Serum-deprived Conditions Research Articles

Abstract 5030: Serum deprivation induces Ewing Sarcoma cells to adopt a more invasive phenotype and to upregulate RHO-MKL transcriptional activity.

Abstract Background: Ewing sarcoma (ES) is an aggressive and metastatic tumor of bone and soft tissue that primarily affects children and adolescents. The primary cause of tumor-related death in patients with ES is metastatic disease. In epithelial-derived cancers metastasis is dependent on phenotypic transition of cells from epithelial- to mesenchymal-like states (EMT). In addition, Rho signaling pathways are primary mediators of cancer cell motility and can induce cell migration and invasion, in part through transcriptional activation of MKL/SRF target genes. The mechanisms of metastasis in non-epithelial tumors such as ES remain to be elucidated. Objective: We sought to determine whether phenotypic plasticity between non-invasive and invasive states is also a feature of ES and to determine the molecular mechanism underlying this transition. Methods: ES cell lines were subjected to serum deprivation and phenotypic differences measured using scratch assays, transwell studies, and the xCelligence system. qRT-PCR, Western blot and immunofluorescence were used to quantitate or visualize changes in gene and protein expression, respectively. Results: Serum deprivation induced ES cells to rapidly adopt a more aggressive phenotype, reflected by changes in cell shape and increased motility, while proliferation was inhibited. Serum replenishment rapidly reverted these phenotypic changes. Mechanistically, markers of EMT were largely unaffected indicating that the observed phenotypic plasticity in ES cells was not mediated by classic EMT-MET transitions. Instead, activation of Rho-MKL signaling was apparent as evidenced by nuclear localization of MKL and upregulation of the metastasis-associated MKL/SRF target gene MYL9 in serum-deprived conditions. Exposure of ES cells to a small molecule antagonist of Rho-MKL signaling inhibited ES cell motility. Conclusions: Serum deprivation causes reversible transformation of ES cells to a more migratory phenotype. This is associated with nuclear translocation of MKL and induction of MYL9, a gene that encodes a regulatory light chain required for myosin stability. We propose a model of ES metastasis in which changes to the microenvironment that result in growth-factor deprivation (e.g. increased tumor size or cytotoxic therapy) induce expression of Rho-MKL transcriptional targets that contribute to cell migration and invasion. The Rho-MKL axis may thus be a novel therapeutic target for prevention of ES metastasis. Citation Format: Merlin Airik, Melanie Krook, Christopher Scannell, Andrew Haak, Richard Neubig, Elizabeth Lawlor. Serum deprivation induces Ewing Sarcoma cells to adopt a more invasive phenotype and to upregulate RHO-MKL transcriptional activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5030. doi:10.1158/1538-7445.AM2013-5030

Comparing the angiogenic potency of naïve marrow stromal cells and Notch-transfected marrow stromal cells

BackgroundAngiogenesis is a critical part of the endogenous repair process in brain injury and disease, and requires at least two sequential steps. First, angiogenic sprouting of endothelial cells occurs, which entails the initial proliferation of endothelial cells and remodeling of the surrounding extracellular matrix. Second, vessel stabilization is necessary to prevent vascular regression, which relies on vascular smooth muscle recruitment to surround the young vessels. Marrow stromal cells (MSCs) have been shown to promote revascularization after hindlimb ischemia, cardiac ischemia, and stroke. SB623 cells are derived from marrow stromal cells by transfection with a Notch1 intracellular domain (NICD)-expressing plasmid and are known to elicit functional improvement in experimental stroke. These cells are currently used in human clinical testing for treatment of chronic stroke. In the current study, the angiogenic property of SB623 cells was investigated using cell-based assays.MethodsAngiogenic paracrine factors secreted by SB623 cells and the parental MSCs were identified using the Qantibody Human Angiogenesis Array. To measure the angiogenic activity of conditioned medium from SB623 cells and MSCs, endothelial tube formation in the human umbilical vein endothelial cell (HUVEC) assay and endothelial cell sprouting and branching in the rodent aortic ring assay were quantified. To validate the angiogenic contribution of VEGF in conditioned medium, endothelial cells and aortic rings were treated with SU5416, which inhibits VEGFR2 at low dose.ResultsConditioned medium from SB623 cells promoted survival and proliferation of endothelial cells under serum-deprived conditions and supports HUVEC vascular tube formation. In a rodent aortic ring assay, there was enhanced endothelial sprouting and branching in response to SB623-derived conditioned medium. SU5416 treatment partially reversed the effect of conditioned medium on endothelial cell survival and proliferation while completely abrogate HUVEC tube formation and endothelial cell sprouting and branching in aortic ring assays.ConclusionsThese data indicate that SB623 cell-secreted angiogenic factors promoted several aspects of angiogenesis, which likely contribute to promoting recovery in the injured brain.

