Maja Grabacka

Peroxisome Proliferator–Activated Receptor α Activation Decreases Metastatic Potential of Melanoma Cells In vitro via Down-Regulation of Akt

Purpose: Peroxisome proliferator-activated receptors (PPAR) regulate lipid and glucose metabolism but their anticancer properties have been recently studied as well. We previously reported the antimetastatic activity of the PPARα ligand, fenofibrate, against melanoma tumors in vivo. Here we investigated possible molecular mechanisms of fenofibrate anti metastatic action. Experimental Design: Monolayer cultures of mouse (B16F10) and human (SkMell88) melanoma cell lines, soft agar assay, and cell migration assay were used in this study. In addition, we analyzed PPARα expression and its transcriptional activity in response to fenotibrate by using Western blots and liciferase-based reporter system. Results: Fenofibrate inhibited migration of B16F10 and SkMel188 cells in Transwell chambers and colony formation in soft agar. These effects were reversed by PPAR inhibitor, GW9662. Western blot analysis revealed time-dependent down-regulation of Akt and extracellular signal–regulated kinase l/2 phosphorylation in fenofibrate-treated cells. A B16F10 cell line stably expressing constitutively active Akt mutant was resistant to fenofibrate. In contrast, Akt gene silencing with siRNA mimicked the fenofibrate action and reduced the migratory ability of B16F1O cells. In addition, fenofibrate strongly sensitized BI6FIO cells to the proapoptotic drug staurosporine, further supporting the possibility that fenofibrate-induced down-regulation of Akt function contributes to fenofibrate-mediated inhibition of metastatic potential in this experimental model. Conclusions: Our results show that the PPAR-dependent antimetastatic activity of fenofibrate involves down-regulation of Akt phosphorylation and suggest that supplementation with this drug may improve the effectiveness of melanoma chemotherapy.

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.

Activation of PPARα inhibits IGF-I-mediated growth and survival responses in medulloblastoma cell lines

Recent studies suggest a potential role of lipid lowering drugs, fibrates and statins, in anticancer treatment. One candidate for tumor chemoprevention is fenofibrate, which is a potent agonist of peroxisome proliferator activated receptor alpha (PPARα). Our results demonstrate elevated expression of PPARα in the nuclei of neoplatic cells in 12 out of 13 cases of medulloblastoma, and of PPARγ in six out of 13 cases. Further analysis demonstrated that aggressive mouse medulloblastoma cells, BsB8, express PPARα in the absence PPARγ, and human medulloblastoma cells, D384 and Daoy, express both PPARα and PPARγ. Mouse and human cells responded to fenofibrate by a significant increase of PPAR‐mediated transcriptional activity, and by a gradual accumulation of cells in G1 and G2/M phase of the cell cycle, leading to the inhibition of cell proliferation and elevated apoptosis. Preincubation of BsB8 cells with fenofibrate attenuated IGF‐I‐induced IRS‐1, Akt, ERKs and GSK3β phosphorylation, and inhibited clonogenic growth. In Daoy and D384 cells, fenofibrate also inhibited IGF‐I‐mediated growth responses, and simultaneous delivery of fenofibrate with low dose of the IGF‐IR inhibitor, NVP‐AEW541, completely abolished their clonogenic growth and survival. These results indicate a strong supportive role of fenofibrate in chemoprevention against IGF‐I‐induced growth responses in medulloblastoma.

Integrins mediate adhesion of medulloblastoma cells to tenascin and activate pathways associated with survival and proliferation

Medulloblastoma spreads by leptomeningeal dissemination rather than by infiltration that characterizes other CNS tumors, eg, gliomas. This study represents an initial attempt to identify both the molecules that mediate medulloblastoma adhesion to leptomeninges and the pathways that are key to survival and proliferation of tumor following adhesion. As a first step in molecule identification, we produced adhesion of D283 medulloblastoma cells to the extracellular matrix (ECM) of H4 glioma cells in vitro. Within this context, D283 cells preferentially expressed the α9 and β1 integrin subunits; antibody and disintegrin blockade of α9 and β1 binding eliminated the adhesion. The H4 ECM was enriched in tenascin, a binding partner for the α9β1 integrin heterodimer. Purified tenascin-C supported D283 cell adhesion. The adhesion was blocked by antibodies to α9 and β1 integrin. In vivo data were similar; immunohistochemistry of primary human medulloblastomas with leptomeningeal extension demonstrated increased expression of α9 and β1 integrins as well as tenascin at the interface of brain and leptomeningeal tumor. These data suggest that tumor-cell expressions of α9 and β1 integrins in combination with extracellular tenascin are necessary for medulloblastoma adhesion to the leptomeninges. As a first step in the identification of pathways that mediate survival and proliferation of tumor following adhesion, we demonstrated that adhesion to H4 ECM was associated with survival and proliferation of D283 cells as well as activation of the MAPK pathway in a growth factor deficient environment. Antibody blockade of α9 and β1 integrin binding that eliminated adhesion also eliminated the in vitro survival benefit. These data suggest that adhesion of medulloblastoma to the meninges is necessary for the survival and proliferation of these tumor cells at the secondary site.

