Peroxisome proliferator‐activated receptor γ upregulates galectin‐9 and predicts prognosis in intestinal‐type gastric cancer

The importance of PPARγ (peroxisome proliferator‐activated receptor γ) in gastric cancer (GC) is unclear. We investigated the role of PPARγ in GC cell lines and an animal model, and its prognostic significance of PPARγ in GC patients. We controlled PPARγ and galectin‐9 expression by using siRNAs and lentiviral constructs. Interaction between PPARγ and galectin‐9 was evaluated using luciferase and chromatin immunoprecipitation assays. PPARγ expression in GCs was determined by immunohistochemical staining of tissue microarrays and survival analysis was done. Overexpression of PPARγ was accompanied by increased galectin‐9. Enhanced PPARγ or galectin‐9 expression increased E‐cadherin expression; decreased expression of N‐cadherin, fibronectin, snail, twist and slug and reduced cell invasion and migration. PPARγ bound to the galectin‐9 promoter region. Galectin‐9 activity increased in PPARγ‐overexpressing cells but decreased in PPARγ siRNA‐treated cells. In a zebrafish xenograft model, the number of migrated cancer cells and number of fish with AGS cells in the tail vein were reduced in PPARγ‐overexpressing GC cells. PPARγ was expressed in 462 of the 688 patients (69.2%) with GC. In 306 patients with intestinal‐type GC, those with PPARγ‐positive tumors had lower overall and cancer‐specific mortalities than those with PPARγ‐negative tumors. PPARγ expression was an independent prognostic factor for overall and GC‐specific mortality in patients with intestinal‐type GC (adjusted hazard ratio, 0.42; 95% CI, 0.22–0.81). PPARγ inhibits cell invasion, migration and epithelial–mesenchymal transition through upregulation of galectin‐9 in vitro and in vivo.     

Loss of PPARγ expression in mammary secretory epithelial cells creates a pro‐breast tumorigenic environment

Breast cancer is the leading cause of new cancer diagnoses among women. Using peroxisome proliferator‐activated receptor (PPAR)γ^(+/?) mice, we showed normal expression of PPARγ was critical to stop 7,12‐dimethylbenz[a]anthracene (DMBA)‐induced breast tumorigenesis. PPARγ is expressed in many breast cell types including mammary secretory epithelial (MSE) cells. MSEs proliferate as required during pregnancy, and undergo apoptosis or reversible transdifferentiation during involution once lactation is complete. Thus, MSE‐specific loss of PPARγ was hypothesized to enhance DMBA‐mediated breast tumorigenesis. To test this, MSE cell‐specific PPARγ knockout (PPARγ‐MSE KO) and control (PPARγ‐WT) mice were generated, mated and allowed to nurse for three days. One week after involution, dams were treated with DMBA to initiate breast tumors, and randomized on week 7 to continue receiving a normal chow diet (DMBA Only: PPARγ‐WT, n = 15; PPARγ‐MSE KO, n = 25) or one supplemented with a PPARγ activating drug (DMBA + ROSI: PPARγ‐WT, n = 17; PPARγ‐MSE KO, n = 24), and monitored for changes in breast tumor outcomes. PPARγ‐MSE KOs had significantly lower overall survival and decreased mammary tumor latency as compared to PPARγ‐WT controls. PPARγ activation significantly reduced DMBA‐mediated malignant mammary tumor volumes irrespective of genotype. MSE‐specific PPARγ loss resulted in decreased mammary gland expression of PTEN and Bax, increased superoxide anion production, and elevated serum eotaxin and RANTES, creating a protumorigenic environment. Moreover, PPARγ activation in MSEs delayed mammary tumor growth in part by down‐regulating Cox‐1, Cox‐2 and cyclin D1. Collectively, these studies highlight a protective role of MSE‐specific PPARγ during breast tumorigenesis, and support a novel chemotherapeutic role of PPARγ activation in breast cancer.     

