Pro-apoptotic Bcl-2 Family Proteins Research Articles

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.

Abstract 5344: Novel thioxodihydroquinazolinone small molecules for combination with platinum drugs to reverse platinum resistance through inducing mitochondrial apoptosis independent of Bax and Bak

Abstract Platinum drugs are among the most effective anticancer drugs against various types of cancers, and the effective treatment of tumors resistant to platinum drugs-based anticancer therapies (either intrinsic or acquired during treatment) is a critical challenge in the current clinic. Bax and Bak, which are the proapoptotic Bcl-2 family proteins, are essential for cisplatin-induced apoptosis. Unfortunately, Bax and its upstream apoptotic signaling pathways are often altered in cancer cells. Strategies that induce apoptosis bypassing Bax- and Bak-dependent pathway will thus provide new opportunities to overcome platinum drug resistance. We have identified the thioxodihydroquinazolinone, mdivi-1, as a member of a novel class of small molecules that when combined with cisplatin are able to induce Bax- and Bak-independent mitochondrial outer membrane permeabilization and subsequent apoptosis in platinum resistant tumor cells. In order to identify the pharmacophore that is minimally required for enhancing the efficacy of platinum drugs and bax/bak-independent mitochondrial apoptosis, we performed a structure activity relationship (SAR) study of a computationally selected library of mdivi-1 related small molecules through substructure and similarity searches in the small molecule library collection of the University of Pittsburgh Center for Chemical Methodologies and Library Development (UPCMLD). We were able to identify that a thiourea function is necessary but not sufficient for the synergism of this class of thioxodihydroquinazolinones with cisplatin. We have also identified more potent mdivi-1 analogs through this SAR study. These more potent novel thioxodihydroquinazolinone small molecules, when combined with cisplatin, induce a synergistic increase of replication stress and DNA damage, upregulation of a pro-apoptotic Bcl-2 family protein Noxa, and substantial mitochondrial dysfunction including mitochondrial uncoupling. Importantly, the combination of these thioxodihydroquinazolinones with cisplatin leads to efficient apoptosis in cells depleted of Bax and Bak. Taken together, we were able to establish a preliminary pharmacophore model of this class of thioxodihydroquinazolinones and identify more potent structures that are essential for guiding future compound optimization for preclinical studies. Given the essential roles of Bax and Bak in many different classes of chemotherapeutic and targeted therapeutic drugs, our thioxodihydroquinazolinone / platinum based combination approach provides a novel targeting strategy for a broad spectrum of drug resistant tumors. Citation Format: Wei Qian, Joseph Salamoun, Jingnan Wang, Vera Roginskaya, Bennett Van Houten, Peter Wipf. Novel thioxodihydroquinazolinone small molecules for combination with platinum drugs to reverse platinum resistance through inducing mitochondrial apoptosis independent of Bax and Bak. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5344. doi:10.1158/1538-7445.AM2015-5344

Activation of the Mitochondrial Apoptotic Pathway Produces Reactive Oxygen Species and Oxidative Damage in Hepatocytes That Contribute to Liver Tumorigenesis

Chronic hepatitis, including viral hepatitis and steatihepatitis, is a well-known high-risk condition for hepatocellular carcinoma. We previously reported that continuous hepatocyte apoptosis drives liver tumors in hepatocyte-specific Bcl-xL or Mcl-1 knockout mice. In this study, we further examine the underlying cellular mechanisms of generating tumors in apoptosis-prone liver. In cultured hepatocytes, the administration of ABT-737, a Bcl-xL/-2/-w inhibitor, led to production of reactive oxygen species (ROS) as well as activation of caspases. Mitochondria isolated from murine liver, upon administration of truncated-Bid, a proapoptotic Bcl-2 family protein, released cytochrome c and produced ROS, which was dependent on mitochondrial respiration. Hepatic apoptosis, regeneration, accumulation of oxidative damages, and tumorigenesis observed in hepatocyte-specific Mcl-1 knockout mice were substantially attenuated by further deficiency of Bax or Bid, suggesting that a balance of mitochondrial Bcl-2 family proteins governs generation of oxidative stress and other pathologies. Whole-exome sequencing clarified that C>A/G>T transversion, which is often caused by oxidative DNA damage in proliferating cells, was a frequently observed mutation pattern in liver tumors of Mcl-1 knockout mice. The administration of antioxidant L-N-acetylcysteine did not affect apoptosis, compensatory regeneration, or fibrotic responses but significantly reduced oxidative DNA damage and incidence and multiplicity of live tumors in Mcl-1 knockout mice. In conclusion, activation of the mitochondrial apoptotic pathway in hepatocytes accumulates intracellular oxidative damages, leading to liver tumorigenesis, independently of liver regeneration or fibrosis. This study supports a concept that antioxidant therapy may be useful for suppressing liver carcinogenesis in patients with chronic liver disease.

