Cytoskeletal Integrity Research Articles

Using white noise to gate organic transistors for dynamic monitoring of cultured cell layers.

Impedance sensing of biological systems allows for monitoring of cell and tissue properties, including cell-substrate attachment, layer confluence, and the “tightness” of an epithelial tissue. These properties are critical for electrical detection of tissue health and viability in applications such as toxicological screening. Organic transistors based on conducting polymers offer a promising route to efficiently transduce ionic currents to attain high quality impedance spectra, but collection of complete impedance spectra can be time consuming (minutes). By applying uniform white noise at the gate of an organic electrochemical transistor (OECT), and measuring the resulting current noise, we are able to dynamically monitor the impedance and thus integrity of cultured epithelial monolayers. We show that noise sourcing can be used to track rapid monolayer disruption due to compounds which interfere with dynamic polymerization events crucial for maintaining cytoskeletal integrity, and to resolve sub-second alterations to the monolayer integrity.

Acrylamide toxic effects on mouse oocyte quality and fertility in vivo.

Acrylamide is an industrial chemical that has attracted considerable attention due to its presumed carcinogenic, neurotoxic, and cytotoxic effects. In this study we investigated possible acrylamide reproductive toxic effects in female mice. Mice were fed an acrylamide-containing diet for 6 weeks. Our results showed the following effects of an acrylamide-containing diet. (1) Ovary weights were reduced in acrylamide-treated mice and oocyte developmental competence was also reduced, as shown by reduced GVBD and polar body extrusion rates. (2) Acrylamide feeding resulted in aberrant oocyte cytoskeletons, as shown by an increased abnormal spindle rate and confirmed by disrupted γ-tubulin and p-MAPK localization. (3) Acrylamide feeding resulted in oxidative stress and oocyte early stage apoptosis, as shown by increased ROS levels and p-MAPK expression. (4) Fluorescence intensity analysis showed that DNA methylation levels were reduced in acrylamide-treated oocytes and histone methylation levels were also altered, as H3K9me2, H3K9me3, H3K4me2, and H3K27me3 levels were reduced after acrylamide treatment. (5) After acrylamide feeding, the litter sizes of acrylamide-treated mice were significantly smaller compared to thus of control mice. Thus, our results indicated that acrylamide might affect oocyte quality through its effects on cytoskeletal integrity, ROS generation, apoptosis induction, and epigenetic modifications.

Multiphasic stress relaxation response of freshly isolated and cultured vascular smooth muscle cells measured by quasi-in situ tensile test.

Vascular smooth muscle cells (SMCs) undergo a phenotypic change from a contractile to a synthetic state under pathological conditions, such as atherogenesis and restenosis. Although the viscoelastic properties of SMCs are of particular interest because of their role in the development of these vascular diseases, the effects of phenotypic changes on their viscoelastic properties are unclear at this stage. We performed the stress relaxation test at constant strain (ε=30%) for the freshly isolated contractile SMCs (FSMCs) and the cultured synthetic SMCs (CSMCs) maintaining in situ cell shape and cytoskeletal integrity. We also investigated the effect of extracellular Ca2+ on their viscoelastic behaviors. FSMCs and CSMCs exhibited multiphasic stress relaxation, which consisted of rapid relaxation, occurring on a time scale of several seconds and several 10seconds, and slow relaxation occurring on a time scale of 1000seconds. The estimated elastic modulus of CSMCs was less than one-half that of FSMCs, that was associated with a decreased of amount of actin stress fibers (SFs) during the transition from contractile to synthetic phenotypes. FSMCs showed a conservation of tension with extracellular Ca2+ following rapid stress relaxation. In contrast, CSMCs showed a consecutive decrease in tension independent of Ca2+. This suggests that the decrease in tension in a long time scale may be involved in mechanical remodeling of SFs induced through a Rho-dependent pathway, which is Ca2+-independent and become predominant in the transition from contractile to synthetic phenotypes.

