Pro-apoptotic Bcl-2 Family Proteins Research Articles

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins.

Down-regulation of BNIP3 by olomoucine, a CDK inhibitor, reduces LPS- and NO-induced cell death in BV2 microglial cells

Proinflammatory responses eliciting the microglial production of cytokines and nitric oxide (NO) have been reported to play a crucial role in the acute and chronic pathogenic effects of neurodegeneration. Chemical inhibitors of cyclin-dependent kinases (CDKs) may prevent the progression of neurodegeneration by both limiting cell proliferation and reducing cell death. However, the mechanism underlying the protective effect of CDK inhibitors on microglia remains unexplored. In this study, we found that olomoucine, a CDK inhibitor, alleviated lipopolysaccharide (LPS)-induced BV2 microglial cell death by reducing the generation of NO and inhibiting the gene expression of proinflammatory cytokines. In addition, olomoucine reduced inducible NO synthase promoter activity and alleviated NF-κB- and E2F-mediated transcriptional activation. NO-induced cell death involved mitochondrial disruptions such as cytochrome c release and loss of mitochondrial membrane potential, and pretreatment with olomoucine prior to NO exposure reduced these disruptions. Microarray analysis revealed that olomoucine treatment induced prominent down-regulation of Bcl2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a pro-apoptotic Bcl-2 family protein that is involved in mitochondrial disruption. As BNIP3 knock-down significantly increased the viability of LPS- and NO-treated BV2 cells, we conclude that olomoucine may protect cells by limiting proinflammatory responses, thereby reducing NO generation. Simultaneously, down-regulation of BNIP3 prevents NO stimulation from inducing mitochondrial disruption.

DNA damaging agent-induced apoptosis is regulated by MCL-1 phosphorylation and degradation mediated by the Noxa/MCL-1/CDK2 complex.

Noxa, a BH3-only pro-apoptotic BCL-2 family protein, causes apoptosis by specifically interacting with the anti-apoptotic protein MCL-1 to induce its proteasome-mediated degradation. We show here that the DNA damaging agents cisplatin and etoposide upregulate Noxa expression, which is required for the phosphorylation of MCL-1 at Ser64/Thr70 sites, proteasome-dependent degradation, and apoptosis. Noxa-induced MCL-1 phosphorylation at these sites occurs at the mitochondria and is primarily regulated by CDK2. MCL-1 and CDK2 form a stable complex and Noxa binds to this complex to facilitate the phosphorylation of MCL-1. When Ser64 and Thr70 of MCL-1 are substituted with alanine, the mutated MCL-1 is neither phosphorylated nor ubiquitinated, and becomes more stable than the wild-type protein. As a consequence, this mutant can inhibit apoptosis induced by Noxa overexpression or cisplatin treatment. These results indicate that Noxa-mediated MCL-1 phosphorylation followed by MCL-1 degradation is critical for apoptosis induced by DNA damaging agents through regulation of the Noxa/MCL-1/CDK2 complex.

Helix B Surface Peptide Protects Cardiomyocytes Against Hypoxia/Reoxygenation-induced Apoptosis Through Mitochondrial Pathways.

Helix B surface peptide (HBSP), a newly developed tissue-protective erythropoietin derivative, has beneficial effects on myocardial ischemia. This study aimed to investigate the cardio-protective effects of HBSP against hypoxia/reoxygenation (H/R) injury and its possible mechanism. A rat-derived cardiomyocyte cell line (H9C2 cells) were established and pretreated with HBSP. The pretreated primary cultures were subjected to H/R and monitored for cell viability using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Intracellular reactive oxygen species (ROS) levels, apoptosis, and mitochondrial membrane potential (ΔΨm) were detected by flow cytometry. The expression of cytochrome C and Bcl-2 family proteins, as well as the activities of caspases 3 and 9 were determined by Western blot analysis and a colorimetric method, respectively. HBSP reduced apoptotic cells in cardiomyocytes subjected to H/R. In HBSP-treated cardiomyocytes, the H/R-induced mitochondrial ROS production, ΔΨm collapse, and cytochrome C release from mitochondria to the cytosol significantly decreased. Moreover, HBSP inhibited the activation of caspases 9 and 3, as well as the alteration of Bcl-2 family proteins, which were induced by H/R. These results indicated that HBSP has protective effects against H/R-induced apoptosis by regulating the mitochondrial pathway. This mechanism involves inhibiting mitochondrial ROS generation, inhibiting caspase-3 activity, reducing ΔΨm collapse, reducing cytochrome release, and balancing anti and proapoptotic Bcl-2 family proteins.

