The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

<p>Abstract</p> <p>Background</p> <p>Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway.</p> <p>Methods</p> <p>The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases.</p> <p>Results</p> <p>ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected.</p> <p>Conclusion</p> <p>These results demonstrate that MEKK1 is directly modified and inhibited by ITCs, and that this correlates with inhibition of downstream activation of SAPK. These results support the conclusion that ITCs may carry out many of their actions by directly targeting important cell regulatory proteins.</p

BIM-Mediated AKT Phosphorylation Is a Key Modulator of Arsenic Trioxide-Induced Apoptosis in Cisplatin-Sensitive and -Resistant Ovarian Cancer Cells

Background: Chemo-resistance to cisplatin-centered cancer therapy is a major obstacle to the effective treatment of human ovarian cancer. Previous reports indicated that arsenic trioxide (ATO) induces cell apoptosis in both drug-sensitive and-resistant ovarian cancer cells. Principal Findings: In this study, we determined the molecular mechanism of ATO-induced apoptosis in ovarian cancer cells. Our data demonstrated that ATO induced cell apoptosis by decreasing levels of phosphorylated AKT (p-AKT) and activating caspase-3 and caspase-9. Importantly, BIM played a critical role in ATO-induced apoptosis. The inhibition of BIM expression prevented AKT dephosphorylation and inhibited caspase-3 activation during cell apoptosis. However, surprisingly, gene silencing of AKT or FOXO3A had little effect on BIM expression and phosphorylation. Moreover, the activation of caspase-3 by ATO treatment improved AKT dephosphorylation, not only by cleaving the regulatory A subunit of protein phosphatase 2A (PP2A), but also by increasing its activation. Furthermore, our data indicated that the c-Jun N-terminal kinases (JNK) pathway is involved in the regulation of BIM expression. Conclusions: We demonstrated the roles of BIM in ATO-induced apoptosis and the molecular mechanisms of BIM expression regulated by ATO during ovarian cancer cell apoptosis. Our findings suggest that BIM plays an important role in regulating p-AKT by activating caspase-3 and that BIM mediates the level of AKT phosphorylation to determine th

A review of elliptical and disc galaxy structure, and modern scaling laws

A century ago, in 1911 and 1913, Plummer and then Reynolds introduced their models to describe the radial distribution of stars in `nebulae'. This article reviews the progress since then, providing both an historical perspective and a contemporary review of the stellar structure of bulges, discs and elliptical galaxies. The quantification of galaxy nuclei, such as central mass deficits and excess nuclear light, plus the structure of dark matter halos and cD galaxy envelopes, are discussed. Issues pertaining to spiral galaxies including dust, bulge-to-disc ratios, bulgeless galaxies, bars and the identification of pseudobulges are also reviewed. An array of modern scaling relations involving sizes, luminosities, surface brightnesses and stellar concentrations are presented, many of which are shown to be curved. These 'redshift zero' relations not only quantify the behavior and nature of galaxies in the Universe today, but are the modern benchmark for evolutionary studies of galaxies, whether based on observations, N-body-simulations or semi-analytical modelling. For example, it is shown that some of the recently discovered compact elliptical galaxies at 1.5 < z < 2.5 may be the bulges of modern disc galaxies.Comment: Condensed version (due to Contract) of an invited review article to appear in "Planets, Stars and Stellar Systems"(www.springer.com/astronomy/book/978-90-481-8818-5). 500+ references incl. many somewhat forgotten, pioneer papers. Original submission to Springer: 07-June-201

LY294002 may overcome 5-FU resistance via down-regulation of activated p-AKT in Epstein-Barr virus-positive gastric cancer cells