Dietary tocopherols inhibit cell proliferation,regulate expression of ERα, PPARγ, and Nrf2, and decrease serum inflammatory markers during the development of mammary hyperplasia

Previous clinical and epidemiological studies of vitamin E have used primarily α‐tocopherol for the prevention of cancer. However, γ‐tocopherol has demonstrated greater anti‐inflammatory and anti‐tumor activity than α‐tocopherol in several animal models of cancer. This study assessed the potential chemopreventive activities of a tocopherol mixture containing 58% γ‐tocopherol (γ‐TmT) in an established rodent model of mammary carcinogenesis. Female ACI rats were utilized due to their sensitivity to 17β‐estradiol (E₂) to induce mammary hyperplasia and neoplasia. The rats were implanted subcutaneously with sustained release E₂ pellets and given dietary 0.3% or 0.5% γ‐TmT for 2 or 10 wk. Serum E₂ levels were significantly reduced by the treatment with 0.5% γ‐TmT. Serum levels of inflammatory markers, prostaglandin E₂ and 8‐isoprostane, were suppressed by γ‐TmT treatment. Histology of mammary glands showed evidence of epithelial hyperplasia in E₂‐treated rats. Immunohistochemical analysis of the mammary glands revealed a decrease in proliferating cell nuclear antigen (PCNA), cyclooxygenase‐2 (COX‐2), and estrogen receptor α (ERα), while there was an increase in cleaved‐caspase 3, peroxisome proliferator‐activated receptor γ (PPARγ), and nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) in γ‐TmT‐treated rats. In addition, treatment with γ‐TmT resulted in a decrease in the expression of ERα mRNA, whereas mRNA levels of ERβ and PPARγ were increased. In conclusion, γ‐TmT was shown to suppress inflammatory markers, inhibit E₂‐induced cell proliferation, and upregulate PPARγ and Nrf2 expression in mammary hyperplasia, suggesting that γ‐TmT may be a promising agent for human breast cancer prevention. © 2012 Wiley Periodicals, Inc.     

Insights on distinct pathways of thiazolidinediones (PPARγ ligand)‐promoted apoptosis in TRAIL‐sensitive or ‐resistant malignant urothelial cells

Thiazolidinediones, including rosiglitazone and troglitazone, are insulin‐sensitizing drugs and high‐affinity ligands for the peroxisome proliferator‐activated receptor γ (PPARγ). Apart from their antidiabetic activity, these molecules possess antitumor properties. We investigated their potential apoptotic effects on RT4 (derived from a well‐differentiated Grade I papillary tumor) and T24 (derived from an undifferentiated Grade III carcinoma) bladder cancer cells. Rosiglitazone induced G2/M or G0/G1 phase cell cycle arrest in RT4 and T24 cells, respectively. Only troglitazone triggered apoptosis via extrinsic and intrinsic pathways in both cell lines. Interestingly, rosiglitazone amplified TRAIL‐induced apoptosis in TRAIL‐sensitive RT4 cells or let TRAIL‐resistant T24 cells to respond to TRAIL. Thiazolidinediones acted through PPARγ activation‐independent mechanisms. The underlying mechanisms involved for the first time in cancer cells the upregulation of soluble and/or membrane‐bound TRAIL. This was associated with increased cell surface death receptor 5 expression and c‐FLIP and survivin downregulation, mediated in part through proteasome‐dependent degradation in troglitazone‐promoted cell death. Therefore, the combination of rosiglitazone and TRAIL could be clinically relevant as chemopreventive or therapeutic agents for the treatment of TRAIL‐resistant high‐grade urothelial cancers.     

Lentiviral‐mediated miRNA against liver‐intestine cadherin suppresses tumor growth and invasiveness of human gastric cancer