Abstract 2524: Combination with vorinostat overcomes ABT-263 resistance of small cell lung cancer

Abstract Lung cancer is the most common cause of cancer related mortality worldwide, with small cell lung cancer (SCLC) representing 15% of cases, accounting for 30,000 new cases in the US each year. While SCLC is initially a highly chemotherapy-responsive disease, relapse and progressive development of chemotherapy resistance is the rule. One promising approach to overcome the therapeutic resistance would be to utilize agents targeting molecular abnormalities regulating resistance to apoptosis. Tumor cell death induced by chemotherapy or lack of appropriate cellular survival signals is mediated by the BCL-2 family-dependent mitochondrial apoptotic pathway. We and others have shown that the pro-survival member BCL-2, as well as BCL-XL and MCL-1, is overexpressed in SCLC. BH3 mimetic antagonists have been developed to block the function of pro-survival BCL-2 family members. ABT-737 and its orally available derivative ABT-263 bind to and block BCL-2 and BCL-XL, but not MCL-1 function. Although SCLC is the only non-hematologic malignancy against which ABT-737/ABT-263 are effective as single agents, the sensitivity of SCLC to these drugs varies over a broad range in vitro and in clinical trials. We have shown in a model SCLC system that expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is a critical determinant of ABT-737 sensitivity, demonstrating that Noxa specifically binds to and recruits MCL-1 from the cytosol to the mitochondria. Recent integrative genome analysis in SCLC implicates histone modification as a major feature of SCLC. It has also been demonstrated that Noxa is transcriptionally activated by HDAC inhibitors. Thus, we hypothesized that alteration of histone modification by a HDAC inhibitor, vorinostat could induce Noxa to enhance cell death by ABT-737/263 treatment. We have found that combination of ABT-263 and vorinostat efficiently induces apoptosis in a variety of SCLC cell lines, even in the ABT-263 resistant cells. Cell death induced by combined treatment is Noxa- and/or BIM-dependent in some cell lines but in others appears to be mediated by an alternative mechanism. These results suggest that combination of HDAC inhibitors and BCL-2 inhibitors could be an alternative effective regimen for SCLC treatment. Citation Format: Wataru Nakajima, Mark A. Hicks, Kanika Sharma, Ngoc Le, Geoffrey W. Krystal, Hisashi Harada. Combination with vorinostat overcomes ABT-263 resistance of small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2524. doi:10.1158/1538-7445.AM2015-2524

Abstract 11: Molecular mechanisms of inverse association between cancer and Alzheimer's disease

Abstract Apoptosis has been implicated in both cancer and neurodegenerative disease such as Alzheimer's disease (AD). Defective or inefficient apoptosis is an acquired hallmark of cancer cells, while cytochrome c-dependent apoptosis is a crucial pathway in AD related neuronal cell death. Notably, recent studies have revealed an inverse correlation between development of cancer and AD—AD patients show a reduced risk of cancer, while cancer survivors are less likely to acquire AD. Here we propose that amyloid β (Aβ), a widely accepted pathological culprit of AD, hijacks the intrinsic apoptotic pathway eliciting different symptoms for cancer and AD. By developing an in vitro single-vesicle cytochrome c release assay, we show that even in the absence of death signal the oligomeric form of synthetic Aβ directly interacts with a pro-apoptotic bcl-2 family protein BAK and induces apoptotic pores sufficient for cytochrome c release. Furthermore, Aβ oligomers not only cause neuronal cell death in HT-22 cells but also the apoptotic effect was significantly reduced with suppressed BAK expression via siRNA, further substantiating its apoptotic interaction with BAK. Moreover, a significant reduction of cytochrome c release was also observed with isolated mitochondria from BAK siRNA-treated HT-22 cells which specifically identifies the target machinery. These results demonstrate Aβ oligomers’ efficacy as a potent apoptotic ligand. In collection, we suggest that while Aβ oligomers may cause neuronal cell death in AD it may also ameliorate tumor proliferation in relation to cancer. Thus, providing molecular level evidence for the inverse association reported between cancer and AD patients. Citation Format: Yoosoo Yang, EunJung Lee, In-San Kim. Molecular mechanisms of inverse association between cancer and Alzheimer's disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 11. doi:10.1158/1538-7445.AM2015-11