Overexpression of Hsp70 confers cytoprotection during gliadin exposure in Caco-2 cells.

In Celiac disease (CD), cytoskeletal integrity of intestinal cells is disrupted by gliadin exposure. This study investigates the role of heat shock protein (Hsp)70 during cytoskeletal recovery in CD by assessing its induction and effects on junctional proteins. Using an in-vitro model of CD, cytoskeletal injury and recovery was assessed in gliadin-exposed Caco-2 cells by measuring cellular distribution of ezrin, E-cadherin, and Hsp70 by differential centrifugation. Effects of Hsp70 were tested by an in-vitro repair assay, based on the incubation of injured or recovered cytoskeletal cellular fractions in noncytoskeletal supernatants containing low or high levels of Hsp70, or by transient transfection of Caco-2 cells with Hsp70. Cytoskeletal disruption of ezrin and E-cadherin was demonstrated in gliadin-exposed Caco-2 cells by their significant shift from the cytoskeletal pellet into the noncytoskeletal supernatant fraction. Recovery from gliadin exposure was associated with induction and cytoskeletal redistribution of Hsp70. The in-vitro repair assay delineated direct evidence for HSP-mediated repair by stabilization of junctional proteins by Hsp70. Overexpression of Hsp70 resulted in significantly increased cytoskeletal integrity. Our results establish an essential role of HSP-mediated cytoskeletal repair in Caco-2 cells during recovery from in-vitro gliadin exposure.

Drosophila modelling axonal transport in the face of tau pathology.

Alzheimer’s disease (AD) is a devastating neurodegenerative disease causing irreversible cognitive decline in the elderly. There is no disease-modifying therapy for this condition and the mechanisms underpinning neuronal dysfunction and neurodegeneration are unclear. Compromised cytoskeletal integrity within neurons is reported in AD. This is believed to result from loss-of-function of the microtubule-associated protein tau, which becomes hyper-phosphorylated and deposits into neurofibrillary tangles in AD. We have developed a Drosophila model of tauopathy in which abnormal human tau mediates neuronal dysfunction characterised by microtubule destabilisation, axonal transport disruption, synaptic defects and behavioural impairments. Here we show that a microtubule-stabilising drug, NAPVSIPQ (NAP), prevents as well as reverses these phenotypes even after they have become established. Moreover, it does not alter abnormal tau levels indicating that it by-passes toxic tau altogether. Thus, microtubule stabilisation is a disease-modifying therapeutic strategy protecting against tau-mediated neuronal dysfunction, which holds great promise for tauopathies like AD.

A novel closed-body model of spinal cord injury caused by high-pressure air blasts produces extensive axonal injury and motor impairments

Diffuse axonal injury is thought to be the basis of the functional impairments stemming from mild traumatic brain injury. To examine how axons are damaged by traumatic events, such as motor vehicle accidents, falls, sports activities, or explosive blasts, we have taken advantage of the spinal cord with its extensive white matter tracts. We developed a closed-body model of spinal cord injury in mice whereby high-pressure air blasts targeted to lower thoracic vertebral levels produce tensile, compressive, and shear forces within the parenchyma of the spinal cord and thereby cause extensive axonal injury. Markers of cytoskeletal integrity showed that spinal cord axons exhibited three distinct pathologies: microtubule breakage, neurofilament compaction, and calpain-mediated spectrin breakdown. The dorsally situated axons of the corticospinal tract primarily exhibited microtubule breakage, whereas all three pathologies were common in the lateral and ventral white matter. Individual axons typically demonstrated only one of the three pathologies during the first 24h after blast injury, suggesting that the different perturbations are initiated independently of one another. For the first few days after blast, neurofilament compaction was frequently accompanied by autophagy, and subsequent to that, by the fragmentation of degenerating axons. TuJ1 immunolabeling and mice with YFP-reporter labeling each revealed more extensive microtubule breakage than did βAPP immunolabeling, raising doubts about the sensitivity of this standard approach for assessing axonal injury. Although motor deficits were mild and largely transient, some aspects of motor function gradually worsened over several weeks, suggesting that a low level of axonal degeneration continued past the initial wave. Our model can help provide further insight into how to intervene in the processes by which initial axonal damage culminates in axonal degeneration, to improve outcomes after traumatic injury. Importantly, our findings of extensive axonal injury also caution that repeated trauma is likely to have cumulative adverse consequences for both brain and spinal cord.