Liver‐intestine cadherin (CDH17) represents a novel type of cadherin within the cadherin superfamily, and is distinguished from other cadherins by its distinct structural and functional features. Our previous studies had identified that increased CDH17 was significantly associated with tumor differentiation and lymph node metastasis in gastric cancer. In this study, we tested the hypothesis that CDH17 was associated with proliferation and invasiveness in gastric cancer using recombinant lentivirus‐mediated miRNA targeting to CDH17 both in vitro and in vivo. We also detected the activity of matrix metalloproteinase (MMP)‐2 and MMP‐9 with gelatin zymography to explore the mechanisms underlying the inhibition of the CDH17 gene. Our results showed that a well‐differentiated gastric cancer cell line had higher CDH17 expression. Down‐regulation of CDH17 inhibited proliferation, adherence, and invasion of the poorly differentiated BGC823 gastric cancer cells in vitro, and induced cell cycle arrest. The activities of MMP‐2 and MMP‐9 were lower in the CDH17‐miRNA‐transfected cells compared to the control cells. Using an in vivo tumor growth assay, we confirmed that CDH17 silencing could obviously slow the growth of gastric cancer derived from BGC823 cells. Taken together, we have demonstrated that CDH17 maybe a positive regulator for proliferative, adhesive, and invasive behaviors of gastric cancer. (Cancer Sci 2010)     

Amino‐terminal fragments of laminin γ2 chain retract vascular endothelial cells and increase vascular permeability

Laminin γ2 (Lmγ2) chain, a subunit of laminin‐332, is a typical molecular marker of invading cancer cells, and its expression correlates with poor prognosis of cancer patients. It was previously found that forced expression of Lmγ2 in cancer cells promotes their invasive growth in nude mice. However, the mechanism of the tumor‐promoting activity of Lmγ2 remains unknown. Here we investigated the interaction between Lmγ2 and vascular endothelial cells. When treated with an N‐terminal proteolytic fragment of γ2 (γ2pf), HUVECs became markedly retracted or shrunken. The overexpression of Lmγ2 or treatment with γ2pf stimulated T‐24 bladder carcinoma cells to invade into the HUVEC monolayer and enhanced their transendothelial migration in vitro. Moreover, γ2pf increased endothelial permeability in vitro and in vivo. As the possible mechanisms, γ2pf activated ERK and p38 MAPK but inactivated Akt in HUVECs. Such effects of γ2pf led to prominent actin stress fiber formation in HUVECs, which was blocked by a ROCK inhibitor. In addition, γ2pf induced delocalization of VE‐cadherin and β‐catenin from the intercellular junction. As possible receptors, γ2pf interacted with heparan sulfate proteoglycans on the surface of HUVECs. Moreover, we localized the active site of γ2pf to the N‐terminal epidermal growth factor‐like repeat. These data suggest that the interaction between γ2pf and heparan sulfate proteoglycans induces cytoskeletal changes of endothelial cells, leading to the loss of endothelial barrier function and the enhanced transendothelial migration of cancer cells. These activities of Lmγ2 seem to support the aberrant growth of cancer cells.     

Alpha‐eleostearic acid suppresses proliferation of MCF‐7 breast cancer cells via activation of PPARγ and inhibition of ERK 1 / 2

Alpha‐eleostearic acid (α‐ESA) is known to suppress the growth in cancer cells although its underlying molecular mechanisms have not been fully elucidated. The present study was designed to elucidate and evaluate the anticancer mechanism of α‐ESA on MCF‐7 breast cancer cells. Also, an attempt was made to better understand the anticancer mechanism by which α‐ESA activated PPARγ and attenuated the ERK1/2 MAPK phosphorylation state. The MCF‐7 breast cancer cell‐line and nontumorigenic MCF‐10A human mammary epithelial cells were treated with α‐ESA and compared with negative control (without treatment) and positive control groups (treated with rosiglitazone), and changes of apoptosis‐related molecules, PPARγ and pERK1/2 were examined. In MCF‐7 cells treated with α‐ESA, we found that the expression of p53, p21, and Bax was up‐regulated whereas expression of Bcl‐2 and procaspase‐9 was down‐regulated. Moreover, nuclear translocation of PPARγ by α‐ESA positively correlated with inhibition of ERK1/2 activation. Our data suggest that α‐ESA can be considered to be a PPARγ agonist and thus a candidate for a chemotherapeutic agent against breast cancer. (Cancer Sci 2009; 00: 000–000)     

Extracellular 5′‐nucleotidase (CD73) promotes human breast cancer cells growth through AKT/GSK‐3β/β‐catenin/cyclinD1 signaling pathway

