Presence Of Trails Research Articles

ELF3 plays an important role in defining TRAIL sensitivity in breast cancer by modulating the expression of decoy receptor 2 (DCR2).

TRAIL protein on binding to its cognate death receptors (DR) can induce apoptosis specifically in breast tumor cells sparing normal cells. However, TRAIL also binds to decoy receptors (DCR) thereby inhibiting the apoptotic pathways thus causing TRAIL resistance. Also, one of the barriers due to which TRAIL-based therapy could not become FDA-approved might be because of resistance to therapy. Therefore, in the current study we wanted to explore the role of transcription factors in TRAIL resistance with respect to breast cancer. Microarray data from TRAIL-sensitive (TS) and TRAIL-resistant (TR) MDA-MB-231 cells were reanalyzed followed by validation of the candidate genes using quantitative PCR (qPCR), immunoblotting and immunofluorescence technique. Overexpression of the candidate gene was performed in MDA-MB-231 and MCF7 cells followed by cell viability assay and immunoblotting for cleaved caspase-3. Additionally, immunoblotting for DCR2 was carried out. TCGA breast cancer patient survival was used for Kaplan-Meier (KM) plot. Validation of the candidate gene i.e. ELF3 using qPCR and immunoblotting revealed it to be downregulated in TR cells compared to TS cells. ELF3 overexpression in MDA-MB-231 and MCF7 cells caused reversal of TRAIL resistance as observed using cell viability assay and cleaved caspase-3 immunoblotting. ELF3 overexpression also resulted in DCR2 downregulation in the MDA-MB-231 and MCF7 cells. Furthermore, KM analysis found high ELF3 and low DCR2 expression to show better patient survival in the presence of TRAIL. Our study shows ELF3 to be an important factor that can influence TRAIL-mediated apoptosis in breast cancer. Also, ELF3 and DCR2 expression status should be taken into consideration while designing strategies for successful TRAIL-based therapy.

Spatial distribution of biodiversity citizen science in a natural area depends on area accessibility and differs from other recreational area use

Abstract Opportunistic citizen science produces large amounts of primary biodiversity data but is underutilized in the conservation and management of protected areas despite these areas’ status as citizen science hotspots. Application of these data may be limited by the challenge of understanding sampling patterns associated with opportunistic data at a scale relevant to local area management. An improved understanding of citizen science activity patterns within protected areas could strengthen both data analysis and the local promotion and guidance of citizen science activity. We investigated local‐scale patterns of citizen science activity, using a case study approach to examine citizen science activity in a recreationally popular natural area that serves as a regional citizen science hotspot. We modelled the relationship between local citizen science activity and 10 spatial covariates broadly related to ease of access and natural interest, factors which have been shown to drive citizen science activity at regional scales. We further compared the distribution of citizen science activity with that of professional data collection and recreational visitor activity in the study area. We found that citizen science data largely complement rather than replicate openly available professional data. Citizen science participation was primarily driven by ease of access, especially the presence of trails. However, citizen science use of the trail network differed from other types of recreational trail use, including a weaker preference for well‐established trails and a stronger association with developed areas. This improved understanding of patterns in citizen science participation may be used to better account for spatial biases in citizen science data and to manage natural areas in a way that supports and guides future citizen science activity.

The importance of direct and indirect trophic interactions in determining the presence of a locally rare day-flying moth

Ecosystem engineers affect other organisms by creating, maintaining or modifying habitats, potentially supporting species of conservation concern. However, it is important to consider these interactions alongside non-engineering trophic pathways. We investigated the relative importance of trophic and non-trophic effects of an ecosystem engineer, red deer, on a locally rare moth, the transparent burnet (Zygaena purpuralis). This species requires specific microhabitat conditions, including the foodplant, thyme, and bare soil for egg-laying. The relative importance of grazing (i.e., trophic effect of modifying microhabitat) and trampling (i.e., non-trophic effect of exposing bare soil) by red deer on transparent burnet abundance is unknown. We tested for these effects using a novel method of placing pheromone-baited funnel traps in the field. Imago abundance throughout the flight season was related to plant composition, diversity and structure at various scales around each trap. Indirect effects of red deer activity were accounted for by testing red deer pellet and trail presence against imago abundance. Imago abundance was positively associated with thyme and plant diversity, whilst negatively associated with velvet grass and heather species cover. The presence of red deer pellets and trails were positively associated with imago abundance. The use of these sites by red deer aids the transparent burnet population via appropriate levels of grazing and the provision of a key habitat condition, bare soil, in the form of deer trails. This study shows that understanding how both trophic and non-trophic interactions affect the abundance of a species provides valuable insights regarding conservation objectives.

