Cellular Inhibitor Of Apoptosis Protein Research Articles

Proteasome inhibitor PS-341 limits macrophage necroptosis by promoting cIAPs-mediated inhibition of RIP1 and RIP3 activation

Apoptotic and necrotic macrophages have long been known for their existence in atherosclerotic lesions. However, the mechanisms underlying the choice of their death pattern have not been fully elucidated. Here, we report the effects of PS-341, a potent and specific proteasome inhibitor, on the cell death of primary bone marrow-derived macrophages (BMDMs) in vitro. The results showed that PS-341 could not induce macrophage apoptosis or promote TNF-induced macrophage apoptosis, on the other hand, PS-341 could significantly inhibit macrophage necroptosis induced by TNF and pan-caspase inhibitor z-VAD treatment. Remarkably, high-dose of PS-341 showed similar inhibitory effects on macrophage necroptosis comparable to that of kinase inhibition of RIP1 through specific inhibitor Nec-1 or inhibition of RIP3 via specific genetical ablation. Furthermore, the degradation of cellular inhibitor of apoptosis proteins (cIAPs) was suppressed by PS-341, which could antagonize the activation of RIP1 kinase via post-translational mechanism. Further evidences demonstrated reduced levels of both RIP1 and RIP 3 upon PS-341 treatment, concomitantly, a more strong association of RIP1 with cIAPs and less with RIP3 was found following PS-341 treatment, these findings suggested that PS-341 may disrupt the formation of RIP1-RIP3 complex (necrosome) through stabilizing cIAPs. Collectively, our results indicated that the proteasome-mediated degradation of cIAPs could be inhibited by PS-341 and followed by limited RIP1 and RIP3 kinase activities, which were indispensable for necroptosis, thus eliciting a significant necroptosis rescue in BMDMs in vitro. Overall, our study has identified a new role of PS-341 in the cell death of BMDMs and provided a novel insight into the atherosclerotic inflammation caused by proteasome-mediated macrophage necroptosis.

Synergistic cytotoxicity of BIIB021 with triptolide through suppression of PI3K/Akt/mTOR and NF-κB signal pathways in thyroid carcinoma cells.

The effec.t of BIIB021, a novel heat shock protein 90 (hsp90) inhibitor, on survival of thyroid carcinoma cells has not been evaluated. In this study, the impact of BIIB021 alone or in combination with the histone acetyltransferase inhibitor triptolide on survival of thyroid carcinoma cells was identified. In 8505C and TPC-1 thyroid carcinoma cells, BIIB021 caused cell death in conjunction with alterations in expression of hsp90 client proteins. Cotreatment of both BIIB021 and triptolide, compared with treatment of BIIB021 alone, decreased cell viability, and increased the percentage of dead cells and cytotoxic activity. All of the combination index values were lower than 1.0, suggesting synergistic activity of BIIB021 with triptolide in induction of cytotoxicity. In treatment of both BIIB021 and triptolide, compared with treatment of BIIB021 alone, the protein levels of total and phospho-p53, and cleaved caspase-3 were elevated, while those of total Akt, phospho-mTOR, phospho-4EBP1, phospho-S6K, phospho-NF-κB, survivin, X-linked inhibitor of apoptosis protein (xIAP), cellular inhibitor of apoptosis protein (cIAP) and acetyl. histone H4 were reduced. These results suggest that BIIB021 has a cytotoxic activity accompanied by regulation of hsp90 client proteins in thyroid carcinoma cells. Moreover, the synergism between BIIB021 and triptolide in induction of cytotoxicity is associated with the inhibition of PI3K/Akt/mTOR and NF-κB signal pathways, the underexpression of survivin and the activation of DNA damage response in thyroid carcinoma cells.

Helicobacter pylori Eradication Downregulates Cellular Inhibitor of Apoptosis Protein 2 in Gastric Carcinogenesis.