To identify the role and to explore the mechanism of extracellular 5′‐nucleotidase (CD73) in human breast cancer growth, CD73 expression was measured firstly in breast cancer tissues and cell lines, and then interfered with or over‐expressed by recombinant lentivirus in cell lines. Impacts of CD73 on breast cancer cell proliferation and cell cycle were investigated with colony formation assay, CCK‐8 and flow cytometry. The relationship between CD73 and AKT/GSK‐3β/β‐catenin pathway was assessed with adenosine, adenosine 2A receptor antagonist (SCH‐58261), adenosine 2A receptor agonist (NECA), CD73 enzyme inhibitor (APCP) and Akt inhibitor (MK‐2206). Moreover, the effect of CD73 on breast cancer growth in vivo was examined with human breast cancer transplanting model of nude mice. The results showed that the expression of CD73 was high in breast cancer tissues and increased with advanced tumor grades and lympho‐node status. CD73 expression was higher in more malignant cells, and CD73 overexpression promoted breast cancer cell proliferation in both in vivo and in vitro. It activated AKT/GSK‐3β/β‐catenin/cyclinD1 signaling pathway through CD73 enzyme activity and other mechanism.     

Peroxisome proliferator-activated receptor γ-dependent activity of indole ring-substituted 1,1-bis(3′-indolyl)-1-(p-biphenyl)methanes in cancer cells

Purpose

1,1-Bis(3-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) substituted in the phenyl ring with a para-, t-butyl, trifluoromethyl (DIM-C-pPhCF₃) or phenyl (DIM-C-pPhC₆H₅) group activate peroxisome proliferator-activated receptor γ (PPARγ) in several cancer cell lines, and DIM-C-pPhCF₃ also activates the orphan receptor Nur77. In this study, we have examined the effects of 5,5′-dihydroxy, 5,5′-dimethyl, 5,5′-dibromo, 5,5′-dinitro and 5,5′-dimethoxyindole ring-substituted analogs of DIM-C-pPhC₆H₅ on their activity as PPARγ agonists.

Methods

Various substituted C-DIM analogs were used to investigate their growth-inhibitory activities and activation of PPARγ-mediated transactivation in colon and pancreatic cancer cells. Their structure-dependent induction of putative PPARγ-responsive genes/proteins including p21, KLF-4 and caveolin1 were also determined by Western and Northern blot analysis.

Results

Introduction of the 5,5′-dihydroxy and 5,5′-dimethyl substituents enhanced activation of PPARγ in colon and pancreatic cancer cells. However, activation of p21 in Panc28 pancreatic cancer cells and induction of caveolin-1 and KLF4 in colon cancer cells by the C-DIM compounds were structure- and cell context-dependent.

Conclusions

The results demonstrate that DIM-C-pPhC₆H₅ and indole ring-substituted analogs are selective PPARγ modulators.     

过氧化物酶体增殖物激活受体γ在大肠肿瘤中的表达及意义

目的 在蛋白及mRNA水平确定过氧化物酶体增殖物激活受体γ（peroxiome proliferator activated receptor γ, PPARγ）在大肠肿瘤中的表达。方法 结肠镜下活检标本,分为正常大肠粘膜组、大肠腺瘤组及大肠癌组,免疫组化及RT PCR方法检测各组PPARγ的表达。结果 正常大肠粘膜中表达PPARγ蛋白的细胞主要是接近肠腔,分化良好的腺上皮细胞;而在大肠腺瘤中,PPARγ阳性的细胞呈弥漫分布。PPARγ蛋白及mRNA在大肠癌中的表达显著增加,明显高于大肠腺瘤和正常大肠粘膜(P<0.05)。PPARγ蛋白及mRNA在正常大肠粘膜及腺瘤中的表达无差异（P>0.05）。PPARγ mRNA在重度异型增生腺瘤中的表达高于正常大肠粘膜(P<0.05),接近于在大肠癌中的表达（P>0.05）。结论 大肠腺瘤最先发生PPARγ表达部位的变化,PPARγ在重度异型增生的大肠腺瘤及大肠癌中的表达水平较高,表明PPARγ可能在早期即参与大肠癌的发生发展。     

