as a novel mechanism behind neuroprotective activity of phytochemicals in aging and age-associated neurodegenerative disorders, and also behind dual functions of phytochemicals in neuronal and cancer cells.

ITOC2 – 018. Hyperthermia induced immunogenic cell-death

<h3>Background</h3> Some hyperthermia studies in oncology deals with connected immune-effects. Modulated electro-hyperthermia (oncothermia, Szasz et al. (2010) [1]) is an emerging curative treatment method. It is selectively focused in the malignant cells without harming the non-malignant surrounding tissues Andocs et al. (2009) [2]. <h3>Aim</h3> Our objective is studying the cell-death process caused by the treatment. <h3>Method</h3> HT29 human colorectal xenograft and C26 mouse colorectal allograft models were studied <i>in vivo</i>, on in time-course investigations. We apply 13.56MHz radiofrequency (RF) signal modulated by 1/f noise pattern. In silico models were performed visualising the selection process. Samples were analysed by various histomorphological and immunhistochemical analyses. <h3>Results</h3> A massive TUNEL positivity was produced. Up regulation of TRAILR2 (DR5), FAS and FADD was observed. AIF nuclear translocalisation together with mitochondrial pore formation and cytochrome-C release, as well as DNA fragmentation was measured. A damage associated molecular pattern (DAMP) was formed, which are probably parts of the immunogenic cell-death (ICD) of the selected malignant cells. <h3>Conclusion</h3> The DAMP associated, induced ICD can be a good basis for hyperthermia and immunotherapy combination.

Binding of Pro-Apoptotic Protein Bax to Cytoprotective UDCA and TUDCA

Hydrophobic bile acids, such as deoxycholic acid (DCA), strongly induce apoptosis in both hepatic and non-hepatic cells while hydrophilic bile acids, like ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA), are cytoprotective and inhibit cell death. The mechanisms associated with these distinct effects are not entirely clear. However, the effect of hydrophilic bile acids seems to be related with the blockage of a series of processes that converge on mitochondrial damage. Bax is a pro-apoptotic protein that belongs to the superfamily of the Bcl-2 proteins and is involved in mitochondrial pore formation. Submicellar concentrations of cytoprotective bile acids have been shown to modulate Bax concentration in mitochondria, suggesting that these molecules may interact directly with the protein. In this study, our objective was to evaluate the affinity of bile acids to recombinant Bax protein, making use of fluorescence spectroscopy (FRET and fluorescence anisotropy), as well as Fluorescence Correlation Spectroscopy (FCS). Our results show that the cytoprotective bile acids UDCA and TUDCA associate with recombinant Bax protein with high affinity, while the cytotoxic bile acid DCA only seems to be able to adsorb to the protein with much lower affinity. Notably, the binding site for UDCA seems to be located in a hydrophobic pocket of the protein. This interaction could be responsible for the disruption of Bax translocation to the mitochondrial outer membrane in the presence of UDCA and/or TUDCA.Supported from FCT/Portugal (Projects PTDC/QUI-BIQ/119494/2010 and RECI/CTM-POL/0342/2012).T.S. and F.F. acknowledges FCT grants SFRH/BD/92398/2013 and SFRH/BPD/64320/2009

Mitochondria express several nicotinic acetylcholine receptor subtypes to control various pathways of apoptosis induction

Nicotinic acetylcholine receptors control survival, proliferation and cytokine release in non-excitable cells. Previously we reported that α7 nicotinic receptors were present in the outer membranes of mouse liver mitochondria to regulate mitochondrial pore formation and cytochrome c release. Here we used a wide spectrum of nicotinic receptor subunit-specific antibodies to show that mitochondria express several nicotinic receptor subtypes in a tissue-specific manner: brain and liver mitochondria contain α7β2, α4β2 and less α3β2 nicotinic receptors, while mitochondria from the lung express preferentially α3β4 receptor subtype; all of them are non-covalently connected to voltage-dependent anion channels and control cytochrome c release. By using selective ligands of different nicotinic receptor subtypes (acetylcholine (1μM) or dihydro-β-erythroidine (1μM) for α4β2), conotoxin MII (1nM) for α3β2, MLA (50nM) for α7β2 and acetylcholine (10μM) for all subtypes) and apoptogenic agents triggering different mitochondrial signaling pathways (1μM wortmannin, 90μM Ca2+ or 0.5mM H2O2) it was found that α7β2 receptors affect mainly PI3K/Akt pathway, while α3β2 and α4β2 nAChRs also significantly influence CaKMII- and Src-dependent pathways. It is concluded that cholinergic regulation in mitochondria is realized through multiple nicotinic receptor subtypes, which control various pathways inducing mitochondrial type of apoptosis.

