Produce Cell Death Research Articles

Pulmonary pathology of infection by SARS-CoV-2, what we have learnt through post-mortem studies and pathophysiological considerations

Background: Since the declaration of Coronavirus Disease 2019 (COVID-19) caused the by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a pandemic, millions of people throughout the world have contracted the disease and more than 500,000 have died. While this terrible disease has brought devastation to many countries, it has also generated the fastest growing amount of research output that science has seen during the last decades. Objectives: The objectives of this work were to review the pulmonary pathology from post-mortem studies and case reports of patients who succumbed to COVID-19, perform clinicopathologic correlation and hypothesize about pathogenetic mechanisms, and translate this understanding into better prevention, management and outcomes in the battle against COVID-19. Method: I performed a literature search through PubMed. Results: The main histopathologic findings in COVID-19 pneumonia are diffuse alveolar damage, organizing pneumonia, acute fibrinous and organizing pneumonia, interstitial pneumonia, endotheliitis, thromboembolism that correlates with respiratory failure and manifestations of thrombotic microangiopathy and hypercoagulable state. Immunohistochemical and transmission electron microscopic studies demonstrate viral proteins and viral structures within epithelial and endothelial cells. Reverse transcriptase polymerase chain reaction demonstrates the presence of viral RNA in nasopharynx and lung tissue post-mortem. Conclusions: SARS-CoV-2 is a respiratory virus that infects cells by binding to angiotensin-converting enzyme II (ACE2) receptors, produces cell death and ACE2 downregulation, which, in conjunction with dysregulated immune response, orchestrates a proinflammatory and prothrombic state that results in respiratory and multiple organ failure.

Praziquantel-Clays as Accelerated Release Systems to Enhance the Low Solubility of the Drug.

Praziquantel is an antiparasitic drug indicated for the treatment of the schistosomiasis disease. This drug has very low aqueous solubility, requiring high oral doses for its administration which gives rise to side effects, therapeutic noncompliance and the appearance of resistant forms of the parasite. Clay minerals, like sepiolite and montmorillonite, are innocuous, non-toxic, biocompatible and low-cost excipients. Additionally, clays have high adsorbent properties that allow them to encapsulate drugs in nanometric spaces present in the channels in the case of the sepiolite or between the layers in the case of the montmorillonite. The interactions between the drug and clay minerals are studied experimentally with the strategy for preparing interactions products in organic solvents (ethanol, acetonitrile and dichloromethane) so that the interaction will be more effective and will be enhanced the aqueous solubility of praziquantel. The results showed that in the interaction products, the drug interacted with both clay minerals, which produced the loss of the crystallinity of the drug demonstrated by different techniques. This led to a significant increase in the dissolution rate of the praziquantel in all the interaction products in the simulated gastrointestinal tract media, except for the praziquantel–montmorillonite product prepared in dichloromethane that presented a controlled release in acid medium. Moreover, in vitro cytotoxicity and cell cycle studies were performed in the interaction products prepared with ethanol. The interaction product with sepiolite was biocompatible with the HTC116 line cells, and it did not produce alterations in the cell cycle. However, interaction products with montmorillonite did not produce cell death, but they showed affectation and damage of cells in the cell cycle study at the highest concentration tested (20–100 µM). Therefore, the different organic solvents used are adequate for the improvement of the biopharmaceutical profile of praziquantel. Drug–clay interaction products, specifically with sepiolite, showed very promising results in which new accelerated oral release systems of the praziquantel were obtained.

A Method for the Evaluation of Site-Specific Nephrotoxic Injury in the Intact Rat Kidney.

In a previously published report we detailed an in situ method to quantify cell death in the renal cortex by perfusing the cell membrane impermeable fluorochrome, ethidium homodimer in situ. The objective of the present study was to use this in situ viability assay to examine cell death following the administration of nephrotoxic drugs known to produce cell death and/or injury in specific segments of the nephron. Male Sprague/Dawley rats were treated with the following nephrotoxicants: Gentamicin, amphotericin-B, and indomethacin. Results of the in situ viability assay indicated that gentamicin and amphotericin-B treatment caused cell death localized in the kidney cortex and medulla, respectively. The urinary biomarker kidney injury molecule—1 (Kim-1) showed significant increases in both gentamicin (20 fold increase) and amphotericin-B-treated (9.2 fold increase) animals. Urinary alpha glutathione-S-transferase (GST) showed significant increases for gentamicin (6.2 fold increase) only and mu GST for amphotericin-B-treated (19.1 fold increase) animals only. These results show that this in situ viability assay provides a sensitive method to identify cell death in different regions of the kidney. Furthermore, urinary alpha GST and mu GST are specific for proximal and distal tubule injury, respectively; urinary Kim-1 demonstrated greater sensitivity to both proximal and distal tubule injury.

