Intrinsic Apoptotic Signaling Research Articles

Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway

We aimed to investigate the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human breast cancer cells. The molecular mechanisms involved in the apoptotic activity exerted by nimbolide were studied on the estrogen dependent (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cell lines. The growth inhibitory effect of nimbolide was assessed by MTT assay. Apoptosis induction by nimbolide treatment was determined by JC-1 mitochondrial membrane potential staining, cytochrome c release, caspase activation, cleavage of PARP and AO/EtBr dual staining. The modulation of apoptotic proteins (intrinsic pathway: Bax, bad, Bcl-2, Bcl-xL, Mcl-1, XIAP-1 and caspase-3, 9; extrinsic pathway: TRAIL, FasL, FADDR and Caspase-8) were studied by western blot and real time PCR analysis. Treatment with nimbolide resulted in dose and time-dependent inhibition of growth of MCF-7 and MDA-MB-231 cells. The occurrence of apoptosis in these cells was indicated by JC-1 staining, modulation of both intrinsic and extrinsic apoptotic signaling molecules expression and further apoptosis was confirmed by AO/EtBr dual staining. These events were associated with: increased levels of proapoptotic proteins Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c and reduced levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP-1. Nimbolide induces the cleavage of pro-caspase-8, pro-caspase-3 and PARP. The above data suggest that nimbolide induces apoptosis by both the intrinsic and extrinsic pathways. With evidence of above data it is suggested that nimbolide exhibit anticancer effect through its apoptosis-inducing property. Thus, nimbolide raises new hope for its use in anticancer therapy.

Equine arteritis virus induced cell death is associated with activation of the intrinsic apoptotic signalling pathway

Equine arteritis virus (EAV) causes a respiratory and reproductive disease in horses, equine viral arteritis. Though cell death in infection with EAV is considered to occur by apoptosis, the underlying molecular mechanism has not been extensively elucidated. We investigated the expression of mRNA of pro-apoptotic and caspase genes during EAV infection in BHK21 cells, a well-established cell type for EAV replication. Using a SYBR Green real-time PCR, mRNA of p53, Bax, caspase 3 and caspase 9 were found up-regulated in a time dependent manner in EAV infected cells. Western blot analysis for caspase 3 and caspase 9 showed expression of cleaved forms of these proteins during EAV infection. In addition, a luminescence-based cell assay for caspase 3/7 activation as a hallmark in apoptosis confirmed apoptotic cell death. The findings demonstrate that cell death in EAV infected BHK21 cells results from apoptosis mediated through the intrinsic signalling pathway.

Human Bop is a novel BH3-only member of the Bcl-2 protein family

One group of Bcl-2 protein family, which shares only the BH3 domain (BH3-only), is critically involved in the regulation of programmed cell death. Herein we demonstrated a novel human BH3-only protein (designated as Bop) which could induce apoptosis in a BH3 domain-dependent manner. Further analysis indicated that Bop mainly localized to mitochondria and used its BH3 domain to contact the loop regions of voltage dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane. In addition, purified Bop protein induced the loss of mitochondrial transmembrane potential (Δψm) and the release of cytochrome c. Furthermore, Bop used its BH3 domain to contact pro-survival Bcl-2 family members (Bcl-2, Bcl-X(L), Mcl-1, A1 and Bcl-w), which could inhibit Bop-induced apoptosis. Bop would be constrained by pro-survival Bcl-2 proteins in resting cells, because Bop became released from phosphorylated Bcl-2 induced by microtubule-interfering agent like vincristine (VCR). Indeed, knockdown experiments indicated that Bop was partially required for VCR induced cell death. Finally, Bop might need to function through Bak and Bax, likely by releasing Bak from Bcl-X(L) sequestration. In conclusion, Bop may be a novel BH3-only factor that can engage with the regulatory network of Bcl-2 family members to process intrinsic apoptotic signaling.

