Our presented work is an in vitro study in which we evaluate the involvement of IGF-IR and ROS in fenofibrate/PPARα -mediated inhibition of Glioma cell motility. Our experimental setting is based on human glioma cell model obtained from ATCC, and although our results cannot be directly extrapolated the existing mechanisms that control Glioblastoma invasiveness in vivo, we have shown their potential usefulness for future clinical research studies.
Here, we have evaluated cellular and molecular responses of Glioma cells to fenofibrate, and we attempt to discuss its potential use as a new therapeutic agent against Glioblastoma. In this respect our preliminary studies (not shown) demonstrate elevated levels of PPARα in multiple Glioblastoma clinical samples. Interestingly, PPARα was detected preferentially in the cytoplasm of the tumor cells, and nuclear PPARα was found only in restricted areas of the tumor adjacent to the necrotic tumor tissue. This prominent presence of cytosolic PPARα, which belongs to the family of nuclear steroid receptors, may indicate that its transcriptional activity in Glioblastomas is low in comparison to the nuclear PPARα detected in the control normal brain tissues in which both neurons and astrocytes were positive (preliminary observations). This may also suggest that Glioblastoma cells require exogenous stimulation to activate/translocate PPARα to the nucleus. Indeed, the results in Fig. 1D confirmed enhanced PPARα transcriptional activity following fenofibrate treatment, which was accompanied by increased detection of PPARα in the nuclei of LN-229 Glioma cells (Fig. 1C).
Since in our previous studies fenofibrate attenuated IGF-IR in Medulloblastoma cell lines , we are asking here if fenofibrate could compromise this signaling pathway in human Glioma cell lines. We have selected LN-229 and T98G human Glioma cell lines, which express high and low levels of the IGF-IR, respectively (Fig. 1A). In contrast to T98G, LN-229 cells responded to IGF-I stimulation by elevated cell proliferation (data not shown), and increased cell motility (Fig. 2B). Since, these responses of LN-229 cells were effectively blocked by fenofibrate, we suspected first that fenofibrate-mediated attenuation of the IGF-IR signaling is responsible for its inhibitory action. Interestingly, fenofibrate also inhibited serum-induced cell motility not only in IGF-I sensitive LN-229 cells, but also in T98G cells, which do not respond well to IGF-I stimulation. Surprisingly, serum-stimulated LN-229 and T98G cells were both resistant to small molecular weight IGF-IR inhibitor, NVP-AEW541, which effectively inhibited growth and survival of several other tumor cell lines including Medulloblastoma, colon and prostate cancer [36, 42, 43]. These minimal effects of IGF-IR inhibition on Glioma cell motility could explain only moderate clinical results obtained in the treatment of malignant astrocytomas using antisense strategies . Antisense strategies in which immune response rather than IGF-IR or TGFβ inhibition per se were suggested are more effective [6, 45]. Further, we speculate that although IGF-I contributes to the malignant spread of LN-229 cells, NVP-AEW541 was not effective since other growth promoting mechanism/s, in addition to the IGF-IR, could be involved in supporting dissemination of these tumor cells. Despite of this resistance to IGF-IR inhibition, fenofibrate effectively inhibited Glioma cell motility in the presence of 10% FBS. Further experiments pointed to the accumulation of reactive oxygen species (ROS) as a possible mechanism of the fenofibrate action, since the ROS scavenger, NAC, effectively restored LN-229 cell motility, improved mitochondrial potential and enhanced ATP production in fenofibrate treatment cultures of LN-229 cells.
Another aspect of IGF-IR function is its role in protecting tumor cells from apoptosis [46–48]. Indeed, different strategies aiming against the IGF-IR were often associated with apoptotic death of different types of tumor cells, including Gliomas  and Medulloblastomas [36, 49]. Note however, pro-apoptotic effects of IGF-IR inhibition were observed either when tumor cells were cultured in the condition of anchorage-independence [36, 50] or when IGF-IR inhibition was used to sensitize tumor cells to other anticancer treatments [51–53]. In our experimental setting the treatment of Glioma cells by fenofibrate, which attenuates IGF-IR signaling was applied to monolayer cultures, the condition in which tumor cells are quite resistant to apoptosis. Indeed, we did not observed any significant increase in Glioma apoptotic cell death even in the presence of 50 μM fenofinrate, the concentration, which effectively inhibited both cell motility and IGF-I -mediated phosphorylations (Fig. 2).