Ring A/B-Modified 17β-Hydroxywithanolide Analogues as Antiproliferative Agents for Prostate Cancer and Potentiators of Immunotherapy for Renal Carcinoma and Melanoma.

Physachenolide C (1) is a 17β-hydroxywithanolide natural product with a unique anticancer potential, as it exhibits potent and selective in vitro antiproliferative activity against prostate cancer (PC) cells and promotes TRAIL-induced apoptosis of renal carcinoma (RC) and poly I:C-induced apoptosis of melanoma cells. To explore the effect of ring A/B modifications of physachenolide C (1) on these biological activities, 23 of its natural and semisynthetic analogues were evaluated. Analogues 4-23 were prepared by chemical transformations of a readily accessible compound, physachenolide D (2). Compound 1 and its analogues 2-23 were evaluated for their antiproliferative activity against PC (LNCaP and 22Rv1), RC (ACHN), and melanoma (M14 and SK-MEL-28) cell lines and normal human foreskin fibroblast (HFF) cells. Most of the active analogues had selective and potent activity in reducing cell number for PC cell lines, some showing selectivity for androgen-independent and enzalutamide-resistant 22Rv1 cells compared to androgen-dependent LNCaP cells. Analogues with IC50s below 5.0 μM against ACHN cells, when tested in the presence of TRAIL, showed a significantly increased ability to reduce cell number, and those analogues active against the M14 and SK-MEL-28 cell lines exhibited enhanced activity when combined with poly I:C. These data provide additional structure-activity relationship information for 17β-hydroxywithanolides and suggest that selective activities of some analogues may be exploited to develop natural products-based tumor-specific agents for cancer chemotherapy.

Assessing the role of habitat and species interactions in the population decline and detection bias of Neotropical leaf litter frogs in and around La Selva Biological Station, Costa Rica

Worldwide, amphibian populations have been declining rapidly. This decline can be attributed to many factors including climate change, pesticide exposure, and emerging infectious diseases, among other important factors, but few studies have examined the influence of species interactions. In this study, we examined how habitat factors and co-occurring avian and mammalian species, as well as humans, exert direct and indirect effects on Neotropical amphibian population dynamics. We further examined how these habitat and species interactions could affect our ability to reliably detect amphibian presence to robustly estimate population trends. We conducted amphibian visual encounter surveys at 26 randomly selected sites in the La Selva Biological Station, in northeastern Costa Rica, as well as 26 sites across five additional forest fragments in the region. Furthermore, we used camera traps to collect data on avian and mammalian communities and human visitation at those amphibian survey plots. From these data, we were able to estimate species occupancy probabilities for leaf litter frogs across sites and their relationships to habitat and interspecific species interaction covariates. We also conducted an experiment with plastic model frogs to estimate detection probabilities when a population is known to occur at a site with certainty. Our results suggested that strawberry poison dart frog (Oophagapumilio) occupancy was positively related to secondary forest and their detection was negatively related to increasing air temperatures at the times of the surveys. Leaf litter frog occupancy was negatively related to core La Selva sites and human detections at sites, yet their detection was positively related to human trail presence, which might be related to reduced leaf litter cover due to heavy trampling. Our experimental surveys suggested that Neotropical leaf litter frog communities are difficult to detect when present and future studies should explicitly account for this detection bias to effectively monitor population trends.

Morniga-G, a T/Tn-Specific Lectin, Induces Leukemic Cell Death via Caspase and DR5 Receptor-Dependent Pathways.