S100A4 expression is closely linked to genesis and progression of glioma by regulating proliferation, apoptosis, migration and invasion.

The calcium-binding S100A4 protein is involved in epithelial to mesenchymal transition, oncogenic transformation, angiogenesis, cytoskeletal integrity, mobility and metastasis of cancer cells. This study aimed to clarify the roles of S100A4 in genesis and progression of glioma. S100A4 expression was examined by real-time RT-CPR and Western blot in glioma and paired normal brain tissue (n=69), and compared with clinicopathological parameters of tumors. In addition, glioma U251 cells transfected with an S100A4-expressing plasmid were examined for proliferation by MTT, apoptosis by Annexin V-FITC, and migration and invasion with Transwell chambers. Increased S100A4 mRNA expression was found in gliomas, compared with paired non-tumor tissue (p<0.001). Gradual elevation of overexpression of S100A4 was observed with increasing glioma grade (p<0.001). Astrocytoma showed lower S100A4 mRNA expression than oligodendrogliomas, with glioblastomas having highest values (p<0.001). Similar results were obtained for S100A4 protein, a positive link being found between mRNA and protein expression in gliomas (p<0.001). There was higher growth, lower apoptosis, stronger migration and invasion of S100A4 transfectants than control and mock transfected cells (p<0.001). These findings indicate that up-regulated S100A4 expression is positively linked to pathogenesis, progression and histogenesis of glioma by modulating proliferation, apoptosis, migration and invasion.

Actin-myosin network influences morphological response of neuronal cells to altered osmolarity.

Acute osmotic fluctuations in the brain occur during a number of clinical conditions and can result in a variety of adverse neurological symptoms. Osmotic perturbation can cause changes in the volumes of intra- and extracellular fluid and, due to the rigidity of the skull, can alter intracranial pressure thus making it difficult to analyze purely osmotic effects in vivo. The present study aims to determine the effects of changes in osmolarity on SH-SY5Y human neuroblastoma cells in vitro, and the role of the actin-myosin network in regulating this response. Cells were exposed to hyper- or hypoosmotic media and morphological and cytoskeletal responses were recorded. Hyperosmotic shock resulted in a drop in cell body volume and planar area, a persisting shape deformation, and increases in cellular translocation. Hypoosmotic shock did not significantly alter planar area, but caused a transient increase in cell body volume and an increase in cellular translocation via the development of small protrusions rich in actin. Disruption of the actin-myosin network with latrunculin and blebbistatin resulted in changes to volume and shape regulation, and a decrease in cellular translocation. In both osmotic perturbations, no apparent disruptions to cytoskeletal integrity were observed by light microscopy. Overall, because osmotically induced changes persisted even after volume regulation occurred, it is possible that osmotic stress may play a larger role in neurological dysfunction than currently believed.

Nuclear export inhibitors avert progression in preclinical models of inflammatory demyelination.