Background/AimsTo evaluate the expression of cellular inhibitor of apoptosis protein 2 (cIAP2) during gastric carcinogenesis after Helicobacter pylori (HP) infection and after HP eradication.MethodsWe divided non-cancer patients into four groups according to the status of HP infection and atrophic gastritis (AG)/intestinal metaplasia (IM). We compared cIAP2 mRNA expression among these four groups and patients with HP-positive early gastric cancer (EGC) by using real-time polymerase chain reaction (PCR). We evaluated the expression of cIAP2 messenger RNA (mRNA)/protein by using real-time PCR/immunohistochemistry and the degree of apoptosis with a terminal deoxynucleotidyl transferase-mediated nick end labeling assay before and 12 months after endoscopic submucosal dissection (ESD) in HP-positive EGC patients, regardless of whether they had undergone eradication therapy.ResultsThe expression of cIAP2 mRNA was significantly higher in the groups with HP(+), AG/IM(+), and HP-positive EGC than in the control, HP(+), and AG/IM(−) groups (p<0.005). In the HP eradication group, the expression of cIAP2 mRNA/protein significantly decreased (p=0.006) and apoptosis increased at the 12-month follow-up after ESD. In the HP noneradication group, the aforementioned changes were not found during the same follow-up period.ConclusionsThe expression of cIAP2 increased during gastric carcinogenesis after HP infection; HP eradication in the patients who had undergone ESD for EGC reversed overexpression of cIAP2 and suppressed cell apoptosis.

Neuron-derived orphan receptor 1 transduces survival signals in neuronal cells in response to hypoxia-induced apoptotic insults.

OBJECT Hypoxia can induce cell death or trigger adaptive mechanisms to guarantee cell survival. Neuron-derived orphan receptor 1 (NOR-1) works as an early-response protein in response to a variety of environmental stresses. In this study, the authors evaluated the roles of NOR-1 in hypoxia-induced neuronal insults. METHODS Neuro-2a cells were exposed to oxygen/glucose deprivation (OGD). Cell viability, cell morphology, cas-pase-3 activity, DNA fragmentation, and cell apoptosis were assayed to determine the mechanisms of OGD-induced neuronal insults. RNA and protein analyses were carried out to evaluate the effects of OGD on expressions of NOR-1, cAMP response element-binding (CREB), and cellular inhibitor of apoptosis protein 2 (cIAP2) genes. Translations of these gene expressions were knocked down using RNA interference. Mice subjected to traumatic brain injury (TBI) and NOR-1 was immunodetected. RESULTS Exposure of neuro-2a cells to OGD decreased cell viability in a time-dependent manner. Additionally, OGD led to cell shrinkage, DNA fragmentation, and cell apoptosis. In parallel, treatment of neuro-2a cells with OGD time dependently increased cellular NOR-1 mRNA and protein expressions. Interestingly, administration of TBI also augmented NOR-1 levels in the impacted regions of mice. As to the mechanism, exposure to OGD increased nuclear levels of the transcription factor CREB protein. Downregulating CREB expression using RNA interference simultaneously inhibited OGD-induced NOR-1 mRNA expression. Also, levels of cIAP2 mRNA and protein in neuro-2a cells were augmented by OGD. After reducing cIAP2 translation, OGD-induced cell death was reduced. Sequentially, application of NOR-1 small interfering RNA to neuro-2a cells significantly inhibited OGD-induced cIAP2 mRNA expression and concurrently alleviated hypoxia-induced alterations in cell viability, caspase-3 activation, DNA damage, and cell apoptosis. CONCLUSIONS This study shows that NOR-1 can transduce survival signals in neuronal cells responsible for hypoxiainduced apoptotic insults through activation of a CREB/cIAP2-dependent mechanism.

