Disruption Of Mitochondrial Membrane Integrity Research Articles

Effective Anticancer Potential of a New Sulfonamide as a Carbonic Anhydrase IX Inhibitor Against Aggressive Tumors.

This study examines efficiency of a newly synthesized sulfonamide derivative 2-bromo-N-(4-sulfamoylphenyl)propanamide (MMH-1) on the inhibition of Carbonic Anhydrase IX (CA IX), which is overexpressed in many solid tumors including breast cancer. The inhibitory potential of MMH-1 compound against its four major isoforms, including cytosolic isoforms hCA I and II, as well as tumor-associated membrane-bound isoforms hCA IX and XII, was evaluated. To this context, the cytotoxic effect of MMH-1 on cancer and normal cells was tested and found to selectively affect MDA-MB-231 cells. MMH-1 reduced cell proliferation by holding cells in the G0/G1 phase (72 %) and slowed the cells' wound healing capacity. MMH-1 inhibited CA IX under both hypoxic and normoxic conditions and altered the morphology of triple negative breast cancer cells. In MDA-MB-231 cells, inhibition of CA IX was accompanied by a decrease in extracellular pH acidity (7.2), disruption of mitochondrial membrane integrity (80 %), an increase in reactive oxygen levels (25 %), and the triggering of apoptosis (40 %). In addition, the caspase cascade (CASP-3, -8, -9) was activated in MDA-MB-231 cells, triggering both the extrinsic and intrinsic apoptotic pathways. The expression of pro-apoptotic regulatory proteins (Bad, Bax, Bid, Bim, Cyt-c, Fas, FasL, TNF-a, TNF-R1, HTRA, SMAC, Casp-3, -8, P21, P27, and P53) was increased, while the expression of anti-apoptotic proteins, apoptosis inhibitor proteins (IAPs), and heat shock proteins (HSPs) (Bcl-2, Bcl-w, cIAP-2, HSP27, HSP60, HSP70, Survivin, Livin, and XIAP) was decreased. These results propose that the MMH-1 compound could triggers apoptosis in MDA-MB-231 cells via the pH/MMP/ROS pathway through the inhibition of CA IX. This compound is thought to have high potential and promising anticancer properties in the treatment of aggressive tumors.

Targeting mitochondrial degradation by chimeric autophagy-tethering compounds.

The ability to regulate mitophagy in a living system with small molecules remains a great challenge. We hypothesize that adding fragments specific to the key autophagosome protein LC3 to mitochondria will mimic receptor-mediated mitophagy, thus engaging the autophagy-lysosome pathway to induce mitochondrial degradation. Herein, we develop a general biochemical approach to modulate mitophagy, dubbed mito-ATTECs, which employ chimera molecules composed of LC3-binding moieties linked to mitochondria-targeting ligands. Mito-ATTECs trigger mitophagy via targeting mitochondria to autophagosomes through direct interaction between mito-ATTECs and LC3 on mitochondrial membranes. Subsequently, autophagosomes containing mitochondria rapidly fuse with lysosomes to facilitate the degradation of mitochondria. Therefore, mito-ATTECs circumvent the detrimental effects related to disruption of mitochondrial membrane integrity by inducers routinely used to manipulate mitophagy, and provide a versatile biochemical approach to investigate the physiological roles of mitophagy. Furthermore, we found that sustained mitophagy lead to mitochondrial depletion and autophagic cell death in several malignant cell lines (lethal mitophagy). Among them, apoptosis-resistant malignant melanoma cell lines are particularly sensitive to lethal mitophagy. The therapeutic efficacy of mito-ATTECs has been further evaluated by using subcutaneous and pulmonary metastatic melanoma models. Together, the mitochondrial depletion achieved by mito-ATTECs may demonstrate the general concept of inducing cancer cell lethality through excessive mitochondrial clearance, establishing a promising therapeutic paradigm for apoptosis-resistant tumors.

Targeting BET Proteins With a PROTAC Molecule Elicits Potent Anticancer Activity in HCC Cells.

