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PubMed2002

Peroxisome proliferator-activated receptor gamma agonist ligands stimulate a Th2 cytokine response and prevent acute colitis.

Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of a nuclear transcription factor family, has been previously demonstrated to have antiinflammatory activity. The effects of PPARgamma activation in the development of an immune response are less well characterized. Through evaluation of PPARgamma heterozygote mice (PPARgamma(+/-) and specific PPARgamma agonist ligand binding, we evaluated the immunologic effects of PPARgamma activation in a well-described model of colitis. Increased susceptibility to dextran sodium sulfate (DSS)-induced colitis as defined by body weights, histologic injury, and survival was observed in the PPARgamma(+/-) mice in comparison to wild-type mice. Three different PPARgamma ligands (troglitazone, pioglitazone, and rosiglitazone) demonstrated beneficial dose-related treatment effects when administered prior to the onset of colitis. However, no protection was observed when PPARgamma ligand activation occurred after the onset of colitis. The reduction in DSS-induced inflammation noted with PPARgamma ligand treatment was associated with decreased interferon-gamma and tumor necrosis factor-alpha and increased interleukin (IL)-4 and IL- 10 levels as assessed by quantitative reverse transcriptase-polymerase chain reaction. Consistent with this shift towards a T helper (Th2) cytokine dominance, PPARgamma ligand treatment stimulated increased GATA-3 expression. These results indicate that the protective effects exhibited by PPARgamma ligands in intestinal inflammation may be due to immune deviation away from Th1 and towards Th2 cytokine production.

Acute Disease↗

PubMed2002

Expression of PPARgamma and its ligand-dependent growth inhibition in human brain tumor cell lines.

Peroxisome proliferator-activated receptor gamma (PPARgamma) belongs to a superfamily of thyroid / steroid hormone receptors and regulates transcription of their target genes in a ligand-dependent manner. Recently, PPARgamma was reported to be expressed in several cell lines derived from breast, colon, stomach and lung cancers. Activation of PPARgamma by its ligand inhibits the growth of these tumor cells, suggesting that PPARgamma ligand is a potential anti-cancer agent in PPARgamma-expressing tumors. However, its expression in brain tumors has not been studied. We thus studied the expression in glioma samples with different pathological stages from 20 patients. It was demonstrated that 95% of the glioma tissue expressed PPARgamma mRNA. The results prompted us to study whether PPARgamma ligand affects the growth of cell lines derived from brain tumors. The receptor expression was studied in 9 cell lines either derived from malignant glioma or neuroblastoma. The expression was detected in a glioma cell line SK-MG-1 and in a neuroblastoma cell line NB-1. Addition of one of the PPARgamma ligands, troglitazone, induced growth inhibition in both cell lines. Further analyses revealed that this growth inhibition is caused by a PPARgamma-mediated induction of apoptosis. These results suggest that PPARgamma ligands could be a potential therapeutic agent for the treatment of the brain tumors expressing this receptor.

Antineoplastic Agents↗

PubMed2005

Importance of chroman ring and tyrosine phosphorylation in the subtype-specific translocation and activation of diacylglycerol kinase alpha by D-alpha-tocopherol.

Diacylglycerol kinase (DGK) has been suggested to be a pharmacological target of D-alpha-tocopherol for diabetic renal dysfunctions. However, the DGK subtypes involved in the D-alpha-tocopherol-induced improvement of diabetic renal dysfunctions and the activation mechanisms of DGK by D-alpha-tocopherol are still unknown. Therefore, using GFP-tagged DGKalpha, beta, gamma, epsilon and zeta, we analyzed their response to D-alpha-tocopherol and its derivatives. Only DGKalpha was translocated from the cytoplasm to the plasma membrane with elevation of kinase activity. In addition, troglitazone and trolox possessing 'chroman ring' similarly to D-alpha-tocopherol, induced the subtype-specific translocation of DGKalpha. Furthermore, the translocation of DGKalpha was abolished by pretreatment with tyrosine kinase inhibitors or by mutation on Tyr-334 of the kinase (YF mutant). D-alpha-tocopheryl succinate enhanced the Src-mediated tyrosine phosphorylation of wild-type DGKalpha but the same reagent did not enhance that of the YF mutant. These results demonstrate that tyrosine phosphorylation on Tyr-334 and chroman ring are important for the D-alpha-tocopherol-induced translocation of DGKalpha.