Efficient protein knockdown of HaloTag-fused proteins using hybrid molecules consisting of IAP antagonist and HaloTag ligand

We previously reported a protein knockdown system for HaloTag-fused proteins using hybrid small molecules consisting of alkyl chloride, which binds covalently to HaloTag, linked to BE04 (2), a bestatin (3) derivative with an affinity for cellular inhibitor of apoptosis protein 1 (cIAP1, a kind of ubiquitin ligase). This system addressed several limitations of prior protein knockdown technology, and was applied to degrade two HaloTag-fused proteins. However, the degradation activity of these hybrid small molecules was not potent. Therefore, we set out to improve this system. We report here the design, synthesis and biological evaluation of novel hybrid compounds 4a and 4b consisting of alkyl chloride linked to IAP antagonist MV1 (5). Compounds 4a and 4b were confirmed to reduce the levels of HaloTag-fused tumor necrosis factor α (HaloTag-TNFα), HaloTag-fused cell division control protein 42 (HaloTag-Cdc42), and unfused HaloTag protein in living cells more potently than did BE04-linked compound 1b. Analysis of the mode of action revealed that the reduction of HaloTag-TNFα is proteasome-dependent, and is also dependent on the linker structure between MV1 (5) and alkyl chloride. These compounds appear to induce ubiquitination at the HaloTag moiety of HaloTag-fused proteins. Our results indicate that these newly synthesized MV1-type hybrid compounds, 4a and 4b, are efficient tools for protein knockdown for HaloTag-fused proteins.

USP11: A key regulator of cIAP2 stability and sensitivity to SMAC mimetics

The critical function of cellular inhibitor of apoptosis proteins (cIAPs) in the protection of cancer cells from numerous apoptotic stimuli prompted the development of second mitochondria-derived activator of caspases (SMAC) mimetics. We recently addressed a novel survival pathway in which cIAP2 is induced by tumor necrosis factor-α and is stabilized by its specific deubiquitylase, USP11, rendering cells resistant to SMAC mimetics.

Development of a peptide-based inducer of nuclear receptors degradation

A peptide-based protein knockdown system for inducing nuclear receptors degradation via the ubiquitin–proteasome system was developed. Specifically, the designed molecules were composed of two biologically active scaffolds: a peptide that binds to the estrogen receptor α (ERα) surface and an MV1 molecule that binds to cellular inhibitors of apoptosis proteins (IAP: cIAP1/cIAP2/XIAP) to induce ubiquitylation of the ERα. The hybrid peptides induced IAP-mediated ubiquitylation followed by proteasomal degradation of the ERα. Those peptides were also applicable for inducing androgen receptor (AR) degradation.

Targeting Inhibitor of Apoptosis Proteins Protects from Bleomycin-Induced Lung Fibrosis.

Accumulation of apoptosis-resistant fibroblasts is a hallmark of pulmonary fibrosis. We hypothesized that disruption of inhibitor of apoptosis protein (IAP) family proteins would limit lung fibrosis. We first show that transforming growth factor-β1 and bleomycin increase X-linked IAP (XIAP) and cellular IAP (cIAP)-1 and -2 in murine lungs and mesenchymal cells. Functional blockade of XIAP and the cIAPs with AT-406, an orally bioavailable second mitochondria-derived activator of caspases (Smac) mimetic, abrogated bleomycin-induced lung fibrosis when given both prophylactically and therapeutically. To determine whether the reduction in fibrosis was predominantly due to AT-406-mediated inhibition of XIAP, we compared the fibrotic response of XIAP-deficient mice (XIAP(-/y)) with littermate controls and found no difference. We found no alterations in total inflammatory cells of either wild-type mice treated with AT-406 or XIAP(-/y) mice. AT-406 treatment limited CCL12 and IFN-γ production, whereas XIAP(-/y) mice exhibited increased IL-1β expression. Surprisingly, XIAP(-/y) mesenchymal cells had increased resistance to Fas-mediated apoptosis. Functional blockade of cIAPs with AT-406 restored sensitivity to Fas-mediated apoptosis in XIAP(-/y) mesenchymal cells in vitro and increased apoptosis of mesenchymal cells in vivo, indicating that the increased apoptosis resistance in XIAP(-/y) mesenchymal cells was the result of increased cIAP expression. Collectively, these results indicate that: (1) IAPs have a role in the pathogenesis of lung fibrosis; (2) a congenital deficiency of XIAP may be overcome by compensatory mechanisms of other IAPs; and (3) broad functional inhibition of IAPs may be an effective strategy for the treatment of lung fibrosis by promoting mesenchymal cell apoptosis.

