Inhibitor Of Apoptosis Protein Family Research Articles

Structural basis of the human NAIP/NLRC4 inflammasome assembly and pathogen sensing.

The NLR family caspase activation and recruitment domain-containing 4 (NLRC4) inflammasome is a critical cytosolic innate immune machine formed upon the direct sensing of bacterial infection and in response to cell stress during sterile chronic inflammation. Despite its major role in instigating the subsequent host immune response, a more complete understanding of the molecular events in the formation of the NLRC4 inflammasome in humans is lacking. Here we identify Bacillus thailandensis type III secretion system needle protein (Needle) as a potent trigger of the human NLR family apoptosis inhibitory protein (NAIP)/NLRC4 inflammasome complex formation and determine its structural features by cryogenic electron microscopy. We also provide a detailed understanding of how type III secretion system pathogen components are sensed by human NAIP to form a cascade of NLRC4 protomer through a critical lasso-like motif, a 'lock-key' activation model and large structural rearrangement, ultimately forming the full human NLRC4 inflammasome. These results shed light on key regulatory mechanisms specific to the NLRC4 inflammasome assembly, and the innate immune modalities of pathogen sensing in humans.

Caffeic acid phenethyl ester: Unveiling its potential as a potent apoptosis inducer for combating hypopharyngeal squamous cell carcinoma.

Hypopharyngeal squamous cell carcinoma (HSCC) is a relatively rare form of head and neck cancer that is notorious for its poor prognosis and low overall survival rate. This highlights the need for new therapeutic options for this malignancy. The objective of the present study was to examine the ability of caffeic acid phenethyl ester (CAPE), which is an active compound found in propolis, to combat HSCC tumor growth. CAPE exerted its tumor‑suppressive activity in HSCC cell lines through the induction of apoptosis. Mechanistically, the CAPE‑mediated apoptotic process was attributed to the perturbation of the mitochondrial membrane potential and the activation of caspase‑9. CAPE also modulated survivin and X‑linked inhibitor of apoptosis, which are potent members of the inhibitors of apoptosis protein family, either through transcriptional or post‑translational regulation, leading to HSCC cell line death. Therefore, the findings of the present study suggested that CAPE is an effective treatment alternative for HSCC via the stimulation of mitochondria‑dependent apoptosis.

Structural basis for flagellin-induced NAIP5 activation.

The NAIP (NLR family apoptosis inhibitory protein)/NLRC4 (NLR family CARD containing protein 4) inflammasome senses Gram-negative bacterial ligand. In the ligand-bound state, the winged helix domain of NAIP forms a steric clash with NLRC4 to open it up. However, how ligand binding activates NAIP is less clear. Here, we investigated the dynamics of the ligand-binding region of inactive NAIP5 and solved the cryo-EM structure of NAIP5 in complex with its specific ligand, FliC from flagellin, at 2.9-Å resolution. The structure revealed a "trap and lock" mechanism in FliC recognition, whereby FliC-D0C is first trapped by the hydrophobic pocket of NAIP5, then locked in the binding site by ID (insertion domain) and C-terminal tail of NAIP5. The FliC-D0N domain further inserts into ID to stabilize the complex. According to this mechanism, FliC triggers the conformational change of NAIP5 by bringing multiple flexible domains together.

Potentiating Tweezer Affinity to a Protein Interface with Sequence-Defined Macromolecules on Nanoparticles.