Chronic treatment with selective I2-imidazoline receptor ligands decreases the content of pro-apoptotic markers in rat brain

Selective I(2)-imidazoline receptor ligands induce neuroprotection through various molecular mechanisms including blockade of N-methyl-D-aspartate (NMDA) receptors. To investigate new neuroprotective mechanisms associated with I(2)-imidazoline receptors, the effects of selective (2-styryl-2-imidazoline (LSL 61122), 2-(2-benzofuranyl)-2-imidazoline (2-BFI), 2-(4,5-dihydroimidazol-2-yl) quinoline hydrochloride (BU-224)) and non-selective (idazoxan) I(2)-drugs on canonical apoptotic pathways were assessed in rat brain cortex. The acute treatment with LSL 61122 (10 mg/kg) reduced the content of mitochondrial (pro-apoptotic) Bax (-33%) and cytochrome c (-31%), which was prevented by idazoxan, an I(2)-receptor antagonist. The sustained stimulation of I(2)-imidazoline receptors with selective drugs (10 mg/kg, every 12 h for seven days) was associated with down-regulation of key components of the extrinsic (Fas receptor: -20%; Fas associated protein with death domain (FADD) adaptor: -47-54%) and/or intrinsic (Bax: -20-23%; cytochrome c: -22-28%) apoptotic signalling and/or up-regulation of survival anti-apoptotic factors (p-Ser194 FADD/FADD ratio: +1.6-2.5-fold; and/or Bcl-2/Bax ratio: +1.5-fold), which in the long-term could dampen cell death in the brain. Similar chronic treatments with LSL 60101 (the imidazole analogue of 2-BFI) and idazoxan (a mixed I(2)/α(2)-ligand) did not induce significant alterations of pro- or anti-apoptotic proteins. The disclosed anti-apoptotic mechanisms of selective I(2)-imidazoline drugs may work in concert with other molecular mechanisms of neuroprotection (e.g. blockade of NMDA receptors) that are engaged by I(2)-ligands.

Emodin induces embryonic toxicity in mouse blastocysts through apoptosis

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin inhibits cell proliferation and induces caspase 3-dependent apoptosis. However, its side-effects, particularly those on embryonic development, have not been well characterized as yet. In the current study, we examined the cytotoxic effects of emodin on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation by embryo transfer. Blastocysts treated with 25–75μM emodin exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with emodin was lower than that of their control counterparts. Moreover, in vitro treatment with 25–75μM emodin was associated with increased resorption of post-implantation embryos and decreased fetal weight. With the aid of an in vivo mouse model, we showed that consumption of drinking water containing emodin led to apoptosis and decreased cell proliferation, and inhibited early embryonic development to the blastocyst stage. Our findings support a degree of selective inhibition of retinoic acid receptors in blastocysts treated with emodin. In addition, emodin appears to induce injury in mouse blastocysts through intrinsic apoptotic signaling processes to impair sequent embryonic development. These results collectively indicate that emodin has the potential to induce embryonic cytotoxicity.

Angiogenin inhibits nuclear translocation of apoptosis inducing factor in a Bcl‐2‐dependent manner

Loss-of-function mutations in angiogenin (ANG) gene were discovered in amyotrophic lateral sclerosis (ALS) patients and ANG has been shown to prevent neuronal death both in vitro and in vivo. The neuro-protective activity of ANG was brought about partially by inhibiting stress-induced apoptosis. ANG attenuates both the extrinsic and the intrinsic apoptotic signals by activating Nf-κb-mediated cell survival pathway and Bcl-2-mediated anti-apoptotic pathway. Here we report that ANG inhibits nuclear translocation of apoptosis inducing factor (AIF), an important cell death-executing molecule known to play a dominant role in neurodegenerative diseases. ANG inhibits serum withdrawal-induced apoptosis by attenuating a series of Bcl-2-dependent events including caspase-3 activation, poly ADP-ribose polymerase-1 (PARP-1) cleavage, and AIF nuclear translocation.

