IAP Family Research Articles

Pachymic Acid Induces Apoptosis of EJ Bladder Cancer Cells by DR5 Up-Regulation, ROS Generation, Modulation of Bcl-2 and IAP Family Members.

Pachymic acid (PA) is a lanostane-type triterpenoid derived from Poria cocos mushroom that possess various biological effects such as anti-cancer, antiinflammatory and anti-metastasis effects. In this study, we investigated the anti-cancer effects of PA in EJ bladder cancer cells. The results showed that PA significantly inhibited proliferation of EJ cells in a dose-dependent manner. PA induced accumulation of sub-G1 DNA content (apoptotic cell population), apoptotic bodies and chromatin condensation and DNA fragmentation in EJ cells in a dose-dependent manner. PA also induces activation of caspase-3, -8 and -9, and subsequent cleavage of poly (ADP-ribose) polymerase, and significantly suppressed the inhibitor of apoptosis protein family proteins in a dose-dependent manner. Furthermore, PA activates Bid and induced the loss of mitochondrial membrane potential (ΔΨm ) with up-regulated pro-apoptotic proteins (Bax and Bad), down-regulated anti-apoptotic proteins (Bcl-2 and Bcl-xL) and cytochrome c release. In turn, PA increased the generation of reactive oxygen species (ROS); also, the ROS production was blocked by N-acetyl-L-cysteine. The expressions of TNF-related apoptosis inducing ligand and death receptor 5 were up-regulated by PA in a dose-dependent manner, suggesting extrinsic pathway also involved in PA-induced apoptosis. This study provides evidence that PA might be useful in the treatment of human bladder cancer.

The flavonoid morin from Moraceae induces apoptosis by modulation of Bcl-2 family members and Fas receptor in HCT 116 cells.

It is evident based on literature that flavonoids from fruit can safely modulate cancer cell biology and induce apoptosis. Therefore, we investigated the anticancer activity of morin, a flavonoid which is plentiful in twigs of mulberry focusing on apoptosis, and its mechanisms. Morin upregulated the Fas receptor, and activates caspase-8, -9 and -3 in HCT-116 cells. Morin also activates Bid, and induced the loss of mitochondrial membrane potential (MMP, ∆Ψm) with Bax protein activation and cytochrome c release. In addition, morin induced ROS generation which was not blocked by N-acetylcysteine. Morin also suppressed Bcl-2 and cIAP-1, anti-apoptotic proteins, which may contribute to augmentation of morin-triggered apoptosis. As an upstream signaling pathway, suppressed Akt activity by morin was associated to apoptosis. This study suggests that morin induces caspase-dependent apoptosis through extrinsic pathway by upregulating Fas receptor as well as through the intrinsic pathway by modulating Bcl-2 and IAP family members, and ROS generation, and that Akt is the critical upstream signaling that regulates the apoptotic effect of morin in human colon cancer HCT-116 cells.

Inhibition of Bcl-2 or IAP proteins does not provoke mutations in surviving cells

Chemotherapy and radiotherapy can cause permanent damage to the genomes of surviving cells, provoking severe side effects such as second malignancies in some cancer survivors. Drugs that mimic the activity of death ligands, or antagonise pro-survival proteins of the Bcl-2 or IAP families have yielded encouraging results in animal experiments and early phase clinical trials. Because these agents directly engage apoptosis pathways, rather than damaging DNA to indirectly provoke tumour cell death, we reasoned that they may offer another important advantage over conventional therapies: minimisation or elimination of side effects such as second cancers that result from mutation of surviving normal cells. Disappointingly, however, we previously found that concentrations of death receptor agonists like TRAIL that would be present in vivo in clinical settings provoked DNA damage in surviving cells. In this study, we used cell line model systems to investigate the mutagenic capacity of drugs from two other classes of direct apoptosis-inducing agents: the BH3-mimetic ABT-737 and the IAP antagonists LCL161 and AT-406. Encouragingly, our data suggest that IAP antagonists possess negligible genotoxic activity. Doses of ABT-737 that were required to damage DNA stimulated Bax/Bak-independent signalling and exceeded concentrations detected in the plasma of animals treated with this drug. These findings provide hope that cancer patients treated by BH3-mimetics or IAP antagonists may avoid mutation-related illnesses that afflict some cancer survivors treated with conventional DNA-damaging anti-cancer therapies.