Low expression of pro-apoptotic Bcl-2 family proteins sets the apoptotic threshold in Waldenström macroglobulinemia.

Waldenström Macroglobulinemia (WM) is a proliferative disorder of IgM secreting, lymphoplasmacytoid cells that inhabit the lymph nodes and bone marrow. The disease carries a high prevalence of activating mutations in MyD88 (91%) and CXCR4 (28%). Because signaling through these pathways leads to Bcl-xL induction, we examined Bcl-2 family expression in WM patients and cell lines. Unlike other B-lymphocyte-derived malignancies, which become dependent on expression of anti-apoptotic proteins to counter expression of pro-apoptotic proteins, WM samples expressed both pro- and anti-apoptotic Bcl-2 proteins at low levels similar to their normal B-cell and plasma cell counterparts. Three WM cell lines expressed pro-apoptotic Bcl-2 family members Bim or Bax and Bak at low levels which determined their sensitivity to inducers of intrinsic apoptosis. In two cell lines, miR-155 upregulation, which is common in WM, was responsible for inhibition of FOXO3a and Bim expression. Both antagonizing miR-155 to induce Bim and proteasome inhibition increased the sensitivity to ABT-737 in these lines indicating a lowering of the apoptotic threshold. In this manner, treatments that increase pro-apoptotic protein expression increase the efficacy of agents treated in combination in addition to direct killing.

Ceramide-induced apoptosis in renal tubular cells: a role of mitochondria and sphingosine-1-phoshate.

Ceramide is synthesized upon stimuli, and induces apoptosis in renal tubular cells (RTCs). Sphingosine-1 phosphate (S1P) functions as a survival factor. Thus, the balance of ceramide/S1P determines ceramide-induced apoptosis. Mitochondria play a key role for ceramide-induced apoptosis by altered mitochondrial outer membrane permeability (MOMP). Ceramide enhances oligomerization of pro-apoptotic Bcl-2 family proteins, ceramide channel, and reduces anti-apoptotic Bcl-2 proteins in the MOM. This process alters MOMP, resulting in generation of reactive oxygen species (ROS), cytochrome C release into the cytosol, caspase activation, and apoptosis. Ceramide regulates apoptosis through mitogen-activated protein kinases (MAPKs)-dependent and -independent pathways. Conversely, MAPKs alter ceramide generation by regulating the enzymes involving ceramide metabolism, affecting ceramide-induced apoptosis. Crosstalk between Bcl-2 family proteins, ROS, and many signaling pathways regulates ceramide-induced apoptosis. Growth factors rescue ceramide-induced apoptosis by regulating the enzymes involving ceramide metabolism, S1P, and signaling pathways including MAPKs. This article reviews evidence supporting a role of ceramide for apoptosis and discusses a role of mitochondria, including MOMP, Bcl-2 family proteins, ROS, and signaling pathways, and crosstalk between these factors in the regulation of ceramide-induced apoptosis of RTCs. A balancing role between ceramide and S1P and the strategy for preventing ceramide-induced apoptosis by growth factors are also discussed.