Animals↗

PubMed2004

Differential effects of peroxisome proliferator activated receptor-gamma (PPAR gamma) ligands in proximal tubular cells: thiazolidinediones are partial PPAR gamma agonists.

UNLABELLED: Background. Peroxisome proliferator activated receptors (PPARs) are ligand-activated transcription factors with multiple effects on target cell function. PPAR gamma activity is regulated by extracellular signal-regulated protein kinase (ERK), mitogen-activated protein (MAP) kinase, and PPAR gamma ligands have varying effects on activity of ERK. Different PPAR gamma ligands have been shown to have both protective and detrimental effects in the kidney. Since transcriptional activation by different PPAR agonists is ligand- and depot-specific PPAR gamma, we have examined the effects of different agonists on PPAR activity in the proximal tubule. METHODS: Opossum kidney cells were used in all experiments, transiently transfected with a PPAR response element luciferase reporter and subject to stimulation with various PPAR ligands. The role of ERK and phosphorylation in PPAR gamma activation were studied, as were the effects of PPAR agonists on ERK activation and cell proliferation. RESULTS: Transcriptional activity of PPAR was not stimulated by PPAR alpha agonists, and only very modestly stimulated by a PPAR beta agonist. The PPAR gamma agonists 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), ciglitazone, and troglitazone stimulated significant transcriptional activation and phosphorylation of PPAR gamma. These effects were more marked with 15d-PGJ(2). Thiazolidinediones attenuated 15d-PGJ(2) evoked PPAR gamma activation and phosphorylation. ERK activity positively regulated PPAR activation. Only 15d-PGJ(2) stimulated ERK activity and cell proliferation, and these effects were also inhibited by thiazolidinediones. CONCLUSION: PPAR gamma agonists exert differential effects in proximal tubule cells with thiazolidinediones behaving as partial agonists.

Animals↗

PubMed2005

Inhibitory effect of PPAR-gamma activator on IL-6 and mPGES protein expression in PBMC induced by homocysteine.

The objective was to investigate the relationship between homocysteine (HCY), peroxisome proliferator-activated receptors (PPAR)-gamma and the expression of interleukin 6 (IL-6) and microsomal prostaglandin E synthase (mPGES) by peripheral blood mononuclear cell (PBMC) culture in vitro, as well as to look at the intervention with HCY and PPAR-gamma activators (troglitazone and rosiglitazone). PBMC were isolated from venous blood of normal volunteers and incubated with HCY and PPAR-gamma activator of varying concentrations for 24 hr. The supernatant of the culture medium was collected in order to determine the IL-6 and mPGES levels by ELISA assay. The results show that the expression of IL-6 and mPGES in the HCY experimental group was significantly higher than that in the control group (p<0.01 and p<0.05, respectively). Additionally, the IL-6 and mPGES levels rose with the increase of HCY in a dose-dependent manner (p<0.01 and p<0.05, respectively). PPAR-gamma activator could significantly inhibit the increase of IL-6 and mPGES induced by 100 micromol/L HCY (p<0.05 and p<0.01, respectively). In conclusion, HCY can increase the expression of IL-6 and mPGES, while PPAR-gamma activator can inhibit the increase of IL-6 and mPGES induced by HCY.

Cells, Cultured↗

PubMed2005

Development of a complex scintillation proximity assay for high-throughput screening of PPARgamma modulators.

AIM: To develop a complex high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel peroxisome proliferator-activated receptor gamma (PPARgamma) modulators. METHODS: Full-length PPARgamma and retinoid X receptor alpha (RXRalpha), biotinylated PPAR response element (PPRE), [3H]BRL49653 and streptavidin-coated FlashPlate or microbead were used to develop an HTS assay based on SPA technology. This 'ABCDE' method was validated against conventional hydroxyapatite (HA) assay and applied to large-scale screening of 16,000 synthetic compounds and natural product extracts. RESULTS: (1) IC50 values of positive control compounds (BRL49653 and troglitazone) obtained from the 'ABCDE' method and HA assay were comparable and consistent with those reported elsewhere; (2) Approximately 178 compounds, showing more than 70% competitive inhibition on BRL49653 binding to PPARgamma, were identified initially by the 'ABCDE' method (microbead); (3) Secondary screening using FlashPlate and cross-reactivity studies with RARalpha, beta, gamma and RXRalpha,beta, gamma confirmed that 12 compounds possessed specific PPARgamma binding properties including 2 with IC50 values less than 0.5 micromol/L and novel chemical structures. CONCLUSIONS: The 'ABCDE' method using either FlashPlate or microbead, is a highly efficient, automatable, and robust tool to screen potential PPARgamma modulators in HTS setting. Its application may be expanded to other nuclear receptors that form heterodimers upon activation.