Survivin, a well-known member of the inhibitor of apoptosis protein family, is upregulated in many cancer cells, which is associated with resistance to chemotherapy. To circumvent this, inhibitors are currently being developed to interfere with the nuclear export of survivin by targeting its protein-protein interaction (PPI) with the export receptor CRM1. Here, we combine for the first time a supramolecular tweezer motif, sequence-defined macromolecular scaffolds, and ultrasmall Au nanoparticles (us-AuNPs) to tailor a high avidity inhibitor targeting the survivin-CRM1 interaction. A series of biophysical and biochemical experiments, including surface plasmon resonance measurements and their multivalent evaluation by EVILFIT, reveal that for divalent macromolecular constructs with increasing linker distance, the longest linkers show superior affinity, slower dissociation, as well as more efficient PPI inhibition. As a drawback, these macromolecular tweezer conjugates do not enter cells, a critical feature for potential applications. The problem is solved by immobilizing the tweezer conjugates onto us-AuNPs, which enables efficient transport into HeLa cells. On the nanoparticles, the tweezer valency rises from 2 to 16 and produces a 100-fold avidity increase. The hierarchical combination of different scaffolds and controlled multivalent presentation of supramolecular binders was the key to the development of highly efficient survivin-CRM1 competitors. This concept may also be useful for other PPIs.

P53 and survivin expression in renal cell carcinoma.

Mutation of p53 is detected in more than 50% of human cancers, expression of p53 has a potential prognostic value in patients with renal cell carcinoma (RCC). Survivin is a member of the inhibitor of apoptosis protein family, its overexpression is observed in many malignancies, including RCC. The aim of the study was to estimate a correlation between survivin and p53 expression in tumor samples and the histologic type of a tumor, tumor stage, tumor grade, and survival of patients. Tumor samples were collected from surgical specimens of 90 patients who underwent radical or partial nephrectomy for RCC between November 2017 and July 2020. Tumors were staged according to the UICC (The Union for International Cancer Control) TNM classification system and histopathologically graded according to Fuhrman nuclear grade system. Histopathological diagnosis was confirmed with standard light microscopic evaluation, using hematoxylin and eosin staining and standard p53 and survivin antibodies. Positive p53 staining was observed in 36.7% of tumor specimens and 24.4% were survivin positive. There was a statistically significant correlation between p53 or survivin expression and histologic subtype of clear cell RCC as well as Type I and II of papillary RCC. There was a statistically significant correlation between p53 expression and tumor size, stage, and grade. The p53 or survivin expression was related to lower overall survival. The results of this study suggest that p53 overexpression and survivin positivity in RCC patients could be associated with poor prognosis. Thus, these proteins could be used as prognostic markers in RCC.

Changes in expression levels of Nod-like receptors in the spleen of ewes.

Nucleotide-binding oligomerization domain receptors (NOD-like receptors, NLRs) have critical effects on interfaces of the immune and reproductive systems, and the spleen plays a key role in both innate and adaptive immune functions. It is hypothesized that NLR family participates in maternal splenic immune regulation during early pregnancy in sheep. In this study, maternal spleens were collected on day 16 of the estrous cycle, and days 13, 16 and 25 of gestation (n = 6 for each group) in ewes. Expression of NLR family, including NOD1, NOD2, class II transactivator (CIITA), NLR family apoptosis inhibitory protein (NAIP), nucleotide-binding oligomerization domain, Leucine rich repeat and Pyrin domain containing 1 (NLRP1), NLRP3 and NLRP7, was analyzed using quantitative real-time PCR, Western blot and immunohistochemistry analysis. The results revealed that expression levels of NOD1, NOD2, CIITA and NLRP3 were downregulated at days 13 and 16 of pregnancy, but expression of NLRP3 was increased at day 25 of pregnancy. In addition, expression values of NAIP and NLRP7 mRNA and proteins were improved at days 16 and 25 of pregnancy, and NLRP1 was peaked at days 13 and 16 of pregnancy in the maternal spleen. Furthermore, NOD2 and NLRP7 proteins were limited to the capsule, trabeculae and splenic cords. In summary, early pregnancy changes expression of NLR family in the maternal spleen, which may be related with the maternal splenic immunomodulation during early pregnancy in sheep.

Modulation of Nod-like Receptor Expression in the Thymus during Early Pregnancy in Ewes.