A Sparse Update Method for Solving Underdetermined Systems of Nonlinear Equations Applied to the Manipulation of Biological Signaling Pathways

We are interested in sparse solutions of underdetermined systems of nonlinear equations and present an iterative method that updates the current iterate by a sparse vector defined as the solution of a constrained $\ell_1$-minimization problem. Local quadratic convergence of the iterates toward a solution of the underdetermined system of nonlinear equations is guaranteed under standard differentiability assumptions. Our work is motivated by the pharmaceutical modulation of defective biological signaling pathways as linked to various pathological conditions. Based on differential equation models of signal transduction networks the inverse problem of correcting qualitative biological behavior such as bistability or oscillation typically leads to an underdetermined system of nonlinear equations. In this context sparse solutions point to a low number of network intervention sites that may serve as manageable drug target candidates. The practicability of our method is demonstrated by means of a defective biological switch associated to the intrinsic apoptotic signaling pathway.

Overexpression of angiopoietin-1 reduces doxorubicin-induced apoptosis in cardiomyocytes

Doxorubicin (Dox) is a major anticancer chemotherapeutic agent. However, it causes cardiomyopathy due to the side effect of cardiomyocyte apoptosis. We have previously reported that angiopoietin-1 significantly reduced myocardial infarction after ischemic injury and protected cardiomyocytes from oxidative stress-induced apoptosis. It is hypothesized that angiopoietin-1 may protect cardiomyocytes from Dox-induced apoptosis. Cardiomyocytes H9C2 were transfected with adenovirus expressing angiopoietin-1 (Ad5-Ang-1) 24 h before the cells were challenged with Dox at a concentration of 2 μmol/L. Ad5-GFP served as the vector control. Cardiomyocyte apoptosis was evaluated using Annexin V-FITC staining and caspase-3 and caspase-8 activity was determined by Western blotting. The results showed that Dox treatment significantly induced cardiomyocyte apoptosis as evidenced by the greater number of Annexin V-FITC stained cells and increases in caspase-3 and caspase-8 activity. In contrast, overexpression of angiopoietin-1 significantly prevented Dox-induced cardiomyocyte apoptosis. To elucidate the mechanisms by which angiopoietin-1 protected cells from Dox-induced apoptosis, we analyzed both extrinsic and intrinsic apoptotic signaling pathways. We observed that angiopoietin-1 prevented Dox-induced activation of both extrinsic and intrinsic apoptotic signaling pathways. Specifically, angiopoietin-1 prevented DOX-induced in-creases in FasL and Bax levels and cleaved caspase-3 and caspase-8 levels in H9C2 cells. In addition, overexpression of angiopoietin-1 also activated the pro-survival phosphoinositide-3 kinase (PI3K)/Akt signaling pathway and decreased Dox-induced nuclear factor-kappaB (NF-κB) activation. Our data suggest that promoting the expression of angiopoietin-1 could be a potential approach for reducing Dox-induced cardiomyocyte cytoxicity.

The molecular basis of retinal ganglion cell death in glaucoma

Glaucoma is a group of diseases characterized by progressive optic nerve degeneration that results in visual field loss and irreversible blindness. A crucial element in the pathophysiology of all forms of glaucoma is the death of retinal ganglion cells (RGCs), a population of CNS neurons with their soma in the inner retina and axons in the optic nerve. Strategies that delay or halt RGC loss have been recognized as potentially beneficial to preserve vision in glaucoma; however, the success of these approaches depends on an in-depth understanding of the mechanisms that lead to RGC dysfunction and death. In recent years, there has been an exponential increase in valuable information regarding the molecular basis of RGC death stemming from animal models of acute and chronic optic nerve injury as well as experimental glaucoma. The emerging landscape is complex and points at a variety of molecular signals - acting alone or in cooperation - to promote RGC death. These include: axonal transport failure, neurotrophic factor deprivation, toxic pro-neurotrophins, activation of intrinsic and extrinsic apoptotic signals, mitochondrial dysfunction, excitotoxic damage, oxidative stress, misbehaving reactive glia and loss of synaptic connectivity. Collectively, this body of work has considerably updated and expanded our view of how RGCs might die in glaucoma and has revealed novel, potential targets for neuroprotection.