8 Therapy resistance in HNSCC – Enhanced levels of the cytoprotective protein survivin is mediated by down-regulation of microRNA-542-3p

Head and neck squamous cell carcinomas (HNSCC) are characterized by overexpression of anti-apoptotic proteins (IAP). Survivin’s dual role as an apoptosis inhibitor and a mitotic effector positioned this smallest member of the IAP family in the front line of cancer research. Survivin plays an essential role in mediating resistance to chemo- and radiation-therapy and thus, has direct clinical relevance. MicroRNAs (miRNAs) are deregulated in a variety of human cancers, including HNSCC. Among several miRNAs, miR-542-3p was found to be down-regulated in HNSCC tumors from patients and established HNSCC tumor cell lines. When analyzing the pathobiological relevance of miR-542 in HNSCC tumor biology, we found that miR-542-3p over-expression reduced cell viability and improved chemotherapy-induced apoptosis in HNSCC tumor cell lines. Upon miR-542-3p over-expression, we observed a dose-dependent down-regulation of Survivin levels, which could be rescued by enforced ectopic Survivin expression but not by a Survivin mutant currently isolated from primary tumor cells. Bioinformatic prediction as well as reporter assays confirmed direct targeting of Survivin by miR-542-3p. Targeting Survivin by siRNA or with miR-542-3p-loaded nanoparticles synergistically sensitized HNSCC tumor cells to irradiation- or chemotherapeutic-induced cell death. Collectively, miR-542-3p seems to exerts its tumor suppressive function in HNSCC by targeting Survivin. Exploiting miR-542-3p re-expression by chemical or genetic decoys could be a promising therapeutic strategy for treating HNSCC.

Retinal ganglion cell apoptotic pathway in glaucoma: Initiating and downstream mechanisms.

Apoptosis of retinal ganglion cells (RGCs) in glaucoma causes progressive visual field loss, making it the primary cause of irreversible blindness worldwide. Elevated intraocular pressure and aging, the main risk factors for glaucoma, accelerate RGC apoptosis. Numerous pathways and mechanisms were found to be involved in RGC death in glaucoma. Neurotrophic factors deprivation is an early event. Oxidative stress, mitochondrial dysfunction, inflammation, glial cell dysfunction, and activation of apoptotic pathways and prosurvival pathways play a significant role in RGC death in glaucoma. The most important among the involved pathways are the MAP-kinase pathway, PI-3 kinase/Akt pathway, Bcl-2 family, caspase family, and IAP family.

Small molecule inhibitor YM155-mediated activation of death receptor 5 is crucial for chemotherapy-induced apoptosis in pancreatic carcinoma.

Despite much effort, pancreatic cancer survival rates are still dismally low. Novel therapeutics may hold the key to improving survival. YM155 is a small molecule inhibitor that has shown antitumor activity in a number of cancers by reducing the expression of survivin. The aim of our study is to understand the mechanisms by which YM155 functions in pancreatic cancer cells. We established the antitumor effect of YM155 with in vitro studies in cultured cells, and in vivo studies using a mouse xenograft model. Our data demonstrated that YM155 reduced the expression of survivin; however, downregulation of survivin itself is insufficient to induce apoptosis in pancreatic cancer cells. We showed for the first time that treatment with YM155 increased death receptor 5 (DR5) expression in pancreatic cancer cells. We found that YM155 induced apoptosis by broad-spectrum inhibition of IAP family member proteins (e.g., CIAP1/2 and FLIP) and induced proapoptotic Bak protein upregulation and activation; the antitumor effect of YM155 treatment with either the DR5 agonist lexatumumab or gemcitabine on pancreatic cancer cells was synergistic. Our data also revealed that YM155 inhibits tumor growth in vivo, without apparent toxicity to the noncancerous human pancreatic ductal epithelial cell line. Together, these findings suggest that YM155 could be a novel therapeutic agent for pancreatic cancer.

An Anti-Apoptotic Molecule, Anamorsin, Is Upregulated in Anemia and Essential for Imperative Erytropoiesis