Morniga-G, the Gal-specific black mulberry (Morus nigra) lectin, displays high affinity for T (CD176) and Tn (CD175) antigens, frequently expressed at the cancer cell surface. The effects of Morniga-G were investigated on a Tn-positive leukemic Jurkat cell line. The lectin, used in a concentration range between 5–20 μg/mL, induced cell death in leukemic Jurkat cells. Microscopic and cytofluorometric analyses indicated that Jurkat cell death was essentially apoptotic, associated with an increase in the ceramide content and a depolarization of the mitochondrial transmembrane potential. This lectin-mediated cell death was inhibited by the pan caspase-inhibitor zVAD. In addition, cleavage of caspases 8, 9, and 3 was observed in Morniga-G-treated Jurkat cells whereas Jurkat cell lines that are deficient in caspase 8–10, caspase 9, or FADD, survived to the lectin-mediated toxicity. Furthermore, in the presence of TRAIL- or DR5-blocking mononoclonal antibodies, Jurkat cells became resistant to Morniga-G, suggesting that the lectin triggers cell death via the TRAIL/DR5 pathway. In silico computer simulations suggest that Morniga-G might facilitate both the DR5 dimerization and the building of TRAIL/DR5 complexes. Finally, upon treatment of Jurkat cells with benzyl-GalNAc, an O-glycosylation inhibitor, a decrease in Tn antigen expression associating with a reduced Morniga-G toxicity, was observed. Taken together, these results suggest that Morniga-G induces the cell death of Tn-positive leukemic cells via concomitant O-glycosylation-, caspase-, and TRAIL/DR5-dependent pathways.

Using social media to understand drivers of urban park visitation in the Twin Cities, MN

Green space and parks in urban environments provide a range of ecosystem services and public benefits. However, planners and park managers can lack tools and resources to gather local information on how parks are used and what makes them desirable places for recreation and a wide variety of uses. Traditional survey methods to monitor park use and user preferences can be costly, time consuming, and challenging to apply at scale. Here, we overcome this limitation by using geotagged social media data to assess patterns of visitation to urban and peri-urban green space across park systems in the metropolitan area of the Twin Cities, Minnesota, USA. We find that parks with nearby water features, more amenities, greater accessibility from the presence of trails, and that are located within neighborhoods with higher population density, are associated with higher rates of visitation. As cities grow and shifts in demographics occur, more responsive management of public green space will become increasingly important to ensure urban parks provide ecosystem services and meet users’ needs. Using social media data to rapidly assess park use at a lower cost than traditional surveys has the potential to inform public green space management with targeted information on user behavior and values of urban residents.

Stay on trails – effects of human recreation on the spatiotemporal behavior of red deer Cervus elaphus in a German national park

Human activities can affect the behavior and well‐being of wildlife, and there is high potential for wildlife disturbance due to human outdoor recreation. Hiking is a popular form of outdoor recreation in many countries, including Germany. In this study, we investigate the effects of hiking and hiking trails on space‐use dynamics in GPS‐collared red deer Cervus elaphus inhabiting the Kellerwald‐Edersee National Park, Germany. Specifically, we 1) experimentally assess the reactions of red deer to hiker on‐ and off‐trail and 2) quantify the effects of hiking trails on daily space‐use patterns of the deer. We found that red deer in the Kellerwald‐Edersee National Park did not show a spatiotemporal reaction to recreational on‐trail hiking, but were sensitive to off‐trail hiking that always induced flight of the animals. With increasing trail densities, the flight initiation distance decreased, while the distance moved during a flight was similar across trail densities. Together, these results suggest that the sensitivity to off‐trail hiking increases in areas with low trail density, but that the flight reaction, once induced, is uniform. We further demonstrate that red deer avoid hiking trails during the day, but not during nighttime, and that a negative relationship exists between the distance the animals keep to trails and the trail density in an individual home range. Our results indicate that off‐trail hiking has the potential to disturb red deer in the Kellerwald‐ Edersee National Park, but that red deer are able to cope with recreational activity on trails as well as with the presence of trails in general. Our findings underline the importance of hiking rules, such as staying on trails, and visitor management focusing on minimizing conflicts between wildlife and human recreation.