A Novel Decalin-Based Bicyclic Scaffold for FKBP51-Selective Ligands.

Selective inhibition of FKBP51 has emerged as possible novel treatment for diseases like major depressive disorder, obesity, chronic pain, and certain cancers. The current FKBP51 inhibitors are rather large, flexible, and have to be further optimized. By using a structure-based rigidification strategy, we hereby report the design and synthesis of a novel promising bicyclic scaffold for FKBP51 ligands. The structure-activity analysis revealed the decalin scaffold as the best moiety for the selectivity-enabling subpocket of FBKP51. The resulting compounds retain high potency for FKBP51 and excellent selectivity over the close homologue FKBP52. With the cocrystal structure of an advanced ligand in this novel series, we show how the decalin locks the key selectivity-inducing cyclohexyl moiety of the ligand in a conformation typical for FKBP51-selective binding. The best compound 29 produces cell death in a HeLa-derived KB cell line, a cellular model of cervical adenocarcinoma, where FKBP51 is highly overexpressed. Our results show how FKBP51 inhibitors can be rigidified and extended while preserving FKBP51 selectivity. Such inhibitors might be novel tools in the treatment of human cancers with deregulated FKBP51.

Cytotoxic saponins and other natural products from flowering tops of Narthecium ossifragum L

For more than four centuries, the intake of Narthecium ossifragum has been associated with poisoning in domesticated animals. Saponins occurring in flowering tops of the plant are considered to cause kidney damage in calves. At present, there are more than 30 papers on the saponins of N. ossifragum in the literature, although the structures of these compounds have hitherto not been determined. Here, we identify the saponins of N. ossifragum as sarsasapogenin, sarsasapogenin-3-O-β-galactopyranoside, sarsasapogenin-3-O-(2′-O-β-glucopyranosyl-β-galactopyranoside) and sarsasapogenin-3-O-(2′-O-β-glucopyranosyl-3′-O-α-arabinopyranosyl-β-galactopyranoside). Moreover, six aromatic natural products were isolated and characterized from the methanolic extract from flowers of N. ossifragum. Five of these aromatic compounds, chrysoeriol 6-C-β-arabinofuranoside-8-C-β-glucopyranoside, chrysoeriol 6-C-β-arabinopyranosyl-8-C-β-glucopyranoside, chrysoeriol 6-C-β-xylopyranosyl-8-C-β-galactopyranoside, chrysoeriol 6-C-β-galactopyranosyl-8-C-β-glucopyranoside and chrysoeriol 6-C-β-glucopyranosyl-8-C-β-galactopyranoside are undescribed. All compounds were tested for cytotoxicity in mammalian cell lines derived from the heart, kidney, and haematological tissues. The saponins exhibited cytotoxicity in the micromolar range, with proportionally increasing cytotoxicity with increasing number of glycosyl substituents. The most potent compound was the main saponin sarsasapogenin-3-O-(2′-O-β-glucopyranosyl-3′-O-α-arabinopyranosyl-β-galactopyranoside), which produced cell death at concentrations below 3–4 μM in all three cell lines tested. This indicates that the saponins are the toxicants mainly responsible for kidney damage observed in cattle after ingestion of N. ossifragum. Our findings also pave the way for analysis of individual compounds isolated during the biopsies of intoxicated animals.