IntroductionAnamorsin (AM) is an anti-apoptotic molecule we originally isolated from hematopoietic cell line, Ba/F3. That is dependent on interleukin-3 (IL-3) stimulation. We showed that AM conferred resistance to apoptosis induced by the lack of IL-3 stimulation. AM has no structural homology to any known anti-apoptotic molecules such as Bcl-2 and IAP family members. AM-deficient mice are embryonic lethal at late gestation due to the defect of definitive hematopoiesis. Its embryos are anemic and very small in size. Thus AM is considered to be crucial in erythropoiesis but the mechanisms how AM works remain unclear. In this study, we tried to clarify the roles of AM in erythropoiesis.Methods and resultsAt first, in order to examine the expression levels of AM in each differential stage of erythroid cells, we sorted those cells from eight-week-old mice bone marrow (n=4) using flow cytometry. The expression levels of AM were evaluated by Immunoblotting. The results showed that the proerythroblast population (CD71high Ter119low) expressed AM higher than any other erythroid cells. The expression of AM in CD71high Ter119 high cells and CD71low Ter119high cells, which correspond to basophilic erythroblasts and orthchromatophilic erythroblasts respectively, were markedly decreased. Next, we analyzed whether AM expression is induced by erythropoietin (EPO). Recombinant human EPO was injected intravenously (0.1mg/kg) with a single treatment (day0) to eight-week-old mice (n=3). The bone marrow and spleen cells were collected seven days after injection and the samples were subjected to immunoblotting. The results showed that EPO treatment led to increased expression of AM in both bone marrow and spleen. At 14 days after EPO injection, the expression of AM returned to the basal. Furthermore, we examined the AM expression in the acute anemia model. Blood of 0.2ml was exsanguinated from each mouse every day until hematocrit reached to the value less than 25 % (day 0).In this phlebotomized mice, the AM expression was strongly induced specially in proerythroblast population on day 1 and returned to the basal after the recovery from anemia. To explore the AM expression under the other stress, 5-FU (150 mg/kg), a chemotherapeutic drug, was administered to mice (n=3) with a single treatment (day 0). The increased expression of AM was also detected when the mice were most severely anemic (day7). To investigate the significance of AM expression induced by anemia, we transplanted 106 14.5 dpc wild-type (AM+/+) and AM-deficient (AM–/–) fetal liver cells into lethally irradiated congenic recipient mice. At 8 weeks after transplantation, we analyzed the response to anemia induced by phlebotomy. The results showed that donor-derived cells from AM+/+ fetal liver cells were detected the smooth recovery on day 3 after phlebotomy. On the other hand, donor-derived cells from AM–/– fetal liver cells showed an approximately 5-fold reduction compared to those from AM+/+fetal liver cells.Conclusion and discussionThe proerythroblast population expressed AM at the highest level among various erythroid lineage populations. The AM expression level was increased by acute anemia induced by phlebotomy and AM-deficiency could show inefficient recovery of erythropoiesis from the induced acute anemia. Previously we reported that AM is essential for Fe/S cluster biogenesis and AM deficiency impaired the iron homeostasis (ASH abs. #610 (2012)). We also reported that the proerythroblast population in AM-deficient fetal liver cell showed more increased labile iron pool and significantly induced apoptosis. From our previous and present data, it is possible that AM may control erythropoiesis by preventing apoptosis when erythrocyte production is needed. DisclosuresNo relevant conflicts of interest to declare.

Oxymatrine Induces Apoptosis in Human Myeloma Cells By Cell Cycle Arrest and Activation of Caspase-Dependent Apoptosis

Background : Multiple myeloma (MM) is a B cell malignant hematologic cancer. Despite the introduction of new drugs and improvement of chemotherapy, MM is still an incurable disease. Oxymatrine (OMT), the active ingredients of traditional Chinese herbal medicine sophora, has been reported to have antitumor activity. This study was to estimate the therapeutic efficacy of OMT in MM.Methods: The growth inhibition of myeloma cell lines (RPMI8226, U266, ARP-1) or primary cells by OMT was assessed by MTT assay. Apoptosis of MM cells was examined by annexin V-FITC using flow cytometry analysis. DNA content was analyzed by flow cytometry. RT-PCR and western-blot analysis were used to assess the expression of Bcl-2 family proteins and the IAP family proteins. Western blotting was also used to elucidate the signaling pathway that may mediate OMT-induced apoptosis of MM cells.Results: OMT treatment resulted in cell growth inhibition and apoptosis in primary MM cells and all tested MM cell lines in a dose-dependent manner (P <0.05). To elucidate OMT -induced MM cell apoptosis, MM cell lines were treated with or without OMT for 24h and assessed for caspase activation and signaling pathway by Western blotting. The results showed the cleavage of PARP, caspase-3, and caspase-9, and p-AKT were down-regulated after OMT treatment. The mRNA expression of survivin and HIAP by RT-PCR was down-regulated. OMT treatment at 5mM for 48h resulted in increased G-phase cells and decreased S-phase cells in MM cell lines (P <0.05). Cell cycle repressor P21 protein was up-regulated while CDK4, CDK6 and CyclinD1 expression was down-regulated. Our finding also showed a synergistic anti-MM activity of OMT and dexamethasone or adriamycin at a low does (CI<1). In addition, LC3-II expression was significantly increased both in RPMI8226 and U266 cells after treatment with OMT. However, treatment with different doses of OMT and 5 mM autophagy inhibitor 3-MA, significant increased cell apoptosis (P <0.05).Conclusion: Our findings demonstrate the anti-MM activity of OMT and indicate that OMT alone or together with other MM chemotherapeutics may be a prospective treatment for MM. DisclosuresNo relevant conflicts of interest to declare.