Chromans↗

PubMed2005

Peroxisome proliferator-activated receptor gamma ligands induce cell cycle arrest and apoptosis in human renal carcinoma cell lines.

AIM: To study the effect of peroxisome proliferator-activated receptor gamma (PPARgamma) ligands on cell proliferation and apoptosis in human renal carcinoma cell lines. METHODS: The expression of PPARgamma was investigated by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry. The effect of thiazolidinedione (TZD) PPARgamma ligands on growth of renal cell carcinoma (RCC) cells was measured by MTT assay and flow cytometric analysis. Cell death ELISA, Hoechst 33342 fluorescent staining and DNA ladder assay were used to observe the effects of PPAR gamma ligands on apoptosis. Regulatory proteins of cell cycle and apoptosis were detected by Western blot analysis. RESULTS: PPARgamma was expressed at much higher levels in renal tumors than in the normal kidney (2.16+/-0.85 vs 0.90+/-0.73; P<0.01). TZD PPARgamma ligands inhibited RCC cell growth in a dose-dependent manner with IC50 values of 7.08 micromol/L and 11.32 micromol/L for pioglitazone, and 5.71 micromol/L and 8.38 micromol/L for troglitazone in 786-O and A498 cells, respectively. Cell cycle analysis showed a G0/G1 arrest in human RCC cells following 24-h exposure to TZD. Analysis of cell cycle regulatory proteins revealed that TZD decreased the protein levels of proliferating cell nuclear antigen, pRb, cyclin D1, and Cdk4 but increased the levels of p21 and p27 in a time-dependent manner. Furthermore, high doses of TZD induced massive apoptosis in renal cancer cells, with increased Bax expression and decreased Bcl-2 expression. CONCLUSION: TZD PPAR gamma ligands showed potent inhibitory effect on proliferation, and could induce apoptosis in RCC cells. These results suggest that ligands for PPAR gamma have potential antitumor effects on renal carcinoma cells.

Antineoplastic Agents↗

PubMed1999

Endrin inhibits adipocyte differentiation by selectively altering expression pattern of CCAAT/enhancer binding protein-alpha in 3T3-L1 cells.

The effects of selected chlorinated cyclodiene pesticides on the adipocyte differentiation process were examined using the 3T3-L1 adipocyte model in vitro. Endrin was found to cause a dose-dependent inhibition of adipocyte differentiation in 3T3-L1 cells. Aldrin and dieldrin were less potent than endrin in interfering with the adipogenic process. Endrin's inhibitory effect was effective only when the pesticide was present in the medium during the first 48 h after exposure of 3T3-L1 cells to adipogenic inducers. Immunoblots analysis revealed that endrin caused a dose-dependent, selective inhibition of the intracellular levels of CCAAT enhancer binding protein (C/EBP)alpha without altering the expression patterns of C/EBPbeta or C/EBPdelta along the differentiation. Supershift analysis showed that DNA-binding capacity of C/EBPalpha was affected most by endrin treatment. Endrin also caused a decrease in the elevation of the adipogenic factor peroxisome proliferator-activated receptor (PPAR)gamma elicited by the adipogenic inducers. However, the cotreatment with troglitazone, a thiazolidinedione known to activate PPARgamma, did not suppress the antiadipogenic action of endrin, indicating that its direct action site is not PPARgamma receptor. Endrin also altered the pattern of activation of nuclear factor-kappaB, a factor activated by 12-O-tetradecanoylphorbol-13-acetate and tumor necrosis factor-alpha, which are known to interfere with adipocyte differentiation. Thus, endrin inhibited the normal decrease in nuclear factor-kappaB-DNA binding observed as cells are acquiring the adipocyte phenotype at a late stage of differentiation. Our results suggest that endrin inhibits adipocyte differentiation through the specific suppression of C/EBPalpha.

