ROCK Inhibitor Y-27632 Research Articles

The flavonoid isoquercitrin promotes neurite elongation by reducing RhoA activity.

BackgroundNeurite formation and synaptic patterning are fundamental to the development of a functional nervous system. Flavonoids are natural molecules known for having beneficial effects on brain health through diverse molecular pathways. Cytoskeletal changes occurring during neuritogenesis and synapse formation often involve Rho GTPases. Here we hypothesized that the flavonoid isoquercitrin promotes neuronal differentiation through Rho signalling.Methodology/Principal FindingsWe performed time lapse imaging of NG108-15 cells during incubation with/without isoquercitrin. Isoquercitrin stimulated extensive neurites enriched in the synaptic vesicle protein synaptotagmin-1. Neurite extension was augmented by the ROCK inhibitor Y-27632 suggesting an inactivation of RhoA/Rho kinase as the mechanism. To test this, we first measured the dose-dependent effect of isoquercitrin on RhoA activity and found a 47% reduction in RhoA activity at concentrations which induced neurites (≥40 µM). Secondly, we tested the ability of isoquercitrin to rescue the neural phenotype in a model of RhoA-induced neurite retraction and found that 40 µM isoquercitrin added to cultures previously treated with the RhoA activator calpeptin produced significantly more neurite length/cell than calpeptin alone. Finally, we tested the hypothesis that isoquercitrin may affect RhoA localization preventing the translocation to the plasma membrane. Unexpectedly, immunolocalization studies showed that RhoA was present in nuclear compartments of control NG108-cells, but underwent translocation to the cytoplasm upon treatment with isoquercitrin. DNA microarrays and reverse transcription - quantitative PCR (RT-qPCR) revealed differences in global gene expression of Rho GTPase family members. These data taken together indicate that isoquercitrin is a potential stimulator of neuronal differentiation, through multiple Rho GTPase mediated mechanisms.Conclusions/SignificanceAs several members of the Rho GTPase family are implicated in human neurological disorders/injuries, our results suggest that isoquercitrin could be used in the treatment of these pathological states through its effect on this family of molecular switches.

Vincristine enhances amoeboid-like motility via GEF-H1/RhoA/ROCK/Myosin light chain signaling in MKN45 cells

BackgroundAnti-cancer drugs are widely used in cancer treatment frequently combined with surgical therapy and/or radiation therapy. Although surgery and radiation have been suggested to facilitate invasion and metastasis of tumor cells in some cases, there is so far little information about the effect of anti-cancer drugs on cellular invasive ability and metastasis. In this study, using four different anti-cancer drugs (vincristine, paclitaxel, cisplatin and etoposide), we examined whether these drugs influence the invasive ability of tumor cells.MethodsHuman gastric adenocarcinoma MKN45 cells were used to evaluate the effect of anti-cancer drugs. After drug treatment, cellular invasive ability was assessed using the Matrigel invasion chamber. Cytoskeletal changes after treatment were examined microscopically with F-actin staining. In addition, we monitored cellular motility in 3D matrigel environment by time-lapse microscopic analysis. The drug-induced activation of RhoA and ROCK was evaluated by pull-down assay and Western blotting using an antibody against phosphorylated myosin light chain (MLC), respectively. Where necessary, a ROCK inhibitor Y27632 and siRNA for guanine nucleotide exchange factor-H1 (GEF-H1) were applied.ResultsAmong all drugs tested, only vincristine stimulated the invasive ability of MKN45 cells. Microscopic analysis revealed that vincristine induced the formation of non-apoptotic membrane blebs and amoeboid-like motility. Vincristine significantly enhanced RhoA activity and MLC phosphorylation, suggesting the involvement of RhoA/ROCK pathway in the vincristine-induced cytoskeletal reorganization and cellular invasion. Furthermore, we found that Y27632 as well as the siRNA for GEF-H1, a RhoA-specific activator, attenuated MLC phosphorylation, the formation of membrane blebs and the invasive ability after vincristine treatment.ConclusionsThese results indicate that vincristine activates GEF-H1/RhoA/ROCK/MLC signaling, thereby promoting amoeboid-like motility and the invasive ability of MKN45 cells.