Overcoming Resistance to Apoptosis in Multiple Myeloma By Simultaneous Inhibition of Bcl2 and IAP Families of Anti-Apoptotic Proteins

Background: Multiple myeloma (MM) cells evade apoptosis through multiple mechanisms thus enabling it to evade therapy. The Bcl2 family of anti-apoptotic proteins is aberrantly expressed in MM cell lines and patient cells. Yet, pharmacological intervention of this family appears to have significant activity only in molecular subgroups of MM patients. This clearly suggests alternate mechanisms of overcoming apoptotic signals in MM cells in addition to the Bcl2 family, through proteins such as IAPs. We have previously shown that simultaneous inhibition of the three major IAP proteins, namely cIAP1, cIAP2 and XIAP is required to induce pronounced apoptosis in MM cells. However, IAP inhibition results in apoptosis in only some MM cell lines and patient cells. Given that levels of Bcl2 family proteins are unaffected by IAP inhibition, we hypothesized that combined inhibition of the IAP proteins using a SMAC mimetic LCL161 and the Bcl2 family proteins using a pan-Bcl2 inhibitor obatoclax (OBX) will lead to more pronounced and synergistic cell death in a broader subgroup of MM patients.Methods: LCL161 was synthesized by Novartis Inc. (Basel, Switzerland). OBX was purchased from Selleckchem (Houston, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drugs in order to study the cell signaling pathways and aResults: LCL161/OBX combination induced synergistic cytotoxicity and anti-proliferative effects on a broad range of human MM cell lines, including drug resistant cell lines like DOX40 and MM1R. Components of the bone marrow microenvironment including bone marrow stromal cells and tumor promoting cytokines (VEGF, IGF and IL6) were unable to protect MM cells from the effects of the drug combination. We saw a time dependent increase in apoptosis, with the combination inducing significantly more apoptosis than either of the single agents alone. Examining the mechanism of action of the drug combination showed clear inhibition of the IAP proteins, activation of caspases 9, 8, 3 and Bid by LCL161 and the combination and up regulation of the pro-apoptotic proteins Bim, Bid, Puma and Noxa and accumulation of LC3-II by OBX and the combination. Using chloroquine along with the OBX, we were able to demonstrate that OBX induced protective autophagy and the addition of LCL161 was able to overcome this protective effect induced after single agent OBX treatment. Since protective autophagy can be induced by the ER stress response, we then examined the expression levels of proteins involved in this pathway. We observed clear induction of ER stress mediated UPR pathway by both the drugs. However, LCL161 and OBX induced different branches of the UPR pathway. OBX activated the ATF6 and pErk/peif2α/ATF4 branches of the UPR, both of which have been implicated in cell survival during ER stress. ATF4 under irrecoverable ER stress can lead to increase in transcription of CHOP and cause apoptosis. We therefore examined levels of CHOP and observed no induction of CHOP post treatment with either of the drugs or the combination. LCL161, however differentially modulated the IRE1 branch of the UPR by down regulating Xbp-1 splicing, which is a pro survival activity of IREI and up regulating pJNK, which indicated a pro-apoptotic activity induced by IRE1 post irrecoverable ER stress This indicated that the ER stress induced apoptosis is triggered by LCL161, which might be important to overcome the ER induced protective effects induced by OBX.Conclusion: Taken together, our studies indicate that LCL161/OBX combination induces synergistic cell death through modulation of apoptosis, authophagy and the ER stress response. DisclosuresNo relevant conflicts of interest to declare.

Targeting FoxM1 by thiostrepton inhibits growth and induces apoptosis of laryngeal squamous cell carcinoma

We have previously reported that forkhead box M1 (FoxM1) transcription factor was overexpressed in laryngeal squamous cell carcinoma (LSCC) and was associated with development of LSCC. However, there are limited studies regarding the functional significance of FoxM1 and FoxM1 inhibitor thiostrepton in LSCC. Therefore, the aim of this study was to examine both in vitro and in vivo activity of FoxM1 inhibitor thiostrepton against LSCC cell line and nude mice. Cell viability was studied by CCK-8 assay. Cell growth was evaluated by CFSE staining and cell cycle analysis. Apoptosis was measured by flow cytometry. The mRNA and protein expression were detected by quantitative real-time RT-PCR, Western blot and immunohistochemical staining. Xenograft model of tumor formation was used to investigate how thiostrepton influences tumorigenesis in vivo. Overexpression of FoxM1 in LSCC cells was down-regulated by thiostrepton in a dose-dependent manner. Thiostrepton caused dose- and time-dependent suppression of cell viability of LSCC. Moreover, thiostrepton induced cell cycle arrest at S phase at early time and inhibited DNA synthesis in LSCC cells in a dose- and time-dependent manner by down-regulation of cyclin D1 and cyclin E1. Thiostrepton also induced dose- and time-dependent apoptosis of LSCC cells by down-regulation of Bcl-2, up-regulation of Bax and p53, and inducing release of cytochrome c accompanied by activation of cleaved caspase-9, cleaved caspase-3 and cleaved PARP. In addition, z-VAD-fmk, a universal inhibitor of caspases, prevented activation of cleavage caspase-3 and abrogates cell death induced by thiostrepton treatment. Furthermore, FADD and cleaved caspase-8 were activated, and expression of cIAP1, XIAP and survivin were inhibited by thiostrepton. Finally, treatment of LSCC cell line xenografts with thiostrepton resulted in tumorigenesis inhibition of tumors in nude mice by reducing proliferation and inducing apoptosis of LSCC cells. Collectively, our finding suggest that targeting FoxM1 by thiostrepton inhibit growth and induce apoptosis of LSCC through mitochondrial- and caspase-dependent intrinsic pathway and Fas-dependent extrinsic pathway as well as IAP family. Thiostrepton may represent a novel lead compound for targeted therapy of LSCC.