Bik Mediates Caspase-Dependent Cleavage of Viral Proteins to Promote Influenza A Virus Infection.

Influenza virus induces apoptosis in infected cells to promote viral replication by manipulating the host cell death signaling pathway. Although some Bcl-2 family proteins play a role in the replication of influenza A virus (IAV), the role of cell death pathways in the viral replication cycle is unclear. We investigated whether deficiency of the proapoptotic Bcl-2 family protein, Bik, plays a role in IAV replication. IAV replication was attenuated in mouse airway epithelial cells (MAECs) from bik(-/-) compared with bik(+/+) mice, as indicated by reduced viral titers. Bik(-/-) MAECs showed more stable transepithelial resistance after infection than did bik(+/+) MAECs, were less sensitive to infection-induced cell death, and released fewer copies of viral RNA. Similar results were obtained when Bik expression was suppressed in human airway epithelial cells (HAECs). Bik(+/+) mice lost weight drastically and died within 8 days of infection, whereas 75% of bik(-/-) mice survived infection for 14 days and were 10-fold less likely to die from infection compared with bik(+/+) mice. IAV infection activated caspase 3 in bik(+/+) but not in bik(-/-) MAECs. Cleavage of viral nucleoprotein and M2 proteins were inhibited in bik(-/-) MAECs and when caspase activation was inhibited in HAECs. Furthermore, Bik deficiency impaired cytoplasmic export of viral ribonucleoprotein. These studies suggest a link between Bik-mediated caspase activation and cleavage of viral proteins. Thus, inhibition of proapoptotic host factors such as Bik and downstream mediators of cell death may represent a novel approach to influenza treatment.

Differential apoptosis sensitivity of human effector CD8+ T cells derived from central versus effector memory subsets in response to IL-2 withdrawal

Abstract CD8+ central memory (CM) and effector memory (EM) T cell subsets exhibit well-established differences in proliferative and protective capacity after infectious challenge. However, their relative sensitivity to apoptosis has been largely overlooked, despite the importance of programmed cell death in regulating effector T cell homeostasis. Here we demonstrate that primary human effector T cells derived from the CD8+ EM subset exhibit significantly higher sensitivity to cytokine withdrawal induced cell death (CWID), a critical intrinsic apoptosis program responsible for culling cells once an infection is cleared and interleukin-2 (IL-2) levels diminish. Interestingly, we found no differences in the expression of IL-2 or IL-2 receptor components in cells originating from either subset. Relative to CM-derived effectors, however, EM-derived T cells display more mitochondrial instability and greater basal caspase activation. Indeed, we found that heightened CWID sensitivity in EM-derived effectors is linked to higher expression of the pro-apoptotic Bcl-2 family protein BIM, both at steady state and with de novo induction following withdrawal of exogenous IL-2. These data point to “imprinted” differences in BIM protein regulation preserved between CD8+ CM and EM progeny that govern their relative sensitivity to CWID. These findings offer new insight into why CM-derived T cells display superior effector cell expansion and more persistent memory responses in vivo relative to EM-derived T cells, based on differential apoptosis sensitivity.

Berberine induces mitochondrial apoptosis of EBV-transformed Bcells through p53-mediated regulation of XAF1 and GADD45α.