FADD regulates NF-κB activation and promotes ubiquitination of cFLIPL to induce apoptosis.

Tumor Necrosis Factor-α canonically induces the activation of NF-κB and associated gene product cellular FLICE-like inhibitory protein (cFLIPL) to promote cell survival. Previously, we demonstrated that ectopic expression of the Fas associated death domain (FADD) diminishes the expression of cFLIPL and transduces caspases-8 mediated apoptosis, independent of FasL stimulation in HEK 293T cells. However, the underlying molecular mechanism of FADD mediated ablation of cFLIP and NF-κB signaling to determining the fate of cell death or survival remains elusive. Here, we explored a novel molecular mechanism of FADD mediated apoptotic cell death that was directed by ubiquitination of cFLIPL and inhibition of NF-κB activation, independent of TNF-α stimulation. We found that induced expression of FADD firmly interacts with procaspase-8 and precludes cFLIPL to from the death inducing signaling complex (DISC). In addition, FADD negatively regulates cellular inhibitor of apoptosis protein 2 (cIAP2) and Bcl-2. Furthermore, FADD restrains cIAP2 expression and interacts with RIP1 and procaspase-8 to accomplish apoptotic cell death signaling. Interestingly, FADD was also found to promote JNK1 mediated activation of E3 ubiquitin ligase ITCH to degrade cFLIPL that may lead to commencement of apoptosis. Thus, FADD is an important regulator for determining the fate of cell death or survival.

Cytotoxic effects of SMAC-mimetic compound LCL161 in head and neck cancer cell lines.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumor entities worldwide. Unfortunately, recent drug developments in other fields of oncology have yielded no efficacy in the treatment of oral squamous cell carcinoma. As a new starting point, we investigated the impact of Fas ligand (FasL) and the SMAC-mimetic compound LCL161 in mono- and combination treatment in HNSCC cell lines. Five different cell lines of HNSCC were treated with FasL and LCL161 in mono- and combination treatment. Cytotoxicity was measured via a crystal violet assay. The cell lines were characterized for CD95 (FasL receptor) expression via flow cytometry. The degradation of cellular inhibitor of apoptosis protein 1 (cIAP1) was detected via Western blot. Incubation with FasL led to a significant decrease in three out of five cell lines. Combination treatment with LCL161 enhanced cytotoxicity significantly. Two cell lines were FasL resistant, but one of them could be resensitized with LCL161. In all cell lines, Western blot analysis showed degradation of cIAP1 after LCL161 application. However, one cell line showed only minor vulnerability to the FasL and LCL161 combination. This is the first study investigating combination treatment of FasL and LCL161 in head and neck cancer cell lines. Pro-apoptotic effects of the combination were detected in the majority of the cell lines. Interestingly, one of two FasL-resistant cell lines was sensitive to the combination therapy with FasL and LCL161. SMAC-mimetic compounds show promising results in the treatment of other tumor entities in vitro and might be useful drugs to improve HNSCC therapy.

PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy.

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.

Abstract A92: Aminomethylphosphonic acid inhibits human prostate xenograft tumor growth through interfering glycine synthesis in the cancer cells