Abstract C38: The decrease of the survivin protein expression induces chromosomal missegregation in colon cancer patients lymphocytes treated with oxaliplatin and vinflunine

Abstract Survivin is a novel member of the IAP family of proteins with a potential dual role in apoptosis inhibition and regulation of mitosis. In addition, survivin is implicated in the regulation of the mitotic spindle checkpoint and the promotion of angiogenesis, and chemoresistance. Survivin is up-regulated in almost all cancers, including colon cancer, but has low or no expression in most normal, differentiated adult tissues. Expression of survivin in cancer cells has been shown to promote tumorigenesis. Besides, the expression levels of survivin strongly correlate with the proliferative activity of the tumours indicating a possible role in cell cycle regulation and cancer progression. However, the effect of a novel chemotherapy such as oxaliplatin and vinflunine on survivin expression has been not well characterized yet. Therefore, the effects of oxaliplatin and vinflunine treatment on survivin protein expression in lymphocytes from colon cancer patients against healthy volunteers were investigated. Treatment with either oxaliplatin or vinflunine significantly increased the formation of micronuclei with p &amp;lt; 0.01 in dose-dependent manner by using a cytokinesis-block micronucleus (CBMN) assay. In addition, treatment with both compounds increased chromosomal missegregation (p&amp;lt;0.01) as investigated by a combination of the CBMN assay with fluorescence in situ hybridisation (MN-FISH). Survivin protein expression in lymphocytes from colon cancer patients treated with oxaliplatin and vinflunine were evaluated by Western blot assay. The expression of survivin was significantly reduced up to 3-fold and 6-fold in survivin expression in all the concentrations of oxaliplatin and vinflunine tested respectively (p &amp;lt; 0.001). In conclusion, significant reduction in survivin protein expression was observed in lymphocytes from colon cancer patients exposed to oxaliplatin or vinflunine. Also evidence for mitotic catastrophe has been found for the first time as one plausible mechanism of oxaliplatin and vinflunine induced lymphocyte death. Oxaliplatin and vinflunine effectively induce DNA damage in vitro leading to aneuploidy in human lymphocytes observed in chromosome missegregation during M-phase. Future work will examine the actual impact of DNA damage to improve chemotherapy treatment or open a new approach in cancer therapy. Citation Format: Amal A. Alotaibi. The decrease of the survivin protein expression induces chromosomal missegregation in colon cancer patients lymphocytes treated with oxaliplatin and vinflunine. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C38. doi:10.1158/1538-7755.DISP13-C38

Abstract B35: Survivin inhibition via EZN-3042 in canine models of lymphoma and osteosarcoma