Knockdown of TNF receptor-associated factor 2 (TRAF2) modulates in vitro growth of TRAIL-treated prostate cancer cells

TNF receptor-associated factor 2 (TRAF2) is documented to regulate tumor development and progression. Currently, the effect of TRAF2 on growth of androgen-refractory prostate cancer in response to TRAIL and the molecular mechanisms are not well understood. Here, we aim to investigate the effect of TRAF2 on in vitro growth of human androgen-insensitive prostate cancer DU-145 cells in the presence of TRAIL. Bioinformatics analysis of the Cancer Genome Atlas (TCGA) data was performed to examine TRAF2 expression and the prognostic value in prostate cancer. Microarray data of GSE21032 dataset were downloaded from Gene Expression Omnibus (GEO) to explore TRAF2 expression in metastatic prostate cancer. Bioinformatics analysis was further conducted to investigate the association of TRAF2 expression with recurrence-free survival in prostate cancer patients. Colony formation, cell viability, and Annexin V/PI apoptosis assays were performed to investigate the effect of TRAF2 on in vitro growth and apoptosis in TRAIL-treated DU-145 cells. The expression levels of mRNA and protein were detected by quantitative RT-PCR and immunoblotting assays. Bioinformatics analysis indicated that TRAF2 expression is significantly upregulated in prostate cancer patients with high Gleason scores (GS>7) compared with those with low Gleason scores (GS≤7). Upregulation of TRAF2 expression is significantly associated with recurrence-free survival in patients. In addition, TRAF2 knockdown can enhance apoptosis and downregulate SIRT1 expression in TRAIL-treated DU-145 cells. In vitro experiments further showed that SIRT1 knockdown can inhibit growth, and promote apoptosis in TRAIL-treated DU-145 cells. Overall, TRAF2 can influence in vitro growth of TRAIL-treated DU-145 cells at least partially via regulating SIRT1 expression, and may be a potentially valuable biomarker predicting recurrence-free survival in prostate cancer patients.

Discordance Between The Perceived And Actual Activity-supportive Built Environment Among College Students

An individual’s environment has a major impact on their activities, travel mode, and health. Thus, to improve physical activity (PA), active transportation, and health, it is important to understand a society’s awareness of their environment. PURPOSE: To evaluate discordance between perceived and actual presence of factors associated with the built environment. METHODS: A survey of 170 college students’ (19±.89 years) familiarity with their environment for PA and PA participation was conducted in Spring 2015. Objectively measured environmental audits of the environment were conducted simultaneously using an environment audit (WASABE). Variables included for the present study include: presence of trails, presence of parks, sidewalk maintenance, and perceived activity of those in the neighborhood. A continuous discordance score (-4 to 4) was created by summing the frequency by which an individual differed from the audit on each variable. A score of -4 represents always perceiving the presence of a variable when there was not one (negative discordance). A score of 4 represents always perceiving the absence of a variable when there was one (positive discordance). Paired-samples t-tests assessed discrepancies between the perceived and actual environment. Pearson correlations examined the relationship between fitness and discordance. A linear regression identified which factors within the individual (i.e., demographics, PA and environmental) most predicted discordance. P values were set at <.05. RESULTS: T-tests indicated significant differences between perceived and actual results for: the presence of bike trails (t=10.69), the presence of parks (t=5.81), and the presence of people being physically active (t=2.49). Positive discordance was correlated with a lack of moderate PA (r=-.27). There were no significant correlations with negative discordance. Predictors of discordance in either direction included: not owning a bike (β=-0.83), rarely using bike trails/routes (β=-.27), and not living near a park (β=-1.33). CONCLUSION: Students are unfamiliar with their environment, which may influence their activities and travel mode. Those who do not participate in activities that would place them in their neighborhood environment are even more unaware of their surroundings than others.

The impacts of the trails on property values surrounding Jejudo in Korea: using quantile regressions

The purpose of this study is to quantitatively analyze the effect of Jeju Olle Trails on nearby land prices. Land sale prices are designated as the dependent variable and zoning type and land category, shape of the land, and the width of bordering roads as independent variables. Land price data for actual sales for seven areas surrounding Olle Trails were examined with a geographic information system program to determine the exact distance of land parcels from nearby trekking courses. A quantile regression model is used to estimate the effect of trail proximity to nearby land prices. Through this quantile regression model, the difference in the effects of trail proximity between low-priced and high-priced land is captured. This study shows no significance for the variable up to quantile 0.6; however, the variable becomes significant from the 0.7 quantile and up. This suggests that plots in the top 30% of land prices are affected by the presence of trails, but the plots in the bottom 70% are not affected by this variable. For every 100 m distance from an Olle Trail, land price falls by 0.054%.

MiR-424 and miR-27a increase TRAIL sensitivity of acute myeloid leukemia by targeting PLAG1.