Inhibition of Rho-Kinase Improves Erectile Function, Increases Nitric Oxide Signaling and Decreases Penile Apoptosis in a Rat Model of Cavernous Nerve Injury

Bilateral cavernous nerve injury results in up-regulation of ROCK signaling in the penis. This is linked to erectile dysfunction in an animal model of post-prostatectomy erectile dysfunction. We evaluated whether daily treatment with the ROCK inhibitor Y-27632 (Tocris Bioscience, Ellisville, Missouri) would prevent erectile dysfunction in a rat model of bilateral cavernous nerve injury. Sprague-Dawley® rats underwent surgery to create sham (14) or bilateral (27) cavernous nerve injury. In the injury group 13 rats received treatment with Y-27632 (5 mg/kg twice daily) and 14 received vehicle. At 14 days after injury, rats underwent cavernous nerve stimulation to determine erectile function. Penes were assessed for neuronal and nitric oxide synthase membrane-endothelial nitric oxide synthase. ROCK2 was assessed by Western blot. Cyclic guanosine monophosphate was determined by enzyme-linked immunosorbent assay. Cavernous homogenates were tested for ROCK and protein kinase G enzymatic activity. Penile apoptosis was evaluated using the Apostain technique (Alexis, San Diego, California). Data were analyzed on ROCK using ANOVA and the t test. While erectile function was decreased in rats with bilateral cavernous nerve injury, daily administration of Y-27632 improved erectile responses. Injury decreased neuronal and nitric oxide synthase membrane-endothelial nitric oxide synthase but ROCK2 was significantly increased. Y-27632 treatment restored neuronal nitric oxide synthase, nitric oxide synthase membrane-endothelial nitric oxide synthase and cyclic guanosine monophosphate levels, and protein kinase G activity. Treatment significantly decreased ROCK2 protein and ROCK activity. There were significantly fewer apoptotic cells after treatment than in injured controls. These results provide evidence for up-regulation of the RhoA/ROCK signaling pathway with detrimental effects on erectile function after bilateral cavernous nerve injury. ROCK inhibition improved erectile dysfunction associated with bilateral cavernous nerve injury by preserving penile nitric oxide bioavailability and decreasing penile apoptosis.

Neurokinin 1 Receptor Mediates Membrane Blebbing and Sheer Stress-Induced Microparticle Formation in HEK293 Cells

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2–10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.

Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis

Inherited mutations in the folliculin (FLCN) gene cause the Birt-Hogg-Dubé syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterized. We identified plakophilin-4 (p0071) as a potential novel folliculin interacting protein by yeast two-hybrid analysis. We confirmed the interaction of folliculin with p0071 by co-immunoprecipitation studies and, in view of previous studies linking p0071 to the regulation of rho-signalling, cytokinesis and intercellular junction formation, we investigated the effect of cell folliculin status on p0071-related functions. Folliculin and p0071 partially co-localized at cell junctions and in mitotic cells, at the midbody during cytokinesis. Previously, p0071 has been reported to regulate RhoA signalling during cytokinesis and we found that folliculin deficiency was associated with increased expression and activity of RhoA and evidence of disordered cytokinesis. Treatment of folliculin-deficient cells with a downstream inhibitor of RhoA signalling (the ROCK inhibitor Y-27632) reversed the increased cell migration phenotype observed in folliculin-deficient cells. Deficiency of folliculin and of p0071 resulted in tight junction defects and mislocalization of E-cadherin in mouse inner medullary collecting duct-3 renal tubular cells. These findings suggest that aspects of folliculin tumour suppressor function are linked to interaction with p0071 and the regulation of RhoA signalling.

Enhanced ROCK1 dependent contractility in fibroblast from chronic obstructive pulmonary disease patients