Growth of v-src-transformed cells in serum-free medium through the induction of growth factors

The v-src oncogene is one of only two oncogenes capable of transforming mouse embryo fibroblasts (MEFs) lacking the IGF-IR gene (R-cells). R-/v-src cells grow robustly in the absence of serum, suggesting the hypothesis that they may produce one or more growth factors that would sustain their ability to proliferate in serum-free condition. Using proteomic approaches on serum-free conditioned media derived from v-src-transformed cells, we have identified two growth promoting factors: ostepontin and proliferin. Subsequent experiments have indicated that osteopontin plays a prevalent role in promoting growth of v-src-transformed cells in serum-deprived condition.

Dermatopontin promotes adhesion, spreading and migration of cardiac fibroblasts in vitro

Dermatopontin (DPT), an extracellular matrix (ECM) protein, has been previously shown to be upregulated in the infarct zone of experimentally induced myocardial infarction (MI) rats. However, the accurate role that DPT exerts in the ventricular remodeling process after MI remains poorly understood. In this study, we evaluated the expression pattern of DPT mRNA and protein as well as its secretion in cultured neonatal rat cardiomyocytes (CMs) and cardiac fibroblasts (CFs) under conditions of hypoxia and serum deprivation (hypoxia/SD). Further, we tested the possible roles of DPT in CFs adhesion, spreading, migration and proliferation, which greatly promote the ventricular remodeling process after MI. Results showed that hypoxia/SD stimulated DPT expression and secretion in CMs and CFs and that DPT promoted adhesion, spreading and migration of CFs whereas had no effect on CFs proliferation. In addition, functional blocking antibodies specific for integrin α3 and β1 significantly reduced CFs adhesion and migration that DPT induced, suggesting that integrin α3β1 is at least one receptor for CFs adhesion and migration to DPT. These results implicated that DPT participates in the ventricular remodeling process after MI and may act as a potential therapeutic target for ventricular remodeling.

P2Y1 nucleotide receptor silencing and its effect on glioma C6 calcium signaling.

In our earlier studies of the signaling cross-talk between nucleotide receptors in an in vitro glioma model (C6 cell line) under prolonged serum deprivation conditions, a growth arrest of the cells and expression shift from P2Y(1) to P2Y(12) receptors was found. The aim of the present work was to test if siRNA silencing of P2Y(1) receptor changes P2Y(12) expression similarly as following the serum deprivation and which physiological downstream pathways it affects. Here we demonstrate for the first time the efficiency of siRNA technology in silencing P2Y nucleotide receptors in glioma C6 cell line. Moreover, P2Y(12) proved to be insensitive to the P2Y(1) receptor silencing. The effect of the P2Y(1) silencing on calcium signaling was less pronounced then the extent of the protein change itself, exactly as was the case for the serum starvation experiments. Phosphorylation of ERK and Akt kinases were studied as the downstream effect of P2Y(1)-evoked signaling and similar effects as in the case of serum deprivation were found for ERK, and even stronger ones for Akt phosphorylation.

Promoter methylation of the SALL2 tumor suppressor gene in ovarian cancers

The SALL2 gene product and transcription factor p150 were first identified in a search for tumor suppressors targeted for inactivation by the oncogenic mouse polyoma virus. SALL2 has also been identified as a cellular quiescence factor, essential for cells to enter and remain in a state of growth arrest under conditions of serum deprivation. p150 is a transcriptional activator of p21Cip1/Waf1 and BAX, sharing important growth arrest and proapoptotic properties with p53. It also acts as a repressor of c-myc. Restoration of SALL2 expression in cells derived from a human ovarian carcinoma (OVCA) suppresses growth of the cells in immunodeficient mice. Here we examine the pattern of p150 expression in the normal human ovary, in OVCA-derived cell lines and in primary ovarian carcinomas. Immunohistochemical staining showed that p150 is highly expressed in surface epithelial cells of the normal human ovary. Expression is exclusively from the P2 promoter governing the E1A splice variant of p150. The P2 promoter is CpG-rich and susceptible to methylation silencing. p150 expression was restored in OVCA cell lines following growth in the presence of 5-azacytidine. In a survey of 210 cases of OVCA, roughly 90% across major and minor histological types failed to show expression of the protein. Immunological and biochemical approaches were used to show hypermethylation of the SALL2 P2 promoter in OVCA-derived cell lines and in a majority of primary tumors. These results bring together molecular biological and clinical evidence in support of a role of SALL2 as a suppressor of ovarian cancers.