Berberine exhibits antiproliferative or cytotoxic effects against various cancers. ROS and wild-type p53 play a critical role in berberine-induced cytotoxic effects. In this study, we investigated the correlation between XAF1 and functional p53 in EBV-transformed Bcells or cancerous Bcells after treatment with berberine. Berberine decreased cell viability and induced apoptosis through a mitochondria-dependent pathway in EBV-transformed Bcells and cancerous Bcells, but not in normal peripheral blood mononuclear cells. Activated p53 and its downstream targets XAF1 and GADD45α interacted with PUMA, Bax, and Bim in mitochondria after treatment with berberine. Blocking phosphorylation of p38/JNK MAPK and treatment with PFT-α, a selective p53 inhibitor, effectively prevented apoptosis and the upregulation of phosphorylated p53, XAF1, and GADD45α. NAC, a ROS scavenger, also suppressed berberine-induced mitochondria disruption and the whole apoptotic process via restoration of p53-related proteins and proapoptotic Bcl-2 family proteins. Taken together, our results suggest that ROS generation might be a predisposing event in berberine-induced mitochondrial apoptosis in EBV-transformed Bcells through the upregulation of XAF1 and GADD45α expression by MAPK and functional p53.

VDAC2-specific cellular functions and the underlying structure

Voltage Dependent Anion-selective Channel 2 (VDAC2) contributes to oxidative metabolism by sharing a role in solute transport across the outer mitochondrial membrane (OMM) with other isoforms of the VDAC family, VDAC1 and VDAC3. Recent studies revealed that VDAC2 also has a distinctive role in mediating sarcoplasmic reticulum to mitochondria local Ca2+ transport at least in cardiomyocytes, which is unlikely to be explained simply by the expression level of VDAC2. Furthermore, a strictly isoform-dependent VDAC2 function was revealed in the mitochondrial import and OMM-permeabilizing function of pro-apoptotic Bcl-2 family proteins, primarily Bak in many cell types. In addition, emerging evidence indicates a variety of other isoform-specific engagements for VDAC2. Since VDAC isoforms display 75% sequence similarity, the distinctive structure underlying VDAC2-specific functions is an intriguing problem. In this paper we summarize studies of VDAC2 structure and functions, which suggest a fundamental and exclusive role for VDAC2 in health and disease. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia.

Mutations in the superoxide dismutase 1 (SOD1) gene contribute to motoneuron degeneration and are evident in 20% of familial amyotrophic lateral sclerosis cases. Mutant SOD1 induces microglial activation through a stimulation of Toll-like receptors 2 and 4 (TLR2 and TLR4). In the present study, we identified the proapoptotic Bcl-2 family protein Bid as a positive regulator of mutant SOD1-induced TLR-nuclear factor-κB (NF-κB) signaling in microglia. bid-deficient primary mouse microglia showed reduced NF-κB signaling in response to TLR4 activation or exposure to conditioned medium derived from SOD1 (G93A) expressing NSC-34 cells. Attenuation of NF-κB signaling in bid-deficient microglia was associated with lower levels of phosphorylated IKKα/β and p65, with a delayed degradation of IκBα and enhanced degradation of Peli1. Upstream of IKK, we found that Bid interacted with, and promoted, the K63-linked polyubiquitination of the E3 ubiquitin ligase tumor necrosis factor receptor associated factor 6 (TRAF6) in microglia. Our study suggests a key role for Bid in the regulation of TLR4-NF-κB proinflammatory signaling during mutant SOD1-induced disease pathology. Bid promotes TLR4-NF-κB signaling by interacting with TRAF6 and promoting TRAF6 K63-linked polyubiquitination in microglia.

