Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, independently of PPARγ in human glioma cells

Peroxisome proliferator-activated receptor γ (PPARγ) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPARγ in CGZ-induced cell death was examined. At concentrations of greater than 30 μM, CGZ, a synthetic PPARγ agonist, activated caspase-3 and induced apoptosis in T98G cells. Treatment of T98G cells with less than 30 μM CGZ effectively induced cell death after pretreatment with 30 μM of the PPARγ antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPARγ was down-regulated cells by siRNA, lower concentrations of CGZ (<30 μM) were sufficient to induce cell death, although higher concentrations of CGZ (≥30 μM) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPARγ. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPARγ in glioma cells, by down-regulating Akt activity and inducing MMP collapse.     

Evodiamine inhibits the proliferation of leukemia cell line K562 by regulating peroxisome proliferators-activated receptor gamma (PPARγ) pathway

Evodiamine, a quinolone alkaloid, is one of the major bioactive compounds of Evodia rutaecarpa Bentham (Rutaceae). It exhibits excellent biological activities, especially the anticancer activity. This study aims to investigate the effect of evodiamine on the proliferation of leukemia cell line K562 and to explore the underlying mechanism. The effect of evodiamine on K562 cells proliferation was analyzed by trypan blue dye exclusion assay and MTT assay. The expression levels of peroxisome proliferators-activated receptor gamma (PPARγ), cyclin D1, and p21 were detected by western blot assay. The results demonstrated that evodiamine inhibited the proliferation and decreased the viability of K562 cells in a dose- and time-dependent manner. 2-Chloro-5-nitro-N-phenylbenzamide (GW9662) and/or PPARγ-siRNA pretreatment alleviated the cell growth suppression triggered by evodiamine. Meanwhile, evodiamine intervention elevated the expression of PPARγ in K562 cells, while pretreatment with GW9662 attenuated the enhanced upregulation of PPARγ expression induced by evodiamine. In addition, GW9662 and PPARγ-siRNA pretreatment also significantly attenuated the downregulation of the cell cycle control protein cyclin D1 and the upregulation of cyclin-dependent kinase inhibitor p21 induced by evodiamine. In conclusion, PPARγ signaling pathway may involve in the proliferation inhibition of evodiamine on K562 cells via inhibiting cylcin D1 and stimulating of p21.     

Dihydromyricetin enhances glucose uptake by inhibition of MEK/ERK pathway and consequent down‐regulation of phosphorylation of PPARγ in 3T3‐L1 cells

Accumulating evidence suggests that inhibition of mitogen‐activated protein kinase signalling can reduce phosphorylation of peroxisome proliferator‐activated receptor γ (PPARγ) at serine 273, which mitigates obesity‐associated insulin resistance and might be a promising treatment for type 2 diabetes. Dihydromyricetin (DHM) is a flavonoid that has many beneficial pharmacological properties. In this study, mouse fibroblast 3T3‐L1 cells were used to investigate whether DHM alleviates insulin resistance by inhibiting PPARγ phosphorylation at serine 273 via the MEK/ERK pathway. 3T3‐L1 pre‐adipocytes were differentiated, and the effects of DHM on adipogenesis and glucose uptake in the resulting adipocytes were examined. DHM was found to dose dependently increase glucose uptake and decrease adipogenesis. Insulin resistance was then induced in adipocytes using dexamethasone, and DHM was shown to dose and time dependently promote glucose uptake in the dexamethasone‐treated adipocytes. DHM also inhibited phosphorylation of PPARγ and ERK. Inhibition of PPARγ activity with GW9662 potently blocked DHM‐induced glucose uptake and adiponectin secretion. Interestingly, DHM showed similar effects to PD98059, an inhibitor of the MEK/ERK pathway. DHM acted synergistically with PD98059 to improve glucose uptake and adiponectin secretion in dexamethasone‐treated adipocytes. In conclusion, our findings indicate that DHM improves glucose uptake in adipocytes by inhibiting ERK‐induced phosphorylation of PPARγ at serine 273.     