Inhibition of Tankyrases Induces Axin Stabilization and Blocks Wnt Signalling in Breast Cancer Cells

Constitutive Wnt signalling is characterized by excessive levels of β-catenin protein and is a frequent occurrence in cancer. APC and Axin are key components of the β-catenin destruction complex that acts to promote β-catenin degradation. The levels of Axin are in turn controlled by tankyrases, members of the PARP-family of poly-ADP-ribosylation enzymes. In colorectal cancer cells, which typically harbor APC mutations, inhibition of tankyrase activity promotes Axin stabilization and attenuates Wnt signalling. Here, we examined the effect of inhibiting tankyrases in breast cancer cells with normal APC. We show that application of the small molecule tankyrase inhibitor, XAV939 or siRNA-mediated abrogation of tankyrase expression increases Axin1 and Axin2 protein levels and attenuates Wnt-induced transcriptional responses in several breast cancer lines. In MDA-MB-231 cells, inhibiton of tankyrase activity also attenuate Wnt3a induced cell migration. Moreover, in both MDA-MB-231 and colorectal cancer cells, XAV939 inhibits cell growth under conditions of serum-deprivation. However, the presence of serum prevents this growth inhibitory effect, although inhibition of Wnt-induced transcriptional and migratory responses was maintained. These results indicate that stabilization of Axin by inhibition of tankyrases alone, may not be an effective means to block tumor cell growth and that combinatorial therapeutic approaches should be considered.

Taurine Provides Neuroprotection against Retinal Ganglion Cell Degeneration

Retinal ganglion cell (RGC) degeneration occurs in numerous retinal diseases leading to blindness, either as a primary process like in glaucoma, or secondary to photoreceptor loss. However, no commercial drug is yet directly targeting RGCs for their neuroprotection. In the 70s, taurine, a small sulfonic acid provided by nutrition, was found to be essential for the survival of photoreceptors, but this dependence was not related to any retinal disease. More recently, taurine deprivation was incriminated in the retinal toxicity of an antiepileptic drug. We demonstrate here that taurine can improve RGC survival in culture or in different animal models of RGC degeneration. Taurine effect on RGC survival was assessed in vitro on primary pure RCG cultures under serum-deprivation conditions, and on NMDA-treated retinal explants from adult rats. In vivo, taurine was administered through the drinking water in two glaucomatous animal models (DBA/2J mice and rats with vein occlusion) and in a model of Retinitis pigmentosa with secondary RGC degeneration (P23H rats). After a 6-day incubation, 1 mM taurine significantly enhanced RGCs survival (+68%), whereas control RGCs were cultured in a taurine-free medium, containing all natural amino-acids. This effect was found to rely on taurine-uptake by RGCs. Furthermore taurine (1 mM) partly prevented NMDA-induced RGC excitotoxicity. Finally, taurine supplementation increased RGC densities both in DBA/2J mice, in rats with vein occlusion and in P23H rats by contrast to controls drinking taurine-free water. This study indicates that enriched taurine nutrition can directly promote RGC survival through RGC intracellular pathways. It provides evidence that taurine can positively interfere with retinal degenerative diseases.

Oxidative stress impairs autophagic flux in prion protein-deficient hippocampal cells

We previously reported that autophagy is upregulated in Prnp-deficient (Prnp0/0) hippocampal neuronal cells in comparison to cellular prion protein (PrPC)-expressing (Prnp+/+) control cells under conditions of serum deprivation. In this study, we determined whether a protective mechanism of PrPC is associated with autophagy using Prnp0/0 hippocampal neuronal cells under hydrogen peroxide (H2O2)-induced oxidative stress. We found that Prnp0/0 cells were more susceptible to oxidative stress than Prnp+/+ cells in a dose- and time-dependent manner. In addition, we observed enhanced autophagy by immunoblotting, which detected the conversion of microtubule-associated protein 1 light chain 3 β (LC3B)-I to LC3B-II, and we observed increased punctate LC3B immunostaining in H2O2-treated Prnp0/0 cells compared with H2O2-treated control cells. Interestingly, this enhanced autophagy was due to impaired autophagic flux in the H2O2-treated Prnp0/0 cells, while the H2O2-treated Prnp+/+ cells showed enhanced autophagic flux. Furthermore, caspase-dependent and independent apoptosis was observed when both cell lines were exposed to H2O2. Moreover, the inhibition of autophagosome formation by Atg7 siRNA revealed that increased autophagic flux in Prnp+/+ cells contributes to the prosurvival effect of autophagy against H2O2 cytotoxicity. Taken together, our results provide the first experimental evidence that the deficiency of PrPC may impair autophagic flux via H2O2-induced oxidative stress.