Tanshinone�IIA induces growth inhibition and apoptosis in gastric cancer in vitro and in vivo

As a phytochemical derived from the roots of Salvia miltiorrhiza Bunge, TanshinoneIIA has been reported to possess anti-inflammatory and antioxidant activity. Studies in breast, colon, prostate and lung cancer indicate that TanshinoneIIA may exhibit a promising antitumor activity. However, systemic studies of the cytotoxic effects of TanshinoneIIA on gastric cancer have not been described. The present study offers a comprehensive evaluation of the antitumor effects of TanshinoneIIA in gastric cancer cells invitro and in a mouse xenograft model. Cell viability and apoptosis invitro were evaluated through the MTT assay and flow cytometry analysis. The results indicate that TanshinoneIIA can induce gastric cancer cell growth inhibition and apoptosis in a time- and concentration-dependent manner. Furthermore, we investigated the mechanism of the apoptotic effects induced by TanshinoneIIA. We found that TanshinoneIIA can not only cause cell cycle arrest in the G2/M phase, but also trigger the intrinsic apoptotic signaling pathway. The results suggest that TanshinoneIIA may serve as an effective adjunctive reagent in the treatment of gastric cancer.

Glycoprotein secreted by Absidia corymbifera is associated with apoptosis of human umbilical vein endothelial cells

Objective To partially purify the toxic factor secreted by A. corymbifera and to analyze the mechanism of A. corymbifera-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Methods Glycoprotein secreted by A. corymbifera was purified by Con A Lectin chromatography. The influence of different protein fractions on HUVEC apoptosis was determined by flow eytometer. Both denaturing and nondenaturing deglycosylation of purified glycoprotein was performed and the ability of the protein moiety and carbohydrate moiety to induce HUVEC apoptosis was evaluated respectively. Activation of related caspases during A. corymbifera-induced apoptosis was analyzed by Western blot. The role of caspase-8 and -9 in HUVEC apoptosis was investigated using caspase inhibitors. Caspase inhibitors were used to stop the suppression of HUVEC viability by XTT assay. Results Flow cytometric analysis shows the total protein as well as the glycoprotein fraction of A. corymbifera may induce HUVEC apoptosis in a dose dependent manner. In contrast, similar activity was not observed in the non-glycoprotein fraction. Neither deglycosylated protein nor carbohydrate moiety is able to induce HUVEC apoptosis alone. In the apoptotic signaling pathway, caspase9, caspase-3 and cytochrome C were activated significantly, except caspase-8. Moreover, caspase-9 inhibitor, instead of caspase-8 inhibitor, completely abrogates A. corymbifera-induced HUVEC apoptosis. Caspase9 and caspase-3 inhibitors completely waived the suppression of HUVEC viability by A. corymbifera. Conclusion Glycoprotein secreted by A. corymbifera is associated with HUVEC apoptosis. Intact glycoprotein is essential for the apoptotic progress. Intrinsic apoptotic signaling pathway mediates A. corymbifera-induced HUVEC apoptosis. Key words: Mucorales; Absidia corymbifera; Glycoprotein; Human umbilical vein endothelial cell; Apoptosis

Embryonic toxicity of sanguinarine through apoptotic processes in mouse blastocysts

In this study, we examined the cytotoxic effects of sanguinarine, a phytoalexin with antimicrobial, anti-oxidant, anti-inflammatory and pro-apoptotic effects, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro and in vivo implantation via embryo transfer. Blastocysts treated with 0.5–2 μM sanguinarine exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with sanguinarine were lower than that of their control counterparts. Moreover, in vitro treatment with 0.5–2 μM sanguinarine was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that sanguinarine induces apoptosis and retards early post-implantation development in vitro and in vivo. In addition, sanguinarine induces apoptotic injury effects on mouse blastocysts through intrinsic and extrinsic apoptotic signaling processes to impair sequent embryonic development. However, the extent to which sanguinarine exerts teratogenic effects on early human development is not known at present, and further studies are required to establish effective protection strategies against its cytotoxic effects.