Cyclophilin 40 alters UVA-induced apoptosis and mitochondrial ROS generation in keratinocytes

The CyP40 protein encoded by PPID gene is a member of the peptidyl-prolyl cis–trans isomerase (PPIase) family. PPIases catalyze the cis–trans isomerization of proline imidic peptide bonds in oligopeptides and accelerate the folding of proteins. The CyP40 protein has been shown to possess PPIase activity and, similar to other family members, can bind to the immunosuppressant drug cyclosporin A (CsA). In this study, we created keratinocyte cell lines with CyP40 being stably knocked down using viral particles containing shRNA for CyP40 which knocked down the expression level of CyP40 transcripts by 90–99%. The proliferation rates of the cell lines with silenced CyP40 were decreased compared to the control cells. After UVA irradiation, the rate of apoptosis was found to be significantly lower in CyP40 silenced cell lines than it was in control cells. Moreover, mitochondrial membrane potential (MMP) was found to be less dissipated and mitochondrial permeability transition pore (MPTP) less active in cells with knocked down CyP40 than in control cells after UVA irradiation. Also, less mitochondrial superoxide was detected in the cells with silenced CyP40 compared to control cells after UVA exposure. Moreover, silencing of CyP40 partially modulates expression of key genes involved in mitochondrial pore formation including CyPD, ANTs and VDAC family members. The ability of CyP40 to regulate UV induced apoptosis implicates this protein as a potential target for therapy in cancer cells.

NOXA as critical mediator for drug combinations in polychemotherapy

During polychemotherapy, cytotoxic drugs are given in combinations to enhance their anti-tumor effectiveness. For most drug combinations, underlying signaling mechanisms responsible for positive drug–drug interactions remain elusive. Here, we prove a decisive role for the Bcl-2 family member NOXA to mediate cell death by certain drug combinations, even if drugs were combined which acted independently from NOXA, when given alone. In proof-of-principle studies, betulinic acid, doxorubicin and vincristine induced cell death in a p53- and NOXA-independent pathway involving mitochondrial pore formation, release of cytochrome c and caspase activation. In contrast, when betulinic acid was combined with either doxorubicine or vincristine, cell death signaling changed considerably; the drug combinations clearly depended on both p53 and NOXA. Similarly and of high clinical relevance, in patient-derived childhood acute leukemia samples the drug combinations, but not the single drugs depended on p53 and NOXA, as shown by RNA interference studies in patient-derived cells. Our data emphasize that NOXA represents an important target molecule for combinations of drugs that alone do not target NOXA. NOXA might have a special role in regulating apoptosis sensitivity in the complex interplay of polychemotherapy. Deciphering the differences in signaling of single drugs and drug combinations might enable designing highly effective novel polychemotherapy regimens.

Possible Effect of Activation of α7-Nicotinic Acetylcholine Receptors in the Mitochondrial Membrane on the Development of Apoptosis

Using flow cytometry and sandwich-immunoenzyme assay, we showed that nicotinic acetylcholine receptors with a subunit α7 (nAChRs α7) expressed in the outer mitochondrial membrane are involved in the control of mitochondria-dependent apoptosis. Pre-incubation of the mitochondria with an nAChRs α7 agonist, choline, decreased dissipation of the membrane potential of these organelles induced by the action of 0.5 mM hydrogen peroxide (H2O2) but did not influence the analogous effect of a high Ca2+ concentration (90 μM). Agonists of nAChRs α7 (choline, acetylcholine, and PNU 282987), or an inhibitor of voltage-dependent anion channels, DIDS, prevented the release of cytochrome c from the intermembrane mitochondrial space under the action of H2O2. In contrast, an antagonist of nAChRs α7, methyllycaconitine, promoted the release of cytochrome c and prevented the effects of agonists. The obtained data confirm the active involvement of nAChRs α7 and voltage-dependent anion channels in the process of formation of mitochondrial pores. In this case, agonists of mitochondrial nAChRs α7 subunits exert an antiapoptotic effect, while antagonists of mitochondrial nAChRs α7 subunits manifest a proapoptotic action.

