Ewa Wybieralska

ROS accumulation and IGF-IR inhibition contribute to fenofibrate/PPARα -mediated inhibition of Glioma cell motility in vitro

BackgroundGlioblastomas are characterized by rapid cell growth, aggressive CNS infiltration, and are resistant to all known anticancer regimens. Recent studies indicate that fibrates and statins possess anticancer potential. Fenofibrate is a potent agonist of peroxisome proliferator activated receptor alpha (PPARα) that can switch energy metabolism from glycolysis to fatty acid β-oxidation, and has low systemic toxicity. Fenofibrate also attenuates IGF-I-mediated cellular responses, which could be relevant in the process of glioblastoma cell dispersal.MethodsThe effects of fenofibrate on Glioma cell motility, IGF-I receptor (IGF-IR) signaling, PPARα activity, reactive oxygen species (ROS) metabolism, mitochondrial potential, and ATP production were analyzed in human glioma cell lines.ResultsFenofibrate treatment attenuated IGF-I signaling responses and repressed cell motility of LN-229 and T98G Glioma cell lines. In the absence of fenofibrate, specific inhibition of the IGF-IR had only modest effects on Glioma cell motility. Further experiments revealed that PPARα-dependent accumulation of ROS is a strong contributing factor in Glioma cell lines responses to fenofibrate. The ROS scavenger, N-acetyl-cysteine (NAC), restored cell motility, improved mitochondrial potential, and increased ATP levels in fenofibrate treated Glioma cell lines.ConclusionsOur results indicate that although fenofibrate-mediated inhibition of the IGF-IR may not be sufficient in counteracting Glioma cell dispersal, PPARα-dependent metabolic switch and the resulting ROS accumulation strongly contribute to the inhibition of these devastating brain tumor cells.

Genetically modified adipose tissue−derived mesenchymal stem cells overexpressing CXCR4 display increased motility, invasiveness, and homing to bone marrow of NOD/SCID mice

OBJECTIVE This study evaluates usefulness of CXCR4 overexpression via retroviral transduction in adipose tissue-derived mesenchymal stem cells (AT-MSCs) as a strategy to increase their migration and engraftment ability. MATERIALS AND METHODS AT-MSCs were isolated from lipoaspirates from human healthy donors with liberase 3. Cells were transduced with retroviral vector carrying either CXCR4 or green fluorescent protein (GFP) complementary DNA, and neo-resistant colonies were selected and used in experiments. Chemotaxis, invasion through Matrigel, motor activity, gene expression, osteodifferentiation potential, and engraftment into bone marrow of nonobese diabetic/severe combined immunodeficient mice were analyzed for CXCR4-overexpressing cells and GFP-control cells. RESULTS Approximately 90% of retrovirus-transduced AT-MSCs expressed CXCR4 or GFP and maintained their ability to differentiate into osteocytes. CXCR4-transduced AT-MSCs displayed enhanced migration and higher invasiveness toward SDF-1 gradient. The upregulation of CXCR4 led to phosphorylation of mitogen-activated protein and AKT kinases and an increase in metalloproteinase expression after SDF-1 stimulation. The transplantation of CXCR4-transduced AT-MSCs into nonobese diabetic/severe combined immunodeficient mice led to increased engraftment into bone marrow in comparison to GFP-transduced AT-MSCs. CONCLUSIONS Adipose tissue is one of the alternative sources of MSCs to bone marrow. We showed that AT-MSCs overexpressing CXCR4 preserve their ability for osteodifferentiation. Enhanced migration and engraftment of the transduced AT-MSCs into bone marrow indicate the usefulness of this strategy in overcoming low engraftment of MSCs in clinical approaches of cellular therapies for bone disorders and can represent a powerful tool in regenerative medicine and gene therapies. Thus, these cells may be used as an alternative to bone marrow-derived MSCs.

Fenofibrate attenuates contact-stimulated cell motility and gap junctional coupling in DU-145 human prostate cancer cell populations

In the present study, we investigated the effects of fenofibrate on the invasive potential of DU-145 human prostate cancer cells in the context of gap junctional intercellular coupling and the formation of reactive oxygen species. Time-lapse analyses of cell motility, accompanied by tests of cell viability, membrane microviscosity, reactive oxygen species accumulation and the function of gap junctional protein connexin 43 were performed in monolayer cultures of DU-145 cells following fenofibrate administration. Fenofibrate inhibited the motility of DU-145 cells and attenuated gap junctional intercellular coupling in a manner independent of its effects on cell viability, PPARα activation and cell membrane micro-viscosity. Instead, N-acetyl-L-cysteine, a scavenger of reactive oxygen species, restored cell motility and gap junctional coupling in fenofibrate-treated DU-145 cell populations. These data indicate that two parameters crucial for cancer cell metastatic potential, i.e. cell motility and gap junctional coupling, are inhibited by fenofibrate. Thus, fenofibrate affects prostate cancer cell invasion via an orchestrated action on versatile cancer cell properties determining this process. A novel mechanism of anti-invasive activity of fenofibrate, which depends on its interference with cell motility and the function of gap junctions regulated by reactive oxygen species, is suggested.