3T3 Cells↗

PubMed2002

Characteristics of the peroxisome proliferator activated receptor gamma (PPARgamma) ligand induced apoptosis in colon cancer cells.

BACKGROUND: Involvement of peroxisome proliferator activated receptor gamma (PPARgamma) in the growth response of colon cancer cells has been suggested. AIMS: To investigate the characteristics of PPARgamma induced apoptosis in colon cancer cells. METHODS: The effects of ligands for each of the PPAR subtypes (alpha, delta, and gamma) on DNA synthesis and cell viability were examined in HT-29 colon cancer cells. Modulation of apoptosis related gene expression by PPARgamma ligands was screened with cDNA arrays, and the results were confirmed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. RESULTS: PPARalpha, PPARdelta, and PPARgamma were all expressed in HT-29 cells. PPARgamma ligands, 15-deoxy-delta(12,)(14)-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ), suppressed DNA synthesis of HT-29 cells whereas ligands for PPARalpha and PPARdelta had no significant effects. Both 15d-PGJ2 and TGZ induced HT-29 cell death in a dose dependent manner which was associated with an increase in fragmented DNA and was sensitive to a caspase inhibitor. Among several genes selected by cDNA array screening, quantitative RT-PCR analysis confirmed downregulation of c-myc expression and upregulation of c-jun and gadd153 expression by 15d-PGJ2 and TGZ. PPARgamma induced apoptosis was antagonised by the presence of serum in the culture medium, and interaction between PPARgamma signalling and cell survival signalling through the phosphatidylinositol 3-kinase pathway was suggested. CONCLUSIONS: As c-myc is an important target gene of the adenomatous polyposis coli (APC)/beta-catenin and/or APC/gamma-catenin pathway, activation of PPARgamma signalling appears to compensate for deregulated c-myc expression caused by mutated APC. The present results suggest the potential usefulness of PPARgamma ligands for chemoprevention and treatment of colon cancers.

Apoptosis↗

PubMed2005

Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells.

We have investigated the molecular mechanism whereby 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits adipogenesis in vitro. 1,25(OH)2D3 blocks 3T3-L1 cell differentiation into adipocytes in a dose-dependent manner; however, the inhibition is ineffective 24-48 h after the differentiation is initiated, suggesting that 1,25(OH)2D3 inhibits only the early events of the adipogenic program. Treatment of 3T3-L1 cells with 1,25(OH)2D3 does not block the mitotic clonal expansion or C/EBPbeta induction; rather, 1,25(OH)2D3 blocks the expression of C/EBPalpha, peroxisome proliferator-activated receptor-gamma (PPARgamma), sterol regulatory element-binding protein-1, and other downstream adipocyte markers. The inhibition by 1,25(OH)2D3 is reversible, since removal of 1,25(OH)2D3 from the medium restores the adipogenic process with only a temporal delay. Interestingly, although the vitamin D receptor (VDR) protein is barely detectable in 3T3-L1 preadipocytes, its levels are dramatically increased during the early phase of adipogenesis, peaking at 4-8 h and subsiding afterward throughout the rest of the differentiation program; 1,25(OH)2D3 treatment appears to stabilize the VDR protein levels. Consistently, adenovirus-mediated overexpression of human (h) VDR in 3T3-L1 cells completely blocks the adipogenic program, confirming that VDR is inhibitory. Inhibition of adipocyte differentiation by 1,25(OH)2D3 is ameliorated by troglitazone, a specific PPARgamma antagonist; conversely, hVDR partially suppresses the transacting activity of PPARgamma but not of C/EBPbeta or C/EBPalpha. Moreover, 1,25(OH)2D3 markedly suppresses C/EBPalpha and PPARgamma mRNA levels in mouse epididymal fat tissue culture. Taken together, these data indicate that the blockade of 3T3-L1 cell differentiation by 1,25(OH)2D3 occurs at the postclonal expansion stages and involves direct suppression of C/EBPalpha and PPARgamma upregulation, antagonization of PPARgamma activity, and stabilization of the inhibitory VDR protein.

3T3-L1 Cells↗

PubMed2003

Thiazolidinediones upregulate impaired fatty acid uptake in skeletal muscle of type 2 diabetic subjects.