Background and Aim: Bromodomain and extraterminal domain (BET) family proteins are epigenetic regulators involved in human malignances. Targeting BET proteins for degradation using proteolysis-targeting chimera (PROTAC) recently has drawn increasing attention in the field of cancer therapeutics. BET proteins have been found to be overexpressed in HCC cells and tumor tissues. However, the biological activity of BET-PROTACs in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated anti-HCC activity of BETd-260, a BET-PROTAC molecule using in vitro and in vivo models.Methods: BETd-260-mediated anti-HCC activity was investigated by cell viability, apoptosis assays. Efficacy was examined with a cell lines-derived HCC xenograft model in mice. Anticancer mechanism was investigated by RT-PCR, western blotting and immunohistochemical staining.Results: BETd-260 potently suppressed cell viability and robustly induced apoptosis in HCC cells. BETd-260 reciprocally modulated the expression of several apoptotic genes in HCC cells, i.e., suppressing the expression of anti-apoptotic Mcl-1, Bcl-2, c-Myc, and X-linked inhibitor of apoptosis (XIAP), whereas increasing the expression of pro-apoptotic Bad. BETd-260 treatment led to disruption of mitochondrial membrane integrity, and triggered apoptosis via intrinsic signaling in HCC cells. BETd-260 triggered apoptosis in HCC xenograft tissue and profoundly inhibited the growth of HCC xenograft tumors in mice.Conclusion: Our data suggest that pharmacological targeting of BET for degradation may be a novel therapeutic strategy for the treatment of HCC.

198 Acute Trimethylamine-N-oxide Depresses Cardiac Function and “stress” Mitochondria into Overdrive

Trimethylamine-N-oxide (TMAO) is a secondary gut metabolite recently linked to cardiovascular disease (CVD). However, whether TMAO concentrations are sufficient to directly drive dysfunction and CVD is unclear. Since detrimental effects on the heart and mitochondria are yet to be detailed, we investigated the impacts of acute TMAO on function and respiration in murine hearts. Isolated hearts from male C57Bl/6 mice were retrogradely perfused at either constant flow or pressure while infused with increasing TMAO concentrations to assess effects on contractile function and coronary flow respectively. Shredded left ventricular (LV) myocardium was also suspended in mitochondrial respiration medium ±10 μM or 100 μM TMAO, and analysed using an Oxygraph-2k instrument. A substrate-uncoupler-inhibitor-titration (SUIT) protocol assessed routine mitochondrial respiration. TMAO appears to be a 'cardiodepressant': LV developed pressure and coronary flow were significantly decreased with ≥3 μM TMAO (p<0.05), with flow and contractile changes correlated (R2=0.59). Unexpectedly, however, mitochondrial Complex I as well as complex I & II linked respiration was significantly increased in response to both 10 μM (p<0.01) and 100 μM (p<0.05) TMAO, as was maximum respiratory capacity (p<0.01). However, significant increases in exogenous cytochrome c dependent respiration with both TMAO concentrations (p<0.01), supports disruption of mitochondrial membrane integrity. Data indicate that acute TMAO, at concentrations reported in cardiac, renal and metabolic diseases, appears to decrease cardiac contractile function while potentially 'stressing' cardiac mitochondria into overdrive. This is congruent with the role of elevated TMAO in CVD pathogenesis.

Tau-induced mitochondrial membrane perturbation is dependent upon cardiolipin

Misfolding and aggregate formation by the tau protein has been closely related with neurotoxicity in a large group of human neurodegenerative disorders, which includes Alzheimer’s disease. Here, we investigate the membrane-active properties of tau oligomers on mitochondrial membranes, using minimalist in vitro model systems. Thus, exposure of isolated mitochondria to oligomeric tau evoked a disruption of mitochondrial membrane integrity, as evidenced by a combination of organelle swelling, efflux of cytochrome c and loss of the mitochondrial membrane potential. Tau-induced mitochondrial dysfunction occurred independently of the mitochondrial permeability transition (mPT) pore complex. Notably, mitochondria were rescued by pre-incubation with 10-N-nonyl acridine orange (NAO), a molecule that specifically binds cardiolipin (CL), the signature phospholipid of mitochondrial membranes. Additionally, NAO prevented direct binding of tau oligomers to isolated mitochondria. At the same time, tau proteins exhibited high affinity to CL-enriched membranes, whilst permeabilisation of lipid vesicles also strongly correlated with CL content. Intriguingly, using single-channel electrophysiology, we could demonstrate the formation of non-selective ion-conducting tau nanopores exhibiting multilevel conductances in mito-mimetic bilayers. Taken together, the data presented here advances a scenario in which toxic cytosolic entities of tau protein would target mitochondrial organelles by associating with their CL-rich membrane domains, leading to membrane poration and compromised mitochondrial structural integrity.