Vaccinia Virus Protein A49 Is an Unexpected Member of the B-cell Lymphoma (Bcl)-2 Protein Family

Vaccinia virus (VACV) encodes several proteins that inhibit activation of the proinflammatory transcription factor nuclear factor κB (NF-κB). VACV protein A49 prevents translocation of NF-κB to the nucleus by sequestering cellular β-TrCP, a protein required for the degradation of the inhibitor of κB. A49 does not share overall sequence similarity with any protein of known structure or function. We solved the crystal structure of A49 from VACV Western Reserve to 1.8 Å resolution and showed, surprisingly, that A49 has the same three-dimensional fold as Bcl-2 family proteins despite lacking identifiable sequence similarity. Whereas Bcl-2 family members characteristically modulate cellular apoptosis, A49 lacks a surface groove suitable for binding BH3 peptides and does not bind proapoptotic Bcl-2 family proteins Bax or Bak. The N-terminal 17 residues of A49 do not adopt a single well ordered conformation, consistent with their proposed role in binding β-TrCP. Whereas pairs of A49 molecules interact symmetrically via a large hydrophobic surface in crystallo, A49 does not dimerize in solution or in cells, and we propose that this hydrophobic interaction surface may mediate binding to a yet undefined cellular partner. A49 represents the eleventh VACV Bcl-2 family protein and, despite these proteins sharing very low sequence identity, structure-based phylogenetic analysis shows that all poxvirus Bcl-2 proteins are structurally more similar to each other than they are to any cellular or herpesvirus Bcl-2 proteins. This is consistent with duplication and diversification of a single BCL2 family gene acquired by an ancestral poxvirus.

Natural Compounds Modulating Mitochondrial Functions.

Mitochondria are organelles responsible for several crucial cell functions, including respiration, oxidative phosphorylation, and regulation of apoptosis; they are also the main intracellular source of reactive oxygen species (ROS). In the last years, a particular interest has been devoted to studying the effects on mitochondria of natural compounds of vegetal origin, quercetin (Qu), resveratrol (RSV), and curcumin (Cur) being the most studied molecules. All these natural compounds modulate mitochondrial functions by inhibiting organelle enzymes or metabolic pathways (such as oxidative phosphorylation), by altering the production of mitochondrial ROS and by modulating the activity of transcription factors which regulate the expression of mitochondrial proteins. While Qu displays both pro- and antioxidant activities, RSV and Cur are strong antioxidant, as they efficiently scavenge mitochondrial ROS and upregulate antioxidant transcriptional programmes in cells. All the three compounds display a proapoptotic activity, mediated by the capability to directly cause the release of cytochrome c from mitochondria or indirectly by upregulating the expression of proapoptotic proteins of Bcl-2 family and downregulating antiapoptotic proteins. Interestingly, these effects are particularly evident on proliferating cancer cells and can have important therapeutic implications.

Effects of E2HSA, a Long-Acting Glucagon Like Peptide-1 Receptor Agonist, on Glycemic Control and Beta Cell Function in Spontaneous Diabetic db/db Mice.

Glucagon like peptide-1 (GLP-1) receptor agonists such as exendin-4 have been widely used but their short half-life limits their therapeutic value. The recombinant protein, E2HSA, is a novel, long-acting GLP-1 receptor agonist generated by the fusion of exendin-4 with human serum albumin. In mouse pancreatic NIT-1 cells, E2HSA activated GLP-1 receptor with similar efficacy as exendin-4. After single-dose administration in ICR mice, E2HSA showed prolonged glucose lowering effects which lasted up to four days and extended inhibition on gastric emptying for at least 72 hours. Chronic E2HSA treatment in db/db mice significantly improved glucose tolerance, reduced elevated nonfasting and fasting plasma glucose levels, and also decreased HbA1c levels. E2HSA also increased insulin secretion and decreased body weight and appetite. Furthermore, immunofluorescence analysis showed that E2HSA increased β-cell area, improved islet morphology, and reduced β-cell apoptosis. In accordance with the promotion of β-cell function and survival, E2HSA upregulated genes such as Irs2, Pdx-1, Nkx6.1, and MafA and downregulated the expression levels of FoxO1 and proapoptotic Bcl-2 family proteins. In conclusion, with prolonged glucose lowering effects and promoting β-cell function and survival, the fusion protein, E2HSA, is a promising new therapeutic for once weekly treatment of type 2 diabetes.

The BH3-only protein Bad is dispensable for TNF-mediated cell death.