Isomenthone protects human dermal fibroblasts from TNF-α-induced death possibly by preventing activation of JNK and p38 MAPK

Cell death evoked by tumor necrosis factor-α (TNF-α) is regulated by the TNF-α receptor-associated death domain containing protein, which interacts with and activates apoptotic proteases triggering cell death. c-Jun N-terminal kinase (JNK) and p38 MAPK, induce the apoptotic program and are indispensible early elements in stress-induced apoptosis that control the release of cytochrome c. Isomenthone is a constituent of the essential oil of Mentha arvensis L. and is used as a fragrance and flavor in the cosmetic, drug, and food industries. In this study, we investigated the protective effects of isomenthone against TNF-α-induced cell death and its mechanism in human dermal fibroblasts. To understand the cytoprotective role of isomenthone, MTT and terminal deoxynucleotidyl transferase dUTP nick end labeling assays for cell viability and enzyme-linked immunosorbent assay analysis for the mechanistic study were performed. We found that isomenthone inhibited the TNF-α-mediated reduction in cell viability and inhibited the increase in apoptosis under a serum-free condition. Isomenthone also blocked the JNK and p38 MAPK pathways and downstream apoptotic events. These results indicate that isomenthone has the potential to protect fibroblasts against TNF-α-induced cell death under a serum-deprived condition by blocking activation of the JNK and p38 MAPK pathways and downstream apoptotic events.

Coexpression of human somatostatin receptor-2 (SSTR2) and SSTR3 modulates antiproliferative signaling and apoptosis

BackgroundSomatostatin (SST) via five Gi coupled receptors namely SSTR1-5 is known to inhibit cell proliferation by cytostatic and cytotoxic mechanisms. Heterodimerization plays a crucial role in modulating the signal transduction pathways of SSTR subtypes. In the present study, we investigated human SSTR2/SSTR3 heterodimerization, internalization, MAPK signaling, cell proliferation and apoptosis in HEK-293 cells in response to SST and specific agonists for SSTR2 and SSTR3.ResultsAlthough in basal conditions, SSTR2 and SSTR3 colocalize at the plasma membrane and exhibit heterodimerization, the cell surface distribution of both receptors decreased upon agonist activation and was accompanied by a parallel increase in intracellular colocalization. Receptors activation by SST and specific agonists significantly decreased cAMP levels in cotransfected cells in comparison to control. Agonist-mediated modulation of pERK1/2 was time and concentration-dependent, and pronounced in serum-deprived conditions. pERK1/2 was inhibited in response to SST; conversely receptor-specific agonist treatment caused inhibition at lower concentration and activation at higher concentration. Strikingly, ERK1/2 phosphorylation was sustained upon prolonged treatment with SST but not with receptor-specific agonists. On the other hand, SST and receptor-specific agonists modulated p38 phosphorylation time-dependently. The receptor activation in cotransfected cells exhibits Gi-dependent inhibition of cell proliferation attributed to increased PARP-1 expression and TUNEL staining, whereas induction of p21 and p27Kip1 suggests a cytostatic effect.ConclusionOur study provides new insights in SSTR2/SSTR3 mediated signaling which might help in better understanding of the molecular interactions involving SSTRs in tumor biology.

Abstract 2075: Overexpression of the peroxiredoxin family of antioxidant proteins in MCF7 breast cancer cells and investigation of possible regulatory mechanisms