Alzheimer’s disease via enhanced calcium signaling caused by the decrease of endoplasmic reticulum–mitochondrial distance

It has long been recognized that Ca2+ dysregulation is relevant to the initiation of Alzheimer’s disease (AD), and most recent works have suggested that increased cross-talk between endoplasmic reticulum (ER) and mitochondria plays an important role in the pathogenesis of the disease. However, the detailed mechanism involved has not been fully elucidated. Owing to its importance in the regulation of Ca2+ signaling, ER–mitochondrial distance in the neurons is tightly controlled in the physiological conditions. When the distance is decreased, Ca2+ overload occurs both in the cytosol and mitochondria. The cytosolic Ca2+ overload can (1) hyperactivate Ca2+-dependent enzymes, which in turn regulate activities of pro-apoptotic BCL-2 family proteins, causing mitochondrial outer membrane permeabilization and thereby resulting in the release of cytochrome c to activate caspase-3; (2) indirectly activate caspase-3 through the activation of caspase-12; and (3) promote the production and aggregation of β-amyloid. The three pathways eventually trigger neuronal apoptotic cell death. The mitochondrial Ca2+ overload can lead to increased generation of reactive oxygen species, inducing the opening of the mitochondrial permeability transition pore and ultimately causing neuronal apoptotic and necrotic cell death. The resultant death of neurons which are responsible for memory and cognition would contribute to the pathogenesis of AD. Therefore, we propose that the reduction in the distance between ER and mitochondria may be implicated in AD pathology by enhanced Ca2+ signaling, which provides a more complete picture of the Ca2+ hypothesis of AD.

Cdk2 phosphorylation of Bcl-xL after stress converts it to a pro-apoptotic protein mimicking Bax/Bak.

Apoptosis is a regulated form of cell death that proceeds by defined biochemical pathways. Most apoptosis is controlled by interactions between pro-survival and pro-apoptotic Bcl-2 family proteins in which death is often the consequence of permeabilization of the mitochondrial outer membrane. Many drugs affect this equilibrium to favor apoptosis but this process is not completely understood. We show that the chemotherapeutic drug cisplatin initiates an apoptotic pathway by phosphorylation of a pro-survival Bcl-2 family member, Bcl-xL, by cyclin-dependent kinase 2. The phosphorylation occurred at a previously unreported site and its biologic significance was demonstrated by a phosphomimetic modification of Bcl-xL that was able to induce apoptosis without addition of cisplatin. The mechanism of cell death induction was similar to that initiated by pro-apoptotic Bcl-2 family proteins, that is, phosphorylated Bcl-xL translocated to the mitochondrial membrane, and formed pores in the membrane. This initiated cytochrome c release and caspase activation that resulted in cell death.

Crucial Residues Modulating Interface of hBcl-B - hBaxBH3 Heterodimer as Probed by Computational Methods

Cancerous cells develop resistance to cell death by over expression of anti-apoptotic proteins, which are specific to interact with pro-apoptotic and BH3-only proteins of Bcl-2 family. Delineating crucial residues mediating the heterodimer complexes (anti-apoptotic proteins - pro-apoptotic/BH3-only proteins) is indispensable to develop specific antagonists to anti-apoptotic proteins. In these backgrounds, we have herein reported crucial residues of hBaxBH3 and hBcl-B (an anti-apoptotic protein specifically interacts with human Bax but does not interact with human Bak) for hetero dimerization of the polypeptides and as well validated the structural determinants of the polypeptides through variety of virtual 'alanine mutants' and 'switch mutants' by using an array of computational methods. Residues such as D53, S60, E61, K64, E69 and D71 of hBaxBH3 and R45, H50, F53, F54, Y57, M71, S74, V75, R86, V88, T89, F93 and F159 of hBcl-B were found to be crucial residues of the polypeptides for intermolecular interaction leading hetero dimerization. Moreover, 'pharmacophoric residues' for the hBaxBH3 and hBcl-B have also been figured out and rationalized.