Abstract Rapidly proliferating cancer cells consume more glycine than rapidly proliferating normal cells, which offers an opportunity to target glycine metabolism in the cancer cells while avoiding causing severe side effects in the normal cells. Two-thirds of glycine consumed is synthesized intracellularly, e.g., through conversion of serine into glycine by serine hydroxymethyltransferase (SHMT). Aminomethylphosphonic acid (AMPA, C3H3NO5P) is an analog to glycine, which inhibits SHMT enzyme activity, thus blocking conversion of serine into glycine. Our previous in-vitro studies have shown that AMPA inhibited cell growth in six cancer cell lines (including four human prostate cancer cell lines PC-3, DU-145, LNCaP, and C4-2B), while had little effects on two human normal immortalized prostatic epithelial cell lines (RWPE-1 and pRNS-1-1). The purpose of the present study was to determine if AMPA could inhibit PC-3 xenograft tumor growth in nude mice. PC-3-LacZ-luc cells (stably expressing LacZ and luciferase for staining and in-vivo imaging) were first implanted subcutaneously to form tumors. Then, the tumors were cut into 2-mm pieces and surgically implanted orthotopically in the prostates of 39 six-week-old athymic nude male mice that were castrated during the same surgery. One week later, the prostate tumor sizes were measured using D-luciferin and IVIS® Lumina XRMS imaging system (PerkinElmer, Inc.). The animals were randomized into three treatment groups: 1) saline as control (n = 14); 2) 400 mg/kg/day of AMPA (n= 10); and 3) 800 mg/kg/day of AMPA (n = 15). The treatment was administrated intraperitoneally once a day until animal death. Animal body weight measurement and in-vivo imaging were performed once every 5 to 7 days. Upon animal death, tumors were harvested and weighed. Pathologic examination, immunohistochemical staining, and Western blot analyses were performed using the tumors. We found that the tumor size rapidly increased in the control group, whereas the tumor size increased only slightly in the two AMPA-treated groups (p &amp;lt; 0.05). Animal survival time was significantly longer in the AMPA-treated groups than the control group (p &amp;lt; 0.05) and the high-dose group had slightly longer survival time than the low-dose group (p = 0.087). The average final tumor weight was significantly less in the high-dose group than the control group (p &amp;lt; 0.05). The levels of cellular inhibitor of apoptosis protein 1 (C-IAP1) and cyclin D1 were dramatically reduced in the tumors from the two AMPA-treated groups compared to the control group. We also observed that the two AMPA-treated groups had much less intra-abdominal metastases compared to the control group. Further in-vitro studies found that AMPA inhibited migration and invasion of PC-3 cells using Transwell assays. In conclusion, we found that AMPA inhibits human prostate xenograft tumor growth through inducing apoptosis and inhibiting cellular proliferation, which suggests that AMPA may be developed into a new treatment for prostate cancer. Citation Format: Keshab R. Parajuli, Qiuyang Zhang, Sen Liu, Zongbing You. Aminomethylphosphonic acid inhibits human prostate xenograft tumor growth through interfering glycine synthesis in the cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A92.

Targeting the X-Linked Inhibitor of Apoptosis Protein (XIAP) Can Promote Tumor Cell Death, and Increase the Cytotoxic Effects of Chemotherapy Agents in in Vitro and In Vivo Models of Rituximab-Resistant Lymphoma

In diffuse large B-cell lymphoma (DLBCL) the addition of rituximab to front line multi-agent therapy has changed the biology and clinical outcome of patients with relapsed/refractory disease. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines, which also display significant resistance to a wide range of chemotherapy agents. These therapy resistant cell lines (TRCL)s have multiple deregulations the apoptotic response pathway, including a loss of Bak and Bax. We previously demonstrated that small molecule mimetics of the second mitochondrial activator of caspases (SMAC), which antagonizes the inhibitor of apoptosis (IAP) family of proteins, could boost response to chemotherapy agents and extend survival in TRCL xenograft animal models. To investigate how SMAC mimetics overcome chemotherapy resistance in TRCL (RL-4RH and Raji-4RH) models we used RNA interference to transiently knockdown expression of individual IAPs (cIAP1, cIAP2, livin, survivin, and XIAP), and then exposed those cells to a panel of chemotherapy agents. Apoptosis induction was analyzed by flow cytometry staining for annexin V and Sytox blue (a DNA stain). While SMAC mimetics can decrease levels of the cellular inhibitor of apoptosis proteins (cIAP1/cIAP2), siRNA knockdown of cIAP1/cIAP2 did not increase chemotherapy-induced apoptosis. On the other hand, siRNA knockdown of XIAP in TRCLs substantially increased rates of apoptosis compared to cells transfected with non-targeting siRNA (5% in controls vs. 22% in XIAP knockdown RL-4RH cells). An additional 24% of XIAP knockdown RL-4RH cells showed signs of necrosis compared to 0.5% in control RL-4RH. Results were confirmed by western blot (PARP cleavage). In addition, siRNA XIAP knockdown re-sensitized TRCLs (Raji-4RH and RL-4H) to the cytotoxic effects of multiple chemotherapy agents (gemcitabine, etoposide, and vincristine). To investigate if XIAP knockdown could improve chemotherapy response in vivo we created a TRCL (Raji-4RH) that stably expresses XIAP targeting shRNA along with luciferase to enable in vivo imaging. SCID mice were inoculated with either the Raji-4RH XIAP knockdown or Raji-4RH non-targeting shRNA control cell line (10x106 cells per animal given i.v.). Tumors were allowed to engraft for 7 days prior to treatment administration. Animal were either given no treatment, or rituximab in combination with ifosfamide, carboplatin, etoposide (RICE) and mesna. Following treatment animals were imaged on a weekly basis to track tumor progression. At the time of the submission disease progression was present in animals in the observation groups, and in animals inoculated with non-targeting control shRNA Raji-4RH treated with RICE. InterestingHowever, animals inoculated with XIAP knockdown Raji-4RH and treated with RICE therapy remain tumor free with no detectable disease. In summary, XIAP acts as a key cell survival regulator in rituximab-chemotherapy resistant lymphoma models. Downregulation of XIAP can enhance chemotherapy activity and promote tumor cell death. Additionally, XIAP knockdown appears to enhance the activity of RICE, a commonly used regiment in the relapsed/refractory setting for DLBCL patients. Our results support the hypothesis that selective XIAP inhibitors, currently in development, may be highly active against relapsed/refractory B-cell lymphoma. DisclosuresCzuczman:Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board; MorphoSys: Consultancy; Celgene: Employment.

