Targeting Mcl-1 for Radiosensitization of Pancreatic Cancers

In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-X_L, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells.     

Increased FGF1-FGFRc expression in idiopathic pulmonary fibrosis

Background

Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts.

Methods

FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed.

Results

Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects.

Conclusions

Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0242-2) contains supplementary material, which is available to authorized users.     

Yeast ubiquitin ligase Rsp5 contains nuclear localization and export signals

The Rsp5 ubiquitin ligase regulates numerous cellular processes. Rsp5 is mainly localized to the cytoplasm but nuclear localization was also reported. A potential nuclear export signal was tested for activity by using a GFP(2) reporter. The 687-LIGGIAEIDI-696 sequence located in the Hect domain was identified as a nuclear export signal active in a Crm1-dependent manner, and its importance for the localization of Rsp5 was documented by using fluorescence microscopy and a lacZ-based reporter system. Analysis of the cellular location of other Rsp5 fragments fused with GFP(2) indicated two independent potential nuclear localization signals, both located in the Hect domain. We also uncovered Rsp5 fragments that are important to targeting/tethering Rsp5 to various regions in the cytoplasm. The presented data indicate that Rsp5 ligase is a shuttling protein whose distribution within the cytoplasm and partitioning between cytoplasmic and nuclear locations is determined by a balance between the actions of several targeting sequences and domains.     

Ecophysiology of mycolic acid-containing Actinobacteria (Mycolata) in activated sludge foams

Increasing incidences of activated sludge foaming have been reported in the last decade in Danish plants treating both municipal and industrial wastewaters. In most cases, foaming is caused by the presence of Actinobacteria; branched mycolic acid-containing filaments (the Mycolata) and the unbranched Candidatus'Microthix parvicella'. Surveys from wastewater treatment plants revealed that the Mycolata were the dominant filamentous bacteria in the foam. Gordonia amarae-like organisms and those with the morphology of Skermania piniformis were frequently observed, and they often coexisted. Their identity was confirmed by FISH, using a new permeabilization procedure. It was not possible to identify all abundant Mycolata using existing FISH probes, which suggests the presence of currently undetectable and potentially undescribed populations. Furthermore, some Mycolata failed to give any FISH signal, although substrate uptake experiments with microautoradiography revealed that they were physiologically active. Ecophysiological studies were performed on the Mycolata identified by their morphology or FISH in both foams and mixed liquors. Large differences were seen among the Mycolata in levels of substrate assimilation and substrate uptake abilities in the presence of different electron acceptors. These differences were ascribed mainly to the presence of currently undescribed Mycolata species and/or differences in foam age.     

Ex vivo measurement of calpain activation in human peripheral blood lymphocytes by detection of immunoreactive products of calpastatin degradation

Limited proteolysis of multiple intracellular proteins by endogenous Ca-dependent cysteine proteases--calpains--is an important regulatory mechanism for cell proliferation, apoptosis etc. Its importance for cellular functions is stressed by existence of endogenous calpain inhibitors--calpastatins. The calpain-calpastatin system within living cells is in a fragile balance, which depends on both partners. The interdependence of calpain--a protease--and calpastatin--an endogenous inhibitor and at the same time a substrate for this enzyme makes any assessment of actual activity of this enzyme in the cells very difficult. In this work we made an attempt to estimate and compare the activity of calpain in human peripheral blood lymphocytes by assessing the levels of limited proteolysis of calpastatin in these cells by western blot, while at the same time the levels of calpain protein inside these cells was measured by flow cytometry. Our results indicate that it is possible to compare (semi-quantitatively) the activities of calpain in peripheral blood CD4+ and CD19+ lymphocytes from various donors that way. Preliminary results showed that calpain activity is increased in the CD4+ T cells isolated from peripheral blood of rheumatoid arthritis patients as compared to control lymphocytes. Extremely high intrinsic activity of calpain was detected in chronic lymphocytic leukemia (CD19+) cells. All this confirms the detection of immunoreactive products of calpastatin as a good maker of endogenous calpain activity.     

Structure-based design, synthesis and biochemical testing of novel and potent Smac peptido-mimetics

Structure-based design, chemical synthesis and biochemical testing of a series of novel Smac peptido-mimetics as inhibitors of XIAP protein are described. The most potent compound, 6j, has a binding affinity (K(i) value) of 24 nM to XIAP BIR3 protein and is 24 times more potent than the native Smac AVPI peptide. Further optimization of these potent Smac mimetics may ultimately lead to the development of a novel class of anticancer drugs for the treatment of human cancer by overcoming apoptosis-resistance of cancer cells through targeting the inhibitor of apoptosis proteins.     