Although microRNAs have been elaborated to participate in various physiological and pathological processes, their functions in TRAIL resistance of acute myeloid leukemia (AML) remain obscure. In this study, we detected relatively lower expression levels of miR-424&27a in TRAIL-resistant and semi-resistant AML cell lines as well as newly diagnosed patient samples. Overexpression of miR-424&27a, by targeting the 3′UTR of PLAG1, enhanced TRAIL sensitivity in AML cells. Correspondingly, knockdown of PLAG1 sensitized AML cells to TRAIL-induced apoptosis and proliferation inhibition. We further found that PLAG1 as a transcription factor could reinforce Bcl2 promoter activity, causing its upregulation at the mRNA level. Both downregulated PLAG1 and elevated expression of miR-424&27a led to Bcl2 downregulation and augmented cleavage of Caspase8, Caspase3 and PARP in the presence of TRAIL. Restoration of Bcl2 could eliminate their effects on AML TRAIL sensitization. Overall, we propose that miR-424&27a and/or PLAG1 might serve as novel therapeutic targets in AML TRAIL therapy.

Gli2 silencing enhances TRAIL-induced apoptosis and reduces tumor growth in human hepatoma cells in vivo

Our previous studies have showed that Gli2 played a predominant role in proliferation and apoptosis resistance to TRAIL in hepatoma cells. The purpose of this study was to explore whether Gli2 silencing enhances efficiency of TRAIL for hepatoma in vivo. SMMC-7721-shRNA cells were implanted subcutaneously into nude mices and TRAIL was injected into the peritoneal space. TUNEL assay was used to detect apoptosis of tumor cells. The expression of Gli2, c-FLIPL, c-FLIPS, and Bcl-2 protein was determined by immunohistochemistry, respectively. Apoptosis and the level of caspases proteins in SMMC-7721 and HepG2 cells were detected by Flow cytometry and Western blot. Transcriptional activity of c-FLIP induced by Gli2 was measured by luciferase reporter gene assay. The results showed that lower volumes and weights of tumor were found in mice xenografted with SMMC-7721-shRNA cells as compared with control cells in the presence of TRAIL (P < 0.05). TUNEL assay showed significantly higher apoptosis index (AI) in the SMMC-7721-shRNA group than in the control groups (P < 0.05). There were remarkable positive correlations between Gli2 and c-FLIPL, c-FLIPS, Bcl-2 protein expression. Over-expression of c-FLIP or Bcl-2 in HepG2 cells attenuated TRAIL-induced apoptosis via suppression of caspase-8 or caspase-9 activity, respectively. Luciferase reporter gene assay found a regulatory sequence by which Gli2 activated transcription between -1007 to -244 in the c-FLIP promoter region. This study demonstrates that Gli2 showed regulatory activity on transcription of c-FLIP gene, and Gli2 silencing enhances TRAIL-induced apoptosis via down-regulation of c-FLIP and Bcl-2 in human hepatoma cells in vivo.

Bioactive heterocyclic natural products from actinomycetes having effects on cancer-related signaling pathways.

During recent studies on the search for bioactive natural products from various types of organisms, the research group of the author has focused on targeting signaling molecules related to cancer-related biological pathways such as TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand), Wnt, and hedgehog signaling. For the screening studies conducted, natural product extracts including those of unexplored myxomycetes and marine nudibranches as well as medicinal plants collected from Thailand and Bangladesh have been evaluated. In addition, bioactive metabolites of actinomycete strains collected in several locations but mainly from the Chiba region of Japan were investigated, leading to the isolation of a series of new bioactive aromatic heterocyclic natural products, including izumiphenazines A-D from the cultured broth of Streptomyces sp. IFM 11204, izuminosides A-C from Streptomyces sp. IFM 11260, new pyranonaphthoquinones such as (+)-deacetylgriseusin A, yorophenazine, and yoropyrazone from Streptomyces sp. IFM 11307, and katorazone from Streptomyces sp. IFM 11299. The structures of these new compounds were elucidated on the basis of spectroscopic data interpretation, and their effects on cancer-related signaling pathways were evaluated. As a result, (+)-deacetylgriseusin A at 0.1 μM proved to be the most active in TRAIL-resistance overcoming activity, with 33 % decrease in cell viability in the presence of TRAIL (100 ng/cm3). Also isolated were fuzanins A-H, which are new carbamate- or pyridine-containing alkaloids, from Kitasatospora sp. IFM 10917, and a new tyrosine derivative, prenyltyrosine and its acetamide, with TRAIL-resistance overcoming activity from Streptomyces sp. IFM 10937. A screening study targeting enhancement of death-receptor 5 (DR5) promoter activity led to the identification of a known indole alkaloid, teleocidin A-2, which showed a 4.7-fold enhancement in DR5 promoter activity at 0.1 μM. Nonactins, griseoviridin, and nocardamines were isolated from the author’s collection of actinomycete strains using a screening procedure targeting Wnt signal inhibitory activity.