BackgroundDuring wound healing processes fibroblasts account for wound closure by adopting a contractile phenotype. One disease manifestation of COPD is emphysema which is characterized by destruction of alveolar walls and our hypothesis is that fibroblasts in the COPD lungs differentiate into a more contractile phenotype as a response to the deteriorating environment.MethodsBronchial (central) and parenchymal (distal) fibroblasts were isolated from lung explants from COPD patients (n = 9) (GOLD stage IV) and from biopsies from control subjects and from donor lungs (n = 12). Tissue-derived fibroblasts were assessed for expression of proteins involved in fibroblast contraction by western blotting whereas contraction capacity was measured in three-dimensional collagen gels.ResultsThe basal expression of rho-associated coiled-coil protein kinase 1 (ROCK1) was increased in both centrally and distally derived fibroblasts from COPD patients compared to fibroblasts from control subjects (p < 0.001) and (p < 0.01), respectively. Distally derived fibroblasts from COPD patients had increased contractile capacity compared to control fibroblasts (p < 0.01). The contraction was dependent on ROCK1 activity as the ROCK inhibitor Y27632 dose-dependently blocked contraction in fibroblasts from COPD patients. ROCK1-positive fibroblasts were also identified by immunohistochemistry in the alveolar parenchyma in lung tissue sections from COPD patients.ConclusionsDistally derived fibroblasts from COPD patients have an enhanced contractile phenotype that is dependent on ROCK1 activity. This feature may be of importance for the elastic dynamics of small airways and the parenchyma in late stages of COPD.

Erythropoietin promotes axonal regeneration after optic nerve crush in vivo by inhibition of RhoA/ROCK signaling pathway

We investigated whether the RhoA/ROCK pathway was involved in the effect of erythropoietin (EPO) to promote retinal ganglion cells (RGCs) axonal regeneration in a rat optic nerve crush (ONC) model. We demonstrated that both EPO and ROCK inhibitor Y-27632 significantly enhanced RGCs survival and axon regeneration in vivo, and the effects of these agents were additive. Expression of active-RhoA was decreased after EPO or Y-27632 per pull down assay and affinity precipitation. Administration of EPO and Y-27632 cocktail resulted in even more RhoA inactivation, decreased expression of ROCK-1 and ROCK-2, and increased expression of growth associated protein-43 (GAP-43) protein per immunohistochemistry and western blot analysis. Down-regulation of active-RhoA, ROCK-1, and ROCK-2 expression by EPO coincided with the appearance of larger numbers of regenerating axons. In conclusion, the RhoA/ROCK signaling pathway was involved in the EPO effect to promote RGCs axon regeneration after ONC.

ROCK Inhibitor Converts Corneal Endothelial Cells into a Phenotype Capable of Regenerating In Vivo Endothelial Tissue

Corneal endothelial dysfunction accompanied by visual disturbance is a primary indication for corneal transplantation. We previously reported that the adhesion of corneal endothelial cells (CECs) to a substrate was enhanced by the selective ROCK inhibitor Y-27632. It is hypothesized that the inhibition of ROCK signaling may manipulate cell adhesion properties, thus enabling the transplantation of cultivated CECs as a form of regenerative medicine. In the present study, using a rabbit corneal endothelial dysfunction model, the transplantation of CECs in combination with Y-27632 successfully achieved the recovery of corneal transparency. Complications related to cell injection therapy, such as the abnormal deposition of the injected cells as well as the elevation of intraocular pressure, were not observed. Reconstructed corneal endothelium with Y-27632 exhibited a monolayer hexagonal cell shape with a normal expression of function-related markers, such as ZO-1, and Na(+)/K(+)-ATPase, whereas reconstruction without Y-27632 exhibited a stratified fibroblastic phenotype without the expression of markers. Moreover, transplantation of CECs in primates in the presence of the ROCK inhibitor also achieved the recovery of long-term corneal transparency with a monolayer hexagonal cell phenotype at a high cell density. Taken together, these results suggest that the selective ROCK inhibitor Y-27632 enables cultivated CEC-based therapy and that the modulation of Rho-ROCK signaling activity serves to enhance cell engraftment for cell-based regenerative medicine.

High Glucose Enhances Responsiveness of Human Airways Smooth Muscle via the Rho/ROCK Pathway