Axonal damage has been associated with aberrant protein trafficking. This study characterizes a novel class of compounds targeting nucleo-cytoplasmic shuttling, by binding to the catalytic groove of the nuclear export protein XPO1/CRM1 (chromosome region maintenance protein1). Oral administration of novel reversible CRM1 inhibitors in preclinical murine models of demyelination significantly attenuated disease progression, even when started after the onset of paralysis. Clinical efficacy was associated with decreased proliferation of immune cells, characterized by nuclear accumulation of cell cycle inhibitors, and preservation of cytoskeletal integrity even in demyelinated axons. Neuroprotection was not limited to models of demyelination, but observed also in other mouse models of axonal damage (i.e. kainic acid injection) and detected in cultured neurons after knockdown of Xpo1, the gene encoding for CRM1. A proteomic screen for target molecules revealed that CRM1 inhibitors in neurons prevented nuclear export of molecules associated with axonal damage while retaining transcription factors modulating neuroprotection.

Effects of nanosecond pulse electric fields on cellular elasticity

We investigated the effects of a single 60 nanosecond pulsed electric field (nsPEF) of low (15kV/cm) and high (60kV/cm) field strengths on cellular morphology and membrane elasticity in Jurkat cells using fluorescent microscopy and atomic force microscopy (AFM). We performed force displacement measurements on cells using AFM and calculated the Young's modulus for membrane elasticity. Differential effects were observed depending upon pulsing conditions. We found that a single nsPEF of low field strength did not induce any apparent cytoskeletal breakdown and had minor morphological changes. Interestingly, force measurements and calculation of Young's modulus showed a significant decrease in membrane elasticity. A single nsPEF of high field strength induced stark morphological changes due to disruption of the actin cytoskeleton and a marked decrease in elasticity likely caused by irreversible membrane damage. We suggest that the cellular morphology is mainly dependent on stabilization by the actin cytoskeleton, while the elasticity changes are partially dependent on the cytoskeletal integrity.

Crk and CrkL are required for cell transformation by v-fos and v-ras.

Crk and CrkL are SH2- and SH3-containing cytosolic adaptor proteins that can induce anchorage-independent growth of fibroblasts. Crk and CrkL play key roles in maintaining cytoskeletal integrity, cell motility and migration. We investigated the role of these two proteins in oncogenic transformation induced by v-fos and v-ras oncogenes using cell lines and fibroblasts carrying conditional alleles of Crk or CrkL. Transformation was assessed by cell morphology, saturation density and anchorage-independent growth in soft agar. We found that cell lines expressing v-fos or v-ras in the absence of Crk or CrkL displayed no evident morphological alterations and reduced anchorage-independent growth compared to those retaining Crk and CrkL. Similarly, overexpression of v-fos in mouse embryonic fibroblasts conferred a growth advantage and induced morphological changes, both of which were abrogated in the absence of either Crk or CrkL. In contrast, Crk, but not CrkL, contributed to v-ras-induced transformation of embryonic fibroblasts. These results suggest that both Crk and CrkL are required for the acquisition of cellular transformation by v-fos, whereas Crk plays a more prominent role than CrkL in v-ras-induced transformation.

Abstract B57: Distinct interactions of Ras superfamily members with common protein targets

