Chimeric antigen receptor dendritic cells targeted delivery of a single tumoricidal factor for cancer immunotherapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells have been used to treat blood cancers by producing a wide variety of cytokines. However, they are not effective in treating solid cancers and can cause severe side-effects, including cytokine release syndrome. TNFα is a tumoricidal cytokine, but it markedly increases the protein levels of cIAP1 and cIAP2, the members of inhibitor of apoptosis protein (IAP) family of E3 ubiquitin ligase that limits caspase-induced apoptosis. Degradation of IAP proteins by an IAP antagonist does not effectively kill cancer cells but enables TNFα to strongly induce cancer cell apoptosis. It would be a promising approach to treat cancers by targeted delivery of TNFα through an inactive adoptive cell in combination with an IAP antagonist. METHODS: Human dendritic cells (DCs) were engineered to express a single tumoricidal factor, TNFα, and a membrane-anchored Mucin1 antibody scFv, named Mucin 1 directed DCs expressing TNFα (M-DCsTNF). The efficacy of M-DCsTNF in recognizing and treating breast cancer was tested in vitro and in vivo. RESULTS: Mucin1 was highly expressed on the surface of a wide range of human breast cancer cell lines. M-DCsTNF directly associated with MDA-MB-231 cells in the bone of NSG mice. M-DCsTNF plus an IAP antagonist, SM-164, but neither alone, markedly induce MDA-MB-231 breast cancer cell apoptosis, which was blocked by TNF antibody. Importantly, M-DCsTNF combined with SM-164, but not SM-164 alone, inhibited the growth of patient-derived breast cancer in NSG mice. CONCLUSION: An adoptive cell targeting delivery of TNFα combined with an IAP antagonist is a novel effective approach to treat breast cancer and could be expanded to treat other solid cancers. Unlike CAR-T cell, this novel adoptive cell is not activated to produce a wide variety of cytokines, except for additional overexpressed TNF, and thus could avoid the severe side effects such as cytokine release syndrome.

IAPs and RIPK1 mediate LPS-induced cytokine production in healthy subjects and Crohn's disease.

Innate immune activity fuels intestinal inflammation in Crohn's disease (CD), an inflammatory bowel disease. Identification and targeting of new molecular regulators of the innate activity are warranted to control the disease. Inhibitor of apoptosis proteins (IAPs) regulate both cell survival and inflammatory signaling. We investigated the effects of IAP inhibition by second mitochondria-derived activator of caspases (SMAC) mimetics (SMs) on innate responses and cell death to pathogen-associated molecular patterns in peripheral blood mononuclear cells (PBMCs) and monocytes. IAPs inhibited lipopolysaccharide (LPS)-induced expression of proinflammatory interleukin (IL)-1ß, IL-6. Likewise, LPS (but not muramyl dipeptide or Escherichia coli) induced TNF-α was inhibited in CD and control PBMCs. The SM effect was partially reversed by inhibition of receptor-interacting serine/threonine-protein kinase 1 (RIPK1). The effect was mainly cell death independent. Thus, IAP inhibition by SMs leads to reduced production of proinflammatory cytokines and may be considered in the efforts to develop new therapeutic strategies to control CD.

Birinapant selectively enhances immunotoxin-mediated killing of cancer cells conditional on the IAP protein levels within target cells.

Immunotoxins (ITs) target cancer cells via antibody binding to surface antigens followed by internalization and toxin-mediated inhibition of protein synthesis. The fate of cells responding to IT treatment depends on the amount and stability of specific pro-apoptotic and pro-survival proteins. When treated with a pseudomonas exotoxin-based immunotoxin (HB21PE40), the triple-negative breast cancer (TNBC) cell line MDA-MB-468 displayed a notable resistance to toxin-mediated killing compared to the epidermoid carcinoma cell line, A431, despite succumbing to the same level of protein synthesis inhibition. In a combination screen of ~1912 clinically relevant and mechanistically annotated compounds, we identified several agents that greatly enhanced IT-mediated killing of MDA-MB-468 cells while exhibiting only a modest enhancement for A431 cells. Of interest, two Smac mimetics, birinapant and SM164, exhibited this kind of differential enhancement. To investigate the basis for this, we probed cells for the presence of inhibitor of apoptosis (IAP) proteins and monitored their stability after the addition of immunotoxin. We found that high levels of IAPs inhibited immunotoxin-mediated cell death. Further, TNFα levels were not relevant for the combination's efficacy. In tumor xenograft studies, combinations of immunotoxin and birinapant caused complete regressions in MDA-MB-468tumor-bearing mice but not in mice with A431 tumors. We propose that IAPs constitute a barrier to immunotoxin efficacy which can be overcome with combination treatments that include Smac mimetics.