We examined the regulation of free fatty acid (FFA, palmitate) uptake into skeletal muscle cells of nondiabetic and type 2 diabetic subjects. Palmitate uptake included a protein-mediated component that was inhibited by phloretin. The protein-mediated component of uptake in muscle cells from type 2 diabetic subjects (78 +/- 13 nmol. mg protein-1. min-1) was reduced compared with that in nondiabetic muscle (150 +/- 17, P < 0.01). Acute insulin exposure caused a modest (16 +/- 5%, P < 0.025) but significant increase in protein-mediated uptake in nondiabetic muscle. There was no significant insulin effect in diabetic muscle (+19 +/- 19%, P = not significant). Chronic (4 day) treatment with a series of thiazolidinediones, troglitazone (Tgz), rosiglitazone (Rgz), and pioglitazone (Pio) increased FFA uptake. Only the phloretin-inhibitable component was increased by treatment, which normalized this activity in diabetic muscle cells. Under the same conditions, FFA oxidation was also increased by thiazolidinedione treatment. Increases in FFA uptake and oxidation were associated with upregulation of fatty acid translocase (FAT/CD36) expression. FAT/CD36 protein was increased by Tgz (90 +/- 22% over control), Rgz (146 +/- 42%), and Pio (111 +/- 37%, P < 0.05 for all 3) treatment. Tgz treatment had no effect on fatty acid transporter protein-1 and membrane-associated plasmalemmal fatty acid-binding protein mRNA expression. We conclude that FFA uptake into cultured muscle cells is, in part, protein mediated and acutely insulin responsive. The basal activity of FFA uptake is impaired in type 2 diabetes. In addition, chronic thiazolidinedione treatment increased FFA uptake and oxidation into cultured human skeletal muscle cells in concert with upregulation of FAT/CD36 expression. Increased FFA uptake and oxidation may contribute to lower circulating FFA levels and reduced insulin resistance in skeletal muscle of individuals with type 2 diabetes following thiazolidinedione treatment.

Adult↗

PubMed2005

PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is considered to be one of the master regulators of adipocyte differentiation. PPARgamma2 is abundantly expressed in mature adipocytes and is elevated in the livers of animals that develop fatty livers. The aim of this study was to determine the ability of PPARgamma2 to induce lipid accumulation in hepatocytes and to delineate molecular mechanisms driving this process. The hepatic cell line AML-12 was used to generate a cell line stably expressing PPARgamma2. Oil Red O staining revealed that PPARgamma2 induces lipid accumulation in hepatocytes. This phenotype is accompanied by a selective upregulation of several adipogenic and lipogenic genes including adipose differentiation-related protein (ADRP), adipocyte fatty acid-binding protein 4, sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), and acetyl-CoA carboxylase, genes whose expression levels are known to increase in steatotic livers of ob/ob mice. Furthermore, the PPARgamma2-regulated induction of both SREBP-1 and FAS parallels an increase in de novo triacylglycerol synthesis in hepatocytes. Triacylglycerol synthesis and lipid accumulation are further enhanced by culturing hepatocytes with troglitazone in the absence of exogenous lipids. These results correspond with an increase in the lipid droplet protein, ADRP, and the data demonstrate that ADRP functions to coat lipid droplets in hepatocytes as observed by confocal microscopy. Taken together, these observations propose a role for PPARgamma2 as an inducer of steatosis in hepatocytes and suggest that this phenomenon occurs through an induction of pathways regulating de novo lipid synthesis.

Acetyl-CoA Carboxylase↗

PubMed2001

Regulation of cardiac and skeletal muscle malonyl-CoA decarboxylase by fatty acids.

Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, an important modulator of fatty acid oxidation. We hypothesized that increased fatty acid availability would increase the expression and activity of heart and skeletal muscle MCD, thereby promoting fatty acid utilization. The results show that high-fat feeding, fasting, and streptozotocin-induced diabetes all significantly increased the plasma concentration of nonesterified fatty acids, with a concomitant increase in both rat heart and skeletal muscle MCD mRNA. Upon refeeding of fasted animals, MCD expression returned to basal levels. Fatty acids are known to activate peroxisome proliferator-activated receptor-alpha (PPARalpha). Specific PPARalpha stimulation, through Wy-14643 treatment, significantly increased the expression of MCD in heart and skeletal muscle. Troglitazone, a specific PPARgamma agonist, decreased MCD expression. The sensitivity of MCD induction by fatty acids and Wy-14643 was soleus > extensor digitorum longus > heart. High plasma fatty acids consistently increased MCD activity only in solei, whereas MCD activity in the heart actually decreased with high-fat feeding. Pressure overload-induced cardiac hypertrophy, in which PPARalpha expression is decreased (and fatty acid oxidation is decreased), resulted in decreased MCD mRNA and activity, an effect that was dependent on fatty acids. The results suggest that fatty acids induce the expression of MCD in rat heart and skeletal muscle. Additional posttranscriptional mechanisms regulating MCD activity appear to exist.