<p>Abstract</p> <p>Background</p> <p>As EBV-associated gastric cancer has unique features that are different from EBV (-) gastric cancer, EBV is considered to have a key role in gastric carcinogenesis. It has been reported that viral latent membrane protein 2A (LMP2A) in EBV-transformed tumor cells activates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which provides a survival signal and chemo-resistance to cytotoxic anti-cancer drugs. This study was to evaluate anti-proliferative effect and cell cycle change when 5-FU and LY294002 (LY), a selective inhibitor of PI3K, were treated separately or combined with different schedules in EBV positive gastric cancer cell line, SNU-719.</p> <p>Methods</p> <p>After single treatment and sequential combination of 5-FU and LY, cytotoxic activity was measured by MTS assay. When 5-FU and LY were treated in single and sequential combinations, the expression of p-AKT, p-NFkB, p-p53 and bcl-2 was observed on different concentrations by Western blot analysis. We also investigated the effect on apoptosis and cell cycle distribution using flow cytometry. The LMP2A siRNA inhibition was done to confirm the reversal of decreased 5-FU activity and p-AKT.</p> <p>Results</p> <p>When 5-FU was sequentially combined with LY, the combination index (CI) value indicated synergistic anti-proliferative effect. The expression of p-AKT and p-NFκB was upregulated by 5-FU alone but sequential treatment of 5-FU and LY decreased the expression of both p-AKT and p-NFκB. When 5-FU was combined with LY, G0/G1 and sub G1 cell population (%) increased. When 5-FU was added to the cells transfected with LMP2A siRNA, its anti-proliferative effect increased and the expression of p-AKT decreased. In sequential combination of 5-FU and LY, the expression of p-p53 was increased and bcl-2 expression was diminished compared to 5-FU alone.</p> <p>Conclusion</p> <p>These data suggest that sequential combination of 5-FU and LY induce synergistic cytotoxicity and overcome intrinsic and acquired resistance of 5-FU via downregulation of activated p-AKT and mitochondria-dependent apoptosis in EBV gastric cancer cell line, SNU-719.</p

Analyses of apoptotic regulators CASP9 and DFFA at 1P36.2, reveal rare allele variants in human neuroblastoma tumours

The genes encoding Caspase-9 and DFF45 have both recently been mapped to chromosome region 1p36.2, that is a region alleged to involve one or several tumour suppressor genes in neuroblastoma tumours. This study presents an update contig of the ‘Smallest Region of Overlap of deletions’ in Scandinavian neuroblastoma tumours and suggests that DFF45 is localized in the region. The genomic organization of the human DFF45 gene, deduced by in-silico comparisons of DNA sequences, is described for the first time in this paper. In the present study 44 primary tumours were screened for mutation by analysis of the genomic sequences of the genes. In two out of the 44 tumours this detected in the DFFA gene one rare allele variant that caused a non-polar to a polar amino acid exchange in a preserved hydrophobic patch of DFF45. One case was hemizygous due to deletion of the more common allele of this polymorphism. Out of 194 normal control alleles only one was found to carry this variant allele, so in respect of it, no healthy control individual out of 97 was homozygous. Moreover, our RT–PCR expression studies showed that DFF45 is preferably expressed in low-stage neuroblastoma tumours and to a lesser degree in high-stage neuroblastomas. We conclude that although coding mutations of Caspase-9 and DFF45 are infrequent in neuroblastoma tumours, our discovery of a rare allele in two neuroblastoma cases should be taken to warrant further studies of the role of DFF45 in neuroblastoma genetics

Apoptosis Induced by Cytoskeletal Disruption Requires Distinct Domains of MEKK1

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the MAPK JNK and is required for microtubule inhibitor-induced apoptosis in B cells. Here, we find that apoptosis induced by actin disruption via cytochalasin D and by the protein phosphatase 1/2A inhibitor okadaic acid also requires MEKK1 activation. To elucidate the functional requirements for activation of the MEKK1-dependent apoptotic pathway, we created mutations within MEKK1. MEKK1-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug. We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption. Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD

LBH589, a deacetylase inhibitor, induces apoptosis in adult T-cell leukemia/lymphoma cells via activation of a novel RAIDD-caspase-2 pathway

