Fabric response to stress probing in granular materials:Two-dimensional, anisotropic systems

The microstructure of granular materials has a significant influence on their macroscopic quasi-static strength and deformational behaviour. This microstructure is often quantified by a second-order fabric tensor that describes the primary orientational statistics of interparticle contacts. Here, it is investigated how the fabric tensor changes when samples are subjected to small (strain) loadings with different ‘directions’, i.e. probes. This is accomplished by the analysis of extensive sets of Discrete Element Method (DEM) simulations for various anisotropic, pre-peak two-dimensional samples, where both in-plane (i.e. coaxial with the current stress and fabric tensor) and out-of-plane, noncoaxial probes are considered. The results of DEM simulations show that the in-plane and out-of-plane fabric responses are effectively decoupled, i.e. they are only dependent on the in-plane and out-of-plane strain increment, respectively. The out-of-plane fabric increment is proportional to the out-of-plane strain increment whereas the in-plane fabric increment is linearly dependent on the in-plane strain increment. An accurate theoretical description (with a modest number of model parameters) has been developed that describes the fabric response to the imposed, in-plane as well out-of-plane, strain increments for the considered systems

BFD-22 a new potential inhibitor of BRAF inhibits the metastasis of B16F10 melanoma cells and simultaneously increased the tumor immunogenicity

Benzofuroxan is an interesting ring system, which has shown a wide spectrum of biological responses against tumor cell lines. We investigated, herein, the antitumor effects of benzofuroxan derivatives (BFDs) in vitro and in a melanoma mouse model. Cytotoxic effects of twenty-two BFDs were determined by MTT assay. Effects of BFD-22 in apoptosis and cell proliferation were evaluated using Annexin V-FITC/PI and CFSE staining. In addition, the effects in the cell cycle were assessed. Flow cytometry, western blot, and fluorescence microscopy analysis were employed to investigate the apoptosis-related proteins and the BRAF signaling. Cell motility was also exploited through cell invasion and migration assays. Molecular docking approach was performed in order to verify the BFD-22 binding mode into the ATP catalytic site of BRAF kinase. Moreover, the BFD-22 antitumor effects were evaluated in a melanoma murine model using B16F10. BFD-22 was identified as a potential hit against melanoma cells. BFD-22 induced apoptosis and inhibited cell proliferation of B16F10 cells. BFD-22 has suppressed, indeed, the migratory and invasive behavior of B16F10 cells. Cyclin D1 and CDK4 expression were reduced leading to cell cycle arrest at G0/G1 phase. Of note, phosphorylation of BRAF at Ser338 was strongly down-regulated by BFD-22 in B16F10 cells. The accommodation/orientation into the binding site of BRAF was similar of BAY43-9006 (co-crystallized inhibitor of BRAF, sorafenib). Importantly, BFD-22 presented in vivo antimetastatic effects and showed better therapeutic efficacy than sorafenib and taxol. BFD-22 can be considered as a new lead compound and, then, can be helpful for the designing of novel drug candidates to treat melanoma

MiR-221/222 promote epithelial-mesenchymal transition by targeting Notch3 in breast cancer cell lines

Basal-like breast cancer (BLBC) is an aggressive subtype with a strong tendency to metastasize. Due to the lack of effective chemotherapy, BLBC has a poor prognosis compared with luminal subtype breast cancer. MicroRNA-221 and -222 (miR-221/222) are overexpressed in BLBC and associate with metastasis as well as poor prognosis; however, the mechanisms by which miR-221/222 function as oncomiRs remain unknown. Here, we report that miR-221/222 expression is inversely correlated with Notch3 expression in breast cancer cell lines. Notch3 is known to be overexpressed in luminal breast cancer cells and inhibits epithelial to mesenchymal transition (EMT). We demonstrate that miR-221/222 target Notch3 by binding to its 3' untranslated region and suppressing protein translation. Ectopic expression of miR-221/222 significantly promotes EMT, whereas overexpression of Notch3 intracellular domain attenuates the oncogenic function of miR-221/222, suggesting that miR-221/222 exerts its oncogenic role by negatively regulating Notch3. Taken together, our results elucidated that miR-221/222 promote EMT via targeting Notch3 in breast cancer cell lines suggesting that miR-221/222 can serve as a potential therapeutic target in BLBC