Fenofibrate enhances barrier function of endothelial continuum within the metastatic niche of prostate cancer cells

Objective: Extravasation of circulating cancer cells is an important step of the metastatic cascade and a potential target for anti-cancer strategies based on vasoprotective drugs. Reports on anti-cancer effects of fenofibrate (FF) prompted us to analyze its influence on the endothelial barrier function during prostate cancer cell diapedesis. Research design and methods: In vitro co-cultures of endothelial cells with cancer cells imitate the ‘metastatic niche’ in vivo. We qualitatively and quantitatively estimated the effect of 25 μM FF on the events which accompany prostate carcinoma cell diapedesis, with the special emphasis on endothelial cell mobilization. Results: Fenofibrate attenuated cancer cell diapedesis via augmenting endothelial cell adhesion to the substratum rather than through the effect on intercellular communication networks within the metastatic niche. The inhibition of endothelial cell motility was accompanied by the activation of PPARα-dependent and PPARα-independent reactive oxygen species signaling, Akt and focal adhesion kinase (FAK) phosphorylation, in the absence of cytotoxic effects in endothelial cells. Conclusions: Fenofibrate reduces endothelial cell susceptibility to the paracrine signals received from prostate carcinoma cells, thus inhibiting endothelial cell mobilization and reducing paracellular permeability of endothelium in the metastatic niche. Our data provide a mechanistic rationale for extending the clinical use of FF and for the combination of this well tolerated vasoactive drug with the existing multidrug regimens used in prostate cancer therapy.

DU-145 PROSTATE CARCINOMA CELLS THAT SELECTIVELY TRANSMIGRATE NARROW OBSTACLES EXPRESS ELEVATED LEVELS OF Cx43

The formation of aqueous intercellular channels mediating gap junctional intercellular coupling (GJIC) is a canonical function of connexins (Cx). In contrast, mechanisms of GJIC-independent involvement of connexins in cancer formation and metastasis remain a matter of debate. Because of the role of Cx43 in the determination of carcinoma cell invasive potential, we addressed the problem of the possible Cx43 involvement in early prostate cancer invasion. For this purpose, we analysed Cx43-positive DU-145 cell subsets established from the progenies of the cells most readily transmigrating microporous membranes. These progenies displayed motile activity similar to the control DU-145 cells but were characterized by elevated Cx43 expression levels and GJIC intensity. Thus, apparent links exist between Cx43 expression and transmigration potential of DU-145 cells. Moreover, Cx43 expression profiles in the analysed DU-145 subsets were not affected by intercellular contacts and chemical inhibition of GJIC during the transmigration. Our observations indicate that neither cell motility nor GJIC determines the transmigration efficiency of DU-145 cells. However, we postulate that selective transmigration of prostate cancer cells expressing elevated levels of Cx43 expression may be crucial for the “leading front” formation during cancer invasion.

Topographical control of prostate cancer cell migration

Tumour cells can efficiently respond to numerous factors affecting their motility. However, the role of substrata topography in the regulation of cancer cell motility has been quantitatively studied in only a few cases. We demonstrated that human (DU-145) and rat (MAT-LyLu and AT-2) prostate cancer cells are efficiently contact guided by underlying normal cells when invading surrounding tissues and forming metastases. Prostate cancer cells moving on the surface of fibroblasts displayed significantly greater cell displacement than those moving on plastic substrata. This effect was correlated with the polarization (contact guidance) and increased speed of cell movements. We subsequently verified the hypothesis that the observed contact guidance of prostate cancer cells migrating on the surface of fibroblasts results from their reaction to the microtopography of normal cells. The responses of cells to multiple grooved substrata of a size corresponding to the dimensions of a compact monolayer culture of human skin fibroblasts were studied, and the migration of prostate cancer cells appeared to be efficiently affected by topographical features of the substratum. In contrast to random movement under control conditions, all investigated prostate cancer cell lines grown on patterned substrata migrated mainly along artificial grooves and covered, as a result of contact guidance, a longer distance than cells on plain substrata. Moreover, the reaction to microtopography was correlated with the metastatic activity of prostate cancer cells. In conclusion, our results show that grooved substrata have a substantial effect on prostate cancer migration. Since all types of tissue show some kind of patterning and alignment, topographic factors may be crucial for the effective migration of prostate cancer cells during the metastatic process.