Proteasomal Degradation of TRAF2 Mediates Mitochondrial Dysfunction in Doxorubicin-Cardiomyopathy.

BACKGROUND: Cytokines such as tumor necrosis factor-α (TNFα) have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). Although TNFα can elicit different cellular responses, including survival or death, the mechanisms underlying these divergent outcomes in the heart remain cryptic. The E3 ubiquitin ligase TRAF2 (TNF receptor associated factor 2) provides a critical signaling platform for K63-linked polyubiquitination of RIPK1 (receptor interacting protein 1), crucial for nuclear factor-κB (NF-κB) activation by TNFα and survival. Here, we investigate alterations in TNFα-TRAF2-NF-κB signaling in the pathogenesis of DOX cardiotoxicity. METHODS: Using a combination of in vivo (4 weekly injections of DOX 5 mg·kg-1·wk-1) in C57/BL6J mice and in vitro approaches (rat, mouse, and human inducible pluripotent stem cell-derived cardiac myocytes), we monitored TNFα levels, lactate dehydrogenase, cardiac ultrastructure and function, mitochondrial bioenergetics, and cardiac cell viability. RESULTS: In contrast to vehicle-treated mice, ultrastructural defects, including cytoplasmic swelling, mitochondrial perturbations, and elevated TNFα levels, were observed in the hearts of mice treated with DOX. While investigating the involvement of TNFα in DOX cardiotoxicity, we discovered that NF-κB was readily activated by TNFα. However, TNFα-mediated NF-κB activation was impaired in cardiac myocytes treated with DOX. This coincided with loss of K63- linked polyubiquitination of RIPK1 from the proteasomal degradation of TRAF2. Furthermore, TRAF2 protein abundance was markedly reduced in hearts of patients with cancer treated with DOX. We further established that the reciprocal actions of the ubiquitinating and deubiquitinating enzymes cellular inhibitors of apoptosis 1 and USP19 (ubiquitin-specific peptidase 19), respectively, regulated the proteasomal degradation of TRAF2 in DOX-treated cardiac myocytes. An E3-ligase mutant of cellular inhibitors of apoptosis 1 (H588A) or gain of function of USP19 prevented proteasomal degradation of TRAF2 and DOX-induced cell death. Furthermore, wild-type TRAF2, but not a RING finger mutant defective for K63-linked polyubiquitination of RIPK1, restored NF-κB signaling and suppressed DOX-induced cardiac cell death. Last, cardiomyocyte-restricted expression of TRAF2 (cardiac troponin T-adeno-associated virus 9-TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX. CONCLUSIONS: Our findings reveal a novel signaling axis that functionally connects the cardiotoxic effects of DOX to proteasomal degradation of TRAF2. Disruption of the critical TRAF2 survival pathway by DOX sensitizes cardiac myocytes to TNFα-mediated necrotic cell death and DOX cardiotoxicity.

The expanded inhibitor of apoptosis gene family in oysters possesses novel domain architectures and may play diverse roles in apoptosis following immune challenge.