Nucleotide-binding oligomerization domain receptors (NOD-like receptors, NLRs) are involved in modulating the innate immune responses of the trophoblast and the placenta in normal pregnancy. The thymus participates in regulation of innate and adaptive immune responses. However, it is unclear whether expression of NLR is modulated in the maternal thymus during early pregnancy. In this study, thymuses were sampled at day 16 of the estrous cycle, and at days 13, 16 and 25 of gestation (n = 6 for each group) from ewes after slaughter. Different stages were chosen because the maternal thymus was under the different effects of interferon-tau and/or progesterone or not. RT-qPCR, Western blot and immunohistochemistry analysis were used to analyze the expression of the NLR family, including NOD1; NOD2; major histocompatibility complex class II transactivator (CIITA); NLR family apoptosis inhibitory protein (NAIP); nucleotide-binding oligomerization domain and Leucine-rich repeat and Pyrin domain containing protein 1 (NLRP1), NLRP3 and NLRP7. The results showed that expression level of NOD1 was changed with the pregnancy stages, and expression levels of NOD2, CIITA, NAIP, NLRP1, NLRP3 and NLRP7 mRNA and proteins were peaked at day 13 of pregnancy. The levels of NOD2 and CIITA were increased during early pregnancy. The stainings for NOD2 and NLRP7 proteins were located in epithelial reticular cells, capillaries and thymic corpuscles. In summary, pregnancy stages changed expression of NLR family in the maternal thymus, which may be related to the modulation of maternal thymic immune responses, and beneficial for normal pregnancy in sheep.

Association between septic patient mortality and blood survivin concentrations

ObjectiveSurvivin is a member of inhibitors of apoptosis proteins family. There are not data about the association between mortality of septic patients and blood survivin concentrations. Therefore, the objective of this study was to determine whether exist that association. DesignObservational and prospective study. SettingThree Spanish Intensive Care Units. PatientsPatients with sepsis or septic shock according to Sepsis-3 Consensus criteria. InterventionsSerum survivin concentrations were determined at moment of sepsis diagnosis. Main variable of interestMortality at 30 days. ResultsA total of 204 patients were included in the study, of which 75 (36.8%) died in the first 30 days. Lower age (p<0.001), serum lactic acid levels (p=0.001), rate of septic shock (p=0.001) and SOFA (p<0.001), and higher serum survivin levels (p=0.001) exhibited surviving (n=129) than non-surviving patients (n=75). We found in multiple logistic regression analysis an association between serum survivin concentrations and mortality independently of SOFA, lactic acid, age, INR, activated partial thromboplastin time (aPTT) and empiric antimicrobial treatment adequate (OR=0.968; 95% CI=0.946–0.990; p=0.005), and also independently of APACHE-II, lactic acid, platelet, INR, aPTT and empiric antimicrobial treatment adequate (OR=0.966; 95% CI=0.943–0.989; p=0.004). ConclusionsThere is an association between septic patient mortality and low blood survivin concentrations.

A novel inhibitor of apoptosis protein (IAPs) from Crassostrea gigas is involved in anti-apoptosis and innate defense role under Vibrio alginolyticus stimulation

Apoptosis is an essential biological process in vertebrates and invertebrates for the development and maintenance of homeostasis. Inhibition of apoptosis can help some pathogens survive in host cells and escape from immune surveillance. Importantly, it also plays an important role in immune signaling regulation from insects to mammals. In this study, we identified a new inhibitor of the apoptosis protein gene (IAP), CgIAP3 from the Pacific oyster C. gigas, which contained one BIR domain and one RING domain. Blast analysis showed that CgIAP3 share the highest sequence identity of 65.68% with C. virginica IAP8, and clustered with other shellfish IAP in the phylogenetic tree, indicating that CgIAP3 belonged to a member of the IAPs family of shellfish. Homology comparison showed that the BIR domain of CgIAP3 was highly conserved with the BIR3 domain of human IAP. The tissue distribution of CgIAP3 in healthy C. gigas showed that CgIAP3 is ubiquitously expressed in all analyzed tissues, with the highest level of expression in the gills and digestive gland than the adductor muscle and was rapidly up-regulated after V. alginolyticus stimulation. Moreover, cell experiments showed CgIAP3 could inhibit apoptosis by maintaining cell morphology and cell viability. The result of subcellular localization showed that CgIAP3 is widely distributed in cytoplasm and nucleus and only expressed in A549 cells with damaged nucleus. Taken together, we speculated that CgIAP3 might be involved in the innate immune process of the Pacific oyster against V. alginolyticus through maintaining host cell morphology and viability.