Ascorbic acid inhibits the migration of walker 256 carcinosarcoma cells

The results of several experimental studies have shown that ascorbic acid inhibits tumor growth and metastasis. Ascorbic acid is an antioxidant that acts as a scavenger for a wide range of reactive oxygen species (ROS). Both tumour metastasis and cell migration have been correlated with the intracellular ROS level, so it was postulated that the inhibitory effect of ascorbic acid derivatives on cell motility may be caused by scavenging of ROS. Time-lapse analyses of Walker 256 carcinosarcoma cell migration showed that both the speed of movement and the cell displacement were inhibited by ascorbic acid applied in concentrations ranging from 10 to 250 μM. This effect correlated with a reduction in the intracellular ROS level in WC 256 cells, suggesting that ROS scavenging may be a mechanism responsible for the inhibition of WC 256 cell migration. However, another potent antioxidant, N-acetyl-L-cysteine, also efficiently decreased the intracellular ROS level in WC 256 cells, but did not affect the migration of the investigated cells. These results demonstrate that intact, unmodified ascorbic acid applied in physiologically relevant and nontoxicconcentrations exerts an inhibitory effect on the migration of WC 256 carcinosarcoma cells, and that this may be one of the factors responsible for the anti-metastatic activity of vitamin C. However, our data does not support the hypothesis that the scavenging of intracellular ROS is the main mechanism in the inhibition of cancer cell migration by ascorbic acid.

Solute-dependent activation of cell motility in strongly hypertonic solutions in Dictyostelium discoideum, human melanoma HTB-140 cells and walker 256 carcinosarcoma cells

Published data concerning the effects of hypertonicity on cell motility have often been controversial. The interpretation of results often rests on the premise that cell responses result from cell dehydration, i.e. osmotic effects. The results of induced hypertonicity on cell movement of Dictyostelium discoideum amoebae and human melanoma HTB-140 cells reported here show that: i) hypertonic solutions of identical osmolarity will either inhibit or stimulate cell movement depending on specific solutes (Na+ or K+, sorbitol or saccharose); ii) inhibition of cell motility by hypertonic solutions containing Na+ ions or carbohydrates can be reversed by the addition of calcium ions; iii) various cell types react differently to the same solutions, and iv) cells can adapt to hypertonic solutions. Various hypertonic solutions are now broadly used in medicine and to study modulation of gene expression. The observations reported suggest the need to examine whether the other responses of cells to hypertonicity can also be based on the solute-dependent cell responses besides cell dehydration due to the osmotic effects.

Abstract 1204: MET receptor as a key player responsible for malignant properties and intracellular trafficking and fate of CXCR4 in cervical carcinoma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction Cervical carcinoma (CC) is one of the major causes of death among women suffering from tumor especially in low and middle income countries. MET activation is crucial in epithelial EMT, cell motility and invasiveness under both normal and pathological conditions. Another family of receptors that play a critical role in tumor initiation, promotion and progression are chemokine receptors. One of the best studied is a seven - span transmembrane G - protein coupled receptor, CXCR4. CXCR4 promotes metastasis of tumor cells and its blockade inhibits tumor growth. Recently we have shown that HGF/MET axis synergizes with SDF-1/CXCR4 axis in stimulating CC cell chemotaxis and activation of various intracellular pathways. Aim The aim of the study was to evaluate the influence of MET receptor downregulation on cervical carcinoma cells. Materials and methods HTB-35 CC cell line was transduced with MET and LacZ shRNA expressing virus. Morphometry was performed with Nomarski interference contrast optics and MIGRA software. F-actin organization was evaluated using falloidin staining. The level of various genes expression was estimated by real-time RT-PCR Cells were transfected by EGFP-CXCR4 plasmid using lipofectamine and intracellular trafficking of CXCR4 was studied by immunofluorescence staining with confocal microscopy. Results MET receptor downregulation changes the morphology of HTB35 shMET cell line. Cells became smaller than wild type and shLacZ cells. Moreover, F-actin was located under cells membrane and it did not form regular stress fibers as they were observed in control cells. Downregulation of MET receptor influences changes in gene expression responsible for invasive phenotype and EMT. We have observed downregulation of CXCR4 and Slug and upregulation of E-cadherin. In trafficking studies we have observed different localization and polarization of CXCR4 receptor. In MET deficient cells CXCR4 was partially located in Golgi but it did not colocalize with AP1 and Sec-8 protein in exocytosis process. We have not observed recycling back of the CXCR4 receptor with AP2 staining in MET deficient HTB-35 cells. Conclusions The downregulation of MET receptor expression is responsible for a change in the expression of genes correlated with malignant phenotype and involved in EMT of CC cells. The downregulation of MET expression is responsible for more epithelial phenotype of HTB-35 cells. MET receptor downregulation influences intracellular trafficking of CXCR4 what might have a significant clinical implication for the treatment of tumor cells. Acknowledgement This study was supported by research grant from the Polish Ministry of Science and Higher Education NN 401142339, NN 401054839, NN 401010036, grant from Jagiellonian University Collegium Medicum: K/ZDS/002279 and EMBO short term fellowship ASTF 97-2010 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 1204. doi:1538-7445.AM2012-1204