Morphine counteracts the antiviral effect of antiretroviral drugs and causes upregulation of p62/SQSTM1 and histone-modifying enzymes in HIV-infected astrocytes

Accelerated neurological disorders are increasingly prominent among the HIV-infected population and are likely driven by the toxicity from long-term use of antiretroviral drugs. We explored potential side effects of antiretroviral drugs in HIV-infected primary human astrocytes and whether opioid co-exposure exacerbates the response. HIV-infected human astrocytes were exposed to the reverse transcriptase inhibitor, emtricitabine, alone or in combination with two protease inhibitors ritonavir and atazanavir (ERA) with and without morphine co-exposure. The effect of the protease inhibitor, lopinavir, alone or in combination with the protease inhibitor, abacavir, and the integrase inhibitor, raltegravir (LAR), with and without morphine co-exposure was also explored. Exposure with emtricitabine alone or ERA in HIV-infected astrocytes caused a significant decrease in viral replication and attenuated HIV-induced inflammatory molecules, while co-exposure with morphine negated the inhibitory effects of ERA, leading to increased viral replication and inflammatory molecules. Exposure with emtricitabine alone or in combination with morphine caused a significant disruption of mitochondrial membrane integrity. Genetic analysis revealed a significant increase in the expression of p62/SQSTM1 which correlated with an increase in the histone-modifying enzyme, ESCO2, after exposure with ERA alone or in combination with morphine. Furthermore, several histone-modifying enzymes such as CIITA, PRMT8, and HDAC10 were also increased with LAR exposure alone or in combination with morphine. Accumulation of p62/SQSTM1 is indicative of dysfunctional lysosomal fusion. Together with the loss of mitochondrial integrity and epigenetic changes, these effects may lead to enhanced viral titer and inflammatory molecules contributing to the neuropathology associated with HIV.

Mtu1-Mediated Thiouridine Formation of Mitochondrial tRNAs Is Required for Mitochondrial Translation and Is Involved in Reversible Infantile Liver Injury.

Reversible infantile liver failure (RILF) is a unique heritable liver disease characterized by acute liver failure followed by spontaneous recovery at an early stage of life. Genetic mutations in MTU1 have been identified in RILF patients. MTU1 is a mitochondrial enzyme that catalyzes the 2-thiolation of 5-taurinomethyl-2-thiouridine (τm5s2U) found in the anticodon of a subset of mitochondrial tRNAs (mt-tRNAs). Although the genetic basis of RILF is clear, the molecular mechanism that drives the pathogenesis remains elusive. We here generated liver-specific knockout of Mtu1 (Mtu1LKO) mice, which exhibited symptoms of liver injury characterized by hepatic inflammation and elevated levels of plasma lactate and AST. Mechanistically, Mtu1 deficiency resulted in a loss of 2-thiolation in mt-tRNAs, which led to a marked impairment of mitochondrial translation. Consequently, Mtu1LKO mice exhibited severe disruption of mitochondrial membrane integrity and a broad decrease in respiratory complex activities in the hepatocytes. Interestingly, mitochondrial dysfunction induced signaling pathways related to mitochondrial proliferation and the suppression of oxidative stress. The present study demonstrates that Mtu1-dependent 2-thiolation of mt-tRNA is indispensable for mitochondrial translation and that Mtu1 deficiency is a primary cause of RILF. In addition, Mtu1 deficiency is associated with multiple cytoprotective pathways that might prevent catastrophic liver failure and assist in the recovery from liver injury.