β-胡萝卜素抑制MCF-7细胞生长上调过氧化酶体增殖物激活受体γ（PPARγ）表达

为了研究过氧化酶体增殖物激活受体γ（PPARγ）表达在β 胡萝卜素影响乳腺癌MCF 7细胞活力中所起的作用,采用MTT法测定细胞活力、Western 印迹检测细胞中PPARγ的蛋白质水平,用RT-PCR从mRNA水平检测细胞内PPARγ、P21^WAF1/CIP1、COX-2和P27表达.研究发现,β 胡萝卜素显著抑制人乳腺癌细胞株MCF-7细胞的生长,β-胡萝卜素对细胞生长的抑制作用呈现出时间和计量依赖关系;β-胡萝卜素能够呈现时间效应地从mRNA和蛋白质水平显著上调PPARγ的表达,β-胡萝卜素能够通过PPARγ调节P21^WAF1/CIP1和COX-2mRNA水平;PPARγ的抑制剂GW9662和抗氧化剂还原型谷胱甘肽（GSH）都能部分阻止由β-胡萝卜素引起的细胞活力下降.研究结果提示,激活PPARγ途径和调制细胞氧化状态是β 胡萝卜素对乳腺癌细胞MCF-7的生长抑制效应原因之一.     

The environmental obesogen tributyltin chloride acts via peroxisome proliferator activated receptor gamma to induce adipogenesis in murine 3T3-L1 preadipocytes

Obesogens are chemicals that predispose exposed individuals to weight gain and obesity by increasing the number of fat cells, storage of fats into existing cells, altering metabolic rates, or disturbing the regulation of appetite and satiety. Tributyltin exposure causes differentiation of multipotent stromal stem cells (MSCs) into adipocytes; prenatal TBT exposure leads to epigenetic changes in the stem cell compartment that favor the production of adipocytes at the expense of bone, in vivo. While it is known that TBT acts through peroxisome proliferator activated receptor gamma to induce adipogenesis in MSCs, the data in 3T3-L1 preadipocytes are controversial. Here we show that TBT can activate the RXR-PPARγ heterodimer even in the presence of the PPARγ antagonist GW9662. We found that GW9662 has a 10-fold shorter half-life in cell culture than do PPARγ activators such as rosiglitazone (ROSI), accounting for previous observations that GW9662 did not inhibit TBT-mediated adipogenesis. When the culture conditions are adjusted to compensate for the short half-life of GW9662, we found that TBT induces adipogenesis, triglyceride storage and the expression of adipogenic marker genes in 3T3-L1 cells in a PPARγ-dependent manner. Our results are broadly applicable to the study of obesogen action and indicate that ligand stability is an important consideration in the design and interpretation of adipogenesis assays.     

Peroxisome proliferator-activated receptor γ (PPARγ) plays a critical role in the development of TGFβ resistance of H460 cell

The primary goal of the study was to investigate how peroxisome proliferator-activated receptor γ (PPARγ) played a critical role in the protection of H460 cell, one of the non-small cell lung cancer (NSCLC) cells with multidrug resistance, from transforming growth factor β (TGFβ)-mediated mitoinhibition. In the study, TGFβ resistance of H460 cell was first confirmed by analyses of PPARγ expression, its interaction with TGFβ-induced Smad3 and phospho-Smad3 (p-Smad3) and survival of H460. Results showed that enable to escape from G2/M phase arrest, H460 cell had higher resistance to TGFβ-mediated mitoinhibition than CH27 (a drug sensitive control). TGFβ significantly increased PPARγ expression of H460 but not of CH27 cell whereas nuclear accumulation of p-Smad3 was only limited to CH27, the latter was believed to contribute to the induction of P^{21 waf1/cip1} and cyclin B1, cell cycle arrest at G2/M phase and TGFβ-mediated mitoinhibition of CH27 cell. TGFβ-induced PPARγ of H460 cell was further demonstrated to bind to Smad3 and p-Smad3, and GW9662 (PPARγ inhibitor) or PPARγ-specific shRNA could disrupt the binding. GW9662 also increased the nuclear accumulation of p-Smad3 that eventually led to the reduction of TGFβ resistance of H460. A transient knockdown of PPARγ with shRNA revealed a similar effect as GW9662. In addition, activation of P³⁸ instead of ERK played a critical role in TGFβ-induced expression of PPARγ, which subsequently activated RhoA in H460 cell.     