Parthenolide, a Sesquiterpene Lactone, Expresses Multiple Anti-cancer and Anti-inflammatory Activities

Parthenolide, a naturally occurring sesquiterpene lactone derived from feverfew (Tanacetum parthenium), exhibits exceptional anti-cancer and anti-inflammatory properties, making it a prominent candidate for further studies and drug development. In this review, we briefly investigate molecular events and cell-specific activities of this chemical in relation to cytochrome c, nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), signal transduction and activation of transcription (STAT), reactive oxygen species (ROS), TCP, HDACs, microtubules, and inflammasomes. This paper reports that parthenolide shows strong NF-κB- and STAT-inhibition-mediated transcriptional suppression of pro-apoptotic genes. This compound acts both at the transcriptional level and by direct inhibition of associated kinases (IKK-β). Similarly, this review discusses parthenolide-induced ROS-mediated apoptosis of tumor cells via the intrinsic apoptotic signaling pathway. The unique ability of this compound to not harm normal cells but at the same time induce sensitization to extrinsic as well as intrinsic apoptosis signaling in cancer cells provides an important, novel therapeutic strategy for treatment of cancer and inflammation-related disorders.

The Proapoptotic Bcl-2 Protein Bax Plays an Important Role in the Pathogenesis of Reovirus Encephalitis

Encephalitis induced by reovirus serotype 3 (T3) strains results from the apoptotic death of infected neurons. Extrinsic apoptotic signaling is activated in reovirus-infected neurons in vitro and in vivo, but the role of intrinsic apoptosis signaling during encephalitis is largely unknown. Bax plays a key role in intrinsic apoptotic signaling in neurons by allowing the release of mitochondrial cytochrome c. We found Bax activation and cytochrome c release in neurons following infection of neonatal mice with T3 reoviruses. Bax(-/-) mice infected with T3 Abney (T3A) have reduced central nervous system (CNS) tissue injury and decreased apoptosis, despite viral replication that is similar to that in wild-type (WT) Bax(+/+) mice. In contrast, in the heart, T3A-infected Bax(-/-) mice have viral growth, caspase activation, and injury comparable to those in WT mice, indicating that the role of Bax in pathogenesis is organ specific. Nonmyocarditic T3 Dearing (T3D)-infected Bax(-/-) mice had delayed disease and enhanced survival compared to WT mice. T3D-infected Bax(-/-) mice had significantly lower viral titers and levels of activated caspase 3 in the brain despite unaffected transneuronal spread of virus. Cytochrome c and Smac release occurred in some reovirus-infected neurons in the absence of Bax; however, this was clearly reduced compared to levels seen in Bax(+/+) wild-type mice, indicating that Bax is necessary for efficient activation of proapoptotic mitochondrial signaling in infected neurons. Our studies suggest that Bax is important for reovirus growth and pathogenesis in neurons and that the intrinsic pathway of apoptosis, mediated by Bax, is important for full expression of disease, CNS tissue injury, apoptosis, and viral growth in the CNS of reovirus-infected mice.