Glucose moves into airway secretions after a glucose load. Therefore people with diabetes or hyperglycemia spend a significant proportion of each day with glucose in their airways secretions. This study investigated the effects of glucose on isolated human airways and on cultured airway smooth muscle (ASM) cells. Human isolated bronchi were stimulated with acetylcholine, histamine, and transmural stimulation and treated with the selective ROCK inhibitors Y27632 and SB772077B under high-glucose conditions. The effect of high glucose concentrations on intracellular calcium flux and the phosphorylation of MYPT1 in ASM cells was also investigated. High (44 mM for 6 h) glucose, but not mannitol, concentrations led to an enhanced responsiveness of ASM to contractile agents. Y27632 and SB772077B completely abolished (P < 0.05) the enhanced contractile effects with a high-concentration glucose solution, compared with control tissues. In cultured ASM cells, incubation with high glucose concentrations significantly (P < 0.05) enhanced bradykinin-induced intracellular calcium flux and the levels of pMYPT1, which were inhibited by Y27632 (P < 0.05). Our study has demonstrated that high glucose concentrations leads to hyperresponsiveness of human isolated bronchi and enhances intracellular calcium release in cultured ASM cells via a Rho/ROCK- and pMYPT1-dependent pathway, suggesting that this crucial pathway may contribute to the reduced lung function observed in patients with diabetes. These data propose novel targets for the treatment of patients with respiratory diseases that also suffer from diabetes mellitus.

ROCK inhibitor Y-27632 enhances the survivability of dissociated buffalo (Bubalus bubalis) embryonic stem cell-like cells

This study investigated the effects of supplementation of culture medium with 10 μM Y-27632, a specific inhibitor of Rho kinase activity, for 6 days on self-renewal of buffalo embryonic stem (ES) cell-like cells at Passage 50-80. Y-27632 increased mean colony area (P<0.05) although it did not improve their survival. It decreased OCT4 expression (P<0.05), increased NANOG expression (P<0.05), but had no effect on SOX2 expression. It also increased expression of anti-apoptotic gene BCL-2 (P<0.05) and decreased that of pro-apoptotic genes BAX and BID (P<0.05). It increased plating efficiency of single-cell suspensions of ES cells (P<0.05). Following vitrification, the presence of Y-27632 in the vitrification solution or thawing medium or both did not improve ES cell colony survival. However, following seeding of clumps of ES cells transfected with pAcGFP1N1 carrying green fluorescent protein (GFP), Y-27632 increased colony formation rate (P<0.01). ES cell colonies that formed in all Y-27632-supplemented groups were confirmed for expression of pluripotency markers alkaline phosphatase, SSEA-4 and TRA-1-60, and for their ability to generate embryoid bodies containing cells that expressed markers of ectoderm, mesoderm and endoderm. In conclusion, Y-27632 improves survival of buffalo ES cells under unfavourable conditions such as enzymatic dissociation to single cells or antibiotic-assisted selection after transfection, without compromising their pluripotency.

Thrombin Receptor Regulates Hematopoiesis and Endothelial-to-Hematopoietic Transition

Hematopoietic development and vascular development are closely related physiological processes during vertebrate embryogenesis. Recently, endothelial-to-hematopoietic transition (EHT) was demonstrated to be critical for hematopoietic stem and progenitor cell induction, but its underlying regulatory mechanisms remain poorly understood. Here we show that thrombin receptor (F2r), a protease-activated G protein-coupled receptor required for vascular development, functions as a negative regulator during hematopoietic development. F2r is significantly upregulated during hematopoietic differentiation of mouse embryonic stem cells (mESCs) and zebrafish hematopoietic development. Pharmacological or genetic inhibition of F2r promotes hematopoietic differentiation, whereas F2r overexpression shows opposite effects. Further mechanistic studies reveal that F2r-RhoA/ROCK pathway inhibits EHT in vitro and negatively regulates zebrafish EHT and hematopoietic stem cell induction in vivo. Taken together, this study demonstrates a fundamental role of F2r-RhoA/ROCK pathway in vertebrate hematopoiesis and EHT, as well as an important molecular mechanism coordinating hematopoietic and vascular development.

Involvement of microglial RhoA/Rho-Kinase pathway activation in the dopaminergic neuron death. Role of angiotensin via angiotensin type 1 receptors