Zinc deficiency induces apoptosis via mitochondrial p53- and caspase-dependent pathways in human neuronal precursor cells

Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent increases in the pro-apoptotic mitochondrial protein BAX leading to a loss of mitochondrial membrane potential as demonstrated by a 25% decrease in JC-1 red:green fluorescence ratio. Disruption of mitochondrial membrane integrity was accompanied by efflux of the apoptosis inducing factor (AIF) from the mitochondria and translocation to the nucleus with a significant increase in reactive oxygen species (ROS) after 24h of zinc deficiency. Measurement of caspase cleavage, mRNA, and treatment with caspase inhibitors revealed the involvement of caspases 2, 3, 6, and 7 in zinc deficiency-mediated apoptosis. Down-stream targets of caspase activation, including the nuclear structure protein lamin and polyADP ribose polymerase (PARP), which participates in DNA repair, were also cleaved. Transfection with a dominant-negative p53 construct and use of the p53 inhibitor, pifithrin-μ, established that these alterations were largely dependent on p53. Together these data identify a cascade of events involving mitochondrial p53 as well as p53-dependent caspase-mediated mechanisms leading to apoptosis during zinc deficiency.

Curcumin supplementation improves mitochondrial and behavioral deficits in experimental model of chronic epilepsy

The present study was aimed to investigate the potential beneficial effect of curcumin, a polyphenol with pleiotropic properties, on mitochondrial dysfunctions, oxidative stress and cognitive deficits in a kindled model of epilepsy. Kindled epilepsy was induced in rats by administering a sub-convulsive dose of pentylenetetrazole (PTZ, 40mg/kg body weight) every alternate day for 30days. PTZ administered rats exhibited marked cognitive deficits assessed using active and passive avoidance tasks. This was accompanied by a significant decrease in NADH:cytochrome-c reductase (complex I) and cytochrome-c oxidase (complex IV) activities along with an increase in ROS, lipid peroxidation and protein carbonyls. The levels of glutathione also decreased in the cortex and hippocampus. Electron micrographs revealed disruption of mitochondrial membrane integrity with distorted cristae in PTZ treated animals. Histopathological examination showed pyknotic nuclei and cell loss in the hippocampus as well as in the cortex of PTZ treated animals. Curcumin administration at a dose of 100mg/kg, p.o. throughout the treatment paradigm was able to ameliorate cognitive deficits with no significant effect on seizure score. Curcumin was able to restore the activity of mitochondrial complexes. In addition, significant reduction in ROS generation, lipid peroxidation and protein carbonyls was observed in PTZ animals supplemented with curcumin. Moreover, glutathione levels were also restored in PTZ treated rats supplemented with curcumin. Curcumin protected mitochondria from seizure induced structural alterations. Further, the curcumin supplemented PTZ rats had normal cell morphology and reduced cell loss. These results suggest that curcumin supplementation has potential to prevent mitochondrial dysfunctions and oxidative stress with improved cognitive functions in a chronic model of epilepsy.

Disruption of mitochondrial membrane integrity induced by amyloid aggregates arising from variants of SOD1

Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disorder selectively affecting motor neurons; 90% of the total cases are sporadic, but 2% are associated with mutations in the gene coding for copper–zinc superoxide dismutase (SOD1). The causes of motor neuron death in ALS are poorly understood in general, but for SOD1-linked familial ALS (fALS), aberrant oligomerization of SOD1 mutant proteins has been strongly implicated. A growing body of evidence suggests that fALS-causing mutations destabilize the native structure of SOD1, leading to aberrant protein interactions for aggregation. In this work, we show that wild-type human SOD1 and two of its mutants (D101N, G72S) form amyloid like fibrils under destabilizing condition (in the presence of KSCN 0.2M and DTT 50mM) at 37°C, pH 7.4. The formation of the aggregates was monitored by their ability to enhance the fluorescence of Thioflavin T (ThT) and their morphology was assessed by transmission electron microscopy (TEM). Furthermore, interaction of SOD1 aggregates with mitochondrial membrane of rat brain, as an in vitro biological model, with the aim of gaining an insight into possible mechanism of cytotoxicity at the membrane level was verified. Release of mitochondrial enzyme, malate dehydrogenase (MDH), upon exposure to SOD1 aggregates demonstrates that these aggregates could affect membrane permeability.