BACKGROUND: Apoptosis plays important roles in a variety of functions, including immunity and response to environmental stress. The Inhibitor of Apoptosis (IAP) gene family of apoptosis regulators is expanded in molluscs, including eastern, Crassostrea virginica, and Pacific, Crassostrea gigas, oysters. The functional importance of IAP expansion in apoptosis and immunity in oysters remains unknown. RESULTS: Phylogenetic analysis of IAP genes in 10 molluscs identified lineage specific gene expansion in bivalve species. Greater IAP gene family expansion was observed in C. virginica than C. gigas (69 vs. 40), resulting mainly from tandem duplications. Functional domain analysis of oyster IAP proteins revealed 3 novel Baculoviral IAP Repeat (BIR) domain types and 14 domain architecture types across gene clusters, 4 of which are not present in model organisms. Phylogenetic analysis of bivalve IAPs suggests a complex history of domain loss and gain. Most IAP genes in oysters (76% of C. virginica and 82% of C. gigas), representing all domain architecture types, were expressed in response to immune challenge (Ostreid Herpesvirus OsHV-1, bacterial probionts Phaeobacter inhibens and Bacillus pumilus, several Vibrio spp., pathogenic Aliiroseovarius crassostreae, and protozoan parasite Perkinsus marinus). Patterns of IAP and apoptosis-related differential gene expression differed between the two oyster species, where C. virginica, in general, differentially expressed a unique set of IAP genes in each challenge, while C. gigas differentially expressed an overlapping set of IAP genes across challenges. Apoptosis gene expression patterns clustered mainly by resistance/susceptibility of the oyster host to immune challenge. Weighted Gene Correlation Network Analysis (WGCNA) revealed unique combinations of transcripts for 1 to 12 IAP domain architecture types, including novel types, were significantly co-expressed in response to immune challenge with transcripts in apoptosis-related pathways. CONCLUSIONS: Unprecedented diversity characterized by novel BIR domains and protein domain architectures was observed in oyster IAPs. Complex patterns of gene expression of novel and conserved IAPs in response to a variety of ecologically-relevant immune challenges, combined with evidence of direct co-expression of IAP genes with apoptosis-related transcripts, suggests IAP expansion facilitates complex and nuanced regulation of apoptosis and other immune responses in oysters.

The conservation of IAP-like proteins in fungi, and their potential role in fungal programmed cell death.

Programmed cell death (PCD) is a tightly regulated process which is required for survival and proper development of all cellular life. Despite this ubiquity, the precise molecular underpinnings of PCD have been primarily characterized in animals. Attempts to expand our understanding of this process in fungi have proven difficult as core regulators of animal PCD are apparently absent in fungal genomes, with the notable exception of a class of proteins referred to as inhibitors of apoptosis proteins (IAPs). These proteins are characterized by the conservation of a distinct Baculovirus IAP Repeat (BIR) domain and animal IAPs are known to regulate a number of processes, including cellular death, development, organogenesis, immune system maturation, host-pathogen interactions and more. IAP homologs are broadly conserved throughout the fungal kingdom, but our understanding of both their mechanism and role in fungal development/virulence is still unclear. In this review, we provide a broad and comparative overview of IAP function across taxa, with a particular focus on fungal processes regulated by IAPs. Furthermore, their putative modes of action in the absence of canonical interactors will be discussed.

Xevinapant or placebo plus chemoradiotherapy in locally advanced squamous cell carcinoma of the head and neck: TrilynX phase III study design.

Xevinapant is a first-in-class antagonist of inhibitor of apoptosis proteins, which enhances cancer cell sensitivity to chemotherapy and radiotherapy. In a phase II randomized study in patients with unresected locally advanced squamous cell carcinoma of the head and neck (LA SCCHN), xevinapant plus standard-of-care cisplatin-based chemoradiotherapy (CRT) showed superior efficacy versus placebo plus CRT. Here, we describe the design of TrilynX (NCT04459715), a randomized, double-blind, phase III study. In total, 700 patients with unresected LA SCCHN will be randomized 1:1 to receive xevinapant or placebo plus standard-of-care CRT followed by xevinapant monotherapy or placebo. The primary end point is event-free survival by blinded independent review committee. Secondary end points include progression-free survival, locoregional control, overall survival and safety.

Xevinapant is being developed as a new type of cancer treatment. Xevinapant works by enhancing the effects of chemotherapy and radiotherapy (chemoradiotherapy), which are standard anticancer treatments. Researchers are studying whether adding xevinapant to these treatments could be helpful for people with head and neck cancers that have not spread to other parts of the body and cannot be removed by surgery. In a study of 96 people with this disease, those treated with chemoradiotherapy plus xevinapant on average lived longer than people treated with chemoradiotherapy plus placebo (liquid that looked the same but did not contain any medicine). To confirm the results, researchers have started a larger study, called TrilynX, that will compare the same treatments in around 700 people worldwide. This study will show if adding xevinapant to chemoradiotherapy can help to keep the cancer from progressing, control symptoms better and help people live longer. Clinical trial registration: NCT04459715 (ClinicalTrials.gov).