Animals↗

PubMed2002

Increased lymphatic lipid transport in genetically diabetic obese rats.

Otsuka Long-Evans Tokushima fatty (OLETF) rats are a model for noninsulin-dependent diabetes mellitus (NIDDM), which is first manifested at 18 wk of age. We assessed age-related changes in lymphatic lipid transport in the intestine of OLETF rats and compared them with those of control Long-Evans Tokushima Otsuka (LETO) rats. Olive oil was infused into the rats with a mesenteric lymph fistula, which was created under ethrane anesthesia. A significant increase in lymphatic triglyceride (TG) transport in OLETF rats was observed at 18-19 wk compared with under 17 wk, but no age-related change was observed in LETO rats. Food restriction, exercise training, or troglitazone treatment in OLETF rats prevented the age-related increase in lipid transport. Biliary phosphatidylcholine concentration was higher in OLETF rats than in LETO rats, but no difference was seen in bile acid concentrations or the activity of microsomal TG transfer protein between the two strains. This study shows that increased lipid transport in the intestine may occur in NIDDM.

Animals↗

PubMed2004

Effects of PPAR-gamma ligands on vascular smooth muscle marker expression in hypertensive and normal arteries.

Having previously demonstrated that glucose transporter-4 (GLUT4) expression was reduced in aortas and carotid arteries of deoxycorticosterone acetate (DOCA) salt-hypertensive rats, we hypothesized that troglitazone (TG), through activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma), would stabilize GLUT4 expression and possibly preserve the differentiated phenotype in vascular smooth muscle cells. In DOCA salt-hypertensive rats treated with TG (100 mg/day), there was a significant (P < 0.001) decrease in systolic blood pressure (BP; 149.9 +/- 4.4 mmHg) compared with the untreated DOCA salt-hypertensive rats (202.2 +/- 10.34 mmHg). Separate trials with rosiglitazone (RS; 3 mg/day) demonstrated a significant (P < 0.001) decrease in BP (DOCA salt, 164.2 +/- 9.8 vs. DOCA-RS, 124.9 +/- 3.7 mmHg) comparable to that with TG. Expression of GLUT4, h-caldesmon, and smooth muscle myosin heavy chain SM2 was significantly decreased in aortas of DOCA salt-hypertensive rats and was reversed by TG to levels similar to those in aortas of sham-treated rats. TG (50 microM) induced GLUT4 and h-caldesmon expression in 24-h culture of explanted carotid arteries of DOCA salt-hypertensive rats, and the endogenous PPAR-gamma ligand 15-deoxy-Delta(12-14)-prostaglandin J(2) (PGJ(2); 20 microM) and TG (50 microM) similarly increased GLUT4, h-caldesmon, and SM2 protein expression in explanted aortas. The expression of activated, phosphorylated Akt was increased by PGJ(2) and TG with no significant effect on total Akt levels. Inhibition of phosphorylated Akt expression using the phosphatidylinositol 3-kinase inhibitor LY-294002 (16 microM) abrogated the increased expression of h-caldesmon and SM2. These data demonstrate that PPAR-gamma agonists maintain or induce expression of markers of the contractile phenotype independently of their effects on hypertension, and that this effect may be mediated through activation of phosphatidylinositol 3-kinase/Akt.

Animals↗

PubMed2005

Peroxisome proliferator-activated receptor gamma inhibition prevents adhesion to the extracellular matrix and induces anoikis in hepatocellular carcinoma cells.