Abstract Ras superfamily members act as molecular switches to many key signaling pathways, including proliferation cytoskeletal integrity, differentiation and cellular cargo transport. The Ras subfamily, consisting of KRas, NRas and HRas are the most widely studied Ras-family members due to the high instance of Ras activating mutation in human cancers. Rab subfamily members are important regulators of endocytosis, consisting of Rab5 (early endosome), Rab7 (recycling endosome) and Rab11 (late endosome). We have discovered that common binding partners exist between these Ras subfamilies, resulting in distinct interactions and regulatory effects. Two such commonalities are scaffold protein ITSN1 and lipid kinase PI3KC2β. There is evidence that the learning disabilities associated with Down syndrome may be somewhat attributed to deregulated vesicular transport in the brain. DS patients also have a 10-20 fold increased risk of developing acute myeloid leukemia (AML). ITSN1, a regulator of endocytosis and activator of the oncogenic AKT pathway (1), is overexpressed in Down syndrome patients. PI3KC2β is a member of the PI3K family of lipid kinases, which phosphorylate phosphatidylinositol leading to AKT activation. Treatment of neuroblastoma and AML cell lines with PI3KC2β-specific inhibitors greatly decreases proliferation (2) and silencing PI3KC2β in neuroblastoma cell lines results in a 50% reduction in tumor volume in xenograft models. Our previous work demonstrated that nucleotide-free HRas preferentially binds to PI3KC2β, inhibiting its activity (see poster by JP O'Bryan; 3). The addition of ITSN1 disrupts this interaction, presumably restoring the active state of both proteins. In contrast, constitutively activated Rab5 and KRas preferentially interact with PI3KC2β. Interestingly KRas sequesters PI3KC2β to the plasma membrane. The current dogma states that Ras proteins must be GTP-loaded and associated with the plasma membrane to activate their classic effectors suggesting that KRas would be active in this capacity and may therefore positively regulate PI3KC2β activity. Here we present evidence of three Ras superfamily members interacting in distinct ways with the same target protein and provide support for their related yet contrasting roles in disease states. Citation Format: Russell Spencer Smith, Katy A. Wong, Angela Russo, John P. O'Bryan. Distinct interactions of Ras superfamily members with common protein targets. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B57. doi: 10.1158/1557-3125.RASONC14-B57

Binding of WIP to actin is essential for T cell actin cytoskeleton integrity and tissue homing.

The Wiskott-Aldrich syndrome protein (WASp) is important for actin polymerization in T cells and for their migration. WASp-interacting protein (WIP) binds to and stabilizes WASp and also interacts with actin. Cytoskeletal and functional defects are more severe in WIP(-/-) T cells, which lack WASp, than in WASp(-/-) T cells, suggesting that WIP interaction with actin may be important for T cell cytoskeletal integrity and function. We constructed mice that lack the actin-binding domain of WIP (WIPΔABD mice). WIPΔABD associated normally with WASp but not F-actin. T cells from WIPΔABD mice had normal WASp levels but decreased cellular F-actin content, a disorganized actin cytoskeleton, impaired chemotaxis, and defective homing to lymph nodes. WIPΔABD mice exhibited a T cell intrinsic defect in contact hypersensitivity and impaired responses to cutaneous challenge with protein antigen. Adoptively transferred antigen-specific CD4(+) T cells from WIPΔABD mice had decreased homing to antigen-challenged skin of wild-type recipients. These findings show that WIP binding to actin, independently of its binding to WASp, is critical for the integrity of the actin cytoskeleton in T cells and for their migration into tissues. Disruption of WIP binding to actin could be of therapeutic value in T cell-driven inflammatory diseases.

Decreased miR-26a Expression Correlates with the Progression of Podocyte Injury in Autoimmune Glomerulonephritis

MicroRNAs contribute to the pathogenesis of certain diseases and may serve as biomarkers. We analyzed glomerular microRNA expression in B6.MRLc1, which serve as a mouse model of autoimmune glomerulonephritis. We found that miR-26a was the most abundantly expressed microRNA in the glomerulus of normal C57BL/6 and that its glomerular expression in B6.MRLc1 was significantly lower than that in C57BL/6. In mouse kidneys, podocytes mainly expressed miR-26a, and glomerular miR-26a expression in B6.MRLc1 mice correlated negatively with the urinary albumin levels and podocyte-specific gene expression. Puromycin-induced injury of immortalized mouse podocytes decreased miR-26a expression, perturbed the actin cytoskeleton, and increased the release of exosomes containing miR-26a. Although miR-26a expression increased with differentiation of immortalized mouse podocytes, silencing miR-26a decreased the expression of genes associated with the podocyte differentiation and formation of the cytoskeleton. In particular, the levels of vimentin and actin significantly decreased. In patients with lupus nephritis and IgA nephropathy, glomerular miR-26a levels were significantly lower than those of healthy controls. In B6.MRLc1 and patients with lupus nephritis, miR-26a levels in urinary exosomes were significantly higher compared with those for the respective healthy control. These data indicate that miR-26a regulates podocyte differentiation and cytoskeletal integrity, and its altered levels in glomerulus and urine may serve as a marker of injured podocytes in autoimmune glomerulonephritis.

