Caspase-dependent proteolytic cleavage of STAT3alpha in ES cells, in mammary glands undergoing forced involution and in breast cancer cell lines.

BACKGROUND: The STAT (Signal Transducers and Activators of Transcription) transcription factor family mediates cellular responses to a wide range of cytokines. Activated STATs (particularly STAT3) are found in a range of cancers. Further, STAT3 has anti-apoptotic functions in a range of tumour cell lines. After observing a proteolytic cleavage in STAT3alpha close to a potential apoptotic caspase protease cleavage site we investigated whether STAT3alpha might be a caspase substrate. METHODS: STAT3alpha status was investigated in vitro in several cell systems:- HM-1 murine embryonic stem (ES) cells following various interventions; IOUD2 murine ES cells following induction to differentiate along neural or adipocyte lineages; and in a number of breast cancer cell lines. STAT3alpha status was also analysed in vivo in wild type murine mammary glands undergoing controlled, forced involution. RESULTS: Immunoblotting for STAT3alpha in HM-1 ES cell extracts detected amino and carboxy terminal species of approximately 48 kDa and 43 kDa respectively--which could be diminished dose-dependently by cell treatment with the nitric oxide (NO) donor drug sodium nitroprusside (SNP). UV irradiation of HM-1 ES cells triggered the STAT3alpha cleavage (close to a potential caspase protease cleavage site). Interestingly, the pan-caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone (z-VAD-FMK) and the JAK2 tyrosine kinase inhibitor AG490 both inhibited cleavage dose-dependently, and cleavage was significantly lower in a heterozygous JAK2 knockout ES cell clone. STAT3alpha cleavage also occurred in vivo in normal murine mammary glands undergoing forced involution, coinciding with a pulse of phosphorylation of residue Y705 on full-length STAT3alpha. Cleavage also occurred during IOUD2 ES cell differentiation (most strikingly along the neural lineage) and in several human breast cancer cell lines, correlating strongly with Y705 phosphorylation. CONCLUSION: This study documents a proteolytic cleavage of STAT3alpha into 48 kDa amino and 43 kDa carboxyl terminal fragments in a range of cell types. STAT3alpha cleavage occurs close to a potential caspase site, and can be inhibited dose-dependently by SNP, AG490 and z-VAD-FMK. The cleavage seems to be caspase-dependent and requires the phosphorylation of STAT3alpha at the Y705 residue. This highly regulated STAT3alpha cleavage may play an important role in modulating STAT3 transcriptional activity.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines

<p>Abstract</p> <p>Background</p> <p>Curcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin. FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA) cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3. Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells.</p> <p>Methods</p> <p>Human and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT^®), apoptosis (SensoLyte^®Homogeneous AMC Caspase- 3/7 Assay kit, western blotting), STAT3 DNA binding (EMSA), and vascular endothelial growth factor (VEGF), survivin, and matrix metalloproteinase-2 (MMP2) expression (RT-PCR, western blotting) were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting.</p> <p>Results</p> <p>Our data showed that FLLL32 decreased STAT3 DNA binding by EMSA. FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway.</p> <p>Conclusions</p> <p>These data demonstrate that the novel curcumin analog FLLL32 has biologic activity against OSA cell lines through inhibition of STAT3 function and expression. Future work with FLLL32 will define the therapeutic potential of this compound <it>in vivo</it>.</p

Gene expression analysis of cell death induction by Taurolidine in different malignant cell lines

<p>Abstract</p> <p>Background</p> <p>The anti-infective agent Taurolidine (TRD) has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types.</p> <p>Methods</p> <p>Five different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3) were incubated with TRD (100 μM, 250 μM and 1000 μM). Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the <it>Agilent </it>-microarray platform to indentify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to <it>Ingenuity Pathways Analysis </it>and selected genes were validated by qRT-PCR and Western Blot.</p> <p>Results</p> <p>TRD 250 μM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3) as well as genes involved in the ER stress response (PPP1R15A), in ubiquitination (TRAF6) and mitochondrial apoptotic pathways (PMAIP1).</p> <p>Conclusions</p> <p>This is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis.</p

Evidence of protocarnivory in triggerplants (Stylidium spp.; stylidiaceae)

