Fenofibrate suppresses growth of the human hepatocellular carcinoma cell via PPARα-independent mechanisms

Fenofibrate, a peroxisome proliferator-activated receptor (PPAR) α agonist, is a hypolipidemic drug. Although several studies have explored the fenofibrate-induced antiproliferative effect in cultured human cells, it is not clear which role PPARα plays in this antiproliferative effect. Therefore, we investigated the antiproliferative mechanism of fenofibrate in Huh7 (human hepatoma cell line). Cell viability was measured by the WST-8 assay and cell proliferation was assessed using the BrdU incorporation assay. The cell cycle was analyzed by flow cytometry. The cyclins, tumor suppressor proteins and regulators of the AKT signaling pathway were analyzed by immunoblotting. Using flow cytometry, we showed that fenofibrate blocks entry into the S phase of the cell cycle. We certified that this G1 arrest is caused by the reduction of cyclin A and E2F1 and the accumulation of the cyclin-dependent kinase inhibitor p27. Interestingly, the antiproliferative effect of fenofibrate was not affected by the PPARα antagonist (GW6471) or by PPARα-specific siRNA. These results suggest that fenofibrate suppresses Huh7 cell growth through a PPARα independent mechanism. Furthermore, we showed that treatment of Huh7 cells with fenofibrate leads to suppression of AKT phosphorylation. We also found for the first time that fenofibrate increased the C-terminal modulator protein (CTMP), which inhibits AKT phosphorylation. Our data suggest that fenofibrate inhibits the proliferation of Huh7 cells by blocking Akt activation, and that CTMP is one of the key players for this antiproliferative property of fenofibrate in Huh7 cells.     

Discovery of new PPARγ agonists based on arylopeptoids

In this study we present the design, synthesis and biological evaluation of a small, first-generation library of small molecule aromatic amides based on the arylopeptoid skeleton. The compounds were efficiently synthesized using a highly convenient submonomer solid-phase methodology which potentially allows for access to great product diversity. The synthesized compounds were tested for their ability to activate peroxisome proliferator-activated receptors (PPARs) and they all acted as PPARγ agonists in the μM range spanning from 2.5- to 14.7-fold activation of the receptor. This is the first discovery of bioactive molecules based on the arylopeptoid architecture.     

Ligand entry pathways in the ligand binding domain of PPARγ receptor

To address the question of ligand entry process, we report targeted molecular dynamics simulations of the entry of the flexible ionic ligand GW0072 in the ligand binding domain of the nuclear receptor PPARγ. Starting with the ligand outside the receptor the simulations led to a ligand docked inside the binding pocket resulting in a structure very close to the holo-form of the complex. The results showed that entry process is guided by hydrophobic interactions and that entry pathways are very similar to exit pathways. We suggest that TMD method may help in discriminating between ligands generated by in silico docking.     

Egr-1 is involved in the inhibitory effect of leptin on PPARγ expression in hepatic stellate cell in vitro

AimsHepatic stellate cell (HSC) activation is a key step in the hepatic fibrogenic process. Increasing evidence demonstrates the pro-fibrogenic action of leptin in rodent liver. Peroxisome proliferator-activated receptor-γ (PPARγ) is a potential molecular target for inhibition of HSC activation. Our previous study suggested that leptin markedly down-regulated PPARγ gene expression in HSCs. The aim of this study is to explore the molecular mechanisms underlying the inhibitory effect of leptin on PPARγ expression in rat HSCs in vitro.Main methodsThe effects of leptin on the expression and trans-activation activity of early growth response-1 (Egr-1) are examined by using real-time PCR, Western blotting analysis, transient transfection, and electrophoretic mobility shift assay. The role of Egr-1 in PPARγ gene expression is demonstrated by co-transfection approach, Western blotting analysis and real-time PCR.Key findingsWe document that leptin increases Egr-1 expression at protein and mRNA levels, and significantly stimulates Egr-1 trans-activation activity. Moreover, leptin induces the expression and activity of Egr-1 through activation of extracellular signal-regulated kinase (ERK) or phosphatidylinositol 3-kinase/AKT signaling (PI-3K/AKT) pathway. Further investigation reveals that Egr-1 exerts a clear inhibitory effect on the promoter activity and expression of PPARγ gene and demonstrates that Egr-1 increases the expression of HSC activation markers and promotes HSC growth. Taken together, these findings suggest that Egr-1 is involved in the inhibitory effect of leptin on PPARγ expression in rat HSCs in vitro.SignificanceOur results provide novel insights into the mechanisms of leptin-induced inhibition of PPARγ expression in HSCs in vitro.     