Concurrent acetylation of FoxO1/3a and p53 due to sirtuins inhibition elicit Bim/PUMA mediated mitochondrial dysfunction and apoptosis in berberine-treated HepG2 cells

Post-translational modifications i.e. phosphorylation and acetylation are pivotal requirements for proper functioning of eukaryotic proteins. The current study aimed to decode the impact of acetylation/deacetylation of non-histone targets i.e. FoxO1/3a and p53 of sirtuins (NAD+ dependent enzymes with lysine deacetylase activity) in berberine treated human hepatoma cells. Berberine (100μM) inhibited sirtuins significantly (P<0.05) at transcriptional level as well as at translational level. Combination of nicotinamide (sirtuin inhibitor) with berberine potentiated sirtuins inhibition and increased the expression of FoxO1/3a and phosphorylation of p53 tumor suppressor protein. As sirtuins deacetylate non-histone targets including FoxO1/3a and p53, berberine increased the acetylation load of FoxO1/3a and p53 proteins. Acetylated FoxO and p53 proteins transcriptionally activate BH3-only proteins Bim and PUMA (3.89 and 3.87 fold respectively, P<0.001), which are known as direct activator of pro-apoptotic Bcl-2 family protein Bax that culminated into mitochondria mediated activation of apoptotic cascade. Bim/PUMA knock-down showed no changes in sirtuins' expression while cytotoxicity induced by berberine and nicotinamide was curtailed up to 28.3% (P<0.001) and it restored pro/anti apoptotic protein ratio in HepG2 cells. Sirtuins inhibition was accompanied by decline in NAD+/NADH ratio, ATP generation, enhanced ROS production and decreased mitochondrial membrane potential. TEM analysis confirmed mitochondrial deterioration and cell damage. SRT-1720 (1–10μM), a SIRT-1 activator, when pre-treated with berberine (25μM), reversed sirtuins expression comparable to control and significantly restored the cell viability (P<0.05). Thus, our findings suggest that berberine mediated sirtuins inhibition resulting into FoxO1/3a and p53 acetylation followed by BH3-only protein Bim/PUMA activation may in part be responsible for mitochondria-mediated apoptosis.

Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation

We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins.

Combination with vorinostat overcomes ABT-263 (navitoclax) resistance of small cell lung cancer

ABSTRACTSmall cell lung cancer (SCLC) is an aggressive tumor type with high mortality. One promising approach for SCLC treatment would be to utilize agents targeting molecular abnormalities regulating resistance to apoptosis. BH3 mimetic antagonists, such as ABT-737 and its orally available derivative ABT-263 (navitoclax) have been developed to block the function of pro-survival BCL-2 family members. The sensitivity of SCLC to these drugs varies over a broad range in vitro and in clinical trials. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is a critical determinant of sensitivity to ABT-737. Thus, pharmacological up-regulation of Noxa could enhance cell death induced by the BH3 mimetics. We find that the combination of ABT-263 and a HDAC inhibitor, vorinostat, efficiently induces apoptosis in a variety of SCLC cell lines, including ABT-263 resistant cell lines. Cell death induced by combined treatment is Noxa- and/or BIM-dependent in some cell lines but in others appears to be mediated by down-regulation of BCL-XL and release of BAK from BCL-XL and MCL-1. These results suggest that combination of HDAC inhibitors and BCL-2 inhibitors could be an alternative and effective regimen for SCLC treatment.

Knockdown of miR-221 promotes the cisplatin-inducing apoptosis by targeting the BIM-Bax/Bak axis in breast cancer.

Accumulating evidence shows that microRNAs (miRNAs) have a critical role in the initiation and progression of types of human cancer, including breast cancer. Recent research indicated that miRNAs are also related with the chemotherapy on cancers. In this study, the expression of miR-221 in breast cancer (BC) patients' serum and cancer tissues was found to be significantly up-regulated. The results of in vitro MTT assay indicated that although the anti-miR-221 oligonucleotide alone did not influence the viability of BC cell lines markedly, it significantly promoted the cytotoxicity of cisplatin (DDP) to BC cells. Mechanistic studies demonstrated that the gene of BIM (Bcl-2 interacting mediator of cell death), a pro-apoptotic Bcl-2 family protein, was up-regulated by the knockdown of miR-221. We found that the synergetic effect of anti-miR-221 on increasing the sensitivity of MDA-MB-231 was BIM dependant. Furthermore, results of immunoprecipitation showed the up-regulated BIM directly combined with the Bax and Bak, leading to mitochondrial dysfunction. Our results suggest the anti-miR-221 could promote the cisplatin-inducing apoptosis by targeting the Bim-Bax/Bak axis in breast cancer.