Australian triggerplants (Stylidium spp.; Stylidiaceae) trap small insects using mucilage-secreting glandular hairs held at various points on their inflorescence stems and flower parts. Triggerplants are generally found in habitats also containing genera of plants already accepted as carnivorous, two of which (Drosera, Byblis) use the same basic mechanism as Stylidium to trap their prey. In the herbarium, sheets of triggerplants and of accepted groups of carnivorous plants held similar numbers of trapped insects, and in the field, trapping of small prey per unit of glandular surface area was the same at a given site for triggerplants and for nearby carnivorous plants at three sites in northern Australia. Even more important, protease activity was produced by glandular regions of both triggerplants and Drosera after induction with yeast extract. A panel of negative and positive controls, including use 1) of plants grown in tissue culture, which therefore lack surface microorganisms, and 2) of protease inhibitors, shows that this activity 1) is generated by the glandular regions of the triggerplant itself, not by organisms that might reside on the surface of the plants, and 2) is due to proteases. All of this evidence taken together provides strong evidence of protocarnivory in Stylidium, something not previously suggested in the scientific literature, though the insect trapping has been noted informally. Experiments remain to be done to determine nutrient uptake, so triggerplants may well be fully carnivorous

Bortezomib inhibits nuclear factor-kappaB dependent survival and has potent in vivo activity in mesothelioma.

Purpose: Purpose of this study has been the assessment of nuclear factor-\u3baB (NF-\u3baB) as a survival factor in human mesothelial cells (HMC), transformed HMC and malignant mesothelioma (MMe) cells. We aimed at verifying whether the proteasome inhibitor Bortezomib could abrogate NF-\u3baB activity in MMe cells, leading to tumor cell death and may be established as a novel treatment for this aggressive neoplasm. Experimental Design: In HMC and MMe cells, NF-\u3baB nuclear translocation and DNA binding were studied by electrophoretic mobility shift assay, following treatment with tumor necrosis factor-\u3b1 (TNF-\u3b1). The IKK inhibitor Bay11-7082 was also tested to evaluate its effects on HMC, transformed HMC, and MMe cell viability upon exposure to asbestos fibers. Following Bortezomib treatment, cytotoxicity of MMe cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, whereas apoptosis and cell-cycle blockade were investigated by high-content analysis. Bortezomib was also given to mice bearing i.p. xenografts of MMe cells, and its effects on tumor growth were evaluated. Results: Here, we show that NF-\u3baB activity is a constitutive survival factor in transformed HMC, MMe cells, and acts as a survival factor in HMC exposed to asbestos fibers. Bortezomib inhibits NF-\u3baB activity in MMe cells and induces cell cycle blockade and apoptosis in vitro as well as tumor growth inhibition in vivo. Conclusions: Inhibition of NF-\u3baB constitutive activation in MMe cells by Bortezomib resulted in in vitro cytotoxicity along with apoptosis and in vivo tumor regression. Our results support the use of Bortezomib in the treatment of MMe and has led to a phase II clinical trial currently enrolling in Europe

Size matters: trap size primarily determines prey spectra differences among sympatric species of carnivorous sundews

Even though carnivorous plants (CPs) are a popular focus of ecological research, surprisingly few studies have investigated their prey spectra (the number and composition of captured prey). This knowledge gap has important implications for our understanding of sympatric speciation processes in CPs and may potentially hinder effective conservation and ecological restoration efforts. We applied a novel photography-based analysis method to characterize the in situ prey spectra of eight species from Drosera sect. Arachnopus, including five species that were studied across multiple populations in northern Australia. The prey spectra of all studied species predominantly comprised of flying insects, with small Nematocera (Diptera) being the most common prey group across all species. While the prey spectra of most species varied significantly among locations, differences in prey spectra among sympatric species were most strongly determined by trap size. The number of prey captured per plant and per centimeter of trapping leaf was strongly associated with increasing leaf length, and species with larger trapping leaves also captured comparatively greater numbers of large prey items than species producing smaller trapping leaves. Although niche segregation in prey spectra was not observed at any of the study sites, at one location D. fragrans (a species producing a strong, honey-like scent from trapping leaves) was found to capture significantly more winged Hymenoptera than the unscented sympatric D. aquatica, potentially indicating selective prey attraction in D. fragrans. Small species (such as D. nana) captured a disproportionally low amount of prey, despite being relatively widespread over large areas of northern Australia. Results indicate that carnivory may not have been a primary driver of diversification in D. sect. Arachnopus