Adult T-cell leukemia/lymphoma (ATLL), an aggressive neoplasm etiologically associated with human T-lymphotropic virus type-1 (HTLV-1), is resistant to treatment. In this study, we examined the effects of a new inhibitor of deacetylase enzymes, LBH589, on ATLL cells. LBH589 effectively induced apoptosis in ATLL-related cell lines and primary ATLL cells and reduced the size of tumors inoculated in SCID mice. Analyses, including with a DNA microarray, revealed that neither death receptors nor p53 pathways contributed to the apoptosis. Instead, LBH589 activated an intrinsic pathway through the activation of caspase-2. Furthermore, small interfering RNA experiments targeting caspase-2, caspase-9, RAIDD, p53-induced protein with a death domain (PIDD) and RIPK1 (RIP) indicated that activation of RAIDD is crucial and an event initiating this pathway. In addition, LBH589 caused a marked decrease in levels of factors involved in ATLL cell proliferation and invasion such as CCR4, IL-2R and HTLV-1 HBZ-SI, a spliced form of the HTLV-1 basic zipper factor HBZ. In conclusion, we showed that LBH589 is a strong inducer of apoptosis in ATLL cells and uncovered a novel apoptotic pathway initiated by activation of RAIDD

Caspase-3 and caspase-8 expression in breast cancer: caspase-3 is associated with survival

Impaired apoptosis is one of the hallmarks of cancer. Caspase-3 and -8 are key regulators of the apoptotic response and have been shown to interact with the calpain family, a group of cysteine proteases, during tumorigenesis. The current study sought to investigate the prognostic potential of caspase-3 and -8 in breast cancer, as well as the prognostic value of combinatorial caspase and calpain expression. A large cohort (n = 1902) of early stage invasive breast cancer patients was used to explore the expression of caspase-3 and -8. Protein expression was examined using standard immunohistochemistry on tissue microarrays. High caspase-3 expression, but not caspase-8, is significantly associated with adverse breast cancer-specific survival (P = 0.008 and P = 0.056, respectively). Multivariate analysis showed that caspase-3 remained an independent factor when confounding factors were included (hazard ratio (HR) 1.347, 95% confidence interval (CI) 1.086–1.670; P = 0.007). The analyses in individual subgroups demonstrated the significance of caspase-3 expression in clinical outcomes in receptor positive (ER, PR or HER2) subgroups (P = 0.001) and in non-basal like subgroup (P = 0.029). Calpain expression had been previously assessed. Significant association was also found between high caspase-3/high calpain-1 and breast cancer-specific survival in the total patient cohort (P = 0.005) and basal-like subgroup (P = 0.034), as indicated by Kaplan–Meier analysis. Caspase-3 expression is associated with adverse breast cancer-specific survival in breast cancer patients, and provides additional prognostic values in distinct phenotypes. Combinatorial caspase and calpain expression can predict worse prognosis, especially in basal-like phenotypes. The findings warrant further validation studies in independent multi-centre patient cohorts

Circadian Phase Resetting via Single and Multiple Control Targets

Circadian entrainment is necessary for rhythmic physiological functions to be appropriately timed over the 24-hour day. Disruption of circadian rhythms has been associated with sleep and neuro-behavioral impairments as well as cancer. To date, light is widely accepted to be the most powerful circadian synchronizer, motivating its use as a key control input for phase resetting. Through sensitivity analysis, we identify additional control targets whose individual and simultaneous manipulation (via a model predictive control algorithm) out-perform the open-loop light-based phase recovery dynamics by nearly 3-fold. We further demonstrate the robustness of phase resetting by synchronizing short- and long-period mutant phenotypes to the 24-hour environment; the control algorithm is robust in the presence of model mismatch. These studies prove the efficacy and immediate application of model predictive control in experimental studies and medicine. In particular, maintaining proper circadian regulation may significantly decrease the chance of acquiring chronic illness