PPAR γ regulates MITF and β-catenin expression and promotes a differentiated phenotype in mouse melanoma S91

Melanoma represents one of the most rapidly metastasizing, hence deadly tumors due to its high proliferation rate and invasiveness, characteristics of undifferentiated embryonic tissues. Given the absence of effective therapy for metastatic melanoma, understanding more fully the molecular mechanisms underlying melanocyte differentiation may provide opportunities for novel therapeutic intervention. Here we show that in mouse melanoma S91 cells activation of the peroxisome proliferator activated receptor (PPAR) γ induces events resembling differentiation, such as growth arrest accompanied by apoptosis, spindle morphology and enhanced tyrosinase expression. These events are preceded by an initial transient increase in expression from the Microphthalmia‐associated transcription factor gene, (MITF) promoter, whereas exposure to a PPAR γ ligand‐ ciglitazone that exceeds 8 h, causes a gradual decrease of MITF, until by 48 h MITF expression is substantially reduced. Beta‐catenin, an MITF transcriptional activator, shows a similar pattern of decline during ciglitazone treatment, consistent with previous reports that activated PPAR γ inhibits the Wnt/β‐catenin pathway through induction of β‐catenin proteasomal degradation. We suggest that the PPAR γ‐mediated β‐catenin down‐regulation is likely to be responsible for changes in MITF levels. The data suggest that PPAR γ, besides its well‐established role in mesenchymal cell differentiation towards adipocytes, might regulate differentiation in the melanocytic lineage.

Peroxisome proliferator activated receptor α ligands as anticancer drugs targeting mitochondrial metabolism.

Tumor cells show metabolic features distinctive from normal tissues, with characteristically enhanced aerobic glycolysis, glutaminolysis and lipid synthesis. Peroxisome proliferator activated receptor α (PPAR α) is activated by nutrients (fatty acids and their derivatives) and influences these metabolic pathways acting antagonistically to oncogenic Akt and c-Myc. Therefore PPAR α can be regarded as a candidate target molecule in supplementary anticancer pharmacotherapy as well as dietary therapeutic approach. This idea is based on hitting the cancer cell metabolic weak points through PPAR α mediated stimulation of mitochondrial fatty acid oxidation and ketogenesis with simultaneous reduction of glucose and glutamine consumption. PPAR α activity is induced by fasting and its molecular consequences overlap with the effects of calorie restriction and ketogenic diet (CRKD). CRKD induces increase of NAD+/NADH ratio and drop in ATP/AMP ratio. The first one is the main stimulus for enhanced protein deacetylase SIRT1 activity; the second one activates AMP-dependent protein kinase (AMPK). Both SIRT1 and AMPK exert their major metabolic activities such as fatty acid oxidation and block of glycolysis and protein, nucleotide and fatty acid synthesis through the effector protein peroxisome proliferator activated receptor gamma 1 α coactivator (PGC-1α). PGC-1α cooperates with PPAR α and their activities might contribute to potential anticancer effects of CRKD, which were reported for various brain tumors. Therefore, PPAR α activation can engage molecular interplay among SIRT1, AMPK, and PGC-1α that provides a new, low toxicity dietary approach supplementing traditional anticancer regimen.

Anticancer Properties of PPARα-Effects on Cellular Metabolism and Inflammation

Peroxisome proliferator-activated receptors (PPARs) have lately attracted much attention as therapeutic targets. Previously, PPAR ligands were associated with the treatment of diabetes, hyperlipidemia and cardiovascular diseases, as they modulate the expression of genes regulating glucose and lipid metabolism. Recently, PPAR ligands have been also considered as potential anticancer agents, with relatively low systemic toxicity. The emerging evidence for antiproliferative, proapoptotic, antiinflammatory and potential antimetastatic properties of PPARα ligands prompted us to discuss possible roles of PPARα in tumor suppression. PPARα activation can target cancer cells energy balance by blocking fatty acid synthesis and by promoting fatty acid β-oxidation. In the state of limited nutrient availability, frequently presents in the tumor microenvironment, PPARα cooperates with AMP-dependent protein kinase in: (i) repressing oncogenic Akt activity, (ii) inhibiting cell proliferation, and (iii) forcing glycolysis-dependent cancer cells into “metabolic catastrophe.” Other potential anticancer effects of PPARα include suppression of inflammation, and upregulation of uncoupling proteins (UCPs), which attenuates mitochondrial reactive oxygen species production and cell proliferation. In conclusion, there are strong premises that the low-toxic and well-tolerated PPAR ligands should be considered as new therapeutic agents to fight disseminating cancer, which represents the major challenge for modern medicine and basic research.