HSF-1-mediated cytoskeletal integrity determines thermotolerance and life span.

The conserved heat shock transcription factor-1 (HSF-1) is essential to cellular stress resistance and life-span determination. The canonical function of HSF-1 is to regulate a network of genes encoding molecular chaperones that protect proteins from damage caused by extrinsic environmental stress or intrinsic age-related deterioration. In Caenorhabditis elegans, we engineered a modified HSF-1 strain that increased stress resistance and longevity without enhanced chaperone induction. This health assurance acted through the regulation of the calcium-binding protein PAT-10. Loss of pat-10 caused a collapse of the actin cytoskeleton, stress resistance, and life span. Furthermore, overexpression of pat-10 increased actin filament stability, thermotolerance, and longevity, indicating that in addition to chaperone regulation, HSF-1 has a prominent role in cytoskeletal integrity, ensuring cellular function during stress and aging.

LIMK1 Regulates Human Trophoblast Invasion/Differentiation and Is Down-Regulated in Preeclampsia

Successful human pregnancy requires extensive invasion of maternal uterine tissues by the placenta. Invasive extravillous trophoblasts derived from cytotrophoblast progenitors remodel maternal arterioles to promote blood flow to the placenta. In the pregnancy complication preeclampsia, extravillous trophoblasts invasion and vessel remodeling are frequently impaired, likely contributing to fetal underperfusion and maternal hypertension. We recently demonstrated in mouse trophoblast stem cells that hypoxia-inducible factor-2 (HIF-2)-dependent Lim domain kinase 1 (LIMK1) expression regulates invasive trophoblast differentiation by modulating the trophoblast cytoskeleton. Interestingly, in humans, LIMK1 activity promotes tumor cell invasion by modulating actin and microtubule integrity, as well as by modulating matrix metalloprotease processing. Here, we tested whether HIF-2α and LIMK1 expression patterns suggested similar roles in the human placenta. We found that LIMK1 immunoreactivity mirrored HIF-2α in the human placenta in utero and that LIMK1 activity regulated human cytotrophoblast cytoskeletal integrity, matrix metallopeptidase-9 secretion, invasion, and differentiation in vitro. Importantly, we also found that LIMK1 levels are frequently diminished in the preeclampsia setting in vivo. Our results therefore validate the use of mouse trophoblast stem cells as a discovery platform for human placentation disorders and suggest that LIMK1 activity helps promote human placental development in utero.