Characterization of Cholix Toxin-induced Apoptosis in HeLa Cells

Cholix toxin (Cholix) is a novel ADP-ribosylating cytotoxin produced by Vibrio cholerae, which utilizes eukaryotic elongation factor 2 as a substrate and acts by a mechanism similar to that of diphtheria toxin and Pseudomonas exotoxin A. First it was found that Cholix-treated HeLa cells exhibited caspase-dependent apoptosis, whereas intestinal cells such as Caco-2, HCT116, and RKO did not. Here we investigated Cholix-induced cell death signaling pathways in HeLa cells. Cholix-induced cytochrome c release into cytosol was initiated by specific conformational changes of pro-apoptotic Bak associated with Bax. Silencing of bak/bax genes or bak gene alone using siRNA significantly suppressed cytochrome c release and caspase-7 activation, but not activation of caspases-3 and -9. Although pretreatment with a caspase-8 inhibitor (Z-IETD-FMK) reduced Cholix-induced cytochrome c release and activation of caspases-3, -7, and -9, cytotoxicity was not decreased. Pretreatment with Z-YVAD-FMK, which inhibits caspase-1, -4, and -5, suppressed not only cytochrome c release, activation of caspase-3, -7, -8, or -9, and PARP cleavage, but also cytotoxicity, indicating that caspase-1, -4, and -5 activation is initiated at an early stage of Cholix-induced apoptosis and promotes caspase-8 activation. These results show that the inflammatory caspases (caspase-1, -4, and -5) and caspase-8 are responsible for both mitochondrial signals and other caspase activation. In conclusion, we showed that Cholix-induced caspase activation plays an essential role in generation of apoptotic signals, which are mediated by both mitochondria-dependent and -independent pathways.

Ursodeoxycholic acid in cholestasis: linking action mechanisms to therapeutic applications

UDCA (ursodeoxycholic acid) is the therapeutic agent most widely used for the treatment of cholestatic hepatopathies. Its use has expanded to other kinds of hepatic diseases, and even to extrahepatic ones. Such versatility is the result of its multiple mechanisms of action. UDCA stabilizes plasma membranes against cytolysis by tensioactive bile acids accumulated in cholestasis. UDCA also halts apoptosis by preventing the formation of mitochondrial pores, membrane recruitment of death receptors and endoplasmic-reticulum stress. In addition, UDCA induces changes in the expression of metabolizing enzymes and transporters that reduce bile acid cytotoxicity and improve renal excretion. Its capability to positively modulate ductular bile flow helps to preserve the integrity of bile ducts. UDCA also prevents the endocytic internalization of canalicular transporters, a common feature in cholestasis. Finally, UDCA has immunomodulatory properties that limit the exacerbated immunological response occurring in autoimmune cholestatic diseases by counteracting the overexpression of MHC antigens and perhaps by limiting the production of cytokines by immunocompetent cells. Owing to this multi-functionality, it is difficult to envisage a substitute for UDCA that combines as many hepatoprotective effects with such efficacy. We predict a long-lasting use of UDCA as the therapeutic agent of choice in cholestasis.