Abstract Canine lymphoma (LSA) and osteosarcoma (OS) have high mortality rates and remain in need of more effective therapeutic approaches. Due to the strong similarities between canine and human LSA and OS, canine LSA and OS are excellent models for the human disease. Survivin, an IAP family member protein that inhibits apoptosis and drives cell proliferation, is commonly elevated in human and canine cancer. Survivin expression is a negative prognostic factor in dogs and humans with LSA and OS. The objective of our research was to determine the effects of survivin inhibition using a locked nucleic acid antisense oligonucleotide (EZN-3042) on canine LSA and canine OS cell lines, with respect to cell proliferation, apoptosis, and chemosensitivity in vitro. Furthermore, we sought to determine the efficacy of EZN-3042 on inhibition of survivin transcription, survivin protein production, and tumor growth in subcutaneous and orthotopic canine OS xenografts. We inhibited survivin using EZN-3042 in two canine LSA and two canine OS cell lines. Survivin inhibition was confirmed by qRT-PCR and western blot. Percent dead and total cell number was assessed by manual cell counting with trypan blue. Growth inhibition was confirmed with a bioreductive fluorometric assay. A caspase-3/7 assay was used to determine levels of apoptosis in EZN-3042 treated cells compared to controls. Cells were then treated with antineoplastic drug doxorubicin (DOX) +/- EZN-3042 and assays repeated. In vivo, nude mice with subcutaneous and orthotopic OS xenografts were given 100 mg/kg EZN3042 intraperitoneally. Survivin inhibition was confirmed with Immunohistochemistry and qRT-PCR analysis. Survivin inhibition in canine LSA and OS cells via EZN-3042 resulted in 34-72% decrease in survivin protein expression and a 1.3-3.4 fold decrease in endogenous survivin mRNA expression. When EZN-3042 treated cells were compared to controls, total and viable cell numbers were decreased, and apoptosis was increased. Survivin inhibition via EZN-3042 enhanced canine LSA and OS cell lines sensitivity to DOX. IHC and qRT-PCR analysis of subcutaneous and orthotopic canine OS xenografts confirmed decreased tumor survivin expression in EZN-3042 treated mice. Mice treated with EZN-3042 in combination with DOX had significantly decreased tumor growth when compared to single agent treatment and control groups. These results demonstrate that survivin inhibition via EZN-3042 decreased LSA and OS cell proliferation, and increased cellular apoptosis and chemosensitivity to DOX, and that EZN-3042 treatment inhibited tumor survivin expression in vivo and significantly decreased tumor growth when combined with DOX. Survivin-directed therapies may be highly effective in treatment of both canine and human LSA and OS, and spontaneous canine cancer may be a valuable model for the evaluation of survivin-targeted treatment. Citation Format: Jenette K. Shoeneman, Eugene J. Ehrhart, III, Joseph B. Charles, Douglas H. Thamm. Survivin inhibition via EZN-3042 in canine models of lymphoma and osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B35.

Doxorubicin에 의한 내인성 산화질소가 인간 대장암 세포주에서의 세포사멸에 미치는 효과

G -monomethyl-L-arginine (NMA). Doxorubicin differentially inhibited the growth of both the HCT116 (p53-WT) and HT29 (p53-MUT) cells, which was mitigated by cotreatment with NMA. Further studies revealed that inhibition of endogenous NOmitigated doxorubicin-induced apoptosis in the HCT116 and HT29 cells, as evidenced by apoptotic DNA fra gmentation and the sub-G1 peak of apoptotic markers. Apoptosis was delayed in the HT29 cells, and its magnitude was greatly re- duced, underscoring the importance of the modulation of p53 in the response. RT-PCR analysis re- vealed that doxorubicin down-regulated levels of inhibitors of the apoptosis family (cellular IAP-1 and -2). Collectively, these data show that induction of apoptosis by doxorubicin in human colon cancer cells is possibly related to modulation of endogenous NO• , the expression of the IAP family of genes, and the status of p53. The underlying mechanisms may represent potential targets for adjuvant strat- egies to improve the efficacy of chemotherapy for colon cancer.

Synergistic anti-tumor activity and inhibition of angiogenesis by cotargeting of oncogenic and death receptor pathways in human melanoma.

Improving treatment of advanced melanoma may require the development of effective strategies to overcome resistance to different anti-tumor agents and to counteract relevant pro-tumoral mechanisms in the microenvironment. Here we provide preclinical evidence that these goals can be achieved in most melanomas, by co-targeting of oncogenic and death receptor pathways, and independently of their BRAF, NRAS, p53 and PTEN status. In 49 melanoma cell lines, we found independent susceptibility profiles for response to the MEK1/2 inhibitor AZD6244, the PI3K/mTOR inhibitor BEZ235 and the death receptor ligand TRAIL, supporting the rationale for their association. Drug interaction analysis indicated that a strong synergistic anti-tumor activity could be achieved by the three agents and the AZD6244–TRAIL association on 20/21 melanomas, including cell lines resistant to the inhibitors or to TRAIL. Mechanistically, synergy was explained by enhanced induction of caspase-dependent apoptosis, mitochondrial depolarization and modulation of key regulators of extrinsic and intrinsic cell death pathways, including c-FLIP, BIM, BAX, clusterin, Mcl-1 and several IAP family members. Moreover, silencing experiments confirmed the central role of Apollon downmodulation in promoting the apoptotic response of melanoma cells to the combinatorial treatments. In SCID mice, the AZD6244–TRAIL association induced significant growth inhibition of a tumor resistant to TRAIL and poorly responsive to AZD6244, with no detectable adverse events on body weight and tissue histology. Reduction in tumor volume was associated not only with promotion of tumor apoptosis but also with suppression of the pro-angiogenic molecules HIF1α, VEGFα, IL-8 and TGFβ1 and with inhibition of tumor angiogenesis. These results suggest that synergistic co-targeting of oncogenic and death receptor pathways can not only overcome melanoma resistance to different anti-tumor agents in vitro but can also promote pro-apoptotic effects and inhibition of tumor angiogenesis in vivo.