It has recently been shown that the dopaminergic cell loss induced by neurotoxins is enhanced by brain angiotensin II (AII) via type 1 receptors (AT1). However, the mechanisms involved in the dopaminergic degeneration and the brain inflammatory effects of AII have not been clarified. The RhoA–Rho-Kinase (ROCK) pathway may play a critical role in the inflammatory and oxidative effects of AII. In the substantia nigra of mice, administration of the dopaminergic neurotoxin MPTP induced an increase in the expression of RhoA and ROCK II mRNA levels and ROCK activity, which were inhibited by AT1 receptor deletion (i.e., in AT1a null mice treated with MPTP). Administration of the ROCK inhibitor Y-27632 or AT1 deletion induced a significant decrease in MPTP-induced microglial activation and dopaminergic cell death. In rat primary mesencephalic cultures treated with MPP+, the increase in dopaminergic cell loss induced by AII administration was also inhibited by treatment with Y27632. Intense expression of ROCK II was observed in the microglial cells in the substantia nigra of mice treated with MPTP, and the major role of the microglial ROCK was confirmed by comparing mesencephalic cultures with and without microglia. Activation of the RhoA/ROCK pathway is involved in the MPTP-induced dopaminergic degeneration, and in the enhancing effect of AII/AT1 activation on the microglial response and dopaminergic degeneration. ROCK inhibitors and AT1 receptor antagonists may provide new neuroprotective strategies against the progression of Parkinson's disease.

Abstract LB-145: Folliculin interacts with p0071 (Plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis

Abstract Inherited mutations in the folliculin gene (FLCN) cause the Birt-Hogg-Dubé syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterised. We identified plakophilin-4 (PKP4, p0071) as a potential novel folliculin interacting protein by yeast-2-hybrid analysis. We confirmed the interaction of folliculin with p0071 by co-immunoprecipitation studies and, in view of previous studies linking p0071 to regulation of rho-signalling, cytokinesis and intercellular junction formation, we investigated the effect of cell folliculin status on p0071-related functions. Folliculin and p0071 partially colocalised at cell junctions and in mitotic cells, at the midbody during cytokinesis. Previously, p0071 has been reported to regulate RhoA signalling during cytokinesis and we found that folliculin deficiency was associated with increased expression and activity of RhoA and evidence of disordered cytokinesis. Treatment of folliculin deficient cells with a downstream inhibitor of RhoA signalling (the ROCK inhibitor Y-27632) reversed the increased cell migration phenotype observed in folliculin deficient cells. Deficiency of folliculin and of p0071 resulted in tight junction defects and mislocalisation of E-cadherin in IMCD-3 renal tubular cells. These findings suggest that aspects of folliculin tumour suppressor function are linked to interaction with p0071 and regulation of RhoA signalling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-145. doi:1538-7445.AM2012-LB-145

Abstract 1444: Stathmin suppression influences ROCK signaling and reduces cell invasion and metastasis in neuroblastoma

Abstract Neuroblastoma is a highly metastatic childhood cancer of the sympathetic nervous system. Stathmin is a microtubule destabilizing protein highly expressed in neuroblastoma though studies addressing its functional role in this malignancy have been limited. The Rho- Rho-associated coiled-coil forming kinase (Rho-ROCK) signaling pathway has been implicated in metastasis through its regulation of the cell cytoskeleton namely the actin remodeling proteins, cofilin and myosin light chain (MLC), and microtubule dynamics. The aims of this study were to determine whether stathmin influenced the Rho-ROCK signaling pathway and metastasis in neuroblastoma. Methods: SiRNA-mediated stathmin suppression was confirmed in 2 independent neuroblastoma cell lines, BE(2)-C and SY5Y, by qPCR and western blot. The cell cytoskeleton of siRNA-transfected cells was visualized by staining with phalloidin (actin filaments) and β-tubulin (microtubules) and tubulin polymer levels were examined by western blot. The expression of cofilin and MLC in siRNA-transfected cells, following treatment with the ROCK inhibitor Y-27632, were analyzed by western blot. SiRNA-transfected cells were subjected to chemotaxis transwell migration and invasion assays. To assess stathmin's role in neuroblastoma metastasis, two million control or stathmin shRNA/luciferase-expressing neuroblastoma cells [SK-N-BE(2)/TGL] were injected into the left adrenal fat pad of 8 week old SCID-Beige mice. Tumor growth was monitored weekly using the Xenogen IVIS System. Mice were sacrificed when primary tumors reached 1500mm3, 38 days post-injection or upon health decline. Ex vivo imaging and IHC confirmed neuroblastoma cells in the lungs of mice. Results: SiRNA-mediated stathmin suppression significantly reduced neuroblastoma cell migration by 44-47% [SY5Y cells 44% reduction (p&amp;lt;0.05) and BE(2)C cells 47% reduction (p&amp;lt;0.001)] and invasion through an extra-cellular matrix by 55-62% [SY5Y cells 55% reduction (p&amp;lt;0.05) and BE(2)C cells 62% reduction (p&amp;lt;0.05)]. Stathmin suppression also altered neuroblastoma cell morphology and this was associated with changes in the cytoskeleton including increased tubulin polymer levels and the phosphorylation of cofilin and MLC. Treatment of stathmin-suppressed neuroblastoma cells with Y-27632 ablated MLC phosphorylation, and returned the level of cofilin phosphorylation and cell invasion back to that of untreated control cells. This highlights a novel link between stathmin expression and ROCK signaling. In vivo, shRNA-mediated stathmin suppression did not influence neuroblastoma tumor growth. In contrast, stathmin suppression significantly reduced metastatic neuroblastoma tumor burden in the lungs by 71% (p&amp;lt;0.01) compared to controls. Conclusion: Stathmin is a novel mediator of metastasis in neuroblastoma and potential therapeutic target for this deadly disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1444. doi:1538-7445.AM2012-1444