VSV Oncolysis in Combination With the BCL-2 Inhibitor Obatoclax Overcomes Apoptosis Resistance in Chronic Lymphocytic Leukemia

In chronic lymphocytic leukemia (CLL), overexpression of antiapoptotic B-cell leukemia/lymphoma 2 (BCL-2) family members contributes to leukemogenesis by interfering with apoptosis; BCL-2 expression also impairs vesicular stomatitis virus (VSV)-mediated oncolysis of primary CLL cells. In the effort to reverse resistance to VSV-mediated oncolysis, we combined VSV with obatoclax (GX15-070)—a small-molecule BCL-2 inhibitor currently in phase 2 clinical trials—and examined the molecular mechanisms governing the in vitro and in vivo antitumor efficiency of combining the two agents. In combination with VSV, obatoclax synergistically induced cell death in primary CLL samples and reduced tumor growth in severe combined immunodeficient (SCID) mice-bearing A20 lymphoma tumors. Mechanistically, the combination stimulated the mitochondrial apoptotic pathway, as reflected by caspase-3 and -9 cleavage, cytochrome c release and BAX translocation. Combination treatment triggered the release of BAX from BCL-2 and myeloid cell leukemia-1 (MCL-1) from BAK, whereas VSV infection induced NOXA expression and increased the formation of a novel BAX-NOXA heterodimer. Finally, NOXA was identified as an important inducer of VSV-obatoclax driven apoptosis via knockdown and overexpression of NOXA. These studies offer insight into the synergy between small-molecule BCL-2 inhibitors such as obatoclax and VSV as a combination strategy to overcome apoptosis resistance in CLL.

When life comes to an end: lessons from microbial aging models

Aging of biological systems ultimately leads to death of the individual. In humans, organ failure as the result of functional impairments after stroke, cardio-vascular disease, tumor development, neurodegeneration and other diseases are certainly crucial in bringing life to an end. But what happens in individuals with no obvious disease or disorders? In simple microorganisms like the single cellular fungus Saccharomyces cerevisiae (yeast) very basic cellular processes, resembling mammalian programmed cell death (PCD) as they are important for development and protection against cancer, lead to death of the individuum [1,2]. In the last several years, PCD was also demonstrated to occur in multicellular fungi [2]. In the filamentous fungus Podospora anserina, a well established experimentally tractable short-lived aging model with a small genome, a role of PCD was recently demonstrated to be involved in aging and lifespan control [3-5]. Although differing in complexity, the molecular pathways controlling PCD in the different groups of organisms are conserved [6,7]. In a recent study we addressed the role of PaCYPD (cyclophilin D) a P. anserina [8], a mitochondrial matrix protein previously found to increase in abundance in mitochondria during aging [9]. Cyclophilin D is a peptidyl prolyl-cis,trans-isomerase located in the mitochondrial matrix that is known to be involved in mitochondrial permeability pore transition (mPTP) [10], a process leading to opening of a membrane pore and to entrance of low molecular weight solutes into the mitochondrial matrix. The process leads to swelling of mitochondria, rupture of the outer membrane and the release of apoptogens (e.g., cytochrome c) from the intermembrane space and the subsequent induction of PCD. We now show that over-expression of PaCypD results in accelerating organismal aging. Chronological young PaCypD overexpressors display a ‘premature aging' phenotype with pronounced mitochondrial dysfunction, markers of senescence and apoptosis, and reduced stress tolerance. Treatment with the CYPD inhibitor cyclosporin A leads to correction of mitochondrial function and lifespan to that of the wild-type. It should be emphasized here that the induction of PCD as a final execution program in the life cycle of the fungus appears to result from complex aging processes. In P. anserina, a number of different pathways including those leading to the generation and scavenging of reactive oxygen species [11-14], protein quality control [15], mitochondrial dynamics [16], and others [3,4] are known to be part of the complex network governing aging. It is now the challenge to identify other components of mitochondrial pore formation, characterize the pore biochemically, and to elucidate the detailed role of PCD in aging also in mammalian systems. Interestingly, an age-related increase of CYPD, as in P. anserina, was recently reported in rat gastrocnemius muscle and in brain tissue of mice and humans [17,18]. It is thus likely that the induction of PCD, at least in particular tissues and organs, also in human aging plays a more important role than it is currently anticipated. If so, it will be of special interest to investigate the potential of components like CSA, a cyclic peptide approved for human therapy in transplantation medicine or for treatment of Ullrich congenital muscular dystrophy, in being effective in postponing basic aging processes and using this knowledge to develop efficient strategies to intervene into biological aging.