Catalytic-independent inhibition of cIAP1-mediated RIP1 ubiquitination by EGLN3

EGLN3 belongs to the EGLN family of prolyl hydroxylases that are able to catalyze the hydroxylation of proteins such as the α subunits of hypoxia-inducible factor. We and others have shown that EGLN3 negatively regulates the canonical NFκB pathway. Mechanistically, we demonstrated that EGLN3 inhibits ubiquitination of IKKγ (the regulatory subunit of IκB kinase complex) which is vitally important for NFκB activation. Polyubiquitination of the RIP1 (receptor-interacting protein 1) kinase is important for NFκB activation triggered by tumor necrosis factor α. It remains to be determined whether EGLN3 is able to modulate RIP1 ubiquitination catalyzed by cIAP1 (cellular inhibitor of apoptosis protein 1). This study shows that EGLN3 interacts with cIAP1 and suppresses cIAP1-mediated RIP1 ubiquitination via the C-terminal region. The hydroxylase activity is not required for the ability of EGLN3 to restrain RIP1 ubiquitination. Furthermore, EGLN3 is a novel binding protein of RIP1. The C-terminal region of EGLN3 is responsible for its interaction with RIP1. EGLN3 hydroxylase activity is not essential for the EGLN3–RIP1 interaction. EGLN3 interferes with the association between RIP1 and cIAP1, and attenuates RIP1-induced NFκB activation. This study provides novel insight into the mechanism underlying EGLN3 inhibition of NFκB signaling and sheds light on the regulation of RIP1 ubiquitination.

Autophagy-Mediated Degradation of IAPs and c-FLIP(L) Potentiates Apoptosis Induced by Combination of TRAIL and Chal-24.

Combination chemotherapy is an effective strategy for increasing anticancer efficacy, reducing side effects and alleviating drug resistance. Here we report that combination of the recently identified novel chalcone derivative, chalcone-24 (Chal-24), and TNF-related apoptosis-inducing ligand (TRAIL) significantly increases cytotoxicity in lung cancer cells. Chal-24 treatment significantly enhanced TRAIL-induced activation of caspase-8 and caspase-3, and the cytotoxicity induced by combination of these agents was effectively suppressed by the pan-caspase inhibitor z-VAD-fmk. Chal-24 and TRAIL combination suppressed expression of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein large (c-FLIP(L)) and cellular inhibitor of apoptosis proteins (c-IAPs), and ectopic expression of c-FLIP(L) and c-IAPs inhibited the potentiated cytotoxicity. In addition, TRAIL and Chal-24 cooperatively activated autophagy. Suppression of autophagy effectively attenuated cytotoxicity induced by Chal-24 and TRAIL combination, which was associated with attenuation of c-FLIP(L) and c-IAPs degradation. Altogether, these results suggest that Chal-24 potentiates the anticancer activity of TRAIL through autophagy-mediated degradation of c-FLIP(L) and c-IAPs, and that combination of Chal-24 and TRAIL could be an effective approach in improving chemotherapy efficacy.