Tumor necrosis factor (TNF) is a key signaling molecule orchestrating immune and inflammatory responses and possesses the capacity to trigger apoptotic as well as necroptotic cell death. Apoptotic cell death elicited by TNF has been demonstrated to engage pro-apoptotic Bcl-2 family proteins, most prominently the BH3-only protein Bid, a key substrate of caspase-8, the key effector protease downstream of TNF receptor I. Most recently, the BH3 domain-containing protein Bad (Bcl-2-antagonist of cell death) has been shown to be rate limiting for TNF-mediated cell death, suggesting possible synergy with Bid, but genetic analyses presented here demonstrate that it is dispensable for this process.

Detection of Bax Microsatellite Mutations and BaxΔ2 Isoform in Human Buccal Cells.

Loss of the pro-apoptotic Bcl-2 family protein Bax occurs in ~50% of hereditary nonpolyposis colorectal cancer (HNPCC) due to microsatellite instability (MSI). Recently, we found that some of the “Bax-negative” MSI tumor cells contain a functional Bax isoform, BaxΔ2, which sensitizes cells to selective chemotherapeutics. Here we show the detection of Bax microsatellite mutations and expression of BaxΔ2 proteins in human buccal cells. Our study provides a sensitive and non-invasive approach and a potential clinical application in diagnosis and treatment of MSI colon cancer patients.

The combination of thioxodihydroquinazolinones and platinum drugs reverses platinum resistance in tumor cells by inducing mitochondrial apoptosis independent of Bax and Bak

The effective management of tumors resistant to platinum drugs-based anticancer therapies is a critical challenge in current clinical practices. The proapoptotic Bcl-2 family proteins Bax and Bak are essential for cisplatin-induced apoptosis. Unfortunately, Bax and its related upstream endogenous apoptotic signaling pathways are often dysregulated in cancer cells. Strategies that are able to bypass Bax- and Bak-dependent apoptotic pathways will thus provide opportunities to overcome platinum drug resistance. We have identified the thioxodihydroquinazolinone mdivi-1 as a member of a novel class of small molecules that are able to induce Bax- and Bak-independent mitochondrial outer membrane permeabilization when combined with cisplatin, thereby efficiently triggering apoptosis in platinum-resistant tumor cells. In the present structure activity relationship (SAR) study of a computationally selected library of mdivi-1 related small molecules, we established a pharmacophore model that can lead to the enhancement of platinum drug efficacy and Bax/Bak-independent mitochondrial apoptosis. Specifically, we found that a thiourea function is necessary but not sufficient for the synergism of this class of thioxodihydroquinazolinones with cisplatin. We were also able to identify more potent mdivi-1 analogs through this SAR study, which will guide future designs with the goal to develop novel combination regimens for the treatment of platinum- and multidrug-resistant tumors.

Oridonin induces apoptosis in uveal melanoma cells by upregulation of Bim and downregulation of Fatty Acid Synthase

Oridonin is an orally available drug isolated from Traditional Chinese Medicine. Previous studies with oridonin have demonstrated broad-spectrum anticancer activity in a variety of cancer types. However, the effect of oridonin in uveal melanoma has not been addressed. In this study, we aimed to investigate whether oridonin elicited anticancer activity and its underlying mechanism in human uveal melanoma cells. We demonstrated that oridonin potently reduced cell viability, induced apoptosis and inhibited clonogenic survival and growth with single digit micromolar concentrations in uveal melanoma OCM-1 and MUM2B cell lines. We found that oridonin markedly increased the expression of proapoptotic Bcl-2 family protein Bim in uveal melanoma cells, and knockdown Bim by small interfering RNA significantly attenuated oridonin-induced cell death, indicating an essential role of Bim in oridonin-mediated anticancer activity. Additionally, we observed that oridonin suppressed Fatty Acid Synthase (FAS) expression in uveal melanoma cells, and enforced FAS expression by insulin partially rescued the cells from oridonin-induced apoptosis, showing that inhibition of FAS also contributed to oridonin-mediated apoptosis. Taken together, we reported that oridonin displays potent anticancer effect against uveal melanoma cells through upregulation of Bim and inhibition of FAS. Since oridonin is a popular anticancer agent, our study therefore may have translational implication on the management of patients with uveal melanoma.