Abstract 2016: Mst2 is required for tumor progression and Yap activity

Abstract The survival of patients with head and neck cancer has remained unchanged for decades [1]. Novel therapeutic approaches are needed. The transcription co-activator Yap is known to initiate proliferation and migration programs in response to cytoskeletal cues [2]. As evidence of the relevance of Yap in cancer increases, the lack of pharmacological strategies to inhibit it hinders the possibility of clinical testing. The main Yap regulatory signals in mammals are cytoskeletal integrity and cell shape [2]. Interestingly, Mst2 regulates microtubule coordination in parallel to kinesin Eg5 motor activity[3] and actin polymerization through Rac1 activation [4]. We set out to explore the role of Mst2 in cancer biology and the influence of its cytoskeletal effects in Yap activity using silencing and overexpression systems in early passage oral squamous cell cancer cell lines. In clinically annotated databases, high Mst2 expression correlated with poor prognosis in lung, breast and head and neck cancer. Standard in vitro experiments showed that Mst2 was required and promoted cell migration and invasion. Immunofluorescence staining quantification revealed that Mst2 was necessary for actin polymerization, microtubule targeting to the focal adhesions and microtubule distribution. These effects increased cell height and reduced cell spreading. In keeping with cytoskeletal and cell shape changes, in luciferase reporter assays Mst2 was required for Yap activation, and inhibition of kinesin Eg5 with Monastrol reduced Yap activity in an Mst2 dependent manner. Confocal imaging quantification of 3D organotypic tumors revealed that Mst2 was required for primary tumor invasion into human dermis. Consistently, in an orthotopic xenograft model, tumors with lower Mst2 expression showed a significant reduction in circulating tumor DNA and lung metastasis formation. Although further preclinical studies are warranted, our results support the hypothesis that kinesin Eg5 inhibitors may be used to target Yap activity in oral squamous cell cancers with high Mst2 expression. 1. CRUK. CRUK Oral Cancer Statistics. 2009 [cited 2013; Available from: http://www.cancerresearchuk.org/cancer-info/cancerstats/types/oral/]. 2. Pan, D., The hippo signaling pathway in development and cancer. Dev Cell, 2010. 19(4): p. 491-505. 3. Mardin, B.R., et al., Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction. Nat Cell Biol, 2010. 12(12): p. 1166-76. 4. Mou, F., et al., The Mst1 and Mst2 kinases control activation of rho family GTPases and thymic egress of mature thymocytes. J Exp Med, 2012. 209(4): p. 741-59. Citation Format: Carles Escriu, Klaas Mulder, Samuel Woodhouse, Nathan Benaich, Simon Broad, Frances Richards, Duncan Jodrell, Fiona Watt. Mst2 is required for tumor progression and Yap activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2016. doi:10.1158/1538-7445.AM2014-2016

Abstract 5540: Preclinical investigation of the novel histone deacetylase (HDAC) inhibitor AR-42 in the treatment of cancer-induced cachexia

Abstract Background: Cachexia occurs in more than 50% of cancer patients. Cachexia is characterized by severe loss of weight and skeletal muscle that is not reversed by nutritional support, and contributes significantly to morbidity and mortality. The development of effective therapies for cancer cachexia is clearly warranted. AR-42 is a novel class I/IIB HDAC inhibitor that was developed in our laboratories and currently in Phase I/IB trials at The Ohio State University. Here, we report the anti-cachectic activity of AR-42 in two murine models of cancer cachexia. Methods: Experiments were conducted using the colon-26 adenocarcinoma (C-26) and Lewis lung carcinoma (LLC) tumor mice models of cancer cachexia. AR-42 was administered by oral gavage at 50 mg/kg every other day starting at day 6 after tumor cell inoculation. Serum samples (n = 3) and muscle tissues (n = 8) from each group were sent for RNAseq and small RNAseq (OSU), cytokine/chemokine profiling assays [Eve Technologies, Alberta, Canada] and metabolomic analyses [Metabolon, Research Triangle Park, NC]. Results: In the C-26 model, AR-42 significantly attenuated cachexia-induced losses of skeletal muscle mass and body weight, with minimal effects on C-26 tumor growth, and prolonged survival time relative to mice treated with vehicle only or with other HDAC inhibitors (vorinostat and romidepsin). Metabolomic and gene expression analyses revealed that the effects of AR-42 were associated with its ability to maintain metabolic and gene expression profiles in skeletal muscle to levels comparable to those in muscle from tumor-free mice. Analysis of RNAseq data of relevant genes suggests that AR-42 modulates major pathways involved in muscle wasting, mitochondrial function, lipolysis, and cytoskeletal integrity. The main mechanism of action is thought to be through down-regulating FOXO1 (&amp;gt; 5-fold) leading to suppression of MuRF1 and Atrogin-1. Additional mechanistic validation is underway and full results will be available at the meeting. AR-42-induced abrogation of cachexia and rescue of muscle weight was additionally confirmed in the LLC model. Conclusion: Our results suggest AR-42 induces unique protective effects on cancer-induced muscle wasting and lipolysis, and our findings support further evaluation of AR-42 as a potential treatment for cancer cachexia. Citation Format: Yu-Chou Tseng, Samuel Kulp, I-Lu Lai, Wei He, Ralf Bundschuh, David Frankhouser, Pearlly Yan, Denis Guttridge, Guido Marcucci, Ching-Shih Chen, Tanios Bekaii-Saab. Preclinical investigation of the novel histone deacetylase (HDAC) inhibitor AR-42 in the treatment of cancer-induced cachexia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5540. doi:10.1158/1538-7445.AM2014-5540