Involvement of Akt/NF‐κB pathway in N6‐isopentenyladenosine‐induced apoptosis in human breast cancer cells

N⁶‐isopentenyladenosine (i6A) inhibits the tumor cell growth by inducing cell apoptosis in various cancer cell lines. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. In this study, we further explored the molecular mechanisms of i6A as an anticancer agent on a human breast cancer cell line MDA MB 231. Treatment with i6A decreased the cell proliferation of MDA MB 231 cells in a dose‐dependent manner by arresting the cells at G₀/G₁ phase. This effect was strongly associated with concomitant decrease in the level of cyclin D1, cyclin E, cdk2, and increase of p21waf1 and p27kip. In addition i6A also induced apoptotic cell death by increasing the expression of Bax, and decreasing the levels of Bcl‐2 and Bcl‐xL, and subsequently triggered mitochondria apoptotic pathway (release of cytochrome c and activation of caspase‐3). We observed that i6A suppressed the nuclear factor kappaB (NF‐κB) pathway and inhibited the Akt activation. The results of this study indicate that i6A decreases cell proliferation and induces apoptotic cell death in human breast cancer cells, possibly by decreasing signal transduction through the Akt/NF‐κB cell survival pathway. © 2010 Wiley‐Liss, Inc.     

Loss of XIAP sensitizes colon cancer cells to PPARγ independent antitumor effects of troglitazone and 15-PGJ2

We investigated whether the anticancer effect of a combination of XIAP down-regulation and PPAR γ activation on colon cancer is PPARγ receptor dependent. HCT116-XIAP^+/+ cells and HCT116-XIAP^−/− cells were treated with troglitazone or 15-deoxy-Δ^12,14-prostaglandin J2 (15-PGJ2) with or without prior exposure to PPARγ inhibitor GW9662. Cell proliferation and apoptosis was evaluated. Athymic mice carrying HCT116-XIAP^−/− cells-derived tumors were treated with troglitazone in the presence or absence of GW9662. Inhibition of cell proliferation and induction of apoptosis by troglitazone and 15-PGJ2 were more prominent in HCT116-XIAP^−/− cells. PPARγ ligand-induced growth inhibition, apoptosis, caspase and PARP cleavage could not be blocked by GW9662. Troglitazone significantly retarded growth of xenograft tumors and this effect was not blocked by GW9662. Marked apoptosis and an up-regulation of E-cadherin were observed in xenograft tumor tissues, and GW9662 did not affect these effects. Thus, a combination of XIAP down-regulation and PPARγ ligands exert a significant anticancer effect in colon cancer via a PPARγ independent pathway.     

Bidirectional regulation between TMEFF2 and STAT3 may contribute to Helicobacter pylori‐associated gastric carcinogenesis

The transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is a single‐pass transmembrane protein, and it is downregulated in human gastric cancer and levels correlate with tumor progression and time of survival. However, the mechanism of its dysregulation in gastric cancer is little known. Here we investigate its regulatory mechanism and the bidirectional regulation between TMEFF2 and STAT3 in gastric carcinogenesis. TMEFF2 expression was decreased after Helicobacter pylori (H. pylori) infection in vivo and in vitro. STAT3 directly binds to the promoter of TMEFF2 and regulates H. pylori‐induced TMEFF2 downregulation in normal gastric GES‐1 cells and gastric cancer AGS cells. Conversely, TMEFF2 may suppress the phosphorylation of STAT3 and TMEFF2‐induced downregulation of STAT3 phosphorylation may depend on SHP‐1. A highly inverse correlation between the expression of TMEFF2 and pSTAT3 was also revealed in gastric tissues. We now show the deregulation mechanism of TMEFF2 in gastric carcinogenesis and identify TMEFF2 as a new target gene of STAT3. The phosphorylation of STAT3 may be negatively regulated by TMEFF2, and the bidirectional regulation between TMEFF2 and STAT3 may contribute to H. pylori‐associated gastric carcinogenesis.© 2014 UICC