Sulforaphane blocks hypoxia-mediated resistance to TRAIL-induced tumor cell death

Hypoxia occurs frequently in various solid tumors and elicits a cellular response designed to improve cell survival through adaptive processes, thereby accelerating cancer progression and the development of chemotherapy resistance. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, leads to tumor cell death via both intrinsic and extrinsic apoptotic signaling pathways. Hypoxia inhibits TRAIL-mediated apoptosis and attenuates the therapeutic activity of TRAIL in cancer management. Hypoxia-inducible factor-1α (HIF-1α) plays a central role in tumor hypoxia by up-regulating gene expression related to angiogenesis, cancer invasion and anti-apoptosis. Sulforaphane (SFN), a phenethyl isothiocyanate, elicits HIF-1α inactivation under hypoxia. This study investigated whether hypoxic inhibition of TRAIL-mediated tumor cell death is increased by SFN-mediated HIF-1α instability. SFN induced cell death in various tumor cells, including SK-N-SH, SNU-638, HeLa and A549 cells, and showed cell cytotoxicity in hypoxia-exposed tumor cells. Western blot analysis showed that SFN treatment increased p53 and activated caspase-3 proteins, and decreased HIF-1α activation under hypoxia. Under low-oxygen conditions, TRAIL-treated cells displayed inhibited apoptosis, while SFN-pre-treated cells exhibited stronger sensitization to TRAIL under the hypoxic conditions. SFN treatment enhanced TRAIL-induced activation of proteins, including caspase-3 and p53. SFN dose-dependently decreased HIF-1α protein levels in cancer cells, which was mediated by decreased protein stability. This study demonstrated that SFN recovered hypoxia-mediated resistance to TRAIL via instability of HIF-1α, and also suggests that combination therapy with SFN and TRAIL may provide a novel strategy for treating hypoxic solid tumors.

Regulation of Cell Survival Mechanisms in Alzheimer′s Disease by Glycogen Synthase Kinase‐3

A pivotal role has emerged for glycogen synthase kinase-3 (GSK3) as an important contributor to Alzheimer's disease pathology. Evidence for the involvement of GSK3 in Alzheimer's disease pathology and neuronal loss comes from studies of GSK3 overexpression, GSK3 localization studies, multiple relationships between GSK3 and amyloid β-peptide (Aβ), interactions between GSK3 and the microtubule-associated tau protein, and GSK3-mediated apoptotic cell death. Apoptotic signaling proceeds by either an intrinsic pathway or an extrinsic pathway. GSK3 is well established to promote intrinsic apoptotic signaling induced by many insults, several of which may contribute to neuronal loss in Alzheimer's disease. Particularly important is evidence that GSK3 promotes intrinsic apoptotic signaling induced by Aβ. GSK3 appears to promote intrinsic apoptotic signaling by modulating proteins in the apoptosis signaling pathway and by modulating transcription factors that regulate the expression of proteins involved in apoptosis. Thus, GSK3 appears to contribute to several neuropathological mechanisms in Alzheimer's disease, including apoptosis-mediated neuronal loss.