Minireview: PPARγ as the target of obesogens

The peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of adipogenesis and is medically important for its connections to obesity and the treatment of type II diabetes. Activation of this receptor by certain natural or xenobiotic compounds has been shown to stimulate adipogenesis in vitro and in vivo. Obesogens are chemicals that ultimately increase obesity through a variety of potential mechanisms, including activation of PPARγ. The first obesogen for which a definitive mechanism of action has been elucidated is the PPARγ and RXR activator tributyltin; however, not all chemicals that activate PPARγ are adipogenic or correlated with obesity in humans. There are multiple mechanisms through which obesogens can target PPARγ that may not involve direct activation of the receptor. Ligand-independent mechanisms could act through obesogen-mediated post-translational modification of PPARγ which cause receptor de-repression or activation. PPARγ is active in multipotent stem cells committing to the adipocyte fate during fat cell development. By modifying chromatin structure early in development, obesogens have the opportunity to influence the promoter activity of PPARγ, or the ability of PPARγ to bind to its target genes, ultimately biasing the progenitor pool towards the fat lineage. Obesogens that act by directly or indirectly activating PPARγ, by increasing the levels of PPARγ protein, or enhancing its recruitment to promoters of key genes in the adipogenic pathway may ultimately play an important role in adipogenesis and obesity.     

Combined anti-tumor effects of IFN-α and sorafenib on hepatocellular carcinoma in vitro and in vivo

Hepatocellular carcinoma (HCC) is among the most common and aggressive cancers worldwide, and novel therapeutic strategies are urgently required to improve clinical outcome. Interferon-alpha (IFN-α) and sorafenib are widely used as anti-tumor agents against various malignancies. In this study, we investigated the combined effects of IFN-α and sorafenib against HCC. We demonstrated that the combination therapy synergistically suppressed HCC cellular viability, arrested cell cycle propagation and induced apoptosis in HCC cells. Further research revealed that IFN-α and sorafenib collaboratively regulated the expression levels of cell cycle-related proteins Cyclin A and Cyclin B as well as the pro-survival Bcl-2 family proteins Mcl-1, Bcl-2 and Bcl-X(L). Moreover, sorafenib inhibited IFN-α induced oncogenic signaling of STAT3, AKT and ERK but not the activation of the tumor suppressor STAT1. Xenograft experiments also confirmed the combined effects of IFN-α and sorafenib on tumor growth inhibition and apoptosis induction in vivo. In conclusion, these results provide rationale for the clinical application of IFN-α and sorafenib combination therapy in HCC treatment.     

Possible Involvement of PPAR-γ in the Anticonvulsant Effect of Aegle marmelos (L.) Correa

Aegle marmelos is well documented for antihyperglycemic effect and PPAR-γ activation has been suggested to be the molecular mechanism of its action. Also, the plant has been used in Ayurveda as a brain tonic and has been postulated to have antidepressant activities. The present study was designed to investigate the anticonvulsant effects of A. marmelos leaf extract (AME) in pentylenetetrazole and maximal electroshock induced convulsions; involvement of PPAR-γ, nitric oxide pathway and effect of chronic AME treatment on post-ictal depression. AME was administered at doses of 50, 100 and 200 mg kg^?1 in PTZ and MES model. Severity of convulsions was noted in both the models. Pretreatment with bisphenol A diglycidyl ether (BADGE) was used to study the involvement of PPAR-γ and l-arginine and N-nitro-l-arginine methyl ester hydrochloride (l-NAME) to study the involvement of nitric oxide (NO). Chronic treatment with AME interspersed with sub maximal doses of PTZ (50 mg kg^?1) on every fifth day up to 15 days was given to study post-ictal depression using forced swimming and actophotometer. AME showed significant increase in the onset time and decrease in the duration of convulsions in PTZ and MES models dose dependently. In MES a dose of 100 mg kg^?1 had effect comparable to phenytoin. Pretreatment with BADGE and l-arginine reversed the protective effect while l-NAME did not alter the protective effect, thereby indicating possible involvement of PPAR-γ and inhibition of NO. Chronic AME treatment ameliorated the post-seizure depression significantly as evidenced by increase in the locomotor activity and decrease in the immobility time.     

Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks

The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs)—a highly cytotoxic DNA lesion—activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28γ (REGγ; PSME3) as a novel DDR player. PA28γ depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28γ deficiency abrogates the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair. Furthermore, PA28γ is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28γ-proteasome axis of the DDR that is required for timely coordination of DSB repair.