Therapeutic strategies involving survivin inhibition in cancer.

Survivin is a small protein that belongs to the inhibitor of apoptosis protein family. It is abundantly expressed in tumors compared with adult differentiated tissues, being associated with poor prognosis in many human neoplasms. This apoptotic inhibitor has a relevant role in both the promotion of cancer cell survival and in the inhibition of cell death. Consequently, aberrant survivin expression stimulates tumor progression and confers resistance to several therapeutic strategies in a variety of tumors. In fact, efficient survivin downregulation or inhibition results in spontaneous apoptosis or sensitization to chemotherapy and radiotherapy. Therefore, all these features make survivin an attractive therapeutic target to treat cancer. Currently, there are several survivin inhibitors under clinical evaluation, although more specific and efficient survivin inhibitors are being developed. Moreover, novel combination regimens targeting survivin together with other therapeutic approaches are currently being designed and assessed. In this review, recent progress in the therapeutic options targeting survivin for cancer treatment is analyzed. Direct survivin inhibitors and their current development status are explored. Besides, the major signaling pathways implicated in survivin regulation are described and different therapeutic approaches involving survivin indirect inhibition are evaluated. Finally, promising novel inhibitors under preclinical or clinical evaluation as well as challenges of developing survivin inhibitors as a new therapy for cancer treatment are discussed.

Differentiated macrophages acquire a pro-inflammatory and cell death-resistant phenotype due to increasing XIAP and p38-mediated inhibition of RipK1.

Monocytes differentiate into macrophages, which deactivate invading pathogens. Macrophages can be resistant to cell death mechanisms in some situations, and the mechanisms involved are not clear. Here, using mouse immune cells, we investigated whether the differentiation of macrophages affects their susceptibility to cell death by the ripoptosome/necrosome pathways. We show that treatment of macrophages with a mimetic of second mitochondrial activator of caspases (SMAC) resulted in ripoptosome-driven cell death that specifically depended on tumor necrosis factor α (TNFα) expression and the receptor-interacting serine/threonine protein kinase 1 (RipK1)-RipK3-caspase-8 interaction in activated and cycling macrophages. Differentiation of macrophages increased the expression of pro-inflammatory cytokines but reduced RipK1-dependent cell death and the RipK3-caspase-8 interaction. The expression of the anti-apoptotic mediators, X-linked inhibitor of apoptosis protein (XIAP) and caspase-like apoptosis regulatory protein (cFLIPL), also increased in differentiated macrophages, which inhibited caspase activation. The resistance to cell death was abrogated in XIAP-deficient macrophages. However, even in the presence of increased XIAP expression, inhibition of the mitogen-activated protein kinase (MAPK) p38 and MAPK-activated protein kinase 2 (MK2) made differentiated macrophages susceptible to cell death. These results suggest that the p38/MK2 pathway overrides apoptosis inhibition by XIAP and that acquisition of resistance to cell death by increased expression of XIAP and cFLIPL may allow inflammatory macrophages to participate in pathogen control for a longer duration.

Copper-imidazo[1,2-a]pyridines induce intrinsic apoptosis and modulate the expression of mutated p53, haem-oxygenase-1 and apoptotic inhibitory proteins in HT-29 colorectal cancer cells.

Metastatic colorectal cancer responds poorly to treatment and is a leading cause of cancer related deaths. Worldwide, chemotherapy of metastatic colorectal cancer remains plagued by poor efficacy, development of resistance and serious adverse effects. Copper-imidazo[1,2-a]pyridines were previously shown by our group to be selectively active against several cancer cell lines, with three complexes, JD46(27), JD47(29), and JD88(21), showing IC50 values between 0.8 and 1.8 µM against HT-29 cells. Here, we report that treatment with the copper complexes resulted in fragmented nuclei suggestive of apoptotic cell death, which was confirmed by increased annexin V binding and caspase-3/7 activity. The copper complexes caused a loss of mitochondrial membrane potential and increased caspase-9 activity. The absence of caspase-8 activity indicated activation of the intrinsic pathway. Proteomic analysis revealed that copper-imidazo[1,2-a]pyridines decreased the expression of phosphorylated forms of p53 [phospho-p53(S15), phospho-p53(S46) and phospho-p53(S392)]. The expression of inhibitor of apoptosis proteins, XIAP, cIAP1, livin, and the antiapoptotic proteins, Bcl-2 and Bcl-x, was decreased. HO/HMOX/HSP32, expression was notably increased, which suggested the accumulation of reactive oxygen species. Increased expression of TRAIL-R2/DR5 death receptor indicated the possible dual activation of both the extrinsic and intrinsic apoptotic pathways; however, caspase-8 activation could not be demonstrated. In conclusion, the copper-imidazo[1,2-a]pyridines were effective inducers of apoptotic cell death at low micromolar concentrations and changed the expression levels of proteins important for cell survival and cell death. These copper complexes may be useful tools to better understand the complexity of signalling networks in cancer cell death in response to cell stress.