Role of PPARγ in the salutary effects of 17β‐estradiol on kupffer cell cytokine production following trauma‐hemorrhage

Studies have shown that administration of 17β‐estradiol prevents trauma‐hemorrhage‐induced increase in proinflammatory cytokine production by Kupffer cells and associated multiple organ injury. Since activation of peroxisome proliferator‐activated receptor γ (PPARγ) following ischemic conditions has been shown to be protective, we examined if PPARγ plays any role in the salutary effects of 17β‐estradiol on Kupffer cell cytokine production following trauma‐hemorrhage. Male mice underwent trauma‐hemorrhage (mean blood pressure 40 mmHg for 90 min, then resuscitation). 17β‐estradiol (50 µg/kg) or vehicle with or without PPARγ antagonist GW9662 was injected subcutaneously at the middle of resuscitation. At 2 h after trauma‐hemorrhage, plasma interleukin (IL)‐6 and tumor necrosis factor (TNF)‐α levels, Kupffer cell IL‐6 and TNF‐α production and mRNA expression, and PPARγ, nuclear factor (NF)‐κB and activator protein (AP)‐1 DNA binding activity were determined. Kupffer cell IL‐6 and TNF‐α production, as well as plasma IL‐6 and TNF‐α levels, increased following trauma‐hemorrhage. Moreover, NF‐κB and AP‐1 DNA binding activity and IL‐6 and TNF‐α mRNA expression were also enhanced under such conditions. However, 17β‐estradiol administration normalized all these parameters. Although PPARγ activity decreased after trauma‐hemorrhage, administration of 17β‐estradiol following trauma‐hemorrhage elevated PPARγ activity above the normal level. Inhibition of PPARγ by co‐administration of GW9662, however, abolished the salutary effects of 17β‐estradiol on plasma cytokine and Kupffer cells. Thus, activation of PPARγ appears to play an important role in mediating the salutary effects of 17β‐estradiol on plasma cytokine levels and Kupffer cell cytokine production after trauma‐hemorrhage, which are likely mediated via NF‐κB and AP‐1. J. Cell. Physiol. 226: 205–211, 2010. © 2010 Wiley‐Liss, Inc.     

PPARγ antagonist GW9662 induces functional estrogen receptor in mouse mammary organ culture: potential translational significance

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays a central role in regulating metabolism, including interaction with the estrogen receptor-α (ERα). Significantly, PPARγ activity can be modulated by small molecules to control cancer both in vitro and in vivo (Yin et al., Cancer Res 69:687–694, 2009). Here, we evaluated the effects of the PPARγ agonist GW7845 and the PPARγ antagonist GW9662 on DMBA-induced mammary alveolar lesions (MAL) in a mouse mammary organ culture. The results were as follows: (a) the incidence of MAL development was significantly inhibited by GW 7845 and GW 9662; (b) GW9662 but not GW7845, in the presence of estradiol, induced ER and PR expression in mammary glands and functional ERα in MAL; (c) while GW9662 inhibited expression of adipsin and ap2, GW 7845 enhanced expression of these PPARγ-response genes; and (d) Tamoxifen caused significant inhibition of GW9662 treated MAL, suggesting that GW9662 sensitizes MAL to antiestrogen treatment, presumably through rendering functional ERα and induction of PR. The induction of ERα by GW9662, including newer analogs, may permit use of anti-ER strategies to inhibit breast cancer in ER? patients.     