Cytotoxic activity of the seaweed compound fucosterol, alone and in combination with 5-fluorouracil, in colon cells using 2D and 3D culturing

ABSTRACTColorectal cancer (CRC) is one of the most frequently occurring carcinomas which require effective therapies. Fucosterol is a sterol present in marine brown seaweeds with several biological activities. However, the influence of fucosterol in CRC remains to be determined. Thus, the aim of this study was to examine the anticancer activity of fucosterol alone and in combination with 5-fluorouracil (5-Fu) on two human CRC cell lines (HCT116 and HT29) and compared with cytotoxicity in one normal colon fibroblast cell line (CCD-18co) in monolayer (2D). The effect of fucosterol alone or in combination with 5-Fu was further assessed using HT29 multicellular spheroids (3D). Data demonstrated that fucosterol alone or combined with 5-Fu decreased cell viability in HT29 cells in 2D cultures without inducing cytotoxic in normal colon cells. The combination, fucosterol, and 5-Fu, also inhibited cell proliferation, clonogenic potential and cell migration without producing cell death in 2D. In multicellular spheroids, the combination fucosterol plus 5-Fu at the same concentrations used in 2D was not effective demonstrating that under the tested conditions the 3D model was more resistant than the 2D model. Taken together, these findings suggest that fucosterol might be a promising alternative to enhance the cytotoxic and anti-invasive actions of 5-Fu in colon cancer cells without consequent major adverse effects in normal cells. Our results also reinforce the need to include more complex 3D culture models in the initial stages of drug screening.

DnaJ molecules as potential effectors in Meloidogyne arenaria. An unexplored group of proteins in plant parasitic nematodes

ABSTRACTPlant pathogenic organisms secrete proteins called effectors that recognize, infect and promote disease within host cells. Bacteria, like Pseudomona syringae, use effectors with DnaJ function to disrupt plant defenses. DnaJ proteins (also called Hsp40) are a group of co-chaperone molecules, which assist in the folding of proteins. Despite the described role of DnaJs as effectors in several groups of pathogens, this group of proteins has never been correlated with the infection process in plant parasitic nematodes. In this study, we analyze the importance of DnaJ for plant parasitic nematodes. To do that, we compare the number of DnaJ proteins in nematodes with different lifestyles. Then, we predict the secreted DnaJ proteins in order to detect effector candidates. We found that Meloidogyne species have more secreted DnaJs than the rest of the nematodes analyzed in the study. Particularly, M. arenaria possess the highest proportion of secreted DnaJ sequences in comparison to total DnaJ proteins. Furthermore, we found in this species at least five sequences with a putative nuclear localization signal, three of them with a serine rich region with an unknown function. Then, we chose one of these sequences (MG599854) to perform an expression analysis. We found that MG599854 is over-expressed from 3 days post inoculation onwards in tomato plants. Moreover, MG599854 seems to be enough to produce cell death in Nicotiana benthamiana under transient expression conditions. In concordance with our results, we propose that DnaJ proteins are a potential source of effector proteins in plant parasitic nematodes.

The PDE6 mutation in the rd10 retinal degeneration mouse model causes protein mislocalization and instability and promotes cell death through increased ion influx

The retinal degeneration model rd10 contains a missense mutation of the catalytic PDE6 β subunit, which hydrolyzes cGMP in response to light. This model produces cell death more slowly than others caused by PDE6 loss of function, making it of particular interest for studying potential therapeutics. We used morphology, biochemistry, and single-cell physiology to examine the mechanism of rd10 degeneration. Our results show that the mutation produces no alteration of Pde6b RNA but does dramatically decrease maximal and basal PDE6 activity, apparently caused by a decrease in protein stability and transport. The enzymatic properties of the remaining mutant PDE6 appear to be nearly normal. We demonstrate that an increase in free cGMP, which would result from decreased PDE6 activity and serve to increase opening of the cGMP-gated channels and calcium influx, is an underlying cause of cell death: degeneration of rd10/Cngb1-/- double mutants is slower than the parent rd10 line. Paradoxically, degeneration in rd10/Cngb1-/- is also slower than in Cngb1-/- This rescue is correlated with a lowering of cGMP content in Cngb1-/- retinas and suggests that it may be caused by mislocalization of active PDE6. Single-cell recordings from rd10 rods show that the rates of rise and decay of the response are significantly slower; simulations indicate that these changes are primarily the result of the decrease in PDE6 concentration and rod collecting area. Together, these results provide insights into the complex mechanisms that underlie rd10-mediated retinal degeneration and a cautionary note for analysis of therapeutic interventions.