Fenofibrate-induced nuclear translocation of FoxO3A triggers Bim-mediated apoptosis in glioblastoma cells in vitro

Anti-neoplastic potential of calorie restriction or ligand-induced activation of peroxisome proliferator activated receptors (PPARs) has been demonstrated in multiple studies; however, mechanism(s) by which tumor cells respond to these stimuli remain to be elucidated. One of the potent agonists of PPARα, fenofibrate, is a commonly used lipid-lowering drug with low systemic toxicity. Fenofibrate-induced PPARα transcriptional activity is expected to shift energy metabolism from glycolysis to fatty acid β-oxidation, which in the long-term, could target weak metabolic points of glycolysis-dependent glioblastoma cells. The results of this study demonstrate that 25 μM fenofibrate can effectively repress malignant growth of primary glial tumor cells and glioblastoma cell lines. This cytostatic action involves G1 arrest accompanied by only a marginal level of apoptotic cell death. Although the cells treated with 25 μM fenofibrate remain arrested, the cells treated with 50 μM fenofibrate undergo massive apoptosis, which starts after 72 h of the treatment. This delayed apoptotic event was preceded by FoxO3A nuclear accumulation, FoxO3A phosphorylation on serine residue 413, its elevated transcriptional activity and expression of FoxO-dependent apoptotic protein, Bim. siRNA-mediated inhibition of FoxO3A attenuated fenofibrate-induced apoptosis, indicating a direct involvement of this transcription factor in the fenofibrate action against glioblastoma. These properties of fenofibrate, coupled with its low systemic toxicity, make it a good candidate in support of conventional therapies against glial tumors.

Molecular mechanisms of fenofibrate-induced metabolic catastrophe and glioblastoma cell death.

ABSTRACT Fenofibrate (FF) is a common lipid-lowering drug and a potent agonist of the peroxisome proliferator-activated receptor alpha (PPARα). FF and several other agonists of PPARα have interesting anticancer properties, and our recent studies demonstrate that FF is very effective against tumor cells of neuroectodermal origin. In spite of these promising anticancer effects, the molecular mechanism(s) of FF-induced tumor cell toxicity remains to be elucidated. Here we report a novel PPARα-independent mechanism explaining FFs cytotoxicity in vitro and in an intracranial mouse model of glioblastoma. The mechanism involves accumulation of FF in the mitochondrial fraction, followed by immediate impairment of mitochondrial respiration at the level of complex I of the electron transport chain. This mitochondrial action sensitizes tested glioblastoma cells to the PPARα-dependent metabolic switch from glycolysis to fatty acid β-oxidation. As a consequence, prolonged exposure to FF depletes intracellular ATP, activates the AMP-activated protein kinase–mammalian target of rapamycin–autophagy pathway, and results in extensive tumor cell death. Interestingly, autophagy activators attenuate and autophagy inhibitors enhance FF-induced glioblastoma cytotoxicity. Our results explain the molecular basis of FF-induced glioblastoma cytotoxicity and reveal a new supplemental therapeutic approach in which intracranial infusion of FF could selectively trigger metabolic catastrophe in glioblastoma cells.

Involvement of α1β1 integrin in insulin-like growth factor-1-mediated protection of PC12 neuronal processes from tumor necrosis factor-α-induced injury

Insulin‐like growth factor 1 receptor (IGF‐1R) supports neuronal survival against a wide variety of insults. This includes tumor necrosis factor‐α (TNFα)‐mediated neuronal damage, which represents one of the factors suspected to play a role in HIV‐associated dementia (HAD). PC12 neurons engineered to express human IGF‐1R (PC12/IGF‐1R) maintain neuronal processes on collagen IV for several weeks. However, prolonged treatment with TNFα caused degeneration of neuronal processes, with no apparent signs of apoptosis. In this process, TNFα did not affect IGF‐1‐mediated phosphorylation of IRS‐1, IRS‐2, Akt, or Erks. In addition, PC12/IGF‐1R cells were found to express predominantly α1β1 integrin, which has high affinity to collagen IV. The treatment of PC12/IGF‐1R neurons with a specific α1β1 integrin inhibitor, obtustatin, also caused loss of neuronal processes, accompanied by a quick cell detachment and extensive apoptosis. In the presence of IGF‐1, both TNFα‐induced and obtustatin‐induced degeneration of neuronal processes were effectively inhibited. Furthermore, TNFα‐mediated neuronal degeneration correlated with decreased attachment of PC12/IGF‐1R cells to collagen IV and with a reduced level of α1β1 integrin, consistent with a role for this surface protein in the maintenance of neuronal processes. Thus the neuroprotective effects of IGF‐1 are not restricted to its antiapoptotic properties but also involve an additional neuroprotective mechanism, by which IGF‐1 counteracts the negative effect of TNFα on α1β1 integrin‐mediated attachment to collagen IV.