Abstract 1155: Chemoresistance acquisition by ovarian adenocarcinoma cells due to microenvironment

Abstract Epithelial Ovarian Carcinoma is characterized by high frequency of recurrence (70% of patients) and carboplatin resistance acquisition. We recently showed that Carcinoma-Associated-Mesenchymal Stem Cells (CA-MSC) are involved in ovarian tumor growth via the facilitation of angiogenesis in the tumor site as well as in ovarian cancer chemoresistance acquisition. Our aim is to identify the mechanisms by which CA-MSC activate tumor cells signaling pathway for both effects. First we showed that factors released by CA-MSC are able to induce angiogenic cytokines (IL-6, IL-8 and VEGF) synthesis by tumor cells in a cell line specific way. Second, CA-MSC are able to attract and activate macrophages into a M2 TAM like phenotype and allow them to secrete a huge quantity of pro-angiogenic cytokines, favorable to tumor progression of all the associated ovarian adenocarcinoma cells tested. Third, factors released by CA-MSC protect adenocarcinoma cells from carboplatin-induced apoptosis by inhibiting the activation of effector caspases 3 and 7, inducing the activation of PI3K/Akt pathway signalling and the phosphorylation of the downstream target XIAP (caspase inhibitor from IAP family). XIAP depletion by siRNA strategy or inhibitors permitted to restore carboplatin-induced apoptosis in ovarian cancer cells stimulated by CA-MSC conditioned medium . Our results suggested targeting factors released by CA-MSC could be of interest in ovarian cancer therapy. Note: This abstract was not presented at the meeting. Citation Format: Benoit Thibault, Magali Castells, Delphine Mihas, Ludivine Genre, Cecile Gandy, Eliane Mery, Jean Pierre Delord, Bettina C. Couderc. Chemoresistance acquisition by ovarian adenocarcinoma cells due to microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1155. doi:10.1158/1538-7445.AM2014-1155

The effects of dexamethasone on the proliferation and apoptosis of human ovarian cancer cells induced by paclitaxel.

BackgroundDexamethasone (DEX) has been routinely used as a pre-treatment in the clinical application of paclitaxel (PTX) to treat ovarian cancer. However, PTX-induced apoptosis might be inhibited by DEX. This study was undertaken to investigate the effects of DEX on the apoptosis induced by PTX.MethodsBoth of SKOV-3 and HO-8910 human ovarian cancer cells were divided into four groups: (1) untreated (Con); (2) treated with DEX (0.1 μM) alone; (3) treated with PTX (50 nM); and (4) pre-treated with DEX (0.1 μM), and 24 h later, treated with PTX (DEX + PTX). Cell proliferation was determined by the 3-(4,5)-dimethylthiahiazo (−z-y1)-3,5-di- phenytetrazoliumromide (MTT) dye uptake method, while cell apoptosis was analyzed by propidium iodide (PI) staining and flow cytometry. Then, reverse transcription polymerase chain reactions (RT-PCRs) were applied to semi-quantitative analysis, followed by western blot analysis. Statistical analysis was performed, with Fisher’s least significant difference test.ResultsOur results demonstrated that DEX can differentially inhibit SKOV-3 and HO-8910 cell proliferation induced by PTX and decrease the apoptosis rates in cancer cells. Pre-treatment with DEX could up-regulate the expressions of members of anti-apoptotic Bcl-2 family (Bcl-2 and Bcl-XL) and members of IAP family (survivin). The expression of cleaved caspase-3 was down-regulated by DEX, shown by semi-quantitative RT-PCRs and western blot analysis.ConclusionsOur data gained invaluable insights of the antagonistic mechanisms of DEX on PTX-induced cancer cell death and may provide new methods of using DEX as antineoplastic drugs or agents in the clinical treatment for ovarian cancer patients.