Targeting Recruitment of Disruptor of Telomeric Silencing 1-like (DOT1L): CHARACTERIZING THE INTERACTIONS BETWEEN DOT1L AND MIXED LINEAGE LEUKEMIA (MLL) FUSION PROTEINS*

The MLL fusion proteins, AF9 and ENL, activate target genes in part via recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like). Here we report biochemical, biophysical, and functional characterization of the interaction between DOT1L and MLL fusion proteins, AF9/ENL. The AF9/ENL-binding site in human DOT1L was mapped, and the interaction site was identified to a 10-amino acid region (DOT1L_865–874). This region is highly conserved in DOT1L from a variety of species. Alanine scanning mutagenesis analysis shows that four conserved hydrophobic residues from the identified binding motif are essential for the interactions with AF9/ENL. Binding studies demonstrate that the entire intact C-terminal domain of AF9/ENL is required for optimal interaction with DOT1L. Functional studies show that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that DOT1L interaction with MLL-AF9 and its recruitment are required for transformation by MLL-AF9. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL fusion protein-associated leukemia.     

In silico discovery of acylated flavonol monorhamnosides from Eriobotrya japonica as natural, small-molecular weight inhibitors of XIAP BIR3

Targeting the baculoviral inhibitor of apoptosis proteins repeat (BIR) 3 of X-linked inhibitor of apoptosis proteins (XIAP) represents an innovative strategy for the design of chemosensitizers. Acylated flavonol monorhamnosides (AFMR) from Eriobotrya japonica Lindl. (Rosaceae) were virtually predicted as ligands of the XIAP BIR3 domain by using a previously generated pharmacophore model. From the methanol leaf extract of E. japonica an enriched mixture of AFMR was obtained showing chemosensitizing potential in combination with etoposide in XIAP-overexpressing Jurkat cells. The HPLC-SPE-NMR hyphenated technique facilitated the structure elucidation of three known and two new natural AFMR. The main constituent and virtual hit, kaempferol-3-O-??-l-(2″,4″-di-E-p-coumaroyl)-rhamnoside (3) was isolated from the enriched fraction. Applying a fluorescence polarization based binding assay, 3 was identified as XIAP BIR3 ligand with a dose-dependent affinity (IC₅₀ 10.4 ??M). Further, 3 induced apoptosis in XIAP-overexpressing Jurkat cells and activated caspase-9 in combination with etoposide. Docking experiments revealed a major impact of the coumaric acid and sugar moieties of 3 on XIAP BIR3 binding, which was experimentally confirmed. To conclude, this study elucidates 3 as natural, small-molecular weight XIAP BIR3 inhibitor using a combination of in silico and HPLC-SPE-NMR hyphenated techniques.     

Risk factors for non-adherence and loss to follow-up in a three-year clinical trial in Botswana

Background

Participant non-adherence and loss to follow-up can compromise the validity of clinical trial results. An assessment of these issues was made in a 3-year tuberculosis prevention trial among HIV-infected adults in Botswana.

Methods and Findings

Between 11/2004–07/2006, 1995 participants were enrolled at eight public health clinics. They returned monthly to receive bottles of medication and were expected to take daily tablets of isoniazid or placebo for three years. Non-adherence was defined as refusing tablet ingestion but agreeing to quarterly physical examinations. Loss to follow-up was defined as not having returned for appointments in ≥60 days. Between 10/2008–04/2009, survey interviews were conducted with 83 participants identified as lost to follow-up and 127 identified as non-adherent. As a comparison, 252 randomly selected adherent participants were also surveyed. Multivariate logistic regression analysis was used to identify associations with selected risk factors. Men had higher odds of being non-adherent (adjusted odds ratio (AOR), 2.24; 95% confidence interval [95%CI] 1.24–4.04) and lost to follow-up (AOR 3.08; 95%CI 1.50–6.33). Non-adherent participants had higher odds of reporting difficulties taking the regimen or not knowing if they had difficulties (AOR 3.40; 95%CI 1.75–6.60) and lower odds associated with each year of age (AOR 0.95; 95%CI 0.91–0.98), but other variables such as employment, distance from clinic, alcohol use, and understanding study requirements were not significantly different than controls. Among participants who were non-adherent or lost to follow-up, 40/210 (19.0%) reported that they stopped the medication because of work commitments and 33/210 (15.7%) said they thought they had completed the study.

Conclusions

Men had higher odds of non-adherence and loss to follow-up than women. Potential interventions that might improve adherence in trial participants may include:targeting health education for men, reducing barriers, clarifying study expectations, educating employers about HIV/AIDS to help reduce stigma in the workplace, and encouraging employers to support employee health.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00164281","term_id":"NCT00164281"}}NCT00164281