Abstract Peroxiredoxins are thiol-specific antioxidant proteins that protect cells from oxidative stress. There are six different peroxiredoxin proteins expressed in mammalian cells, and these proteins have been implicated in the regulation of cell signaling, proliferation and apoptosis. Many recent studies have found elevated peroxiredoxin levels in cancer, suggesting that cancer cells may upregulate peroxiredoxin expression as an adaptation to the cancerous state. We compared the expression of all six peroxiredoxins between MCF7 breast cancer cell line and the non-cancerous MCF-10A cell line. We found significant overexpression of five of the six peroxiredoxins (PrdxI-V) at both the mRNA and protein level. To explore possible mechanisms of peroxiredoxin upregulation, we first compared the lines for levels of reactive oxygen species (ROS) and the activity of particular kinases implicated in cancer biology. We found that MCF-7 cells express markedly higher levels of ROS, and increased activity of ERK 1/2 and JNK 1/2, as compared to MCF-10A cells. We hypothesized that elevated ROS and/or enhanced activity of these signal transduction pathways may mediate peroxiredoxin induction in these cells. MCF-7 cells were cultured under conditions of serum deprivation, ROS reduction, or kinase inhibition to examine the effects on peroxiredoxin expression. After 48 hours of serum deprivation, the expression of Prdx2 was reduced. Using NAC treatment, we inhibited ROS accumulation in MCF-7 cells and found a decrease in expression of both Prdx2 and Prdx3. Inhibition of ERK 1/2 activity by the inhibitor PD 98,059 resulted in little to no change in peroxiredoxin levels. Finally, treatment of MCF-7 cells with hydrogen peroxide led to a transient increase in Prdx1 at 4 hours, which returned to basal levels by 24 hours. Together, these data reveal overexpression of the peroxiredoxin family in MCF-7 cancer cells, and suggest that oxidative stress and redox-sensitive kinases may play a role in mediating the upregulation of particular peroxiredoxins. Studies examining the role of JNK and p38 in peroxiredoxin induction, as well as cell line differences in multimeric Prdx complexes, are currently underway. 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 2075. doi:1538-7445.AM2012-2075

Growth arrest induction of 3T3-L1 preadipocytes by serum starvation and their differentiation by the hormonal adipogenic cocktail

The current methods for differentiation of 3T3-L1 preadipocytes start with growth arrest by contact inhibition. However, on day 6, 3T3-L1 preadipocytes reliably detach and differentiation could no longer proceed. In the present study, we used serum starvation to induce growth arrest in 3T3-L1 preadipocytes and investigated their differentiation by the modified hormonal adipogenic cocktail. About 85% of 3T3-L1 cells were in G0/G1 stage under serum deprivation. There were no significant difference between the percentage of cells in G0/G1 phase under these serum deprivation condition and that of post-confluent cells. Growth assays indicated that cells under serum starvation condition grew slow, two-days later than growing and post-confluent cells. We determined that 0.5% FBS for 48 h was the optimal condition to arrest 3T3-L1 cells at the G0/G1 phase. The grow-arrested 3T3-L1 preadipocytes were induced to differentiation by hormonal adipogenic cocktail. After 19 days of differentiation, over 90% 3T3-L1 cells exhibited adipocyte morphology with ring-like lipid droplet in cytoplasm. The mRNA levels of critical transcriptional factor CCAAT/enhancer binding proteins-α and peroxisome proliferator-activated receptor-γ were determined to validate the differentiation. These results indicated that serum starvation effectively arrested the growth of 3T3-L1 preadipocytes and prevented cells from detachemnt caused by contact inhibition at confluence. Furthermore, growth arrested 3T3-L1 preadipocytes by serum starvation successfully differentiated into adipocytes by the hormonal adipogenic cocktail. Key words: Cell cycle, detachment, differentiation, growth arrest, preadipocytes, serum starvation,

Helical Repeat Structure of Apoptosis Inhibitor 5 Reveals Protein-Protein Interaction Modules

Apoptosis inhibitor 5 (API5) is an anti-apoptotic protein that is up-regulated in various cancer cells. Here, we present the crystal structure of human API5. API5 exhibits an elongated all α-helical structure. The N-terminal half of API5 is similar to the HEAT repeat and the C-terminal half is similar to the ARM (Armadillo-like) repeat. HEAT and ARM repeats have been implicated in protein-protein interactions, suggesting that the cellular roles of API5 may be to mediate protein-protein interactions. Various components of multiprotein complexes have been identified as API5-interacting protein partners, suggesting that API5 may act as a scaffold for multiprotein complexes. API5 exists as a monomer, and the functionally important heptad leucine repeat does not exhibit the predicted a dimeric leucine zipper. Additionally, Lys-251, which can be acetylated in cells, plays important roles in the inhibition of apoptosis under serum deprivation conditions. The acetylation of this lysine also affects the stability of API5 in cells.

Adaptation of retrovirus producer cells to serum deprivation: Implications in lipid biosynthesis and vector production