Induction of Apoptosis by Methyl Alcohol Extract of &lt;i&gt;Enteromorpha linza&lt;/i&gt; (Linnaeus) J Agardh in U937 Human Leukemia Cells

Purpose: To investigate the anti-cancer effect of methyl alcohol extract of Enteromorpha linza (Linnaeus) J. Agardh (MEEL) in U937 human leukemia cells.Methods: Cytotoxicity was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptosis was detected using 4',6-diamidino-2-phenylindole (DAPI) staining, agarose gel electrophoresis, and flow cytometry. Protein levels were determined by Western blot analysis. Caspase activity was measured spectrophotometrically at 405 nm.Results: MEEL inhibited U937 cell proliferation and induced apoptosis through up-regulation of death receptor-related gene expression, caspase-8 activation and truncation of Bid, which was associated with the loss of mitochondrial membrane potential. Subsequently, the levels of anti-apoptotic proteins such as Bcl-2 and Bcl-xL, and IAP family proteins decreased but those of pro-apoptotic proteins including Bax and Bad increased in MEEL-treated U937 cells. MEEL treatment also resulted in activation of caspase-9 and -3 as well as concomitant cleavage of poly(ADP-ribose) polymerase and phopholipase Cγ-1. However, pretreatment of U937 cells with z-VAD-fmk, a pan caspase inhibitor, abrogated chromatin condensation and DNA fragmentation and prevented cell death induced by the MEEL.Conclusion: The findings suggest that MEEL induced apoptosis in U937 cells through a signaling cascade of death-receptor-mediated extrinsic as well as mitochondria-mediated intrinsic pathways, thus raising the possibility that MEEL may be of value in the development of novel therapeutic approaches for treating leukemia.Keywords: Enteromorpha linza, Apoptosis, Caspase, U937 cells

Tazarotene induces apoptosis in human basal cell carcinoma via activation of caspase-8/t-Bid and the reactive oxygen species-dependent mitochondrial pathway.

Previous studies suggest that tazarotene, a new member of the acetylenic class of RARβ/γ selective retinoids which is approved to treat a variety of skin diseases, exhibits an anti-proliferative effect in human basal cell carcinoma (BCC) by triggering caspase-dependent apoptosis. However, the detailed molecular mechanisms underlying the anti-tumor activity of tazarotene are poorly understood. This study aims at investigating the molecular mechanisms of tazarotene-induced apoptosis in human BCC cells. Our results are the first to demonstrate that tazarotene induces mitochondria-dependent cleavage of caspase-9 and -3 and PARP in BCC cells by producing reactive oxygen species (ROS) and activating caspase-8 through both ROS and death receptor signaling. These events are accompanied by a decrease in BCL-2 and BCL-xl anti-apoptotic proteins as well as by survivin and XIAP, two IAP family members. Furthermore, our results presented for the first time that tazarotene triggers a convergence of the intrinsic and extrinsic apoptotic pathways via the caspase-8-truncated Bid signaling pathway. Collectively, these data provide insights into the molecular mechanisms underlying tazarotene-induced apoptosis in human BCC cells, suggesting that this compound is a potential anti-skin cancer drug.

Targeting Antiapoptotic and Proapoptotic Proteins for Novel Chronic Lymphocytic Leukemia Therapeutics

SUMMARY Deficiency in the apoptotic program is one of the hallmarks of chronic lymphocytic leukemia. Defective apoptosis mainly results from the constitutive activation of survival pathways, which leads to the transcription and overexpression of antiapoptotic factors. The latter include proteins of the Bcl-2 family and members of the IAP family. The strategy of inhibiting the expression or activity of these antiapoptotic factors has been extensively investigated. Conversely, upregulation of proapoptotic proteins, notably BH3-only members of the Bcl-2 family (capable of antagonizing their antiapoptotic counterparts) has also been consistently described. Either mechanism can promote apoptosis in chronic lymphocytic leukemia cells ex vivo. The present article recapitulates the mechanistic data and how they contribute to the development of therapeutic agents targeting apoptosis.

404 AMPK Positively Regulates Neurotensin Secretion Through Inhibition of mTORC1 Signaling

from SurviviniΔIEC and control mice. Survivin deletion was performed by addition of tamoxifen. Specimens were investigated by quantitative PCR, immunohistochemistry and western blot analysis. Results: Time course analysis of gene deletion of survivin in IECs revealed an initial pulse of apoptosis at the crypt bottom 48 hours after tamoxifen injection. This did not led to death of SurviviniΔIEC animals. Histological analysis at later time points revealed aberrant chromosome segregation in IECs and the formation of multinucleated cells starting from the crypt bottom. Importantly, these cells did not show signs of cell death but of DNA damage, as confirmed by phopho-H2AX and phospho-p53 positive nuclei. Quantitative PCR showed elevated gene expression levels of p21 and ddit3 (dna-damageinducible-transcript 3) in SurviviniΔIEC mice, suggesting mitotic catastrophe in IEC depleted of survivin. Conclusions: Although survivin belongs to the IAP family, we could not see extensive apoptosis after survivin deletion in IECs. Our data demonstrates that the loss of survivin initially leads to the death of apoptosis prone IECs, followed by the formation of multinucleated cells with signs of DNA damage in apoptosis reluctant IECs. This leads to an alternative cell death pathway, known as mitotic catastrophe.