Ccytosporone-B Induces NR4A1 Meditated Apoptosis In Aggressive Lymphoma Cells

AbstractAbstract 467NR4A1 (also called Nur77, TR3, or NGFI-B) and NR4A3 (Nor-1 or Minor) are two of three members of the orphan nuclear hormone receptor (NR) family referred to as Nur77 family. For both NRs no physiological ligand has been identified. These orphan NRs have been implicated in cell cycle regulation, apoptosis, inflammation, and more recently in carcinogenesis. Their function as critical tumour suppressors genes is demonstrated by the observation that the NR4A1 and NR4A3 double knock out mouse develops rapidly acute myeloid leukemia involving decreased expression of the AP-1 transcription factors JunB and c-Jun and defective extrinsic apoptotic (Fas-L and TRAIL) signalling and their reduced expression in leukemic blasts from human AML patients.NR4A1 and NR4A3 expression analyses in lymphoid malignancies revealed a down regulation of both NRs in all lymphoma cell lines analysed (Ly 1, 3, 4, 7, 8, Karpas 422 and Sc-1), in two of eight “low grade lymphoma” entities (B-CLL and FL II) and in three of five “high grade lymphoma” entities (DLBCL, FL III, MBL) and in ALLs compared to normal controls. Further expression analyses of genes involved in extrinsic (Trail and Fas-L) and intrinsic (Bim and Puma) apoptotic pathways resulted in a uniform regulation (significant positive correlation) of all four apoptotic genes to NR4A1 and NR4A3 expression suggesting that NR4A1 and NR4A3 act as putative tumour suppressor in a substantial number of lymphoma entities and that their abrogation leads to a defective extrinsic and intrinsic apoptotic signalling (1).To investigate whether the reduction of NR4A1 and NR4A3 expression is caused by epigenetic (hypermethylation of CpG islands) or genetic (mutations) aberrations, the regulatory region and coding sequence of both NRs were subjected to a comprehensive methylational and mutational analyses in primary aggressive lymphoma samples by methylation specific PCR and direct sequencing. Whereas no hypermethylation of CpG islands in the regulatory region was detected, a total of four mutations were found in four of 17 aggressive lymphoma samples (1 of 5 FLIII, 2 of 6 DLBCL-GCB and 1 of 6 DLBCL-NGCB). One mutation in the group of the DLBCL-GCB led to an amino acid exchange.To further delineate the function of NR4A1 in aggressive lymphomas, we treated three lymphoma cell lines (Karpas422, SC-1 and Ly8) with Cytosporone B (Csn-B), a natural occurring ligand of NR4A1 known to induce NR4A1 expression (2). Treatment with Csn-B led to an induction of NR4A1 expression accompanied by a marked inhibition of lymphoma cell growth in a dose dependent manner in all three lymphoma cell lines. To investigate whether Csn-B induced growth inhibition is mediated by apoptosis we performed various apoptosis assays, and cell cycle analyses in SC-1 and Ly8 cell lines. After 6h 1.5 ×10−4 M Csn-B treatment cleaved caspase 3 was detected in 91% of Sc-1 cells and in 77% of Ly8 cells as compared to 3% and 23% in untreated controls. Similarly, in the Annexin V assay Csn-B displayed a robust pro-apoptotic activity in both cell lines within 10 h (52 % in SC-1 and 67 % in Ly8 vs 2 % and 14 %) of treatment. By flow cytometry a significant higher sub-G1 DNA content (26 % in SC-1 cells and 45% in Ly8 vs 2% and 9%) could be detected. Induction of apoptosis was accompanied by a significant rise in NR4A1 expression.Our data suggest that NR4A1 is down regulated in a substantial fraction of aggressive lymphomas and that Csn-B induces NR4A1 mediated apoptosis in lymphoma cells. Hence, regulation of NR4A1 by Csn-B is a promising new target for the development of new drugs for lymphoma therapy.1. Deutsch AJA, Beham-Schmid C, Linkesch W, Neumeister P. The Nuclear Orphan Receptors NR4A1 and NR4A3 as Putative Tumour Suppressor Genes in Aggressive Non-Hodgkin's Lymphomas. ASH Annual Meeting Abstracts. 2009 November 20, 2009;114(22):2967.2. Zhan Y, Du X, Chen H, Liu J, Zhao B, Huang D, et al. Cytosporone B is an agonist for nuclear orphan receptor Nur77. Nat Chem Biol. 2008 Sep;4(9):548-56. Disclosures:No relevant conflicts of interest to declare.