Glucocorticoid-dependent expression of IAP participates in the protection against TNF-mediated cytotoxicity in MCF7 cells.

BACKGROUND: Glucocorticoid receptor (GR) activation has been associated with breast cancer cell survival in vitro. Glucocorticoid (GC)-dependent protection against tumor necrosis factor (TNF)-induced cell death has been well characterized in MCF7 luminal A breast cancer cells. The GR activates a variety of protective mechanisms, such as inhibitors of apoptosis proteins (IAPs). However, the relative contribution of the GR-dependent expression of IAPs in the protection of cell death has not, to our knowledge, been evaluated. METHODS: MCF7 cells were used for all experiments. GR was activated with cortisol (CORT) or dexamethasone (DEX) and inhibited with mifepristone (RU486). Cell viability was determined in real-time with the xCELLigence™ RTCA System and at specific endpoints using crystal violet stain. The mRNA levels of the eight members of the IAP family were measured by qRT-PCR. The protein levels of GR, PR, ERα, HER2, PARP1, c-IAP1 and XIAP were evaluated by Western blot analysis. The knockdown of c-IAP1 and XIAP was accomplished via transient transfection with specific siRNAs. GR activation was verified by a gene reporter assay. Via the cBioportal interphase we queried the mRNA levels of GR and IAPs in breast cancer tumors. RESULTS: RU486 significantly inhibited the anti-cytotoxic effect of both GCs. PARP1 processing was diminished in the presence of both GCs. The combined treatments of GCs + TNF increased the relative mRNA levels of Survivin>c-IAP1 > NAIP>Apollon>XIAP>Ts-IAP > ML-IAP > c-IAP2. Additionally, GR mRNA content increased with the combined treatments of GCs + TNF. Sustained levels of the proteins c-IAP1 and XIAP were observed after 48 h of the combined treatments with GCs + TNF. With c-IAP1 and XIAP gene silencing, the GC-mediated protection was diminished. In the breast tumor samples, the GR mRNA was coexpressed with Apollon and XIAP with a Pearson coefficient greater than 0.3. CONCLUSIONS: The effect of GCs against TNF-mediated cytotoxicity involves increased mRNA expression and sustained protein levels of c-IAP1 and XIAP. The antagonist effects of RU486 and the qRT-PCR results also suggest the role of the GR in this process. This finding may have clinical implications because the GR and IAPs are expressed in breast tumor samples.

Cloning, expression, and purification of the recombinant pro-apoptotic dominant-negative survivin T34A-C84A protein in Escherichia coli.

Survivin is a well-known inhibitor-of-apoptosis proteins family member and a promising molecular target for anti-cancer treatment. However, it is widely accepted that survivin is only a "semi-druggable" target and development of survivin-specific small molecule inhibitors has shown to be difficult. In this study, we demonstrated that a histidine-tagged survivin T34A-C84A mutated protein (T34A-C84A-dNSur-His) can be produced using a bacterial recombinant protein expression system [E. coli ArcticExpress (DE3) cells] and solubilized using 1% (w/v) Sarkosyl. In addition, we showed that the purified T34A-C84A-dNSur-His protein formed dimers as predicted by in silico protein structure and molecular dynamics analysis. Importantly, results of the MTT assay revealed that the purified recombinant protein was biologically active in decreasing the viability of the human MDA-MB-231 breast adenocarcinoma and MIA-PaCa pancreatic carcinoma cells in vitro. Furthermore, the purified T34A-C84A-dNSur-His protein, but not of the histidine-peptide, induced apoptosis (i.e. caspase-9 activation and DNA fragmentation) in MDA-MB-231 cells at concentrations from 50 to 400 nM. In conclusion, our study provides a protocol of producing a biologically active survivin-targeting macromolecule, T34A-C84A-dNSur-His, which can be used as a tool for studying the molecular and cellular roles of survivin in cells. T34A-C84A-dNSur-His is also a potential therapeutic agent for augmenting cancer therapy.