Vitamin D receptor enhances NLRC4 inflammasome activation by promoting NAIPs-NLRC4 association.

Inflammasomes are cytosolic multiprotein complexes that initiate host defense against bacterial pathogens. The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family caspase-associated recruitment domain-containing protein 4 (NLRC4) inflammasomes plays a critical role in the inflammatory response against intracellular bacterial infection. The NLR family apoptosis inhibitory proteins (NAIPs) detect Flagellin or type III secretion system (T3SS) microbial components to activate NLRC4 inflammasome. However, the underlying mechanism of NLRC4 inflammasome activation is not completely understood. Here, we show that the vitamin D receptor (VDR) is an essential immunological regulator of the NLRC4 inflammasome. Conditional VDR knockout mice (VDRflox/flox lyz2-Cre) exhibited impaired clearance of pathogens after acute Salmonella Typhimurium infection leading to poor survival. In macrophages, VDR deficiency reduced caspase-1 activation and IL-1β secretion upon S. Typhimurium infection. For NAIPs act as upstream sensors for NLRC4 inflammasome assembly, the further study demonstrated that VDR promoted the NAIP-NLRC4 association and triggered NAIP-NLRC4 inflammasome activation, not NLRP3 activation. Moreover, Lys123 residue of VDR is identified as the critical amino acid for VDR-NLRC4 interaction, and the mutant VDR (K123A) effectively attenuates the NLRC4 inflammasome activation. Together, our findings suggest that VDR is a critical regulator of NAIPs-NLRC4 inflammasome activation, mediating innate immunity against bacterial infection.

A novel survivin dimerization inhibitor without a labile hydrazone linker induces spontaneous apoptosis and synergizes with docetaxel in prostate cancer cells

Survivin, a member of the inhibitor of apoptosis protein family, exists as a homodimer and is aberrantly upregulated in a wide spectrum of cancers. It was thought to be an ideal target due to its lack of expression in most adult normal tissues and importance in cancer cell survival. However, it has been challenging to target survivin due to its “undruggable” nature. We previously attempted to target its dimerization domain with a hypothesis that inhibiting survivin dimerization would promote its degradation in proteasome, which led to identification of a lead small-molecule inhibitor, LQZ-7F. LQZ-7F consists of a flat tetracyclic aromatic core with labile hydrazone linking a 1,2,5-oxadiazole moiety. In this study, we tested the hypothesis that LQZ-7F could be developed as a prodrug because the labile hydrazone linker could be hydrolyzed, releasing the tetracyclic aromatic core. To this end, we synthesized the tetracyclic aromatic core (LQZ-7F1) using reported procedure and tested LQZ-7F1 for its biological activities. Here we show that LQZ-7F1 has a significantly improved potency with submicromolar IC50’s and induces spontaneous apoptosis in prostate cancer cells. It also more effectively inhibits survivin dimerization and induces survivin degradation in a proteasome-dependent manner than LQZ-7F. We also show that the combination of LQZ-7F1 and docetaxel have strong synergism in inhibiting prostate cancer cell survival. Together, we conclude that the hydrazone linker with the oxadiazole tail is dispensable for survivin inhibition and the survivin dimerization inhibitor, LQZ-7F, may be developed as a prodrug for prostate cancer treatment and to overcome docetaxel resistance.

Immunohistochemical expression of survivin in oral epithelial dysplasia and different grades of oral squamous cell carcinoma.