SGN-CD33A: A Novel CD33-Directed Antibody-Drug Conjugate, Utilizing Pyrrolobenzodiazepine Dimers, Demonstrates Preclinical Antitumor Activity Against Multi-Drug Resistant Human AML

AbstractAbstract 3589Outcomes for patients with acute myeloid leukemia (AML) are poor, highlighting the need for novel treatment options. Most AML cells express the myeloid differentiation antigen CD33, making CD33-targeted therapy a potential treatment strategy. Gemtuzumab ozogamicin (GO), an anti-CD33 monoclonal antibody (mAb) conjugated to the cytotoxic agent calicheamicin, has recently been shown to improve survival in newly diagnosed patients with more favorable-risk AML but has insufficient activity in those with poor prognostic features as well as in relapsed disease.Here we report the preclinical testing of a novel CD33-directed antibody-drug conjugate, SGN-CD33A, consisting of a humanized anti-CD33 mAb with 2 engineered cysteine residues through which pyrrolobenzodiazepine (PBD) dimer drug moieties are conjugated via a maleimidocaproyl valine-alanine dipeptide linker. PBD dimers exert their biological activity by covalent binding and interstrand cross-linking of DNA. Fluorescence microscopy studies showed that SGN-CD33A is rapidly internalized and traffics to lysosomes within hours of binding to CD33-positive AML cell lines. Following uptake, SGN-CD33A induces DNA damage as measured by phosphorylation of histone 2AX, subsequently leading to G2-M cell cycle arrest, disruption of mitochondrial membrane integrity, increased caspase-3 activity, formation of cleaved poly ADP-ribose polymerase, DNA fragmentation and cell death.The anti-leukemic activity of SGN-CD33A was assessed in cytotoxicity assays against 12 AML cell lines and 18 primary AML patient samples of mixed cytogenetic origin (favorable, intermediate and unfavorable) and multidrug resistance (MDR) status. SGN-CD33A was highly active against all AML cell lines tested (mean IC50, 22 ng/ml), including 5 of 5 MDR-positive cell lines (mean IC50, 27 ng/mL). In contrast, GO was moderately active in 1 of 5 MDR-positive cell lines (IC50, 227 ng/mL) but inactive against the other 4 (IC50, >1000 ng/mL). SGN-CD33A was also active against 15 of 18 primary samples isolated from untreated AML patients at diagnosis (mean IC50 of responsive samples, 8 ng/mL) and was more potent than GO which was active in 10 of 18 AML samples (mean IC50 of responding samples, 27 ng/mL). The 3 AML specimens that were resistant to SGN-CD33A each had low or absent CD33 expression, as determined by flow cytometry. Cytogenetic abnormalities and MDR activity did not correlate with in vitro SGN-CD33A cytotoxicity.In vivo antitumor activity was evaluated in AML mouse xenograft models established with MDR-negative HL-60 and MDR-positive THP-1 and TF1-α cell lines. SGN-CD33A dosed once at 300 mcg/kg yielded durable complete regressions in THP-1 (Figure A) and TF1-α xenografts. In these models of drug-resistant AML, treatment with a single dose of 100 mcg/kg SGN-CD33A significantly delayed tumor growth compared to untreated and non-binding control ADC-treated mice (p<0.001), whereas GO was inactive even when dosed at 1000 mcg/kg. In the MDR-negative HL-60 model, a single dose of 30 mcg/kg of SGN-CD33A delayed tumor growth and 100 mcg/kg induced complete tumor regression (Figure B). In contrast, GO had minimal activity when dosed at 100 mcg/kg but did result in durable tumor regression when administered at a ten-fold higher dose (Figure B).Together, these data demonstrate that SGN-CD33A exhibits antitumor activity against a broad panel of primary AML samples and results in durable remissions in preclinical models of MDR-positive AML that are characteristically resistant to conventional chemotherapy and GO. CD33-directed delivery of PBD dimers may overcome multidrug resistance and may represent a new strategy for the treatment of patients with AML. Clinical trials are planned to further evaluate SGN-CD33A in AML. [Display omitted] Disclosures:Sutherland:Seattle Genetics, Inc.: Employment. Walter:Seattle Genetics, Inc.: Consultancy, Research Funding. Jeffrey:Seattle Genetics, Inc.: Employment. Burke:Seattle Genetics, Inc.: Employment. Yu:Seattle Genetics, Inc.: Employment. Stone:Seattle Genetics, Inc.: Employment. Ryan:Seattle Genetics, Inc.: Employment. Sussman:Seattle Genetics, Inc.: Employment. Zeng:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc.: Employment. Bernstein:Seattle Genetics, Inc.: Consultancy. Senter:Seattle Genetics, Inc.: Employment. Drachman:Seattle Genetics, Inc.: Employment. McEarchern:Seattle Genetics, Inc.: Employment.