GW1929 inhibits α7 nAChR expression through PPARγ-independent activation of p38 MAPK and inactivation of PI3-K/mTOR: The role of Egr-1

Studies demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) ligands reduce nicotine-induced non small cell lung carcinoma (NSCLC) cell growth through inhibition of nicotinic acetylcholine receptor (nAChR) mediated signaling pathways. However, the mechanisms by which PPARγ ligands inhibited nAChR expression remain elucidated. Here, we show that GW1929, a synthetic PPARγ ligand, not only inhibited but also antagonized the stimulatory effect of acetylcholine on NSCLC cell proliferation. Interestingly, GW1929 inhibited α7 nAChR expression, which was not blocked by GW9662, an antagonist of PPARγ, or by PPARγ siRNA, but was abrogated by the p38 MPAK inhibitor SB239063. GW1929 reduced the promoter activity of α7 nAChR and induced early growth response-1 (Egr-1) protein expression, which was overcame by SB239063, but enhanced by inhibitors of PI3-K and mTOR. Silencing of Egr-1 blocked, while overexpression of Egr-1 enhanced, the effect of GW1929 on α7 nAChR expression and promoter activity. Finally, GW1929 induced Egr-1 bound to specific DNA areas in the α7 nAChR gene promoter. Collectively, these results demonstrate that GW1929 not only inhibits but also antagonizes Ach-induced NSCLC cell growth by inhibition of α7 nAChR expression through PPARγ-independent signals that are associated with activation of p38 MPAK and inactivation of PI3-K/mTOR, followed by inducing Egr-1 protein and Egr-1 binding activity in the α7 nAChR gene promoter. By downregulation of the α7 nAchR, GW1929 blocks cholinergic signaling and inhibits NSCLC cell growth.     

Involvement of PPARα and PPARγ in apoptosis and proliferation of human hepatocarcinoma HepG2 cells

Peroxisome proliferator‐activated receptors (PPARs) mediate the effects of various ligands, known as peroxisome proliferators, a heterogeneous class of compounds including industrial chemicals, pharmaceuticals, and biomolecules such as fatty acids and eicosanoids. Among peroxisome proliferators, fibrate derivatives are considered specific ligands for PPARα, whereas eicosanoids, such as PGJ2, for PPARγ. The study aimed to clarify the relation between PPARs and apoptosis or proliferation on the same type of cells, using clofibrate as specific ligand of PPARα and PGJ2 as specific ligand of PPARγ. The cells used were human hepatocarcinoma HepG2 cells. The results showed that PPARα protein content increased in HepG2 cells treated with clofibrate, causing apoptosis in a time‐ and concentration‐dependent way, as evidenced by the citofluorimetric assay and determination of BAD, myc and protein phosphatase 2A protein content. It also emerged that PPARγ increased in the same cells when treated with a specific ligand of this PPAR; in this case the increase of PPARγ did not cause an increase of apoptosis, but a time‐ and concentration‐dependent inhibition of cell proliferation, evidenced by decreased cell numbers and increased number of cells in the G0/G1 phase of the cycle. It may be concluded that PPARα is chiefly related to apoptosis and PPARγ to cell proliferation. Copyright © 2010 John Wiley & Sons, Ltd.     

PPARγ-dependent pathway in the growth-inhibitory effects of K562 cells by carotenoids in combination with rosiglitazone

Background

Carotenoids have been found to play roles in the prevention and therapy of some cancers which PPARγ was also discovered to be involved in. The present studies were directed to determine the inhibitory effects of carotenoids in combination with rosiglitazone, a synthetic PPARγ agonist, on K562 cell proliferation and elucidate the contribution of PPARγ-dependent pathway to cell proliferation suppression.

Methods

The effects of carotenoid and rosiglitazone combination on K562 cell proliferation were evaluated by trypan blue dye exclusion assay and MTT assay. When PPARγ has been inhibited by GW9662 and siRNA, cycle-related regulator expression in K562 cells treated with carotenoid and rosiglitazone combination was analyzed by Western blotting.

Results

Rosiglitazone inhibited K562 cell proliferation and augmented the inhibitory effects of carotenoids on the cell proliferation greatly. Specific PPARγ inhibition attenuated the cell growth suppression induced by carotenoid and rosiglitazone combination. GW9662 pre-treatment attenuated the enhanced up-regulation of PPARγ expression caused by the combination treatment. Moreover, GW9662 and PPARγ siRNA also significantly attenuated the up-regulation of p21 and down-regulation of cyclin D1 caused by carotenoids and rosiglitazone.