Abstract 1529: Cross-talk between carcinoma-associated fibroblasts and invasive breast cancer cells: Role of RhoA / ROCK signaling

Abstract The high density of collagen and carcinoma-associated fibroblasts (CAFs) correlates with metastasis and reduced survival in breast cancer. CAFs have been shown to contribute to tumor progression by promoting notably cancer cell survival, migration and invasion. Furthermore, in breast cancer patients, the expression of RhoA and its downstream effector ROCK1 has been associated with cancer outgrowth and dissemination. RhoA has been shown to promote the motility and invasion of breast cancer cells. Hence, the main goal of our study is to determine how CAFs affect breast cancer cell survival and invasion, and the role of RhoA/ROCK signaling in these two processes. To study in vitro invasion, we grew MDA-MB-231 cells expressing GFP on a low adhesive substrate (poly-HEMA) in order to produce spheroids, which we then seeded alone or with human mammary CAFs in a collagen I gel. We measured RhoA activation with a pull-down assay. Our findings show that CAFs activate RhoA in MDA-MB-231 cells and promote their invasion, by inducing cellular scattering and a switch from an elongated to an amoeboid mode of migration. In presence of CAFs, the ROCK inhibitor Y27632 inhibited the rounding of cancer cells and significantly reduced their invasion, confirming the role of ROCK in the CAF-promoted invasiveness of cancer cells. Then to determine the role of RhoA signaling in cancer cells in response to CAFs, we used an shRNA construct to downregulate the expression of RhoA. Interestingly, RhoA downregulation prevented cellular aggregation and the formation of spheroids on poly-HEMA, and induced anoikis in MDA-MB-231 cells, thus suggesting that RhoA plays a role in cell-cell adhesion, which can promote the survival of cells with relatively poor matrix adhesion. These results further suggest that the high levels of active-RhoA induced by CAFs – in rounded cancer cells – maybe necessary for the survival of cancer cells with an amoeboid migration phenotype. To determine if CAFs could affect other pathways that would improve the survival of rounded cancer cells, we used a pathway-focused RT-PCR array to analyze apoptosis signaling. Our preliminary results show that CAFs can enhance the expression of several pro-survival genes in cancer cells, including Akt, which has been recently reported to promote anoikis resistance in melanoma cells via RhoA. In conclusion, our results suggest that CAFs-dependent activation of RhoA/ROCK signaling promotes anoikis resistance and amoeboid migration in invasive breast cancer cells. Since the ROCK inhibitor Fasudil has been approved for the treatment of cardiovascular diseases in Japan, our findings may lead to the use of this drug or other ROCK inhibitors in the treatment of invasive breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1529. doi:1538-7445.AM2012-1529

Abstract 226: The focal adhesion kinase is phosphorylated on serine732 by a Growth factor-dependent pathway and contributes to the proliferation of tumor cells