Cytotoxic and Antibacterial Assessment of Stem-Barks of <i>Feretia apodanthera</i> and <i>Erythrophleum ivorense</i>; Two West African Medicinal and Socio-Economic Trees

To assess the antibacterial and cytotoxic properties of stem-barks of Feretia apodanthera and Erythrophleum ivorense extracts from powdered stem-barks of Feretia apodanthera and Erythrophleum ivorense were prepared following standard techniques of marceration, filtration and evaporation. Antibacterial activity was assayed against five pathogenic bacteria strains by the well-diffusion and broth microdilution methods. Cytotoxicity was measured by acute toxicity test on female albino rats and confirmed by cell viability assay using 3T3 cell lines. Phytochemical analysis was performed following standard techniques. The aqueous/alkaloid extracts of Feretia apodanthera and the ethanol extract of Erythrophleum ivorense were active against the five pathogenic bacteria strains tested (diameter zone of inhibition (DZI) ranging from 5.1 to 17.8mm). The Feretia apodanthera extracts were the most active against Staphylococcus aureus (DZI 17.1-17.8mm). The MIC and MBC of the extracts of both plants ranged from 0.094mg/ml to 48mg/ml and 0.047mg/ml to 48mg/ml respectively. Extracts of Feretia. apodanthera at 5000mg/Kg had no effect on the behavioural properties of rats and no death was observed. Incubation with 3T3 cell lines did not produce any cell toxicity up to 20mM and 5mM respectively for the aqueous extract and the alkaloid fraction. Incubation with higher concentrations produced cell death with IC50 of 39.41 ± 0.95mM and 38.45 ± 1.64mM respectively. Phytochemical analysis revealed the presence of various constituents. The results show for the first time that stem-bark extracts of F. apodanthera and E. ivorense possess antibacterial activities against common human pathogenic bacteria and the low/lack of toxicity as demonstrated with the F. apodanthera extracts justify and confirm their safe ethnomedical uses.

Bifunctional Platinum(II) Complexes with Bisphosphonates Substituted Diamine Derivatives: Synthesis and In vitro Cytotoxicity

A series of N,N'-dibisphosphonate-containing 1,3-propanediamine derivatives (L1 - L6) and their corresponding dichloridoplatinum(II) complexes (1 - 6) have been synthesized and characterized by elemental analysis, 1 H-NMR, 13 C-NMR, 31 P-NMR and HR-MS spectra. The in vitro antitumor activities of compounds L1 - L6 and 1 - 6 were tested by WST-8 assay with Cell Counting Kit-8, indicating that platinum-based complexes 1 - 6 showed higher cytotoxicity than corresponding ligands L1 - L6 against A549 and MG-63, especially complex 2 which displayed comparable cytotoxicity to those of cisplatin and zoledronate after 48 h incubation. In addition, complexes 1 - 6 were more active in vitro on osteosarcoma cell line MG-63 than normal osteoblast cell line hFOB 1.19. The structure-activity relationship has been summarized based on the in vitro cytotoxicity of three series of platinum complexes from this and our previous studies. The in vitro bone affinity of platinum complexes was also tested by hydroxyapatite (HAP) chromatography in terms of capacity factor K'. Besides, in this paper, representative complex 2, which has been proved to be a promising antitumor agent with high cytotoxicity and bone HAP binding property, was investigated for its mechanism of action producing cell death against MG-63.

RIPK3 promotes adenovirus type 5 activity

Oncolytic adenoviral mutants infect human malignant cells and replicate selectively within them. This induces direct cytotoxicity that can also trigger profound innate and adaptive immune responses. However, the mechanism by which adenoviruses produce cell death remains uncertain. We previously suggested that type 5 adenoviruses, including the E1A CR2 deletion mutant dl922-947, might induce a novel form of programmed death resembling necroptosis. Here we have investigated the roles of core necrosis proteins RIPK1, RIPK3 and MLKL in the cytotoxicity of dl922-947 and other adenovirus serotypes. By electron microscopy, we show that dl922-947 induces similar necrotic morphology as TSZ treatment (TNF-α, Smac mimetic, zVAD.fmk). However, dl922-947-mediated death is independent of TNF-α signalling, does not require RIPK1 and does not rely upon the presence of MLKL. However, inhibition of caspases, specifically caspase-8, induces necroptosis that is RIPK3 dependent and significantly enhances dl922-947 cytotoxicity. Moreover, using CRISPR/Cas9 gene editing, we demonstrate that the increase in cytotoxicity seen upon caspase inhibition is also MLKL dependent. Even in the absence of caspase inhibition, RIPK3 expression promotes dl922-947 and wild-type adenovirus type 5 efficacy both in vitro and in vivo. Together, these results suggest that adenovirus induces a form of programmed necrosis that differs from classical TSZ necroptosis.