Trail following Learning by Young Myrmica rubra Workers (Hymenoptera, Formicidae)

Ants use chemical trails, laid down on the ground, for recruiting congeners and helping them to return to the nest. The present work shows that young ants, less than one year old, though obviously reacting to the trail pheromone, are unable to efficiently follow a trail. These young ants begin to better walk along a trail when being in presence of trail following older congeners. Later on, they can by themselves rather efficiently follow a trail. Queens removed from their nest correctly move along a trail. The knowledge of the trail pheromone is thus native, while the trail following behavior is both partly native and partly learned. The latter learning is rapid and may be induced and/or enhanced by older trail following nestmates.

Methoxyflavone derivatives modulate the effect of TRAIL-induced apoptosis in human leukemic cell lines

BackgroundTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various tumor cells, but does not affect normal cells or human leukemic cells, such as MOLT-4 and U937 cells, which are relatively resistant to TRAIL. Three flavonoids extracted from the rhizome of K. parviflora were 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF) and 3,5,7,3',4'-pentamethoxyflavone (PMF), and synthetic flavonoids including 5-methoxyflavone (5-MF) and 2'-methoxyflavone (2"-MF) were chosen for testing in this study. The aims of this study were to examine whether the treatment of TRAIL-resistant leukemia MOLT-4 and U937 cells, with methoxyflavone derivatives could enhance the apoptotic response and to identify the mechanism involved.MethodsThe cytotoxic effect of methoxyflavone (MF) derivatives in MOLT-4, U937 and peripheral blood mononuclear cells (PBMCs) was analyzed by the MTT assay. The induction of apoptosis and the reduction of mitochondrial transmembrane potential (ΔΨm) after staining with annexin V FITC and propidium iodide (PI), and 3,3'-dihexyloxacarbocyanine iodide (DiOC6), respectively, were performed using flow cytometry. ROS production was determined by staining with 2',7'-dichlorofluorescin diacetate and processed with a flow cytometer. DR4, DR5, cFLIP, Mcl-1, BAX and Bid expression were demonstrated by immunoblotting. Caspase-8 and -3 activities were determined by using IETD-AFC and DEVD-AFC substrates and the fluorescence intensity was measured.ResultsAll methoxyflavone derivatives were cytotoxic to MOLT-4, U937 cells and PBMCs, except DMF, TMF and PMF were not toxic to PBMCs. All MF derivatives induced human leukemic MOLT-4 cell apoptosis, but not in U937 cells. Percentage of MOLT-4 cells with (ΔΨm) was increased when treated with DMF, TMF, PMF, 5-MF and 2'-MF in the presence of TRAIL. 5-MF and 2'-MF enhanced TRAIL-induced apoptosis through the up-regulation of both DRs and the down-regulation of cFLIP and Mcl-1. Bid was cleaved and BAX was up-regulated, followed by the activation of caspase-8 and -3. Oxidative stress was also increased. 2'-MF gave the same result compared with 5-MF but with a less effect.ConclusionMethoxyflavone derivatives enhanced TRAIL-induced apoptosis in human leukemic MOLT-4 cells through the death receptors and mitochondrial pathways.