Cellular inhibitor of apoptosis proteins prevent clearance of hepatitis B virus.

Hepatitis B virus (HBV) infection can result in a spectrum of outcomes from immune-mediated control to disease progression, cirrhosis, and liver cancer. The host molecular pathways that influence and contribute to these outcomes need to be defined. Using an immunocompetent mouse model of chronic HBV infection, we identified some of the host cellular and molecular factors that impact on infection outcomes. Here, we show that cellular inhibitor of apoptosis proteins (cIAPs) attenuate TNF signaling during hepatitis B infection, and they restrict the death of infected hepatocytes, thus allowing viral persistence. Animals with a liver-specific cIAP1 and total cIAP2 deficiency efficiently control HBV infection compared with WT mice. This phenotype was partly recapitulated in mice that were deficient in cIAP2 alone. These results indicate that antagonizing the function of cIAPs may promote the clearance of HBV infection.

Eliminating hepatitis B by antagonizing cellular inhibitors of apoptosis.

We have shown that cellular inhibitor of apoptosis proteins (cIAPs) impair clearance of hepatitis B virus (HBV) infection by preventing TNF-mediated killing/death of infected cells. A key question, with profound therapeutic implications, is whether this finding can be translated to the development of drugs that promote elimination of infected cells. Drug inhibitors of cIAPs were developed as cancer therapeutics to promote TNF-mediated tumor killing. These drugs are also known as Smac mimetics, because they mimic the action of the endogenous protein Smac/Diablo that antagonizes cIAP function. Here, we show using an immunocompetent mouse model of chronic HBV infection that birinapant and other Smac mimetics are able to rapidly reduce serum HBV DNA and serum HBV surface antigen, and they promote the elimination of hepatocytes containing HBV core antigen. The efficacy of Smac mimetics in treating HBV infection is dependent on their chemistry, host CD4(+) T cells, and TNF. Birinapant enhances the ability of entecavir, an antiviral nucleoside analog, to reduce viral DNA production in HBV-infected animals. These results indicate that birinapant and other Smac mimetics may have efficacy in treating HBV infection and perhaps, other intracellular infections.

Changes in cIAP2, survivin and BimEL expression characterize the switch from autophagy to apoptosis in prolonged starvation.

BACKGROUND: Autophagy is a catabolic process involving the engulfment of cytoplasmic content within autophagosomes followed by their delivery to lysosomes. This process is a survival mechanism, enabling cells to cope with nutrient deprivation by degradation and recycling of macromolecules. Yet during continued stress such as prolonged starvation, a switch from autophagy to apoptosis is often detected. OBJECTIVE: In this work, we characterized the temporal dynamics of the transition from autophagy towards apoptosis with the aim of elucidating the molecular mechanism regulating the switch from survival autophagy to apoptotic cell death. RESULTS AND CONCLUSIONS: We defined an inverse relationship between apoptosis and autophagy spanning a period of 72 h, manifested by the sequential reduction in LC3 lipidation and the activation of caspase-3. The transition to apoptosis correlated with a selective decline in the mRNA and protein levels of two anti-apoptotic IAP family proteins, survivin and cIAP2 and a selective increase in the BH3-only protein, BimEL. This 'molecular signature' was common to several cell lines undergoing the switch from autophagy to apoptosis during prolonged starvation. Mechanistically, the increased BimEL protein levels resulted from its reduced binding to its specific E3 ligase, ßTrCP, leading to protein stabilization. Consistent with this, BimEL showed decreased phosphorylation at critical sites previously reported to be essential for binding to the E3 ligase. The decrease in the anti-apoptotic IAPs and the increase in the pro-apoptotic BimEL may thus constitute a molecular switch from autophagy to apoptosis during prolonged starvation.

Inhibitor of apoptosis proteins (IAPs) may be effective therapeutic targets for treating endometriosis.