Induction of cell death by magnetic particles in response to a gradient magnetic field inside a uniform magnetic field

A new instrument based on a magnetic force produced by an alternating magnetic field gradient, which is obtained through Maxwell coils, inside a constant field magnet has been designed and used to produce cell death. We have determined the interaction of microparticles and cells under different conditions such as incubation time with microparticles, particle size, magnetic field exposition time, and different current waveforms at different frequencies to produce a magnetic field gradient. We determined that the highest rate of cell death occurs at a frequency of 1 Hz with a square waveform and 1 h of irradiation. This method could be of great interest to remove cancer cells due mainly to the alterations in stiffness observed in the membranes of the tumor cells. Cancer cells can be eliminated in response to the forces caused by the movement of magnetic nanoparticles of the appropriate size under the application of a specific magnetic field.

Novel Gold(I) Thiolate Derivatives Synergistic with 5-Fluorouracil as Potential Selective Anticancer Agents in Colon Cancer.

New gold(I) thiolate complexes have been synthesized and characterized, and their physicochemical properties and anticancer activity have been tested. The coordination of PTA derivatives provides optimal hydrophilicity/lipophilicity properties to the complexes, which present high solution stability. Moreover, the complexes show a high anticancer activity against Caco-2 cells, comparable to that of auranofin, and a very low cytotoxic activity against enterocyte-like differentiated cells. Their activity has been shown to produce cell death by apoptosis and arrest of the cell cycle because of interaction with the reductase enzymes and consequent reactive oxygen species production. Some of these new complexes are also able to decrease the necessary dose of 5-fluorouracil, a drug used for the treatment of colon cancer, by a synergistic mechanism.

Discovery and Characterization of Novel Nonsubstrate and Substrate NAMPT Inhibitors.

Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell death when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236-45. ©2017 AACR.

Early Golgi Abnormalities and Neurodegeneration upon Loss of Presynaptic Proteins Munc18-1, Syntaxin-1, or SNAP-25.

The loss of presynaptic proteins Munc18-1, syntaxin-1, or SNAP-25 is known to produce cell death, but the underlying features have not been compared experimentally. Here, we investigated these features in cultured mouse CNS and DRG neurons. Side-by-side comparisons confirmed massive cell death, before synaptogenesis, within 1-4 DIV upon loss of t-SNAREs (syntaxin-1, SNAP-25) or Munc18-1, but not v-SNAREs (synaptobrevins/VAMP1/2/3 using tetanus neurotoxin (TeNT), also in TI-VAMP/VAMP7 knock-out (KO) neurons). A condensed cis-Golgi was the first abnormality observed upon Munc18-1 or SNAP-25 loss within 3 DIV. This phenotype was distinct from the Golgi fragmentation observed in apoptosis. Cell death was too rapid after syntaxin-1 loss to study Golgi abnormalities. Syntaxin-1 and Munc18-1 depend on each other for normal cellular levels. We observed that endogenous syntaxin-1 accumulates at the Golgi of Munc18-1 KO neurons. However, expression of a non-neuronal Munc18 isoform that does not bind syntaxin-1, Munc18-3, in Munc18-1 KO neurons prevented cell death and restored normal cis-Golgi morphology, but not synaptic transmission or syntaxin-1 targeting. Finally, we observed that DRG neurons are the only Munc18-1 KO neurons that do not degenerate in vivo or in vitro In these neurons, cis-Golgi abnormalities were less severe, with no changes in Golgi shape. Together, these data demonstrate that cell death upon Munc18-1, syntaxin-1, or SNAP-25 loss occurs via a degenerative pathway unrelated to the known synapse function of these proteins and involving early cis-Golgi abnormalities, distinct from apoptosis.SIGNIFICANCE STATEMENT This study provides new insights in a neurodegeneration pathway triggered by the absence of specific proteins involved in synaptic transmission (syntaxin-1, Munc18-1, SNAP-25), whereas other proteins involved in the same molecular process (synaptobrevins, Munc13-1/2) do not cause degeneration. Massive cell death occurs in cultured neurons upon depleting syntaxin-1, Munc18-1, and/or SNAP-25, well before synapse formation. This study characterizes several relevant cellular phenotypes, especially early cis-Golgi abnormalities, distinct from abnormalities observed during apoptosis, and rules out several other phenotypes as causal (defects in syntaxin-1 targeting and synaptic transmission). As proteins, such as syntaxin-1, Munc18-1, or SNAP-25, modulate α-synuclein neuropathy and/or are dysregulated in Alzheimer's disease, understanding this type of neurodegeneration may provide new links between synaptic defects and neurodegeneration in humans.