The SMAC Mimetic Lcl-161 Augments the in Vitro and in Vivo anti-Tumor Activity of Rituximab and Chemotherapy in Rituximab Relapse/Refractory Lymphoma Models

The addition of rituximab to front-line therapy regimens in diffuse large B-cell lymphoma (DLBCL) has greatly improved clinical outcomes, but is also associated with a disease that is more resistant to salvage chemotherapy in the second-line setting, reinforcing the need for new therapies targeted at the overlapping resistance pathways between rituximab and chemotherapy. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines which exhibited significant concurrent chemotherapy resistance. These multi-therapy resistant cell lines (TRCL) exhibit decreased expression of the pro-apoptotic Bcl-2 family proteins Bak and Bak, along with over-expression of several anti-apoptotic proteins, including the inhibitor of apoptosis proteins (IAP) survivin and livin (determined by Western blot). High IAP expression has been associated with inferior clinical outcomes in a range of hematological malignancies, and solid tumors. To determine the impact of IAP over-expression on TRCL rituximab/chemotherapy resistance we utilized a transient siRNA knockdown of both survivn and livin. TRCLs with livin knockdown had a statistically significant improvement in response to several chemotherapy agents including doxorubicin, vincristine, and the proteasome inhibitor carfilzomib (measured at 48 hours with the Cell Titer-Glo viability assay). These results support livin over-expression as a key lymphoma therapy resistance mechanism, and establish IAPs as potential therapeutic targets.Small molecule IAP inhibitors, like LCL-161 (obtained from Novartis), are chemical mimetics of the endogenous IAP antagonist termed the second mitochondrial inhibitor of caspases (SMAC). Western blot analysis indicated that TRCLs treated in vitro with LCL-161 exhibited a dose dependent decrease in the expression of several IAPs, including livin. In addition, LCL-161 increased rates of TRCL apoptosis, and produced synergistic anti-tumor activity when combined with cytarabine, gemcitabine, and carfilzomib in vitro. LCL-161 also enhanced the ex vivo anti-tumor activity of carfilzomib against primary tumor cells isolated from lymphoma patients with both de novo, and relapse/refractory disease. Cell viability and apoptosis induction were determined at 48 hours with CellTiter-Glo viability assays and flow cytometry respectively. To evaluate the anti-tumor effect of LCL-161 in vivo severe combined immunodeficiency (SCID) mice were inoculated with the TRCL Raji-4RH via tail vein injection (iv), and assigned to observation or treatment arms 7 days after inoculation. Treatments were LCL-161 alone (60mg/kg), the combination of rituximab: 10mg/kg, gemcitabine: 120mg/kg, and vinorelbine: 8mg/kg (RGV), or LCL-161 and RGV together. LCL-161 was administered on day 7 as one dose given p.o. by gavage; RGV was also administered on day 7 as a single i.v. dose given by tail vein injection. Differences in survival (measured as the time to the development of limb paralysis) were evaluated with the Log-rank, Breslow, and Tarone-Ware tests across treatment arms. As a single agent LCL-161 was ineffective in controlling Raji-4RH tumor growth in vivo. However, the combination of LCL-161 with RGV (median survival 133 days) resulted in a statistically significant (P=0.002 with each test) improvement in overall survival when compared to RGV alone (median survival 53 days).In summary, IAPs, especially livin, contribute to rituximab/chemotherapy resistance in relapse/refractory B-cell lymphoma models. However, the IAP inhibitor LCL-161 can disrupt this resistance and augment the effect of chemotherapy in resistant lymphoma cell line models, as well as relapse/refractory lymphoma patient samples. In addition, LCL-161 can improve the anti-tumor activity of the RGV chemotherapy regimen, and increase overall survival in a mouse in vivo model of human rituximab/chemotherapy resistant lymphoma. Our data supports the continued investigation of LCL-161 as a novel and effective targeted agent for the treatment of aggressive rituximab relapse/refractory B-cell lymphomas. (Supported by a NHI SPORE Lymphoma grant: 5 P50 CA130805-04, a NIH grant R01 CA136907-01A1 and The Eugene and Connie Corasanti Lymphoma Research Fund) DisclosuresNo relevant conflicts of interest to declare.