Survivin, a member of the inhibitor of apoptosis proteins family, is not detectable in most differentiated normal adult tissues but is expressed in a wide range of cancer tissues. Survivin expression in cancer has been associated with poor prognosis, cancer progression, and drug resistance, and the expression levels correlate with more aggressive disease and a poor clinical outcome. To evaluate and compare the immunoexpression of survivin in the normal oral epithelium (NOE), oral epithelial dysplasia (OED), and different grades of oral squamous cell carcinoma (OSCC). The patterns of survivin immunoexpression and immunoreactivity were assessed in previously diagnosed, paraffin-embedded sections of 10 tissues of NOE and 15 tissues each of OED and the three grades of OSCC (well-, moderately-, and poorly-differentiated). The pattern of survivin expression was recorded as cytoplasmic, nuclear, or both. Survivin immunoreactivity was assessed semi-quantitatively as the immunoreactive score (IRS). Analysis of variance and Tukey-HSD tests were employed for statistical analysis. No immunoreactivity for survivin was evident in the NOE tissues. In the OED tissues, the immunoexpression pattern of survivin was predominantly nuclear in the basal cells, and in the OSCC tissues, cytoplasmic and nuclear. IRS was highest among the moderately- differentiated OSCC, followed by poorly- and well-differentiated OSCC and OED, with a statistically significant difference in the IRS scores between the normal and the study groups. Survivin protein expression may be an important early event in oral carcinogenesis and may predict unfavorable prognosis in OSCC.

Targeting inhibitor of apoptosis proteins (IAPs) with IAP inhibitors sensitises malignant rhabdoid tumour cells to cisplatin

Malignant rhabdoid tumour (MRT) is a rare, aggressive paediatric malignancy most commonly diagnosed in those below the age of three. MRTs can arise in soft tissue but are more often associated with the central nervous system or kidney. Unfortunately, the prognosis upon diagnosis with MRT is poor. Given the resistance of MRT to current treatment protocols including cisplatin, and the vulnerability of this young patient population to aggressive therapies, there is a need for novel treatment options. Several members of the inhibitor of apoptosis protein (IAP) family including X‑linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/cIAP2), livin and survivin have been implicated in chemotherapy resistance in various malignancies. We have previously demonstrated expression of these IAP family members in a panel of MRT cell lines. In the present study, sensitivity of this same panel of MRT cell lines to small-molecule mediated inhibition of the IAPs via the survivin inhibitor YM155 and the XIAP/cIAP1/cIAP2 inhibitor BV6 was demonstrated. Additionally, both BV6 and the XIAP inhibitor embelin synergistically enhanced cisplatin mediated apoptotic cell death in MRT cell lines, with enhanced caspase-3 cleavage. Importantly, we have demonstrated, for the first time, expression of XIAP, its target caspase-3 and its endogenous inhibitor SMAC in rhabdoid tumour patient tissue. In conclusion, this study provides pre-clinical evidence that IAP inhibition may be a new therapeutic option in MRT.

DDX17 is an essential mediator of sterile NLRC4 inflammasome activation by retrotransposon RNAs.

Detection of microbial products by multiprotein complexes known as inflammasomes is pivotal to host defense against pathogens. Nucleotide-binding domain leucine-rich repeat (NLR) CARD domain containing 4 (NLRC4) forms an inflammasome in response to bacterial products; this requires their detection by NLR family apoptosis inhibitory proteins (NAIPs), with which NLRC4 physically associates. However, the mechanisms underlying sterile NLRC4 inflammasome activation, which is implicated in chronic noninfectious diseases, remain unknown. Here, we report that endogenous short interspersed nuclear element (SINE) RNAs, which promote atrophic macular degeneration (AMD) and systemic lupus erythematosus (SLE), induce NLRC4 inflammasome activation independent of NAIPs. We identify DDX17, a DExD/H box RNA helicase, as the sensor of SINE RNAs that licenses assembly of an inflammasome comprising NLRC4, NLR pyrin domain–containing protein 3, and apoptosis-associated speck-like protein–containing CARD and induces caspase-1 activation and cytokine release. Inhibiting DDX17-mediated NLRC4 inflammasome activation decreased interleukin-18 release in peripheral blood mononuclear cells of patients with SLE and prevented retinal degeneration in an animal model of AMD. Our findings uncover a previously unrecognized noncanonical NLRC4 inflammasome activated by endogenous retrotransposons and provide potential therapeutic targets for SINE RNA–driven diseases.