STUDY QUESTION: What is the role of the inhibitor of apoptosis proteins (IAPs) in human endometriotic tissues and a mouse model of endometriosis? SUMMARY ANSWER: Four IAP proteins were expressed in endometriotic tissue indicating IAPs may be a key factor in the pathogenesis and progression of endometriosis. WHAT IS KNOWN ALREADY: Overexpression of IAPs protects against a number of proapoptotic stimuli. IAPs (c-IAP1, c-IAP2, XIAP and Survivin) are expressed in human ectopic endometrial stromal cells (ESCs) from ovarian endometriomas. STUDY DESIGN, SIZE, DURATION: Forty-eight women with or without ovarian endometrioma are included in this study. BALB/c mice (n = 24) were used for the mouse endometriosis model. Mice with surgically induced endometriosis were treated with an IAP antagonist (BV6) for 4 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human ectopic endometrial tissues from chocolate cysts and eutopic endometrial tissue were collected. ESCs were enzymatically isolated from these tissues. ESC proliferation was examined by 5-bromo-2'-deoxyuridine-enzyme-linked immunosorbent assay. IAPs expression in tissue derived from eutopic endometria and chocolate cysts was evaluated using real-time RT-PCR and immunohistochemistry. A homologous mouse endometriosis model was established by transplanting donor mouse uterine tissue into the abdominal cavities of recipient mice. After treating the mice with BV6 (i.p. 10 mg/ml), the extent of endometriosis-like lesions in mice was measured and proliferative activity assessed by Ki67 staining. All experiments were repeated a minimum of three times. MAIN RESULTS AND THE ROLE OF CHANCE: IAP (c-IAP1, c-IAP2, XIAP and Survivin) mRNA and protein in human ectopic endometrial tissues were expressed at higher levels than in eutopic endometrial tissues (P < 0.05). All four IAPs proteins were expressed in mouse endometriosis-like implants. BV6 inhibited BrdU incorporation of human ESCs (P < 0.05 versus control). BV6 also decreased the total number, weight, surface area and Ki67 positive cells in the endometriosis-like lesions in the mice (P < 0.05 versus control). LIMITATIONS, REASONS FOR CAUTION: Endometriotic lesions were surgically induced in mice by transplanting mouse uterine tissue only, not human pathological endometriotic tissue. Furthermore, the effects of BV6 on human ESCs and mouse endometriosis-like lesions may differ between the species. WIDER IMPLICATIONS OF THE FINDINGS: Our data support the hypothesis that IAPs are involved in the development of endometriosis, and therefore an inhibitor of IAPs has potential as a novel treatment for endometriosis. STUDY FUNDING/COMPETING INTERESTS: This work was supported by KAKENHI (Japan Society for the Promotion of Science, Grant-in-Aid: to F.T.; 21592098 and to T.H.; 24659731) and Yamaguchi Endocrine Research Foundation. The authors have no conflicts of interest to disclose.

The role of p21 Waf1/Cip1 in large airway epithelium in smokers with and without COPD.

Airway epithelium alterations, including squamous cell metaplasia, characterize smokers with and without chronic obstructive pulmonary disease (COPD). The p21 regulates cell apoptosis and differentiation and its role in COPD is largely unknown. Molecules regulating apoptosis (cytoplasmic p21, caspase-3), cell cycle (nuclear p21), proliferation (Ki67/PCNA), and metaplasia (survivin) in central airways from smokers (S), smokers-COPD (s-COPD) and non-smokers (Controls) were studied. The role of cigarette smoke extracts (CSE) in p21, survivin, apoptosis (caspase-3 and annexin-V binding) and proliferation was assessed in a bronchial epithelial cell line (16HBE). Immunohistochemistry, image analysis in surgical samples and flow-cytometry and carboxyfluorescein succinimidyl ester proliferative assay in 16HBE with/without CSE were applied. Cytoplasmic and nuclear p21, survivin, and Ki67 expression significantly increased in large airway epithelium in S and in s-COPD in comparison to Controls. Caspase-3 was similar in all the studied groups. p21 correlated with epithelial metaplasia, PCNA, and Ki67 expression. CSE increased cytoplasmic p21 and survivin expression but not apoptosis and inhibited the cell proliferation in 16HBE. In large airway epithelium of smokers with and without COPD, the cytoplasmic p21 inhibits cell apoptosis, promotes cell proliferation and correlates with squamous cell metaplasia thus representing a potential pre-oncogenic hallmark.