Antagonizing IAPs by SMAC Mimetic TL32711 Induces Apoptosis in AML Cells Including AML Stem/Progenitor Cells Alone and in Combination with Chemotherapy

Abstract 66The antiapoptotic function of the inhibitors of apoptosis family of proteins (IAPs), including cIAP1, cIAP2, and XIAP, is antagonized by SMAC (second mitochondrial-derived activator of caspases). XIAP directly binds and inhibits caspase-9 and caspase-3 and suppresses both mitochondrion-mediated intrinsic and death receptor-mediated extrinsic apoptosis pathways, while the cIAPs are components of the cytoplasmic signaling complex containing members of TNF receptor associated factors and suppress death receptor/caspase-8 mediated extrinsic pathway activation. SMAC mimetics are a new class of anti-cancer agents that induce rapid degradation of cIAP1, relieve XIAP-mediated caspase repression, and promote TRAIL or TNFa-dependent apoptosis in various malignant cell types.To assess the therapeutic potential of SMAC mimetics in AML, we determined the protein levels of cIAP1 and XIAP, which are targets of SMAC mimetics, and caspase-8, the initiator caspase of the death receptor pathway by reverse phase protein array in blasts obtained from 511 newly diagnosed AML patients and in CD34+38− stem/progenitor cells isolated from blasts of these patients. We found that all three proteins were expressed in AML blasts. Importantly, we observed that the protein levels of cIAP1 and caspase-8 in CD34+38− AML stem/progenitor cells were significantly higher than those in bulk AML cells (P < 0.001).TL32711 (TL) is a highly potent and well-tolerated SMAC mimetic in clinical development that promoted rapid degradation of cIAP1 at low nM concentrations and induced pronounced apoptosis in AML cell lines in the presence of TNFα. Cell death was enhanced in the presence of TRAIL, confirming activation of the death receptor pathway as a significant mechanism of apoptosis induction. Caspase-8 mutant Jurkat cells (JurkatI9.2) were completely resistant to TL, further supporting the critical role of caspase-8 in TL-mediated cell death. TL synergistically enhanced apoptosis when combined with various nucleoside analogues clinically used in AML therapy such as Ara-C, clofarabine, and demethylating agents decitabine and 5-azacytidine (5-AC). Mechanistic studies showed that decitabine and 5-AC increased and activated caspase-8, decreased cFLIP, and induced XAF-1, a XIAP antagonist known to be hypermethylated in various malignant cell types. In addition, TL had single agent activity against blasts from primary AML samples with no toxicity in CD34+ cells from normal bone marrows at doses effective against AML cells. Importantly, TL not only induced apoptosis in bulk AML blast but also in CD34+38− AML stem/progenitor cells.Collectively, we showed that cIAP1 and caspase-8 are overexpressed in AML stem/progenitor cells and that inhibition of IAPs by the novel SMAC mimetic TL32711 synergistically enhances drug induced-death of AML cells and also has the potential to eliminate AML stem/progenitor cells. Disclosures:Weng:Tetralogic Pharmaceuticals: Employment. McKinlay:Tetralogic Pharmaceuticals: Employment.

Verticillin A Overcomes Apoptosis Resistance in Human Colon Carcinoma through DNA Methylation-Dependent Upregulation of BNIP3

Drug resistance is a major cause of failure in cancer chemotherapy. Therefore, identification and combined use of adjuvant compounds that can overcome drug resistance may improve the efficacy of cancer therapy. We screened extracts of Verticillium species-infected mushrooms for antitumor compounds and identified the compound Verticillin A as an inducer of hepatoma cell apoptosis in vitro and an inhibitor of tumor xenograft growth in vivo. Verticillin A exhibited a potent apoptosis-sensitizing activity in human colon carcinoma cells exposed to TRAIL or Fas in vitro. Furthermore, Verticillin A effectively sensitized metastatic human colon carcinoma xenograft to TRAIL-mediated growth inhibition in vivo. At the molecular level, we observed that Verticillin A induces cell-cycle arrest in the G₂ phase of the cell cycle in human colon carcinoma cells, markedly upregulating BNIP3 in both hepatoma and colon carcinoma cells. Notably, silencing BNIP3 decreased the sensitivity of tumor cells to Verticillin A-induced apoptosis in the absence or presence of TRAIL. We found that the BNIP3 promoter is methylated in both human hepatoma and colon carcinoma cells and tumor specimens. Verticillin A upregulated the expression of a panel of genes known to be regulated at the level of DNA methylation, in support of the concept that Verticillin A may act by demethylating the BNIP3 promoter to upregulate BNIP3 expression. Taken together, our findings identify Verticillin A as a potent apoptosis sensitizer with great promise for further development as an adjuvant agent to overcome drug resistance in human cancer therapy.