Regulation of antiapoptotic and cytoprotective pathways in colonic epithelial cells in ulcerative colitis

Ulcerative colitis is an inflammatory bowel disease involving the colon resulting in bloody diarrhea and increased risk of colorectal cancer in certain patient subgroups. Increased apoptosis in the epithelial cell layer causes increased permeability, especially during flares; this leads to translocation of luminal pathogens resulting in a continued inflammatory drive. The present work investigates how epithelial apoptosis is regulated in ulcerative colitis. The main results are that Fas mediated apoptosis is inhibited during flares of ulcerative colitis, probably by an upregulation of cellular inhibitor of apoptosis protein 2 (cIAP2) and cellular FLICE-like inhibitory protein. cIAP2 is upregulated in regenerative epithelial cells both in ulcerative colitis and in experimental intestinal wounds. Inhibition of cIAP2 decreases wound healing in vitro possibly through inhibition of migration. Altogether, it is shown that epithelial cells in ulcerative colitis responds to the hostile microenvironment by activation of cytoprotective pathways that tend to counteract the cytotoxic effects of inflammation. However, the present studies also show that epithelial cells produce increased amounts of reactive oxygen species during stimulation with tumor necrosis factor-α and interferon-γ resulting in DNA instability. The combined effect of increased DNA-instability and decreased apoptosis responses could lead to neoplasia.

Fragment-Based Drug Discovery Targeting Inhibitor of Apoptosis Proteins: Discovery of a Non-Alanine Lead Series with Dual Activity Against cIAP1 and XIAP.

Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis and pro-survival signaling pathways whose deregulation is often associated with tumor genesis and tumor growth. IAPs have been proposed as targets for anticancer therapy, and a number of peptidomimetic IAP antagonists have entered clinical trials. Using our fragment-based screening approach, we identified nonpeptidic fragments binding with millimolar affinities to both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP). Structure-based hit optimization together with an analysis of protein-ligand electrostatic potential complementarity allowed us to significantly increase binding affinity of the starting hits. Subsequent optimization gave a potent nonalanine IAP antagonist structurally distinct from all IAP antagonists previously reported. The lead compound had activity in cell-based assays and in a mouse xenograft efficacy model and represents a highly promising start point for further optimization.

Abstract 2560: The cisplatin and Chal-24 combination synergistically kills cancer cells through IAP and c-FLIP degradation, Ripoptosome formation and autophagy-mediated apoptosis

Abstract Because chemoresistance is the major hurdle of anticancer therapy, sensitizing cancer cells to chemotherapy is a main challenge for improving survival in cancer patients. Combined therapy by using drugs with distinct functioning mechanisms may increase anticancer efficacy. We have recently identified the novel chalcone derivative chalcone-24 (Chal-24) as a potential therapeutic that kills cancer cells through activation of an autophagy-mediated necroptosis pathway. In this study, we investigated if Chal-24 can be combined with the frontline genotoxic anticancer drug cisplatin for cancer therapy. The combination of Chal-24 and cisplatin synergistically induced apoptotic cytotoxicity in lung cancer cell lines, which was dependent on Chal-24-induced autophagy. While cisplatin slightly potentiated the JNK/Bcl2/Beklin1 pathway for autophagy activation, it strongly triggered Chal-24-induced proteasomal degradation of the cellular inhibitor of apoptosis proteins (c-IAPs) and formation of the Ripoptosome complex that contains RIP1, FADD and caspase 8. Furthermore, cisplatin induced dramatic degradation of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein large (cFLIPL) which suppresses Ripoptosome-mediated apoptosis activation. These results establish a dual mechanism for potentiation of anticancer activity with combination of Chal-24 and cisplatin: to enhance apoptosis signaling through Ripoptosome formation and to release the apoptosis brake through c-FLIP degradation. Altogether, our work suggests that combination of Chal-24 and cisplatin could be employed for improving chemotherapy efficacy. (Supported by R01ES017328 (NIEHS/NIH) and DE-FG02-09ER64783 (DOE). Citation Format: Shaoqing Shi, Qiong Wang, Jennings Xu, Mabel T. Padilla, Toru Nyunoya, Chengguo Xing, Lin Zhang, Yong Lin. The cisplatin and Chal-24 combination synergistically kills cancer cells through IAP and c-FLIP degradation, Ripoptosome formation and autophagy-mediated apoptosis. [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 2560. doi:10.1158/1538-7445.AM2015-2560