Interruption of cenph Causes Mitotic Failure and Embryonic Death, and Its Haploinsufficiency Suppresses Cancer in Zebrafish

Kinetochore proteins associate with centromeric DNA and spindle microtubules and play essential roles in chromosome segregation during mitosis. In this study, we uncovered a zebrafish mutant, stagnant and curly (stac), that carries the Tol2 transposon element inserted at the kinetochore protein H (cenph) locus. Mutant embryos exhibit discernible cell death as early as 20 hours postfertilization, extensive apoptosis, and upward curly tail during the pharyngula period and deform around 5 days postfertilization. The stac mutant phenotype can be rescued by cenph mRNA overexpression and mimicked by cenph knockdown with antisense morpholinos, suggesting the responsibility of cenph deficiency for stac mutants. We demonstrate that the intrinsic apoptosis pathway is hyperactivated in stac mutants and that p53 knockdown partially blocks excess apoptosis in stac mutants. Mitotic cells in stac mutants show chromosome missegregation and are usually arrested in G(2)/M phase. Furthermore, compared with wild type siblings, heterozygous stac fish develop invasive tumors at a dramatically reduced rate, suggesting a reduced cancer risk. Taken together, our findings uncover an essential role of cenph in mitosis and embryonic development and its association with tumor development.

The Flavonoid Luteolin Increases the Resistance of Normal, but Not Malignant Keratinocytes, Against UVB-Induced Apoptosis

Adequate protection of skin against the carcinogenic effects of UVB irradiation is essential. Flavonoids may have a conspicuous role in cancer prevention because of their antioxidant, anti-inflammatory, and growth-inhibitory effects. Therefore, we tested the effects of the flavone luteolin (LUT) on selected parameters of the sunburn response in normal human keratinocytes, exposed to physiological doses of UVB. LUT attenuated UVB-induced cell death through delay and inhibition of intrinsic apoptotic signaling. Moreover, LUT not only predominantly affected the mitochondrial apoptosis pathway through its antioxidant capacity, but also changed the balance of Bcl2 (B-cell leukemia/lymphoma 2)-family members. Furthermore, LUT had inhibitory effects on the UVB-induced release of the inflammatory mediators, IL-1alpha and prostaglandin-E(2). Using different cell lines derived from squamous cell carcinomas, we showed that LUT did not increase the resistance of malignant keratinocytes to UVB. Our data suggest that LUT inhibits different aspects of the sunburn response, which results ultimately in an increased survival of normal keratinocytes, whereas the sensitivity of malignant cells to UVB remain unchanged. Hence, LUT might have value in new photoprotective applications or improve existing ones.

Activity of deacetylase inhibitor panobinostat (LBH589) in cutaneous T‐cell lymphoma models: Defining molecular mechanisms of resistance

Panobinostat (LBH589) is a highly potent deacetylase inhibitor that has demonstrated clinical efficacy in patients with advanced cutaneous T-cell lymphoma (CTCL). To gain a better understanding of the compound activity in this tumor type, we investigated the cellular and molecular effects of panobinostat using both in vitro and in vivo models of CTCL. All 4 tested CTCL cell lines exhibited very high sensitivity to panobinostat-induced growth inhibition. However, only 2 of 4 lines exhibited significant response to the cytotoxic activity of panobinostat. In a CTCL xenograft mouse tumor model, panobinostat treatment resulted in complete tumor regression. The difference in cell sensitivity to panobinostat-induced death enabled us to further investigate potential mechanisms responsible for tumor sensitivity or resistance. In CTCL cell lines that were insensitive to panobinostat-induced apoptosis, constitutively activated NF-kappaB and high levels of Bcl-2 were observed. Inhibition of Bcl-2 sensitized cells to the cytotoxic activity of panobinostat. Conversely, knockdown of Bax diminished the CTCL cell sensitivity. Interestingly, panobinostat could induce cytotoxicity in vorinostat-resistant CTCL cells by downregulating phosphorylated STAT3 and STAT5 proteins. These studies suggest distinct mechanisms responsible for resistance to different deacetylase inhibitors. We show that the intrinsic apoptotic signaling plays an essential role in mediating panobinostat anticancer activity. Moreover, cancer cell sensitivity to panobinostat treatment may be further improved by combination with inhibition of anti-apoptotic factors. These data provide preclinical support that panobinostat, as a single agent or in combination with other anticancer agents, is a promising therapy for CTCL.