Antileishmanial activity of cryptolepine analogues and apoptotic effects of 2,7-dibromocryptolepine against Leishmania donovani promastigotes

Cryptolepine (5-methyl-10H-indolo [3, 2-b] quinoline), an indoloquinoline alkaloid (1) isolated from a medicinal plant traditionally used in Western Africa for treatment of malaria, has been shown to possess broad spectrum biological activity in addition to its antiplasmodial effect. Here, the antileishmanial properties of 11 synthetic derivatives of cryptolepine against Leishmania donovani parasites have been evaluated for the first time. 2,7-Dibromocryptolepine (8; IC50 0.5 ± 0.1 μM) was found to be the most active analogue against the promastigote form of a classical L. donovani strain (AG83) in comparison to the natural alkaloid, cryptolepine (1; IC50 1.6 ± 0.1 μM). Further, 8 was found to substantially inhibit the intracellular amastigote forms of two clinical isolates, one of them being an SbV-resistant strain of L. donovani. Moreover, the toxicity of 8 against normal mouse peritoneal macrophage cells was markedly lower than that of 1 (IC50 values: 9.0 ± 1.2 and 1.1 ± 0.3 μM, respectively), indicating 8 to be a prospective “lead” towards novel antileishmanial therapy. This was supported by studies on the mechanism of cytotoxicity induced by 8 in L. donovani promastigotes (AG83), which revealed the cytoplasmic and nuclear features of metazoan apoptosis. Light microscopic observation demonstrated a gradual decline in the motility, cell volume, and survival of the treated parasites with increasing incubation time. Flow cytometric analysis of phosphatidylserine externalization and distribution of cells in different phases of cell cycle confirmed the presence of a substantial percentage of cells in early apoptotic stage. Disruption of mitochondrial membrane integrity in terms of depolarization of membrane potential, and finally degradation of chromosomal DNA into oligonucleosomal fragments - the hallmark event of apoptosis - characterized the mode of cell death in L. donovani promastigotes.

Apoptosis by dietary agents for prevention and treatment of prostate cancer

Accumulating data clearly indicate that induction of apoptosis is an important event for chemoprevention of cancer by naturally occurring dietary agents. In mammalian cells, apoptosis has been divided into two major pathways: the extrinsic pathway, activated by pro-apoptotic receptor signals at the cellular surface; and the intrinsic pathway, which involves the disruption of mitochondrial membrane integrity. This process is strictly controlled in response to integrity of pro-death signaling and plays critical roles in development, maintenance of homeostasis, and host defense in multicellular organisms. For chemoprevention studies, prostate cancer (PCa) represents an ideal disease due to its long latency, its high incidence, tumor marker availability, and identifiable preneoplastic lesions and risk groups. In this article, we highlight the studies of various apoptosis-inducing dietary compounds for prevention of PCa in vitro in cell culture, in preclinical studies in animals, and in human clinical trials.