Abstract Focal adhesion kinase (FAK) is a tyrosine kinase localized at the site of focal adhesions (FA). FAK is identified as a key mediator of signaling by integrins, the major family of cell surface receptors for extracellular matrix, as well as by other receptors in both normal and cancer cells. FAK plays a prominent role in tumor progression and metastasis regulating cellular processes including migration, invasion, epithelial to mesenchymal transition, and angiogenesis. Integrin-FAK signaling has been shown to activate a number of biological mechanisms through phosphorylation and protein-protein interactions promoting tumorigenesis. FAK seems also to have a role in the proliferation of tumor cells by its biochemical and biological association to cytoplasmic kinases such as src and ERK. However, the mechanisms that involve FAK in those processes are not yet clarify. The best characterized FAK phosporylation event is the auto-phosphorylation at the tyrosine397 (Tyr397) which creates a motif that is recognized by various SH2 domain-containing proteins, such as src which phosphorylates FAK on Tyr576. The c-terminal domain of FAK undergoes to several serine-phosphorylation events whose role is not well-understood. Previous studies in neural and endothelial cells (EC) have shown a new role for FAK in the regulation of centrosome functions during mitosis dependent on Ser732 phosphorylation. We have found that EGF stimulation of selected starved melanoma, thyroid and high stage ovarian tumor cells induced, in dividing cells, accumulation of phosphorylated (P-)FAK on Ser732. Inhibition of cell/substrate adhesion with specific anti-integrin antibodies demonstrated that P-FAKSer732 activation is not induced by integrin clustering. Confocal immunofluorescence and immunoprecipitation showed that, in these cells, P-FAKSer732 is localized in a perinuclear site independently from FA and P-FAKTyr397. Treatment of an high proliferative melanoma cell line with the Rock inhibitor Y27632 partially decreased the levels of P-FAKSer732, the cellular proliferation and impaired the mitotic spindle formation. FACS analysis showed that the P-FAKSer732 decreased upon treatment with the MEK inhibitor UO126 in a dose-dependent manner together with the levels of P(Ser 10) -Histone H3 thus demonstrating that FAK is involved in the mitotic process regulated by MEK/ERK activation. Indeed, P-FAKSer732 co-localized with microtubules of the mitotic spindle in proliferating cells and immunoprecipitated with the motor protein dynein and with the acetylated tubulin of polymerized microtubules. The finding of a novel role for FAK in tumor cells might provide the underpinning for therapeutic strategies in selected solid tumors. Partially supported by Italian Ministery of Health and Associazione Italiana per la Ricerca sul Cancro (AIRC). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 226. doi:1538-7445.AM2012-226

One-Step Derivation of Mesenchymal Stem Cell (MSC)-Like Cells from Human Pluripotent Stem Cells on a Fibrillar Collagen Coating

Controlled differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into cells that resemble adult mesenchymal stem cells (MSCs) is an attractive approach to obtain a readily available source of progenitor cells for tissue engineering. The present study reports a new method to rapidly derive MSC-like cells from hESCs and hiPSCs, in one step, based on culturing the cells on thin, fibrillar, type I collagen coatings that mimic the structure of physiological collagen. Human H9 ESCs and HDFa-YK26 iPSCs were singly dissociated in the presence of ROCK inhibitor Y-27632, plated onto fibrillar collagen coated plates and cultured in alpha minimum essential medium (alpha-MEM) supplemented with 10% fetal bovine serum, 50 uM magnesium L-ascorbic acid phosphate and 100 nM dexamethasone. While fewer cells attached on the collagen surface initially than standard tissue culture plastic, after culturing for 10 days, resilient colonies of homogenous spindle-shaped cells were obtained. Flow cytometric analysis showed that a high percentage of the derived cells expressed typical MSC surface markers including CD73, CD90, CD105, CD146 and CD166 and were negative as expected for hematopoietic markers CD34 and CD45. The MSC-like cells derived from pluripotent cells were successfully differentiated in vitro into three different lineages: osteogenic, chondrogenic, and adipogenic. Both H9 hES and YK26 iPS cells displayed similar morphological changes during the derivation process and yielded MSC-like cells with similar properties. In conclusion, this study demonstrates that bioimimetic, fibrillar, type I collagen coatings applied to cell culture plates can be used to guide a rapid, efficient derivation of MSC-like cells from both human ES and iPS cells.