ShRNA Depletion of cIAP1 Sensitizes Human Ovarian Cancer Cells to Anticancer Agent-Induced Apoptosis.

Emerging evidence suggests a potential role of cellular inhibitor of apoptosis protein 1 (cIAP1) in the development of human ovarian cancer. However, its function in the progression of ovarian cancer has not been clearly determined. Our study aimed to investigate the effect of cIAP1 gene depletion on the chemosensitivity of ovarian cancer cells. We developed a novel short hairpin RNA (shRNA) plasmid specifically targeting cIAP1. Cell proliferation, invasion, and apoptosis of the shRNA-transfected cells were evaluated using MTT, Transwell chamber, and flow cytometric assays, respectively. The concentration of MMP-9 in the supernatant was detected by ELISA. Targeted depletion of cIAP1 by shRNA significantly reduced expression levels of cIAP1 mRNA and protein, leading to inhibition of cell proliferation and invasion capability in SKOV3 cells. At the same time, cIAP1 downregulation decreased the secretion of MMP-9. shRNA depletion of cIAP1 enhanced chemosensitivity of ovarian cancer cells to Taxol and carboplatin-induced apoptosis. cIAP1 is associated with tumor progression in human ovarian cancer. Therefore, cIAP1 might be a potential target for therapeutic anticancer drugs.

RUNX3 contributes to carboplatin resistance in epithelial ovarian cancer cells

ObjectiveResistance to platinum-based therapeutic agents represents a major hurdle in the treatment of epithelial ovarian cancer (EOC). There is an urgent need to better understand the underlying mechanisms. Here, we investigated the role of RUNX3 in carboplatin resistance in EOC cells. MethodsExpression of RUNX3 was determined in human EOC cell line A2780s (cisplatin-sensitive) and A2780cp (cisplatin-resistant), human ovarian surface epithelium (OSE) and primary EOC cells. The effects of RUNX3 expression on sensitivity to carboplatin were determined in A2780s and A2780cp cells using neutral red uptake and clonogenic assays. Carboplatin-induced apoptosis was determined by measuring cleaved PARP using Western blotting. The expression of cellular inhibitor of apoptosis protein-2 (cIAP2) and its regulation by RUNX3 were assessed by quantitative RT-PCR and Western blotting. ResultsThe expression of RUNX3 was elevated in A2780cp cells compared to A2780s cells and in EOC tissues from chemoresistant patients compared to those from chemosensitive patients. Overexpression of RUNX3 rendered A2780s cells more resistant to carboplatin, whereas inhibition of RUNX3 increased sensitivity to carboplatin in A2780cp cells. Inhibition of RUNX3 potentiated carboplatin-induced apoptosis in A2780cp cells as demonstrated by more pronounced PARP cleavage. Interestingly, the expression of cIAP2 was elevated in A2780cp cells compared to A2780s cells. Overexpression of RUNX3 increased cIAP2 expression in A2780s cells, whereas inhibition of RUNX3 decreased cIAP2 expression and potentiated carboplatin-induced decrease of cIAP2 in A2780cp cells. ConclusionsRUNX3 contributes to carboplatin resistance in EOC cells and may hold promise as a therapeutic target to treat EOC and/or a biomarker to predict chemoresistance.