Comparison of Cytotoxic Activity in Leukemic Lineages Reveals Important Features of β-Hairpin Antimicrobial Peptides.

Several reports described different modes of cell death triggered by antimicrobial peptides (AMPs) due to direct effects on membrane disruption, and more recently by apoptosis and necrosis-like patterns. Cytotoxic curves of four β-hairpin AMPs (gomesin, protegrin, tachyplesin, and polyphemusin) were obtained from several human leukemic lineages and normal monocytes and Two cell lines were then selected based on their cytotoxic sensitivity. One was sensitive to AMPs (K562) and the other resistant (KG-1) and their effect compared between these lineages. Thus, these lineages were chosen to further investigate biological features related with their cytotoxicities to AMPs. Stimulation with AMPs produced cell death, with activation of caspase-3, in K562 lineage. Increase on the fluidity of plasmatic membrane by reducing cholesterol potentiated cytotoxicity of AMPs in both lineages. Quantification of internal and external gomesin binding to the cellular membrane of both K562 and KG-1 cells showed that more peptide is accumulated inside of K562 cells. Additionally, evaluation of multi-drug resistant pumps activity showed that KG-1 has more activity than K562 lineage. A comparison of intrinsic gene patterns showed great differences between K562 and KG-1, but stimulation with gomesin promoted few changes in gene expression patterns. Differences in internalization process through the plasma membrane, multidrug resistance pumps activity, and gene expression pattern are important features to AMPs regulated cell death. J. Cell. Biochem. 118: 1764-1773, 2017. © 2016 Wiley Periodicals, Inc.

Selective photothermal inactivation of cells labeled with near-infrared croconaine dye.

Photothermal inactivation of cells caused by laser heating of a near-infrared croconaine dye is more effective when the dye is located inside the cell. The cell inactivation is spatially confined - laser irradiation of a mixed population of two different cell lines produces selective inactivation of the cells labeled with croconaine dye and does not harm adjacent unlabeled cells.

Determinants of Anti-Cancer Effect of Mitochondrial Electron Transport Chain Inhibitors: Bioenergetic Profile and Metabolic Flexibility of Cancer Cells.

Recent evidence highlights that energy requirements of cancer cells vary greatly from normal cells and they exhibit different metabolic phenotypes with variable participation of both glycolysis and oxidative phosphorylation (OXPHOS). Interestingly, mitochondrial electron transport chain (ETC) has been identified as an essential component in bioenergetics, biosynthesis and redox control during proliferation and metastasis of cancer cells. This dependence converts ETC of cancer cells in a promising target to design small molecules with anti-cancer actions. Several small molecules have been described as ETC inhibitors with different consequences on mitochondrial bioenergetics, viability and proliferation of cancer cells, when the substrate availability is controlled to favor either the glycolytic or OXPHOS pathway. These ETC inhibitors can be grouped as 1) inhibitors of a respiratory complex (e.g. rotenoids, vanilloids, alkaloids, biguanides and polyphenols), 2) inhibitors of several respiratory complexes (e.g. capsaicin, ME-344 and epigallocatechin-3 gallate) and 3) inhibitors of ETC activity (e.g. elesclomol and VLX600). Although pharmacological ETC inhibition may produce cell death and a decrease of proliferation of cancer cells, factors such as degree of inhibition of ETC activity by small molecules, bioenergetic profile and metabolic flexibility of different cancer types or subpopulations of cells in a particular cancer type, can affect the impact of the anti-cancer actions. Particularly interesting are the adaptive mechanisms induced by ETC inhibition, such as induction of glutamine-dependent reductive carboxylation, which may offer a strategy to sensitize cancer cells to inhibitors of glutamine metabolism.