Plant Food Anthocyanins Induced Platelet Apoptosis Via BCL-2/BCL-XL Pathway

Background: Platelets are versatile cells and play important roles in hemostasis/thrombosis, inflammation, and atherosclerosis. The pathogenesis of cardiovascular diseases (CVDs) is linked to platelet hyperactivity which is considered an independent risk factor for CVDs. Platelets are critical for promoting the progression of CVDs, and platelet apoptosis have been reported to be involved in platelet activation. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies demonstrated that anthocyanins from plant food significantly inhibited platelet activation, adhesion, aggregation and granule secretion, as well as attenuated thrombus growth at both arterial and venous shear stresses in vitro and in vivo, however, the effects of anthocyanin on platelet apoptosis and its mechanisms have not been explored. In the present study, we examined whether anthocyanin Cyanidin-3-glucoside (Cy-3-g) affect platelet apoptosis and the BCL-2/BCL-XL intrinsic apoptotic pathway.Methods: Cy-3-g, the predominant bioactive compound of anthocyanin preparations, was obtained from Polyphenol AS Company in Norway.Purified gel-filtered platelets from healthy volunteers were incubated at 37oC for 40 minutes with different concentrations of Cy-3-g (0.5、5、50μM) or PBS buffer as a control. the activated platelets were triggered with 0.5U thrombin for 15min to induce apoptosis. Mitochondria membrane potential (Δψm) and membrane phospholipid phosphatidylserine (PS) exposure in both activated and resting platelets were assessed by flow cytometry. Cytochrome C release, activation of caspase-3, caspase-8, caspase-9, cleavage of gelsolin, the levels of anti-apoptotic BCL-2 family proteins such as BCL-2, BCL-XL and proapoptotic BCL-2 family proteins Bax, Bak, Bad, Bid and tBid in both activated and resting platelets were measured by western blotting.Results: Cy-3-g at 5μM and 50μM directly induced significant ΔΨm dissipation in activated platelets dose dependently. Correspondingly, 50μM Cy-3-g increased cytochrome C release compared to control. The expression of pro-caspase-8 and pro-caspase-9 decreased, activation of caspase-3, caspase-8 and caspase-9 was induced in activated platelets in both 5μM and 50μM Cy-3-g groups. Both PS exposure and the cleavage of gelsolin increased in activated platelets, however these effects were only observed at Cy-3-g doses as high as 50μM. Cy-3-g did not induce the above changes in resting platelets. The intrinsic apoptotic pathway was initiated by Cy-3-g treatment in activated platelets; Cy-3-g significantly inhibited the expression of BCL-2, BCL-XL and increased the levels of Bax, Bak, Bad and Bid in activated platelets dose dependently. No significant difference was observed in resting platelets.Conclusions: Our data demonstrate for the first time that purified anthocyanin Cy-3-g directly accelerated apoptosis in activated platelets via the BCL-2/BCL-XL pathway. Anthocyanins may possess therapeutic potential for patients suffering from thrombotic conditions. DisclosuresNo relevant conflicts of interest to declare.

Mir-155 Expression Raises the Apoptotic Threshold in Waldenström Macroglobulinemia By Inhibition of FOXO3a and Bim