Anticancer &amp;#945;-Helical Peptides and Structure / Function Relationships Underpinning Their Interactions with Tumour Cell Membranes

Cancer is a major cause of premature death and there is an urgent need for new anticancer agents with novel mechanisms of action. Here we review recent studies on a group of peptides that show much promise in this regard, exemplified by arthropod cecropins and amphibian magainins and aureins. These molecules are alpha-helical defence peptides, which show potent anticancer activity (alpha-ACPs) in addition to their established roles as antimicrobial factors and modulators of innate immune systems. Generally, alpha-ACPs exhibit selectivity for cancer and microbial cells primarily due to their elevated levels of negative membrane surface charge as compared to non-cancerous eukaryotic cells. The anticancer activity of alpha-ACPs normally occurs at micromolar levels but is not accompanied by significant levels of haemolysis or toxicity to other mammalian cells. Structure/function studies have established that architectural features of alpha-ACPs such as amphiphilicty levels and hydrophobic arc size are of major importance to the ability of these peptides to invade cancer cell membranes. In the vast majority of cases the mechanisms underlying such killing involves disruption of mitochondrial membrane integrity and/or that of the plasma membrane of the target tumour cells. Moreover, these mechanisms do not appear to proceed via receptor-mediated routes but are thought to be effected in most cases by the carpet/toroidal pore model and variants. Usually, these membrane interactions lead to loss of membrane integrity and cell death utilising apoptic and necrotic pathways. It is concluded that that alpha-ACPs are major contenders in the search for new anticancer drugs, underlined by the fact that a number of these peptides have been patented in this capacity.

FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity.

Survival signals elicited by cytokines include the activation of phosphatidylinositol 3-kinase (PI3K), which in turn promotes the activation of protein kinase B (PKB). Recently, PKB has been demonstrated to phosphorylate and inactivate forkhead transcription factor FKHR-L1, a potent inducer of apoptosis. To explore the mechanisms underlying the induction of apoptosis after cytokine withdrawal or FKHR-L1 activation, we used a cell line in which FKHR-L1 activity could be specifically induced. Both cytokine withdrawal and FKHR-L1 activation induced apoptosis, which was preceded by an upregulation in p27KIP1 and a concomitant decrease in cells entering the cell cycle. Induction of apoptosis by both cytokine withdrawal and activation of FKHR-L1 correlated with the disruption of mitochondrial membrane integrity and cytochrome c release. This was preceded by upregulation of the pro-apoptotic Bcl-2 family member Bim. Ectopic expression of an inhibitory mutant of FKHR-L1 substantially reduced the levels of apoptosis observed after cytokine withdrawal. Activation of PKB alone was sufficient to promote cell survival, as measured by maintenance of mitochondrial integrity and the resultant inhibition of effector caspases. Furthermore, hematopoietic stem cells isolated from Bim−/− mice exhibited reduced levels of apoptosis upon inhibition of PI3K/PKB signaling.These data demonstrate that activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal. Thus PI3K/PKB–mediated inhibition of this transcription factor likely provides an important mechanism by which survival factors act to prevent programmed cell death.

PK11195, a Ligand of the Mitochondrial Benzodiazepine Receptor, Facilitates the Induction of Apoptosis and Reverses Bcl-2-Mediated Cytoprotection