Timosaponin A3 induces hepatotoxicity in rats through inducing oxidative stress and down-regulating bile acid transporters.

Aim:To investigate the mechanisms underlying the hepatotoxicity of timosaponin A3 (TA3), a steroidal saponin from Anemarrhena asphodeloides, in rats.Methods:Male SD rats were administered TA3 (100 mg·kg−1·d−1, po) for 14 d, and the blood and bile samples were collected after the final administration. The viability of a sandwich configuration of cultured rat hepatocytes (SCRHs) was assessed using WST-1. Accumulation and biliary excretion index (BEI) of d8-TCA in SCRHs were determined with LC-MS/MS. RT-PCR and Western blot were used to analyze the expression of relevant genes and proteins. ROS and ATP levels, and mitochondrial membrane potential (MMP) were measured. F-actin cytoskeletal integrity was assessed under confocal microscopy.Results:TA3 administration in rats significantly elevated the total bile acid in serum, and decreased bile acid (BA) component concentrations in bile. TA3 inhibited the viability of the SCRHs with an IC50 value of 15.21±1.73 μmol/L. Treatment of the SCRHs with TA3 (1–10 μmol/L) for 2 and 24 h dose-dependently decreased the accumulation and BEI of d8-TCA. The TA3 treatment dose-dependently decreased the expression of BA transporters Ntcp, Bsep and Mrp2, and BA biosynthesis related Cyp7a1 in hepatocytes. Furthermore, the TA3 treatment dose-dependently increased ROS generation and HO-1 expression, decreased the ATP level and MMP, and disrupted F-actin in the SCRHs. NAC (5 mmol/L) significantly ameliorated TA3-induced effects in the SCRHs, whereas mangiferin (10–200 μg/mL) almost blocked TA3-induced ROS generation.Conclusion:TA3 triggers liver injury through inducing ROS generation and suppressing the expression of BA transporters. Mangiferin, an active component in Anemarrhena, may protect hepatocytes from TA3-induced hepatotoxicity.

Proteome analysis reveals roles of L-DOPA in response to oxidative stress in neurons.

BackgroundParkinson’s disease (PD) is the second most common neurodegenerative movement disorder, caused by preferential dopaminergic neuronal cell death in the substantia nigra, a process also influenced by oxidative stress. L-3,4-dihydroxyphenylalanine (L-DOPA) represents the main treatment route for motor symptoms associated with PD however, its exact mode of action remains unclear. A spectrum of conflicting data suggests that L-DOPA may damage dopaminergic neurons due to oxidative stress whilst other data suggest that L-DOPA itself may induce low levels of oxidative stress, which in turn stimulates endogenous antioxidant mechanisms and neuroprotection.ResultsIn this study we performed a two-dimensional gel electrophoresis (2DE)-based proteomic study to gain further insight into the mechanism by which L-DOPA can influence the toxic effects of H2O2 in neuronal cells. We observed that oxidative stress affects metabolic pathways as well as cytoskeletal integrity and that neuronal cells respond to oxidative conditions by enhancing numerous survival pathways. Our study underlines the complex nature of L-DOPA in PD and sheds light on the interplay between oxidative stress and L-DOPA.ConclusionsOxidative stress changes neuronal metabolic routes and affects cytoskeletal integrity. Further, L-DOPA appears to reverse some H2O2-mediated effects evident at both the proteome and cellular level.