A Novel Ribonuclease from Rana Chensinensis and Its Potential for the Treatment of Human Breast Cancer.

Onconase, a member of the pancreatic RNAase A superfamily of ribonucleases, is a chemotherapeutic agent, which has demonstrated selective antitumor activity in a variety of human malignancies. However, little is known about the mechanisms of it's action on human breast cancer cells. To investigate a novel Onconase from the frog of Rana chensinensis changbaishanensis on human breast cancer cells and the underlying mechanisms, a novel Onconase named Rdchonc from Rana chensinensis changbaishanensis was cloned by polymerase chain reaction. SDS-PAGE revealed that the Rdchonc had a high heterologous expression in Escherichia coli BL21(DE3). The MTT assay indicated that purified Rdchonc was cytotoxic to human breast cancer MCF-7 and MD-MB-231 cells. Treatment with 20 μg/mL Rdchonc protein significantly reduced the invasive capacities of MCF-7 and MD-MB-231 cells. Interestingly, the authors found that such inhibitory effort on tumor cell growth induced by Rdchonc treatment may be explained by the regulation of proapoptotic Bcl-2 family proteins and inhibition of MEK/ERK phosphorylation.

B5, a thioredoxin reductase inhibitor, induces apoptosis in human cervical cancer cells by suppressing the thioredoxin system, disrupting mitochondrion-dependent pathways and triggering autophagy.

The synthetic curcumin analog B5 is a potent inhibitor of thioredoxin reductase (TrxR) that has potential anticancer effects. The molecular mechanism underlying B5 as an anticancer agent is not yet fully understood. In this study, we report that B5 induces apoptosis in two human cervical cancer cell lines, CaSki and SiHa, as evidenced by the downregulation of XIAP, activation of caspases and cleavage of PARP. The involvement of the mitochondrial pathway in B5-induced apoptosis was suggested by the dissipation of mitochondrial membrane potential and increased expression of pro-apoptotic Bcl-2 family proteins. In B5-treated cells, TrxR activity was markedly inhibited with concomitant accumulation of oxidized thioredoxin, increased formation of reactive oxygen species (ROS), and activation of ASK1 and its downstream regulatory target p38/JNK. B5-induced apoptosis was significantly inhibited in the presence of N-acetyl-l-cysteine. Microscopic examination of B5-treated cells revealed increased presence of cytoplasmic vacuoles. The ability of B5 to activate autophagy in cells was subsequently confirmed by cell staining with acridine orange, accumulation of LC3-II, and measurement of autophagic flux. Unlike B5-induced apoptosis, autophagy induced by B5 is not ROS-mediated but a role for the AKT and AMPK signaling pathways is implied. In SiHa cells but not CaSki cells, B5-induced apoptosis was promoted by autophagy. These data suggest that the anticarcinogenic effects of B5 is mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis and autophagy.

Abstract POSTER-THER-1425: Novel small molecules overcoming platinum drug resistance in ovarian cancer cells