Inflammasome-independent functions of NAIPs and NLRs in the intestinal epithelium.

The gut relies on the complex interaction between epithelial, stromal and immune cells to maintain gut health in the face of food particles and pathogens. Innate sensing by the intestinal epithelium is critical for maintaining epithelial barrier function and also orchestrating mucosal immune responses. Numerous innate pattern recognition receptors (PRRs) are involved in such sensing. In recent years, several Nucleotide-binding-domain and Leucine-rich repeat-containing receptors (NLRs) have been found to partake in pathogen or damage sensing while also being implicated in gut pathologies, such as colitis and colorectal cancer (CRC). Here, we discuss the current literature focusing on NLR family apoptosis inhibitory proteins (NAIPs) and other NLRs that have non-inflammasome roles in the gut. The mechanisms behind NLR-mediated protection often converges on similar signalling pathways, such as STAT3, MAPK and NFκB. Further understanding of how these NLRs contribute to the maintenance of gut homeostasis will be important for understanding gut pathologies and developing new therapies.

The transcriptional activity profile of inhibitor apoptosis protein encoding genes in colon cancer patients: A STROBE-compliant study.

The inhibitor of apoptosis family proteins (IAPs) plays a crucial role in the process of carcinogenesis by regulating apoptosis and maintaining the tissue balance.In this study, a transcriptomic analysis of IAP-encoding genes in colon cancer was performed using oligonucleotide microarrays.Adenocarcinoma and healthy colon tissue samples were collected from 32 patients (16 females and 16 males) who underwent surgery due to colon cancer. The mRNA was extracted from tissue samples and tested using oligonucleotide microarrays (Affymetrix). The results were validated using the qRT-PCR technique. Hierarchical grouping was used to allocate 37 samples of normalized mRNA concentrations into 4 groups, with statistically significant differences in gene expression between these groups. The group of genes associated with colon cancer, including IAP-encoding gene - BIRC5 (Survivin), was selected for further testing.Our study confirmed an increased expression of BIRC5 in colon cancer tissue when compared to the control group. Increased levels of Neuronal Apoptosis Inhibitory Proteins were detected only in low-stage colon cancer, while the expression of Human X Chromosome-Encoded inhibitor of apoptosis family proteins decreased in colon cancer.The transcriptional activity of IAP-encoding genes varied, depending on the severity of colon cancer. The concentration of mRNA, encoding BIRC5 was elevated in samples obtained from more advanced colon cancer. Hence BIRC5 could be used as a complementary parameter for the diagnosis and prognosis of colon cancer.

Livin/BIRC7 gene expression as a possible diagnostic biomarker for endometrial hyperplasia and carcinoma

BackgroundLivin/BIRC7 is a member of the inhibitors of apoptosis proteins family which are implicated in development of cancer through the inhibition of apoptosis process. This case-control study was intended to investigate livin/BIRC7 gene expression in endometrial hyperplasia and carcinoma and its correlation to some oxidative stress markers in addition to its possible diagnostic performance. MethodsThis study included 90 participants [30 endometrial hyperplasia patients, 30 endometrial carcinoma patients, and 30 healthy controls]. Livin/BIRC7 gene expression was analyzed using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Serum catalase activity was assessed by enzyme-linked immunosorbent assay (ELISA) and malondialdehyde level was measured by the colorimetric method. ResultsLivin/BIRC7 gene expression was significantly (p < 0.001) higher in endometrial carcinoma from patients with endometrial hyperplasia when compared to controls. A positive correlation was found between livin/BIRC7 expression and serum catalase activity and malondialdehyde level in endometrial hyperplasia and carcinoma. The detection of livin/BIRC7 in endometrial carcinoma has excellent sensitivity and specificity. ConclusionsLivin/BIRC7 was overexpressed in endometrial carcinoma with excellent power to differentiate endometrial carcinoma from endometrial hyperplasia or healthy subjects, suggesting that it might be a useful molecular marker for endometrial carcinoma diagnosis.