The manufacture of enveloped virus, particularly retroviral (RV) and lentiviral (LV) vectors, faces the challenge of low titers that are aggravated under serum deprivation culture conditions. Also, the scarce knowledge on the biochemical pathways related with virus production is still limiting the design of rational strategies for improved production yields. This work describes the adaptation to serum deprivation of two human RV packaging cell lines, 293 FLEX and Te Fly and its effects on lipid biosynthetic pathways and infectious vector production. Total lipid content as well as cellular cholesterol were quantified and lipid biosynthesis was assessed by (13)C-NMR spectroscopy; changes in gene expression of lipid biosynthetic enzymes were also evaluated. The effects of adaptation to serum deprivation in lipid biosynthesis were cell line specific and directly correlated with infectious virus titers: 293 FLEX cells faced severe lipid starvation-up to 50% reduction in total lipid content-along with a 68-fold reduction in infectious vector titers; contrarily, Te Fly cells were able to maintain identical levels of total lipid content by rising de novo lipid biosynthesis, particularly for cholesterol-50-fold increase-with the consequent recovery of infectious vector productivities. Gene expression analysis of lipid biosynthetic enzymes further confirmed cholesterol production pathway to be prominently up-regulated under serum deprivation conditions for Te Fly cells, providing a genotype-phenotype validation for enhanced cholesterol synthesis. These results highlight lipid metabolism dynamics and the ability to activate lipid biosynthesis under serum deprivation as an important feature for high retroviral titers. Mechanisms underlying virus production and its relationship with lipid biosynthesis, with special focus on cholesterol, are discussed as potential targets for cellular metabolic engineering.

Nutrient Availability Alters the Effect of Autophagy on Sulindac Sulfide-Induced Colon Cancer Cell Apoptosis

Autophagy is a catabolic process by which a cell degrades its intracellular materials to replenish itself. Induction of autophagy under various cellular stress stimuli can lead to either cell survival or cell death via apoptotic and/or autophagic (nonapoptotic) pathways. The NSAID sulindac sulfide induces apoptosis in colon cancer cells. Here, we show that inhibition of autophagy under serum-deprived conditions resulted in significant reductions of sulindac sulfide-induced apoptosis in HT-29 colon cancer cells. In contrast, inhibition of autophagy under conditions where serum is available significantly increased sulindac sulfide-induced apoptosis in HT-29 cells. We previously showed that the apoptosis inhibitor, survivin, plays a role in regulating NSAID-induced apoptosis and autophagic cell death. Here, we show that survivin protein half-life is increased in the presence of autophagy inhibitors under serum-deprived conditions, but not under conditions when serum is available. Thus, the increased levels of survivin may be a factor contributing to inhibition of sulindac sulfide-induced apoptosis under serum-deprived conditions. These results suggest that whether a cell lives or dies due to autophagy induction depends on the balance of factors that regulate both autophagic and apoptotic processes.

The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities

Adipose-derived stem cells (ASCs) have valuable applications in regenerative medicine, but maintaining the stemness of ASCs during in vitro culture is still a challenging issue. In this study, human ASCs spontaneously formed three-dimensional spheroids on chitosan films. Most ASCs within the spheroid were viable, and the cells produced more extracellular molecules, like laminin and fibronectin. Comparing to monolayer culture, ASC spheroids also exhibited enhanced cell survival in serum deprivation condition. Although cell proliferation was inhibited in spheroids, ASCs readily migrated out and proliferated upon transferring spheroids to another adherent growth surface. Moreover, spheroid-derived ASCs exhibited higher expansion efficiency and colony-forming activity. Importantly, we demonstrated that spheroid formation of human ASCs on chitosan films induced significant upregulation of pluripotency marker genes ( Sox-2, Oct-4 and Nanog). By culturing the ASC spheroids in proper induction media, we found that ASC differentiation capabilities were significantly enhanced after spheroid formation, including increased transdifferentiation efficiency into neuron and hepatocyte-like cells. In a nude mice model, we further showed a significantly higher cellular retention ratio of ASC spheroids after intramuscular injection of spheroids and dissociated ASCs. These results suggested that ASCs cultured as spheroids on chitosan films can increase their therapeutic potentials.

CXCR4 Promotes the Tumorogenicity of Multiple Myeloma, Including Increased Motility, Clonogenicity, up-Regulation of VLA-4, Protection From Chemotherapy and Aggressive Tumor Development In Vivo