Waldenström Macroglobulinemia (WM) is a proliferative disorder of lymphoplasmacytic cells in the lymph nodes and bone marrow. Phenotypically, WM cells are CD19+, CD20+, CD22+, CD38+, CD138+/- and are lymphoid or plasmacytic in morphology. The disease is characterized by abundant secretion of monoclonal, IgM which causes much of morbidity associated with WM. The disease carries a high prevalence of activating somatic mutations in MyD88 (91%) and CXCR4 (28%), which have been shown to contribute to poor prognosis. These mutations involve signaling cascades that activate pathways known to enhance survival signaling including Bcl-xL. Generally, upregulation of pro-apoptotic Bcl-2 family proteins is observed as cancer cells break differentiation and proliferation checkpoints. To counter this, it becomes necessary for the cell to increase expression of anti-apoptotic Bcl-2 proteins making it dependent on a particular protein or set of proteins for survival. However, we have previously shown data that Bcl-2 family expression in WM is characterized by low expression of both pro- and anti-apoptotic proteins.To investigate a mechanism for this regulation, we examined the Bcl-2 family expression in three WM cell lines and observed that in two lines, BCWM.1 and MWCL-1, the pro-apoptotic BH3-only protein Bim was expressed at very low levels or absent, respectively, which corresponded with low sensitivity to inducers of Bim-dependent intrinsic apoptosis including ABT-737 and dexamethasone. These cell lines were sensitive to bortezomib which can induce apoptosis independent of Bim via a tBid-dependent mechanism. In the third WM cell line, RPCI-WM1, Bim was expressed at moderate levels but the pro-apoptotic proteins Bak and Bax were underexpressed and absent, respectively, which rendered the cell line completely apoptosis-deficient. Having ruled out genomic copy number variation at the loci corresponding to these genes and finding no evidence of epigenetic silencing by methylation, we examined the expression of microRNAs targeting these genes. We first examined the predicted targets of seven commonly dysregulated microRNAs in WM. Of these only one, miR-494, was found to have a moderately conserved target site in the 3’ UTR of Bim. However, the expression pattern of miR-494 did not correlate with the pattern of Bim expression in the WM cell lines. None of these microRNAs were predicted to target Bax or Bak. Therefore, we examined the expression of the remaining commonly dysregulated microRNAs and found that miR-155 was expressed at much higher levels in BCWM.1 and MWCL-1 than in RPCI-WM1 or the multiple myeloma (MM) cell line MM1.s. miR-155 is known to both directly and indirectly regulate FOXO3a, a transcription factor important in the induction of Bim. Confirming this, we observed low protein expression of FOXO3a in both BCWM.1 and MWCL-1 cells. To test this mechanism we stably expressed an anti-miR that targets miR-155 or a control anti-miR in all three WM cell lines and observed an increase in mRNA for FOXO3a and Bim as well as an increase in Bim protein in BWCM.1 and MWCL-1 cells expressing anti-miR-155, while no effect on Bim was observed in the RPCI-WM1 line that does not express miR-155 at high levels. This corresponded with a two-fold increase in ABT-737-induced apoptosis in both BWCM.1 and MWCL-1 in the absence of any additional death signal. As expected, miR-155 antagonism did not significantly increase bortezomib-induced apoptosis. These data indicate that miR-155 expression raises the apoptotic threshold in WM by limiting FOXO3a-mediated Bim expression.Cancer therapy relies on the ability to kill malignant cells at a lower dose than would kill healthy cells. This therapeutic index relies heavily on what is termed mitochondrial priming which is a measure of the expression of pro-apoptotic proteins in a cell. The malignant cell remains alive due to sequestration of these proteins by anti-apoptotic proteins, yet requires less death signaling to cause release of sufficient quantities of pro-apoptotic proteins to activate apoptosis. The data presented here indicate that increased expression of miR-155 raises the apoptotic threshold of WM cells by inhibiting Bim expression and thereby compromises the therapeutic index of many agents. Therefore, the sensitivity to a variety of apoptosis-inducing therapies would be increased by targeting miR-155 in combination as part of the treatment modality. DisclosuresNo relevant conflicts of interest to declare.

NS5B induces up-regulation of the BH3-only protein, BIK, essential for the hepatitis C virus RNA replication and viral release

Hepatitis C virus (HCV) induces cytopathic effects in the form of hepatocytes apoptosis thought to be resulted from the interaction between viral proteins and host factors. Using pathway specific PCR array, we identified 9 apoptosis-related genes that are dysregulated during HCV infection, of which the BH3-only pro-apoptotic Bcl-2 family protein, BIK, was consistently up-regulated at the mRNA and protein levels. Depletion of BIK protected host cells from HCV-induced caspase-3/7 activation but not the inhibitory effect of HCV on cell viability. Furthermore, viral RNA replication and release were significantly suppressed in BIK-depleted cells and over-expression of the RNA-dependent RNA polymerase, NS5B, was able to induce BIK expression. Immunofluorescence and co-immunoprecipitation assays showed co-localization and interaction of BIK and NS5B, suggesting that BIK may be interacting with the HCV replication complex through NS5B. These results imply that BIK is essential for HCV replication and that NS5B is able to induce BIK expression.