Abstract After stage, platinum drug resistance is the single most important factor in determining prognosis in ovarian cancer. Overcoming platinum drug resistance by pharmacological manipulations represents a major challenge in the current clinical practice for the management of ovarian cancer. We discovered a novel drug combination approach using platinum agents with mdivi-1 (mitochondrial division inhibitor 1) that is able to synergistically induce rapid apoptosis in platinum-resistant ovarian cancer cells, including established cisplatin-resistant ovarian cancer cell lines as well as primary cancer cells derived from ovarian cancer patients. We also identified that the synergism between cisplatin and mdivi-1 is not dependent on Drp1 (a mitochondrial fission protein and the reported target of mdivi-1), as mdivi-1 enhances cisplatin toxicity in Drp1 knockout mouse embryonic fibroblast (MEF) cells. In order to improve the compound properties of mdivi-1, we performed a structure activity relationship (SAR) study of mdivi-1 and assembled and screened a small library of structurally related compounds. We were able to identify more potent novel small molecules that synergize with platinum agents. We found that short-term exposure of cells to the combination of cisplatin and those novel compounds is sufficient to reduce cell viability, while cisplatin alone treatment only slightly inhibited cell proliferation in cisplatin-resistant ovarian cancer cells. We further identified that mdivi-1 and its structurally related novel small molecules, when combined with cisplatin, induce a synergistic increase of replication stress and DNA damage, causing upregulation of a pro-apoptotic Bcl-2 family protein Noxa. These small molecules also repress mitochondrial respiration and the combination with cisplatin triggers substantial mitochondrial dysfunction including mitochondrial uncoupling. DNA replication stress and mitochondrial dysfunction converge on the induction of mitochondrial outer membrane permeabilization (MOMP) proceeding robust mitochondrial apoptotic signaling, bypassing a Bax/Bak-dependent mechanism. Since apoptosis induced by cisplatin alone relies on Bax/Bak-dependent MOMP, and inefficient activation of Bax/Bak-dependent MOMP is often associated with cisplatin resistance, the combination of cisplatin and mdivi-1 family compounds represents a novel strategy in overcoming platinum resistance. We thus present a novel platinum-based combination approach in the treatment of platinum resistant ovarian cancer. Citation Format: Wei Qian, Jingnan Wang, Robert Edwards, Peter Wipf, Bennett Van Houten. Novel small molecules overcoming platinum drug resistance in ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1425.

Autophagy capacity and sub-mitochondrial heterogeneity shape Bnip3-induced mitophagy regulation of apoptosis.

BackgroundMitochondria are key regulators of apoptosis. In response to stress, BH3-only proteins activate pro-apoptotic Bcl2 family proteins Bax and Bak, which induce mitochondrial outer membrane permeabilization (MOMP). While the large-scale mitochondrial release of pro-apoptotic proteins activates caspase-dependent cell death, a limited release results in sub-lethal caspase activation which promotes tumorigenesis. Mitochondrial autophagy (mitophagy) targets dysfunctional mitochondria for degradation by lysosomes, and undergoes extensive crosstalk with apoptosis signaling, but its influence on apoptosis remains undetermined. The BH3-only protein Bnip3 integrates apoptosis and mitophagy signaling at different signaling domains. Bnip3 inhibits pro-survival Bcl2 members via its BH3 domain and activates mitophagy through its LC3 Interacting Region (LIR), which is responsible for binding to autophagosomes. Previously, we have shown that Bnip3-activated mitophagy prior to apoptosis induction can reduce mitochondrial activation of caspases, suggesting that a reduction to mitochondrial levels may be pro-survival. An outstanding question is whether organelle dynamics and/or recently discovered subcellular variations of protein levels responsible for both MOMP sensitivity and crosstalk between apoptosis and mitophagy can influence the cellular apoptosis decision event. To that end, here we undertook a systems biology analysis of mitophagy-apoptosis crosstalk at the level of cellular mitochondrial populations.ResultsBased on experimental findings, we developed a multi-scale, hybrid model with an individually adaptive mitochondrial population, whose actions are determined by protein levels, embedded in an agent-based model (ABM) for simulating subcellular dynamics and local feedback via reactive oxygen species signaling. Our model, supported by experimental evidence, identified an emergent regulatory structure within canonical apoptosis signaling. We show that the extent of mitophagy is determined by levels and spatial localization of autophagy capacity, and subcellular mitochondrial protein heterogeneities. Our model identifies mechanisms and conditions that alter the mitophagy decision within mitochondrial subpopulations to an extent sufficient to shape cellular outcome to apoptotic stimuli.ConclusionOverall, our modeling approach provides means to suggest new experiments and implement findings at multiple scales in order to understand how network topologies and subcellular heterogeneities can influence signaling events at individual organelle level, and hence, determine the emergence of heterogeneity in cellular decisions due the actions of the collective intra-cellular population.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0115-9) contains supplementary material, which is available to authorized users.