One critical step of the apoptotic process is the opening of the mitochondrial permeability transition (PT) pore leading to the disruption of mitochondrial membrane integrity and to the dissipation of the inner transmembrane proton gradient (ΔΨm). The mitochondrial PT pore is a polyprotein structure which is inhibited by the apoptosis-inhibitory oncoprotein Bcl-2 and which is closely associated with the mitochondrial benzodiazepine receptor (mBzR). Here we show that PK11195, a prototypic ligand of the 18-kDa mBzR, facilitates the induction of ΔΨmdisruption and subsequent apoptosis by a number of different agents,including agonists of the glucocorticoid receptor,chemotherapeutic agents (etoposide, doxorubicin),gamma irradiation, and the proapoptotic second messenger ceramide. Whereas PK11195 itself has no cytotoxic effect, it enhances apoptosis induction by these agents. This effect is not observed for benzodiazepine diazepam, whose binding site in the mBzR differs from PK11195. PK11195 partially reverses Bcl-2 mediated inhibition of apoptosis in two different cell lines. Thus, transfection-enforced Bcl-2 overexpression confers protection against glucocorticoids and chemotherapeutic agents, and this protection is largely reversed by the addition of PK11195. This effect is observed at the level of ΔΨmdissipation as well as at the level of nuclear apoptosis. To gain insights into the site of action of PK11195, we performed experiments on isolated organelles. PK11195 reverses the Bcl-2-mediated mitochondrial retention of apoptogenic factors which cause isolated nuclei to undergo apoptosis in a cell-free system. Mitochondria from control cells, but not mitochondria from Bcl-2-overexpressing cells, readily release such apoptogenic factors in response to atractyloside, a ligand of the adenine nucleotide translocator. However, control and Bcl-2-overexpressing mitochondria respond equally well to a combination of atractyloside and PK11195. Altogether, these findings indicate that PK11195 abolishes apoptosis inhibition by Bcl-2 via a direct effect on mitochondria. Moreover, they suggest a novel strategy for enhancing the susceptibility of cells to apoptosis induction and, concomitantly, for reversing Bcl-2-mediated cytoprotection.

Mitochondria in chemotherapy-induced apoptosis: a prospective novel target of cancer therapy (review).

Resistance to apoptosis is a frequent characteristic of cancer cells and participates both in the initial phase of carcinogenesis and in the development of chemotherapy resistance. Recently, it has become clear that a disruption in mitochondrial membrane function is a decisive event of the apoptotic process leading to the disposal of chemotherapy-treated cells. Opening of the mitochondrial megachannel (also called permeability transition pore) is at least in part responsible for the disruption of mitochondrial membrane integrity in apoptosis. The megachannel is regulated by numerous endogenous effectors including members of the Bcl-2/Bax family, the redox status of the cell, cytosolic Ca2+ levels, ceramide, and amphipathic peptides. Chemotherapeutic agents may induce opening of the megachannel by modulating some of these endogenous effectors. The disruption of mitochondrial membrane integrity involves a loss of metabolic functions and the liberation of intermembrane proteins into the cytosol. Such proteins, which normally are well secluded in mitochondria, include cytochrome c and AIF (apoptosis inducing factor), which both activate caspases and endonucleases upon release into the cytosol. Strategies for the development of chemotherapeutic agents acting on mitochondria are discussed.

Effect of phthlate esters on energy coupling and succinate oxidation in rat liver mitochondria

Isolated rat liver mitochondria were exposed to mono- and di- n-butyl phthalate (MBP and DBP) and mono- and di(2-ethylhexyl)phthalate (MEHP and DEHP) and examined for effects on mitochondrial energy-dependent processes, including oxidative phosphorylation and active K + uptake. Additional studies on the effects of these phthalate esters on succinate oxidation and on mitochondrial membrane integrity are also included. DBP and MEHP stimulated succinate state 4 respiration, impaired K +-valinomycin induced swelling with succinate, ascorbate, or ATP as the energy sources, and inhibited succinate state 3 respiration and succinate cytochrome c reductase activity. MEHP was found to act as a non-competitive inhibitor of succinate dehydrogenase activity, with an apparent K i = 2.4 × 10 −4 M. At concentrations which uncouple energy linked reactions, MEHP and DBP produced only slight energy-independent swelling and release of soluble proteins from isolated mitochondria. MBP caused only slight stimulation of state 4 respiration and impairment of K +-valinomycin induced swelling with each of the 3 energy sources, however, of the 4 phthalate esters, it produced the greatest energy-independent swelling and led to the greatest release of soluble mitochondrial proteins. DEHP had no apparent effect on any of these processes except for slight impairment of ATP-dependent K +-valinomycin induced swelling. It is concluded that phthalate ester toxicity in liver mitochondria is due to uncoupling of energy linked reactions and/or inhibition of succinate dehydrogenase activity. Uncoupling by MBP may involve disruption of mitochondrial membrane integrity, while uncoupling by DBP and MEHP is probably due to an increase in membrane permeability to H + and other small ions.