Formation of nanofilms on cell surfaces to improve the insertion efficiency of a nanoneedle into cells

A nanoneedle, an atomic force microscope (AFM) tip etched to 200nm in diameter and 10μm in length, can be inserted into cells with the aid of an AFM and has been used to introduce functional molecules into cells and to analyze intracellular information with minimal cell damage. However, some cell lines have shown low insertion efficiency of the nanoneedle. Improvement in the insertion efficiency of a nanoneedle into such cells is a significant issue for nanoneedle-based cell manipulation and analysis. Here, we have formed nanofilms composed of extracellular matrix molecules on cell surfaces and found that the formation of the nanofilms improved insertion efficiency of a nanoneedle into fibroblast and neural cells. The nanofilms were shown to improve insertion efficiency even in cells in which the formation of actin stress fibers was inhibited by the ROCK inhibitor Y27632, suggesting that the nanofilms with the mesh structure directly contributed to the improved insertion efficiency of a nanoneedle.

Testosterone and farnesoid X receptor agonist INT-747 counteract high fat diet-induced bladder alterations in a rabbit model of metabolic syndrome

In the male, metabolic syndrome (MetS) is associated to an increased risk of benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS). A recently established rabbit model of high fat diet (HFD)-induced MetS showed hypogonadism and the presence of prostate gland alterations, including inflammation, hypoxia and fibrosis. The present study investigated whether HFD-induced MetS might also alter bladder structure and function. Testosterone and the farnesoid X receptor (FXR) agonist INT-747, were evaluated for possible effects on HFD bladder. MetS rabbits develop bladder alterations, including fibrosis (reduced muscle/fiber ratio), hypoxia [2-fold increase as compared to regular diet (RD) group], low-grade inflammation (increased leukocyte infiltration and inflammatory markers) and RhoA/ROCK hyperactivity. Bladder strips from HFD rabbits, pre-contracted with carbachol, showed an overactive response to the selective ROCK inhibitor Y-27632. All these HFD-induced bladder alterations were partially blunted by testosterone and almost completely reverted by INT-747. Both treatments prevented some MetS features (glucose intolerance and visceral fat increase), thus suggesting that their effects on bladder could be ascribed to an improvement of the metabolic and/or hypogonadal state. However, a pathogenetic role for hypogonadism has been ruled out as GnRH analog-induced hypogonadal rabbits, fed a regular diet, did not show any detectable bladder alterations. In addition, INT-747 did not revert the MetS-induced hypogonadal state. FXR mRNA was highly expressed in rabbit bladder and positively associated with visceral fat increase. A direct effect of INT-747 on bladder smooth muscle was further suggested by inhibition of RhoA/ROCK-mediated activity by in vitro experiments on isolated cells. In conclusion, HFD-related MetS features are associated to bladder derangements, which are ameliorated by testosterone or INT-747 administration. INT-747 showed the most marked effects in counteracting MetS-related RhoA/ROCK overactivity, thus opening novel therapeutic opportunities for this drug.

Mesothelioma Cell Proliferation through Autocrine Activation of PDGF- ββ Receptor

Background/Aims: Growth factors play a critical role in proliferation for a variety of cancer cells. The present study was conducted to understand the signaling cascades underlying PDGF-D/PDGF-ββ receptor-mediated proliferation of mesothelioma cells. Methods: Cell growth and cell cycle were analyzed in human non-malignant Met5A cells and malignant mesothelioma cells such as MSTO-211H, NCI-H28, NCI-H2052, and NCI-H2452 cells. Results: Growth of all the cells used here was not affected by PDGF-D, regardless of concentrations (1-30 ng/ml) or treatment time (48-72 h). Spontaneous growth of those cells was significantly inhibited by knocking-down PDGFD or PDGF-ββ receptor, without affecting cell cycling. The cell growth was significantly inhibited by the Akt inhibitor MK2206 and the ROCK inhibitor Y27632 for all the cell types, by the PDK1 inhibitor BX912 for NCI-H28 cells alone, and by the Rac1 inhibitor NSC23766 for NCI-H2052 cells alone, while the PI3 kinase inhibitor wortmannin had no effect. The cell growth, alternatively, was significantly attenuated by MAP kinase kinase inhibitor PD98059 or the ERK1/2 inhibitor FR180204 for all the cell types. Conclusion: The results of the present study show that PDGF-D promotes mesothelioma cell proliferation by targeting ROCK or MAP kinase through autocrine activation of PDGF-ββ receptor.