Activation of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits growth and survival of hepatocellular carcinoma (HCC) cell lines. To further investigate the function of PPARgamma in HCC, PPARgamma expression patterns in primary tumors were examined, and the responses of two HCC cell lines to PPARgamma activation and inhibition were compared. PPARgamma expression was increased in HCC and benign-appearing peritumoral hepatocytes compared with remote benign hepatocytes. Both compound PPARgamma inhibitors and PPARgamma small interfering RNAs prevented HCC cell lines from adhering to the extracellular matrix. Loss of adhesion was followed by caspase-dependent apoptosis (anoikis). PPARgamma inhibitors had no effect on initial beta1 integrin-mediated adhesion, or on total focal adhesion kinase levels but did reduce focal adhesion kinase phosphorylation. The PPARgamma inhibitor T0070907 was significantly more efficient at causing cancer cell death than the activators troglitazone and rosiglitazone. T0070907 caused cell death by reducing adhesion and inducing anoikis, whereas the activators had no direct effect on adhesion and caused cell death at much higher concentrations. In conclusion, PPARgamma overexpression is present in HCC. Inhibition of PPARgamma function causes HCC cell death by preventing adhesion and inducing anoikis-mediated apoptosis. PPARgamma inhibitors represent a potential novel treatment approach to HCC.

Anilides↗

PubMed2005

The anti-invasive activity of cyclooxygenase inhibitors is regulated by the transcription factor ATF3 (activating transcription factor 3).

We previously showed that nonsteroidal anti-inflammatory drugs (NSAID) such as sulindac sulfide, which has chemopreventive activity, modulate the expression of several genes detected by microarray analysis. Activating transcription factor 3 (ATF3) was selected for further study because it is a transcription factor involved in cell proliferation, apoptosis, and invasion, and its expression is repressed in human colorectal tumors as compared with normal adjacent tissue. In this report, we show that ATF3 mRNA and protein expression are up-regulated in HCT-116 human colorectal cancer cells following treatment with NSAIDs, troglitazone, diallyl disulfide, and resveratrol. To ascertain the biological significance of ATF3, we overexpressed full-length ATF3 protein in the sense and antisense orientations. Overexpression of ATF3 in the sense orientation decreased focus formation in vitro and reduced the size of mouse tumor xenografts by 54% in vivo. Conversely, overexpression of antisense ATF3 was protumorigenic in vitro, however, not in vivo. ATF3 in the sense orientation did not modulate apoptosis, indicating another mechanism is involved. With microarray analysis, several genes relating to invasion and metastasis were identified by ATF3 overexpression and were confirmed by real-time reverse transcription-PCR, and several of these genes were modulated by sulindac sulfide, which inhibited invasion in these cells. Furthermore, overexpression of ATF3 inhibited invasion to a similar degree as sulindac sulfide treatment, whereas antisense ATF3 increased invasion. In conclusion, ATF3 represents a novel mechanism in which NSAIDs exert their anti-invasive activity, thereby linking ATF3 and its gene regulatory activity to the biological activity of these compounds.

Activating Transcription Factor 3↗

PubMed2006

Insulin-lowering agents inhibit synthesis of testosterone in ovaries of DHEA-induced PCOS rats.

BACKGROUND: Insulin-lowering agents are reported to be useful in treating polycystic ovary syndrome (PCOS) anovulation. It has been suggested that lower insulin levels secondarily affect ovarian tissue, although the direct mechanism of action has not yet been verified. Here we investigated if these agents directly affect the ovary. METHODS: Thirty female Wister rats were studied. Six control rats were injected subcutaneously with 0.2 ml sesame oil, while 24 rats used as PCOS models were injected subcutaneously with dehydroepiandrosterone (DHEA) and divided into four groups. Six rats were injected with only DHEA, while the remaining 18 rats received metformin, pioglitazone or troglitazone. The ovaries were immunohistochemically stained with anti- testosterone and anti-17beta-HSD antibodies, and then evaluated for morphological changes. RESULTS: In the DHEA administration group, the number of atretic follicles significantly increased compared to that of control rats. The insulin-lowering agents did not improve the multicystic appearance. Serum testosterone concentrations significantly increased with DHEA administration, but the increase was inhibited by oral administration of insulin-lowering agents. Testosterone deposits in ovarian tissue were also reduced by feeding rats insulin-lowering agents. CONCLUSION: Insulin-lowering agents affected ovarian tissue by inhibiting testosterone biosynthesis in vivo.

17-Hydroxysteroid Dehydrogenases↗