Molecular Dynamics simulations of Inhibitor of Apoptosis Proteins and identification of potential small molecule inhibitors.

Chemotherapeutic resistance due to over expression of Inhibitor of Apoptosis Proteins (IAPs) XIAP, survivin and livin has been observed in various cancers. In the current study, Molecular Dynamics (MD) simulations were carried out for all three IAPs and a common ligand binding scaffold was identified. Further, a novel sequence based motif specific to these IAPs was designed. SMAC is an endogenous inhibitor of IAPs. Screening of ChemBank for compounds similar to lead SMAC-non-peptidomimetics yielded a cemadotin related compound NCIMech_000654. Cemadotin is a derivative of natural anti-tumor peptide dolastatin-15; hence these compounds were docked against all three IAPs. Based on our analysis, we propose that NCIMech_000654/dolastatin-15/cemadotin derivatives may be investigated for their potential in inhibiting XIAP, survivin and livin.

The survivin/XIAP suppressant YM155 impairs clonal growth and induces apoptosis in JAK2V617F cells.

The central role of the control of apoptosis in the pathophysiology of Philadelphia chromosome-negative myeloproliferative neoplasms has recently been reinforced in genetic and pharmacological studies. The inhibitor of apoptosis protein family has eight members and plays an important role in apoptosis, with the most studied being survivin (BIRC5) and X-linked inhibitor of apoptosis (XIAP). YM155 is a small molecule with antineoplastic potential that has been described as a suppressant of survivin and XIAP. In the present study, BIRC5 expression was significantly increased in primary myelofibrosis patients compared to healthy donors. On the other hand, XIAP expression was reduced in myeloproliferative neoplasms patients. In JAK2V617F cells, YM155 reduces cell viability and autonomous clonal growth and induces apoptosis, cell cycle arrest, and autophagy. HEL cells that show greater malignancy are more sensitive to the drug than SET2 cells. In the molecular scenario, YM155 modulates apoptosis-, cell cycle-, DNA damage- and autophagy-related genes. Protein expression analysis corroborates the observed cellular phenotype and exploratory gene expression findings. In summary, our results indicate that survivin/BIRC5 and XIAP are differently expressed in myeloproliferative neoplasms and YM155 has multiple antineoplastic effects on JAK2V617F cells suggesting that inhibitor of apoptosis proteins may be a target for pharmacological interventions in the treatment of these diseases.

SMAC Mimetics for the Treatment of Lung Carcinoma: Present Development and Future Prospects.

BACKGROUND: Uncontrolled cell growth and proliferation, which originate from lung tissue often lead to lung carcinoma and are more likely due to smoking as well as inhaled environmental toxins. It is widely recognized that tumour cells evade the ability of natural programmed death (apoptosis) and facilitates tumour progression and metastasis. Therefore investigating and targeting the apoptosis pathway is being utilized as one of the best approaches for decades. OBJECTIVE: This review describes the emergence of SMAC mimetic drugs as a treatment approach, its possibilities to synergize the response along with current limitations as well as future perspective therapy for lung cancer. METHOD: Articles were analysed using search engines and databases namely Pubmed and Scopus. RESULT: Under cancerous circumstances, the level of Inhibitor of Apoptosis Proteins (IAPs) gets elevated, which suppresses the pathway of programmed cell death, plus supports the proliferation of lung cancer. As it is a major apoptosis regulator, natural drugs that imitate the IAP antagonistic response like SMAC mimetic agents/Diablo have been identified to trigger cell death. SMAC i.e. second mitochondria activators of caspases is a molecule produced by mitochondria, stimulates apoptosis by neutralizing/inhibiting IAP and prevents its potential responsible for the activation of caspases. Various preclinical data have proven that these agents elicit the death of lung tumour cells. Apart from inducing apoptosis, these also sensitize the cancer cells toward other effective anticancer approaches like chemo, radio, or immunotherapies. There are many SMAC mimetic agents such as birinapant, BV-6, LCL161, and JP 1201, which have been identified for diagnosis as well as treatment purposes in lung cancer and are also under clinical investigation. CONCLUSION: SMAC mimetics acts in a restorative way in the prevention of lung cancer.