Fanconi anemia genes are highly expressed in primitive CD34(+ )hematopoietic cells

BACKGROUND: Fanconi anemia (FA) is a complex recessive genetic disease characterized by progressive bone marrow failure (BM) and a predisposition to cancer. We have previously shown using the Fancc mouse model that the progressive BM failure results from a hematopoietic stem cell defect suggesting that function of the FA genes may reside in primitive hematopoietic stem cells. METHODS: Since genes involved in stem cell differentiation and/or maintenance are usually regulated at the transcription level, we used a semiquantitative RT-PCR method to evaluate FA gene transcript levels in purified hematopoietic stem cells. RESULTS: We show that most FA genes are highly expressed in primitive CD34-positive and negative cells compared to lower levels in more differentiated cells. However, in CD34(- )stem cells the Fancc gene was found to be expressed at low levels while Fancg was undetectable in this population. Furthermore, Fancg expression is significantly decreased in Fancc -/- stem cells as compared to wild-type cells while the cancer susceptibility genes Brca1 and Fancd1/Brac2 are upregulated in Fancc-/- hematopoietic cells. CONCLUSIONS: These results suggest that FA genes are regulated at the mRNA level, that increased Fancc expression in LTS-CD34(+ )cells correlates with a role at the CD34(+ )differentiation stage and that lack of Fancc affects the expression of other FA gene, more specifically Fancg and Fancd1/Brca2, through an unknown mechanism

Caspase-dependent and -independent suppression of apoptosis by monoHER in Doxorubicin treated cells

Doxorubicin (DOX) is an antitumour agent for different types of cancer, but the dose-related cardiotoxicity limits its clinical use. To prevent this side effect we have developed the flavonoid monohydroxyethylrutoside (monoHER), a promising protective agent, which did not interfere with the antitumour activity of DOX. To obtain more insight in the mechanism underlying the selective protective effects of monoHER, we investigated whether monoHER (1 mM) affects DOX-induced apoptosis in neonatal rat cardiac myocytes (NeRCaMs), human endothelial cells (HUVECs) and the ovarian cancer cell lines A2780 and OVCAR-3. DOX-induced cell death was effectively reduced by monoHER in heart, endothelial and A2780 cells. OVCAR-3 cells were highly resistant to DOX-induced apoptosis. Experiments with the caspase-inhibitor zVAD-fmk showed that DOX-induced apoptosis was caspase-dependent in HUVECs and A2780 cells, whereas caspase-independent mechanisms seem to be important in NeRCaMs. MonoHER suppressed DOX-dependent activation of the mitochondrial apoptotic pathway in normal and A2780 cells as illustrated by p53 accumulation and activation of caspase-9 and -3 cleavage. Thus, monoHER acts by suppressing the activation of molecular mechanisms that mediate either caspase-dependent or -independent cell death. In light of the current work and our previous studies, the use of clinically achievable concentrations of monoHER has no influence on the antitumour activity of DOX whereas higher concentrations as used in the present study could influence the antitumour activity of DOX

Role of Endoplasmic Reticulum Stress in α-TEA Mediated TRAIL/DR5 Death Receptor Dependent Apoptosis

Background -- α-TEA (RRR-α-tocopherol ether-linked acetic acid analog), a derivative of RRR-α-tocopherol (vitamin E) exhibits anticancer actions in vitro and in vivo in variety of cancer types. The objective of this study was to obtain additional insights into the mechanisms involved in α-TEA induced apoptosis in human breast cancer cells. Methodology/Principal Findings -- α-TEA induces endoplasmic reticulum (ER) stress as indicated by increased expression of CCAAT/enhancer binding protein homologous protein (CHOP) as well as by enhanced expression or activation of specific markers of ER stress such as glucose regulated protein (GRP78), phosphorylated alpha subunit of eukaryotic initiation factor 2 (peIF-2α), and spliced XBP-1 mRNA. Knockdown studies using siRNAs to TRAIL, DR5, JNK and CHOP as well as chemical inhibitors of ER stress and caspase-8 showed that: i) α-TEA activation of DR5/caspase-8 induces an ER stress mediated JNK/CHOP/DR5 positive amplification loop; ii) α-TEA downregulation of c-FLIP (L) protein levels is mediated by JNK/CHOP/DR5 loop via a JNK dependent Itch E3 ligase ubiquitination that further serves to enhance the JNK/CHOP/DR5 amplification loop by preventing c-FLIP's inhibition of caspase-8; and (iii) α-TEA downregulation of Bcl-2 is mediated by the ER stress dependent JNK/CHOP/DR5 signaling. Conclusion -- Taken together, ER stress plays an important role in α-TEA induced apoptosis by enhancing DR5/caspase-8 pro-apoptotic signaling and suppressing anti-apoptotic factors c-FLIP and Bcl-2 via ER stress mediated JNK/CHOP/DR5/caspase-8 signaling.The Clayton Foundation for Research, the National Institute of Environmental Health Sciences Center Grant ES007784, the Center for Molecular and Cellular Toxicology at the University of Texas at Austin and a NIEHS/NIH Toxicology Training Grant T32 ES07247. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Biological Sciences, School o