Inhibitors of apoptosis: expression and regulation in the endometrium during the estrous cycle and at the maternal-conceptus interface during pregnancy in pigs.

ObjectiveCaspase-mediated apoptosis plays a crucial role in the regulation of endometrial and placental function in females. Caspase activity is tightly controlled by members of the inhibitors of apoptosis proteins (IAPs) family. However, the expression and regulation of IAPs at the maternal-conceptus interface has not been studied in pigs. Therefore, we determined the expression of IAP family members baculovirus IAP repeat-containing 1 (BIRC1) to BIRC6 at the maternal-conceptus interface in pigs.MethodsWe obtained endometrial tissues from pigs at various stages of the estrous cycle and pregnancy, conceptus tissues during early pregnancy, and chorioallantoic tissues during mid- to late pregnancy and analyzed the expression of IAPs. Furthermore, we determined the effects of the steroid hormones estradiol-17β (E2) and progesterone on the expression of IAPs in endometrial explant tissue cultures.ResultsDuring the estrous cycle, BIRC2 and BIRC5 expression varied cyclically, and during pregnancy, endometrial BIRC1, BIRC2, BIRC3, BIRC4, and BIRC5 expression varied in a stage-specific manner. Conceptus and chorioallantoic tissues also expressed IAPs during pregnancy. The BIRC2 and BIR3 mRNAs were localized to luminal epithelial cells, and BIRC4 proteins to glandular epithelial cells in the endometrium. Exposure of endometrial tissues to E2 increased the expression of BIRC6, while progesterone increased the expression of BIRC1, BIRC4, and BIRC6 in a dose-dependent manner.ConclusionThese results indicated that IAPs were expressed in the endometrium during the estrous cycle and at the maternal-conceptus interface during pregnancy in a stage-specific manner. In addition, steroid hormones were found to be responsible for the expression of some IAPs in pigs. Together, the results suggested that IAPs may play important roles in endometrial and placental functions by regulating caspase action and apoptosis at the maternal-conceptus interface.

SMAC exhibits anti-tumor effects in ECA109 cells by regulating expression of inhibitor of apoptosis protein family.

BACKGROUNDThe poor prognosis and rising incidence of esophageal cancer highlight the need for improved therapeutics that are essential prior to treatment. LCL161 is an SMAC (second mitochondrial activator of caspases) mimic and inhibitor of apoptosis protein (IAP) antagonist which exhibits anti-tumor effects and improves the chemical sensitivity of many cancers.AIMTo ascertain the effects and mechanisms of the SMAC analog LCL161 on esophageal cancer cells.METHODSMTT assay and TUNEL assay were used to detect cell proliferation and apoptosis, respectively. Western blot analysis was used to study the molecular mechanisms of LCL161-induced death of ECA109 cells.RESULTSLCL161 decreased ECA109 cell proliferation in dose- and time-dependent manner and induced apoptosis of ECA109 cells in a dose-dependent manner. Also, LCL161 induced a significant decrease in the expression of the XIAP and significant increase in the expression of Caspase-3. In addition, Bax increased significantly with increasing concentrations of LCL161, and the relative expression of Bax was significantly different between groups.CONCLUSIONThese findings support the hypothesis that LCL161 can inhibit proliferation and induce apoptosis in esophageal cancer cells by regulating the expression of IAP family members, suggesting that it has potential to be an effective treatment for esophageal squamous cell carcinoma.