So far, our results indicate that specific inhibition of the IGF-IR affects only minimally Glioma cell motility (Fig. 2C), which makes them very different from Medulloblastoma cell lines in which inhibition of the IGF-IR was sufficient to attenuate their growth and survival in achorage-independence [12, 36]. Although the mechanism by which fenofibrate attenuates IGF-IR is still under investigation, our preliminary observations suggest that fenofibrate utilizes a PPARα independent mechanism in repressing this tyrosine kinase receptor. In this regard, fenofibrate has been shown to increase plasma membrane rigidity in a manner similar to elevated cholesterol content in cell membranes . In this report, fenofibrate did not change the membrane content of cholesterol, but increased plasma membrane rigidity, altering activities of integral membrane proteins such as the endoplasmic reticulum Ca2+-ATPase and γ-secretase-mediated cleavage of APP . Further experiments are required to determine whether similar fenofibrate-mediated changes in the fluidity of plasma membrane are indeed responsible for attenuation of the ligand-induced clustering of the IGF-IR, a critical step in auto-phosphorylation of the receptor molecules and the initiation of growth promoting signaling cascades.
Despite of our seemingly contradictory findings, i.e., that IGF-I treatment induces Glioma cell motility, however, the same cells are resistant to the specific IGF-IR inhibitor; and that fenofibrate attenuates IGF-IR signaling responses, the fenofibrate treatment was still very effective in compromising glioma cell motility. Therefore, alternative mechanism/s of the fenofibrate action should be considered. One possibility is that fenofibrate anti-cancer action could be associated with an aberrant cancer cell energy metabolism. This idea originates from the pioneering work of Otto Warburg who demonstrated a distinctive dependence of tumor cells from glycolysis, even when there is sufficient amount of oxygen available for much more effective oxidative phosphorylation [38, 55]. Only recently, it has been established that the inclination of tumor cells for glycolysis is mainly driven by mitochondrial dysfunction [56, 57]. A direct link between mitochondrial aerobic respiration and carcinogenesis have been provided by the demonstration that the loss of p53 function, which is the most commonly mutated gene in cancer , including Gliomas, results in the decrease of synthesis of cytochrome C oxidase expression (SCO2) . SCO2 is crucial for the incorporation of mitochondrial DNA-encoded cytochrome C oxidase subunit (MTCO2) into the cytochrome C oxidase complex. The proper assembly of this complex is essential for the mitochondrial respiration. Therefore, SCO2 deficit in p53-deficient cells heavily impairs oxidative phosphorylation and may trigger the switch towards glycolysis .
In respect to the anti-cancer properties of fenofibrate, activated PPARα, which is a transcriptional activator of the fatty acid β-oxidation machinery , could switch energy metabolism towards fatty acid degradation, and decrease glucose uptake by repressing glucose transporter GLUT4 [21, 59]. Additionally, increased rate of oxidation of fatty acids and ketone bodies forces the decline in glucose utilization through the inhibition of glycolytic enzymes [60, 61]. This could be highly relevant to the Glioma cells since their energy metabolism and the ability to migrate is mitochondria independent and strongly relies on glycolysis . Therefore, one could speculate that in glucose-dependent Glioma cells  with partial mitochondrial dysfunction, fenofibrate could force an aberrant mitochondrial oxidative phosphorylation leading to ROS accumulation, oxidative damage, and severe deficit in ATP production.
In this respect our results indicate that indeed treatment with fenofibrate was associated with ROS accumulation (Fig. 3), which could be explained by the aberrant function of the mitochondrial electron respiratory chain at the level of NADH cytochrome C reductase , or elevated xanthine oxidase expression , and cytosolic ROS, by elevated peroxisomal β-oxidation or microsomal ω-oxidation [64, 65].
Fenofibrate is also known to be responsible for a strong PPARα-dependent induction of mitochondrial uncoupling proteins, e.g. UCP2  in various cell models, therefore the decreased mitochondrial membrane potential observed in the fenofibrate treated LN-229 cells might be attributed to this event as well. Since the Glioma cell lines used in this study show much higher levels of PPARα expression than control astrocytes, PPARα driven UCP2 expression is not unlikely. UCP2 acting as a protonophore facilitates passive proton flow through the mitochondrial inner membrane, which results in uncoupling respiration from ATP production. Moreover, UCP2 has been shown to act as a metabolic sensor, which promotes the switch from glucose dependent metabolism towards fatty acid and glutamine oxidation . These two effects may additionally contribute to the Glioma cell energy depletion, which was manifested here by a severe inhibition of cell motility.
Since ROS scavenger, N-acetyl-cysteine (NAC), as well as siRNA against human PPARα prevented ROS accumulation, enhanced ATP production, and restored LN-229 cell motility, we have concluded that PPARα induced metabolic switch towards mitochondria could be the major contributing factor in the observed anti-cancer action of fenofibrate. Therefore, in addition to the impairment of the IGF-IR signaling responses, Glioma cells treated with fenofibrate could be brought to the verge of metabolic dysfunction by forcing mitochondrial oxidative respiration in the tumor cells, which strongly depend on glycolysis. This opens an opportunity for the use of PPARα agonists, including fenofibrate, since it should be selectively toxic for tumor cells and relatively harmless for cells with normal mitochondrial function.