Regulation of Retinoid Receptors by Retinoic Acid and Axonal Contact in Schwann Cells

Background: Schwann cells (SCs) are the cell type responsible for the formation of the myelin sheath in the peripheral nervous system (PNS). As retinoic acid (RA) and other retinoids have a profound effect as regulators of the myelination program, we sought to investigate how their nuclear receptors levels were regulated in this cell type. Methodology/Principal Findings: In the present study, by using Schwann cells primary cultures from neonatal Wistar rat pups, as well as myelinating cocultures of Schwann cells with embryonic rat dorsal root ganglion sensory neurons, we have found that sustained expression of RXR-c depends on the continuous presence of a labile activator, while axonal contact mimickers produced an increase in RXR-c mRNA and protein levels, increment that could be prevented by RA. The upregulation by axonal contact mimickers and the transcriptional downregulation by RA were dependent on de novo protein synthesis and did not involve changes in mRNA stability. On the other hand, RAR-b mRNA levels were only slightly modulated by axonal contact mimickers, while RA produced a strong transcriptional upregulation that was independent of de novo protein synthesis without changes in mRNA stability. Conclusions/Significance: All together, our results show that retinoid receptors are regulated in a complex manner i

Suppression of apoptosis inhibitor c-FLIP selectively eliminates breast cancer stem cell activity in response to the anti-cancer agent, TRAIL

Introduction It is postulated that breast cancer stem cells (bCSCs) mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients. Therapeutic targeting of bCSCs however, is hampered by their heterogeneity and resistance to existing therapeutics. In order to identify strategies to selectively remove bCSCs from breast cancers, irrespective of their clinical subtype, we sought an apoptosis mechanism that would target bCSCs yet would not kill normal cells. Suppression of the apoptosis inhibitor cellular FLICE-Like Inhibitory Protein (c-FLIP) partially sensitizes breast cancer cells to the anti-cancer agent Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL). Here we demonstrate in breast cancer cell lines that bCSCs are exquisitely sensitive to the de-repression of this pro-apoptotic pathway, resulting in a dramatic reduction in experimental metastases and the loss of bCSC self-renewal. Methods Suppression c-FLIP was performed by siRNA (FLIPi) in four breast cancer cell lines and by conditional gene-knockout in murine mammary glands. Sensitivity of these cells to TRAIL was determined by complementary cell apoptosis assays, including a novel heterotypic cell assay, while tumour-initiating potential of cancer stem cell subpopulations was determined by mammosphere cultures, aldefluor assay and in vivo transplantation. Results Genetic suppression of c-FLIP resulted in the partial sensitization of TRAIL-resistant cancer lines to the pro-apoptotic effects of TRAIL, irrespective of their cellular phenotype, yet normal mammary epithelial cells remained refractory to killing. While 10%-30% of the cancer cell populations remained viable after TRAIL/FLIPi treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional bCSC pool, eliminating stem cell renewal. This culminated in an 80% reduction in primary tumours and a 98% reduction in metastases following transplantation. The recurrence of residual tumour initiating capacity was consistent with the observation that post-treated adherent cultures re-acquired bCSC-like properties in vitro. Importantly however this recurrent bCSC activity was attenuated following repeated TRAIL/FLIPi treatment. Conclusions We describe an apoptotic mechanism that selectively and repeatedly removes bCSC activity from breast cancer cell lines and suggest that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in a broad range of breast cancer subtypes. [PROVISIONAL