Single Agent and Synergistic Combinatorial Efficacy of First-in-Class Small Molecule Imipridone ONC201 in Hematological Malignancies In Vitro and In Vivo through Apoptosis Via the Integrated Stress Response Pathway

▪ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies.ONC201 was evaluated in 20 human leukemia cell lines (Figure 1): 13 acute myeloid leukemia (AML), 5 acute lymphoblastic leukemia (ALL) and 2 chronic myelogenous leukemia (CML) cell lines. We observed a dose-dependent decrease in cell viability in AML, ALL and CML cells (GI50 1-8 µM). ONC201 also decreased cell viability in 2 primary chronic lymphocytic leukemia (CLL) patient samples with 48-72h treatment. Dose- and time-dependent induction of apoptosis (caspase-3 cleavage), inhibition of Akt and Foxo3a phosphorylation was observed in AML cells treated with ONC201 for 24-48 hr. Sub-G1 analysis confirmed that ONC201 induced apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. ONC201-mediated apoptosis in ALL cells involved PARP cleavage and caspase-9 activation. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim was upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells, which are known to confer a poor clinical prognosis.Low micromolar concentrations of ONC201 also reduced multiple myeloma (MM) cell viability in a time- and dose-dependent manner. ONC201 reduced cell viability and induced apoptosis in dexamethasone-resistant MM.1R cells with efficacy comparable to dexamethasone-sensitive MM.1S cells (Figure 2). ONC201 caused activation of the integrated stress response (ISR) pathway, as observed with induction of ATF/CHOP in MM cells. XIAP is highly up-regulated in MM cells and sownregulation of IAP family proteins induces apoptosis. ONC201 treatment of MM cells resulted in a dose-dependent decrease in the anti-apoptotic proteins XIAP, cIAP1 and cIAP2.Next, ONC201 was evaluated in 33 human lymphoma cell lines: 3 anaplastic large cell lymphoma (ALCL), 7 Burkitt's lymphoma, 2 cutaneous T-cell lymphoma (CTCL), 8 diffuse large B-cell lymphoma (DLBCL), 1 MCL, 2 nodular sclerosis (NS) and 10 other lymphoma cell lines. ONC201 demonstrated low micromolar range efficacy (GI50 1-6 µM) across all lymphoma subtypes. Low micromolar concentrations of ONC201 reduced cell viability of MCL and DLBCL cell lines and primary cells in a time- and dose-dependent manner (Figure 3). ONC201 caused activation of the ISR pathway, as observed with induction of ATF4 in Pfeiffer DLBCL cells. In patient-derived MCL cells, ONC201 reduced cyclin D1 expression, a diagnostic feature of MCL.We then tested the efficacy of ONC201 in combination with approved therapies in hematological malignancies in vitro and in vivo. ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine confirmed by combination indices in AML cell lines CMK, THP-1, HL60 and ALL Kasumi-2 cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model exhibited superior tumor growth inhibition compared to either agent alone (Figure 4). ONC201 demonstrated synergistic reduction in cell viability in combination with 5-azacytidine that was confirmed by combination indices in AML cell lines HL-60, MOLM14 and MV411. ONC201 synergistically reduced cell viability in combination with dexamethasone or ixazomib in RPMI-8226 MM cells. ONC201 in combination with bortezomib synergistically reduced cell viability in KARPAS 299 ALCL cells and RPMI-8226 MM cells and improved apoptosis induction in UPN2 MCL cells. ONC201 combined with bortezomib was evaluated in a Burkitt's lymphoma xenograft model. Histological evaluation of tumor xenografts revealed a reduced tumor cell density at 3-4 weeks and enhanced CHOP induction at 48 hours with combination treatment compared to either agent alone. The combination treatment did not result in body weight loss.These results serve as a rationale for ongoing ONC201 single agent trials in relapsed/refractory acute leukemia (NCT02392572), non-Hodgkin's lymphoma (NCT02420795), MM (NCT02609230) and combination trial with dexamethasone in MM (NCT02863991). Our findings provide pharmacodynamic biomarkers and identify synergistic combinatorial regimens that are well tolerated and can be explored in the clinic. [Display omitted] DisclosuresPrabhu:Oncoceutics: Employment, Equity Ownership. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Oster:Oncoceutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Allen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc: Equity Ownership, Patents & Royalties.