Abstract 1801 Background:Multiple myeloma (MM) is by large incurable neoplasm of plasma cells, characterized by accumulation in the bone marrow (BM), in close contact to cellular and extracellular matrix (ECM) components. Chemokine receptor CXCR4 is expressed by the majority of patients' MM cells. It promotes myeloma cell migration and homing to the BM compartment, supports the tumor cells survival and protects the myeloma cells from chemotherapy-induced apoptosis. Further investigation is required to define the specific molecular mechanisms regulated by the CXCR4/CXCL12 axis in MM. However, surface CXCR4 is commonly down-regulated in the MM cell lines. In order to overcome this limitation, the aim of the current study was to produce a reliable model for studying the functional role of high CXCR4 in MM by generating MM cell lines with stable expression of surface CXCR4. Results:To over-express CXCR4, we transduced CXCL12-expressing MM cell lines ARH77 and RPMI8226 with lentiviral vector and generated cell lines with high and stable levels of surface CXCR4. Enhanced CXCR4 expression significantly increased the in vitro survival and growth of the 2 MM cell lines in serum-deprivation conditions (p<0.01). Furthermore, elevated expression of surface CXCR4 prominently increased MM cells motility and promoted CXCL12-dependent transwell migration of the transduced MM cell lines. Highly CXCR4-expressing RPMI8226 and ARH77 cells demonstrated 40% migration in response to CXCL12 (50 ng/ml), versus only 0–5% migration of MM cells with low expression of surface CXCR4 (p<0.01). Furthermore, adhesion of MM cells to either ECM proteins or BMSCs localize the malignant PCs within the BM microenvironment, promote growth and survival of MM cells and play a critical role in myeloma bone disease and tumor invasion. In accordance, we observed induced adhesion of the transfected RPMI8226-CXCR4 cells to ECM components fibronectin and laminin and to BM fibroblasts. Moreover, we found that enhanced CXCR4 not only functionally activates, but rather significantly elevates the surface levels of VLA-4 integrin on the RPMI8226 cells. In addition, we found that CXCR4-expressing MM cells were less sensitive to melphalan- and bortezomib-induced apoptosis, when they were co-cultured with BM fibroblasts. Testing the molecular signaling pathways regulated by CXCR4, we found that elevated CXCR4 increased the basic level of pERK1/2 and pAKT in the MM cells, and promoted their prolonged activation in response to CXCL12 stimulation. Finally, the ability to produce colonies in the soft agar semi-solid culture reflects the tumorigenic capacity of cancer cells and cancer stem cells. Differentiated MM cells thus rarely produce colonies in soft agar. Here, we demonstrate that up regulation of CXCR4 promoted ARH77 and RPMI8226 colony formation, significantly increasing colonies number and size. Lastly, we determined the role of CXCR4 in MM tumor development in vivo. CXCR4-expressing ARH77 and RPMI8226 cells were subcutaneously injected into NOD/SCID mice. CXCR4-expressing cells, but not parental cell lines, produced detectable tumors already 10 days after the injection. Rapid tumor growth was further observed in both CXCR4-expressing cell lines. These findings indicate that CXCR4 provided aggressive phenotype and supported MM growth in vivo. Conclusions:Taken together, our findings clearly demonstrate the important pathophysiologic role of CXCR4 in MM development and progression. Furthermore, for the first time, we provide the evidence for CXCR4 oncogenic potential in MM, showing that CXCR4 promotes the clonogenic growth of MM cells. Our model may further serve to elucidate CXCR4-regulated molecular events potentially involved in the pathogenesis of MM, and strongly support targeting CXCR4 as therapeutic tool in MM. Disclosures:No relevant conflicts of interest to declare.

Unique Requirements for Lipoproteins Derived From Human Plasma for Maximal Expansion of Human Erythroid Cells Ex-Vivo Under Humanized Conditions