Abstract 1151: IL-8, VEGF and IGF-1 play the important role in ARHI mediated-tumor dormancy in ovarian cancer

Abstract Ovarian cancer is the most lethal gynecologic malignancy. One of the most important factors contributing to poor outcomes is the ability of drug resistant ovarian cancer cells to remain dormant for years after treatment, only to grow progressively and kill their host. Judging from sites of recurrence, most dormant ovarian cancer cells are found on the surface of the peritoneal cavity, often in small, poorly vascularized collagenous scars observed at “second look” operations. Previously, we reported that more than 80% of persistent, dormant drug resistant ovarian cancers express ARHI and are undergoing autophagy. Autophagy can protect or kill tumor cells depending upon the context. Our group has found that autophagy and tumor dormancy can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), which is downregulated in 60% of ovarian cancers. Re-expression of ARHI in cell culture produces cell death within 72 hours, whereas re-expression of ARHI in xenografts produces cell growth arrest and tumor dormancy. When ARHI levels are reduced after 6 weeks of induction, xenografts grow promptly. Our current experiments were designed to determine whether survival of autophagic cells in vivo requires permissive levels of VEGF, IL-8 and IGF-1 in the tumor microenvironment. In cell culture, co-incubation of VEGF, IGF-1 and IL-8 with SKOv3-ARHI cells, increased AKT activity, reduced ARHI-mediated autophagy formation and decreased autophagic cell death. SKOv3-ARHI cells grown subcutaneously in nude mice exhibited higher levels of AKT activity and lower levels of autophagy than cells grown in cell culture. When cytoplasmic and nuclear fractions were obtained from xenografts, the nuclear fractions showed increased phospho-AKT, and decreased levels of a transcription factor EB (TFEB) and low mRNA expression of LC3, suggesting survival factors in tumor environment may stimulate AKT, downregulate expression of TFEB and inhibit formation of autophagy. Treatment with anti-VEGF, anti-IL-8 and anti-IGF-1 antibodies, individually and in combination, inhibited survival factors-mediated AKT activation and slowed the outgrowth of dormant autophagic ovarian cancer cells. Treatment of dormant xenografts with the same monoclonal antibodies against VEGF, IL-8 and IGFR not only delayed outgrowth when DOX is withdrawn, but cured a fraction of mice. Re-expression of ARHI reduced expression of IGF-1 receptor, but not VEGF and IL8 receptors. Treatment ovarian cancer cells with VEGF, IL-8 and IGF-1 reduced ARHI's ability to induce autophagy and to inhibit AKT. These preclinical studies support that the possibility that treatment with a combination of anti-VEGF, anti-IL8 and anti-IGF-1 antibodies could prevent outgrowth of dormant cells in patients with ovarian cancer. Furthermore, these results suggest that survival factors are required to limit autophagic death. Citation Format: Zhen Lu, Aaron Orozco, Weiqun Mao, Yan Wang, Margie Nicole Sutton, Hailing Yang, Douglas A. Levine, Robert C. Bast Jr.. IL-8, VEGF and IGF-1 play the important role in ARHI mediated-tumor dormancy in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1151.

PGA1-induced apoptosis involves specific activation of H-Ras and N-Ras in cellular endomembranes.

The cyclopentenone prostaglandin A1 (PGA1) is an inducer of cell death in cancer cells. However, the mechanism that initiates this cytotoxic response remains elusive. Here we report that PGA1 triggers apoptosis by a process that entails the specific activation of H- and N-Ras isoforms, leading to caspase activation. Cells without H- and N-Ras did not undergo apoptosis upon PGA1 treatment; in these cells, the cellular demise was rescued by overexpression of either H-Ras or N-Ras. Consistently, the mutant H-Ras-C118S, defective for binding PGA1, did not produce cell death. Molecular analysis revealed a key role for the RAF-MEK-ERK signaling pathway in the apoptotic process through the induction of calpain activity and caspase-12 cleavage. We propose that PGA1 evokes a specific physiological cell death program, through H- and N-Ras, but not K-Ras, activation at endomembranes. Our results highlight a novel mechanism that may be of potential interest for tumor treatment.