Conclusions

PPARγ signaling pathway, via stimulating p21 and inhibiting cyclin D1, may play an important role in the anti-proliferative effects of carotenoid and rosiglitazone combination on K562 cells.

General significance

Carotenoids in combination with rosiglitazone are hopeful to provide attractive dietary or supplementation-based and pharmaceutical strategies to treat cancer diseases.     

大豆异黄酮激活PPARγ抑制人乳腺癌MCF-7细胞增殖

目的：探讨两种大豆异黄酮主要成分染料木黄酮（genistein,GEN）和大豆苷元（daidzein,DAI）抑制人乳腺癌MCF-7细胞增殖的作用与过氧化物酶体增殖激活物受体γ（peroxisome proliferators-activated receptorγ,PPARγ）信号途径的关系。方法：采用免疫细胞化学染色方法观察MCF-7细胞的PPARγ表达情况,PPARγ介导的荧光素酶报告基因检测大豆异黄酮和PPARγ配体罗格列酮（rosiglitazone,ROS）对MCF-7细胞PPARγ的激活作用,MCF-7细胞分别经8×10-5mol/L GEN、DAI和1×10-5mol/L的ROS单独或联合1×10-5mol/L的PPARγ特异性抑制剂GW9662联合处理24、48和72 h后,用CCK-8法检测细胞增殖。结果：MCF-7细胞存在有PPARγ表达,GEN、DAI呈剂量依赖性增强报告基因荧光素酶活性,且这种作用可被GW9662明显阻断;GEN、DAI和ROS呈时间依赖性明显抑制MCF-7细胞增殖（P〈0.05）,而GW9662可以显著削弱GEN、DAI和ROS对MCF-7细胞的增殖抑制作用（P〈0.05）。结论：大豆异黄酮可通过激活乳腺癌MCF-7细胞的PPARγ信号途径抑制其增殖。     

PPARγ up-regulates TGFβ/smad signal pathway repressor c-Ski

TGFβ/smad pathway is recognized as an important signal pathway to promote the pathogenesis of atherosclerosis (AS). Peroxisome proliferator-activated receptor γ (PPARγ) activation is considered to be important in modulating AS. Herein, we investigated the regulation of PPARγ on c-Ski, the repressor of TGFβ/smad pathway, in rat AS model and cultured vascular smooth muscle cells (VSMCs). c-Ski mRNA and protein expression were detected by real-time PCR and Western blot, respectively, in vivo and in vitro with treatment of PPARγ agonist rosiglitazone and antagonist GW9662. The proliferation and collagen secretion of VSMCs after c-Ski transfection were investigated. The underlying mechanism was further investigated by online program NUBIScan and luciferase reporter gene analysis. Results showed that both mRNA and protein expressions of c-Ski in the AS lesions was down-regulated in vivo, while in cultured VSMCs, c-Ski transfection significantly suppressed the proliferation and collagen secretion of rat VSMCs. Rosiglitazone significantly up-regulated mRNA and protein levels of c-Ski in VSMCs, which could be blocked by GW9662. Online NUBIScan analysis suggested possible PPARγ binding sites in the promoter region of c-Ski. In addition, luciferase activity of c-Ski reporter gene was also increased obviously in the presence of rosiglitazone. These results indicate that c-Ski is one of the newly found target genes of PPARγ and thus involved in the anti-AS effect of PPARγ.     

Antagonist of peroxisome proliferator-activated receptor γ induces cerebellar amyloid-β levels and motor dysfunction in APP/PS1 transgenic mice

Recent evidences show that peroxisome proliferator-activated receptor γ (PPARγ) is involved in the modulation of the amyloid-β (Aβ) cascade causing Alzheimer’s disease (AD) and treatment with PPARγ agonists protects against AD pathology. However, the function of PPARγ steady-state activity in Aβ cascade and AD pathology remains unclear. In this study, an antagonist of PPARγ, GW9662, was injected into the fourth ventricle of APP/PS1 transgenic mice to inhibit PPARγ activity in cerebellum. The results show that inhibition of PPARγ significantly induced Aβ levels in cerebellum and caused cerebellar motor dysfunction in APP/PS1 transgenic mice. Moreover, GW9662 treatment markedly decreased the cerebellar levels of insulin-degrading enzyme (IDE), which is responsible for the cellular degradation of Aβ. Since cerebellum is spared from significant Aβ accumulation and neurotoxicity in AD patients and animal models, these findings suggest a crucial role of PPARγ steady-state activity in protection of cerebellum against AD pathology.     