Abstract 1257Ex-vivo expanded erythroid cells (EBs) have been proposed as alternative transfusion products. We have recently formulated humanized media (HEMAdef) that sustain amplification of EBs from cord blood as efficiently as media containing fetal bovine serum (HEMAser) and 10-times greater from adult blood (Migliaccio et al, Cell Transplantation, 2010; 19:453–469). However, while a minority (14.1±5.3%) of EBs generated under HEMAser conditions contain alterations of their membrane which produce cell protrusions (see Figure), membrane abnormalities were observed in 22.7±7.1% of EBs generated under HEMAdef conditions. [Display omitted] The membrane of the red cells consists of a lipid-bilayer attached to the cytoskeleton by integral membrane proteins. The physical-chemical properties of these membranes rely on maintenance of the integrity of the lipid bilayer as disruption of this layer leads to shorten red cell survival or cell removal from the circulation. EBs internalize apolipoprotein-lipid complexes (lipoproteins) present in the environment. Lipoproteins are classified on the basis of floating density into high density lipoproteins [HDL, 1.063<ρ<1.210, ∼50:50 (in dry weight) complex of apoliprotein A-I (apoAI; 32%) and apoAII (10%) with cholesterol esters (15%)], low density lipoproteins [LDL, 1.019<ρ<1.063, ∼20:80 complex of apoB100 (18%) with cholesterol esters (40%)] and very low density lipoproteins [VLDL, 0.950<ρ<1.006, ∼8:82 complex of apoB100 (4%), apoE and apoCI, CII and CIII (4%) with triglycerides (55–65%)]. In addition, all lipoproteins contain 20% of phospholipids. In culture, lipids are provided by lipoproteins present in fetal bovine serum or by synthetic vesicles containing animal (egg cholesterol) and/or vegetal (soybean lecithin) lipids conjugated with albumin (bovine or human). In spite of emerging associations between genetic apolipoprotein gene deficiency and red cell membrane abnormalities, the role played by individual lipoproteins in erythroid cell differentiation is unknown.The aim of this study was to analyze the effect of lipoproteins purified from human plasma on the growth and maturation of EBs generated by mononuclear cells (MNC) from adult blood. HDL, LDL and VLDL were purified according to published methods (Havel RJ et al, J Clin Inv 1955; 34:1345) and their purity assessed on the basis of apoprotein/lipid content analyses. HDL, LDL, VLDL and an artificial mixture of the three fractions (total plasma lipoprotein fraction, TPLF) were used at a concentration of 20 mM/mL which had been previously determined to induce optimal EBs proliferation in preliminary concentration/MTT proliferation curves both under HEMAser and HEMAdef conditions.In semisolid assay, none of the fractions had an effect on the growth of CFU-GM-derived colonies. By contrast, LDL and VLDL both increased by 0.5 and 2-fold, respectively, the numbers of BFU-E derived colonies generated by adult blood MNC in serum-supplemented cultures (78±8 and 113±15 vs 57±9, p<0.01) while only LDL increased the numbers of BFU-E-derived colonies under serum-deprived conditions (64±8 vs 39±5, p<0.05). The various lipoprotein fractions did not improve the overall FI in HEMAser cultures (FI ∼20 by day 14 in all conditions) but increased the frequency of EBs with membrane abnormalities (from 14% to 20–27%). LDL and VLDL also increased by 2-fold the total numbers of progenitor cells observed at day 10 (total progenitor cells/culture 756±104 and 1064±292 vs 542±54, p<0.01–0.05). Under HEMAdef conditions, LDL and VLDL increased by 3-2-fold the FI (95±10 and 64±14 vs 27±6, p<0.01–0.05) by day 14 but did not affect the frequency of EBs with membrane abnormalities which remained high (∼30%). By contrast TPLF modestly increased the FI over those observed in HEMAser cultures (40±12 vs 20±14 in HEMAdef + TPLF vs HEMAser, p<0.05) but greatly reduced the frequency of EBs with membrane abnormalities (14±6% vs 28±7%, p<0.05). The frequency of mature CD36posCD235ahigh EBs observed by day 14 was ∼38–50% in HEMAser and 8–16% in HEMAdef.Human LDL and VLDL increase cloning efficiency and ex-vivo expansion of human adult erythroid progenitors in liquid culture but impair membrane assembly which is improved by TPLF. These results suggest that a balanced combination of lipoproteins, in concentrations still to be identified, may be required to observe maximal expansion of morphologically normal human EBs under HEMAdef conditions. Disclosures:No relevant conflicts of interest to declare.

15-Hydroxyeicosatetraenoic acid (15-HETE) protects pulmonary artery smooth muscle cells from apoptosis via inducible nitric oxide synthase (iNOS) pathway

15-Hydroxyeicosatetraenoic acid (15-HETE), one of many important metabolic products of arachidonic acid (AA) catalyzed by 15-lipoxygenase, plays an important role in pulmonary vascular smooth muscle remodeling. We have previously shown its unsubstituted effects on the apoptotic responses of pulmonary artery smooth muscle cells (PASMCs), but the underlying mechanisms are still poorly manifested. Previous studies have shown that inducible nitric oxide synthase (iNOS) plays an important protective role against sepsis-induced pulmonary apoptosis. Therefore, the purpose of this study is to determine whether 15-HETE anti-apoptotic process is mediated through the iNOS pathway in rat PASMCs. To test this hypothesis, we studied the contribution of iNOS to the 15-HETE induced anti-apoptotic responses using cell viability measurement, Western blot, mitochondrial potential analysis, nuclear morphology determination and TUNEL assay. Our results showed that both exogenous and endogenous 15-HETE up-regulated iNOS protein and mRNA expression and 15-HETE enhanced the cell survival, attenuated mitochondrial depolarization, up-regulated the expression of Bcl-2 and procaspase-3 in PASMCs under serum-deprived condition. These effects were reversed by iNOS inhibitor SMT or l-canavanine. Taken together, our data indicates that iNOS is a novel signaling transduction pathway, which is necessary for the effects of 15-HETE in protection PASMCs from apoptosis and may be an important mechanism underlying the treatment of pulmonary artery hypertension and also provides a novel therapeutic insight in future.