Abstract 754: Development of death receptor agonists for breast cancer therapy

Abstract TRAIL and death receptor (TRAIL-R1 and TRAIL-R2) agonistic antibodies have been shown to induce apoptosis in cancer cells and their clinical trials have produced positive results. However, there are no small organic molecular weight compounds that bind to TRAIL-death receptors and induce apoptosis specifically in cancer cells. The purpose of this study was to identify and characterize death receptor agonists (DRAs)/ small molecular weight organic compounds that induce apoptosis in breast cancer cells. The pharmacokinetics of the DRA with a single oral dose was characterized in nude mice. Blood was collected over 72 hour period after drug administration, and analyzed by HPLC-MS/MS for plasma drug concentration. The Cmax, tmax, and AUC of DRA was measured. DRAs inhibited cell viability and colony formation, and induced apoptosis in estrogen-dependent (MCF-7) and independent (MDA-MB-231 and MDA-MB-468) human breast cancer cell lines, but had no effect on human normal mammary epithelial cells and hepatocytes. The ability of DRAs to induce apoptosis was further enhanced in the presence of TRAIL. DRAs formed active TRAIL-DISC (death-inducing signaling complex) similar to that of TRAIL. DRAs inhibited the growth of orthotopically implanted MDA-MB-231 and MDA-MB-468 tumors in nude mice. Histological examination of tumor tissues derived from DRAs-treated nude mice revealed massive caspase-3 activation and apoptosis. Pharmacokinetic data demonstrate that DRA possesses a long terminal half-life, which is acceptable for further clinical development. In conclusion, we have identified, for the first time, DRAs that can be used in the clinic for the treatment of human breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 754.

Abstract 673: Novel anti-tumor activity of lapatinib derives from off-target up-regulation of TRAIL death receptors

Abstract Lapatinib, a HER2/EGFR inhibitor, is the most recently approved targeted therapy for metastatic breast cancer. Based on clinical evidence that lapatinib enhances efficacy of capecitabine in breast cancer patients, we hypothesized that lapatinib could augment the anti-cancer activity of agents in other tumor types such as colon cancer. Using in vitro cell death and apoptosis assays, we found lapatinib to have minimal effect on chemotherapy-induced cytotoxicity in colon cancer cell lines. However, elevated concentrations of lapatinib (5-10μM) significantly enhanced the pro-apoptotic effects of TRAIL and agonistic TRAIL receptor antibodies. In vivo, xenografted colon tumors from mice treated with lapatinib/TRAIL combinations exhibited more immunostaining for cleaved caspase-8, an apical caspase in the extrinsic cell death pathway, compared to tumors from mice treated with lapatinib or TRAIL alone. Furthermore, the combination therapy suppressed tumor growth, whereas single agent treatments had little effect. Mechanistically, lapatinib up-regulated proapoptotic TRAIL death receptors, DR4 and DR5, leading to more efficient induction of apoptosis in the presence of TRAIL and agonistic TRAIL receptor antibodies. Lapatinib had no appreciable effects on death receptor protein stability, and DR5 - but not DR4 - mRNA levels were increased, suggesting that lapatinib transcriptionally up-regulates DR5. Interestingly, other EGFR and HER2 inhibitors, individually or in combination, failed to increase death receptor expression and TRAIL sensitivity, suggesting that this novel effect resulted from off-target activity. In support of this hypothesis, lapatinib induced death receptor up-regulation in SW620 cells, which lack EGFR expression and detectable levels of HER2 signaling. Lower lapatinib concentrations (1μM), which completely inhibited EGFR and HER2, were not sufficient to induce death receptor expression and increase TRAIL sensitivity, further demonstrating the off-target nature of this effect. High concentrations of lapatinib increased phosphorylation of c-Jun, extracellular signal related kinase (ERK) and c-Jun N-terminal kinase (JNK). RNAi-mediated knockdown experiments proved that c-Jun, but not ERK or JNK, were required for lapatinib-induced DR5 up-regulation. We conclude that high concentrations of lapatinib, through an EGFR/HER2-indpendent mechanism, stimulate c-Jun phosphorylation/transcriptional activity leading to DR5 up-regulation and increased TRAIL sensitivity. From a clinical perspective, therapeutic benefit may be gained from this off-target activity by using high doses of lapatinib in combination with TRAIL-receptor-activating agents. Secondly, death receptor induction and signaling may serve as a biomarker of response following lapatinib treatment to identify patients that have responded most favorably to treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 673.