Peroxisome proliferator-activated receptor γ modulates renal crystal retention associated with high oxalate concentration by regulating tubular epithelial cellular transdifferentiation

The differentiated phenotype of renal tubular epithelial cell exerts significant effect on crystal adherence. Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to be critical for the regulation of cell transdifferentiation in many physiological and pathological conditions; however, little is known about its role in kidney stone formation. In the current study, we found that temporarily high oxalate concentration significantly decreased PPARγ expression, induced Madin Darby Canine Kidney cell dedifferentiation, and prompted subsequent calcium oxalate (CaOx) crystal adhesion in vitro. Furthermore, cell redifferentiation after the removal of the high oxalate concentration, along with a decreasing affinity to crystals, was an endogenic PPARγ-dependent process. In addition, the PPARγ antagonist GW9662, which can depress total-PPARγ expression and activity, enhanced cell dedifferentiation induced by high oxalate concentration and inhibited cell redifferentiation after removal of the high oxalate concentration. These effects were partially reversed by the PPARγ agonist 15d-PGJ2. Similar results were observed in animals that suffered from temporary hyperoxaluria followed by a recovery period. The active crystal-clearing process occurs through the transphenotypical morphology of renal tubular epithelial cells, reflecting cell transdifferentiation during the recovery period. However, GW9662 delayed cell redifferentiation and increased the secondary temporary crystalluria-induced crystal retention. This detrimental effect was partially reversed by 15d-PGJ2. Taken together, our results revealed that endogenic PPARγ activity plays a vital regulatory role in crystal clearance, subsequent crystal adherence, and CaOx stone formation via manipulating the transdifferentiation of renal tubular epithelial cells.     

Trans‐repression of NFκB pathway mediated by PPARγ improves vascular endothelium insulin resistance

Previous study has shown that thiazolidinediones (TZDs) improved endothelium insulin resistance (IR) induced by high glucose concentration (HG)/hyperglycaemia through a PPARγ‐dependent‐NFκB trans‐repression mechanism. However, it is unclear, whether changes in PPARγ expression affect the endothelium IR and what the underlying mechanism is. In the present study, we aimed to address this issue. HG‐treated human umbilical vascular endothelial cells (HUVEC) were transfected by either PPARγ‐overexpressing (Ad‐PPARγ) or PPARγ‐shRNA‐containing (Ad‐PPARγ‐shRNA) adenoviral vectors. Likewise, the rats fed by high‐fat diet (HFD) were infected by intravenous administration of Ad‐PPARγ or Ad‐PPARγ‐shRNA. The levels of nitric oxide (NO), endothelin‐1 (ET‐1) and cytokines (TNFα, IL‐6, sICAM‐1 and sVCAM‐1) and the expression levels of PPARγ, eNOS, AKT, p‐AKT, IKKα/β and p‐IKKα/β and IκBα were examined; and the interaction between PPARγ and NFκB‐P65 as well as vascular function were evaluated. Our present results showed that overexpression of PPARγ notably increased the levels of NO, eNOS, p‐AKT and IκBα as well as the interaction of PPARγ and NFκB‐P65, and decreased the levels of ET‐1, p‐IKKα/β, TNFα, IL‐6, sICAM‐1 and sVCAM‐1. In contrast, down‐expression of PPARγ displayed the opposite effects. The results demonstrate that the overexpression of PPARγ improves while the down‐expression worsens the endothelium IR via a PPARγ‐mediated NFκB trans‐repression dependent manner. The findings suggest PPARγ is a potential therapeutic target for diabetic vascular complications.