Akt Mediates Metastasis-Associated Gene 1 (MTA1) Regulating the Expression of E-cadherin and Promoting the Invasiveness of Prostate Cancer Cells

Human metastasis-associated gene 1 (MTA1) is highly associated with the metastasis of prostate cancer; however, the molecular functions of MTA1 that facilitate metastasis remain unclear. In this study, we demonstrate that the silencing of MTA1 by siRNA treatment results in the upregulation of E-cadherin expression by the phosphorylation of AKT (p-AKT) and decreases the invasiveness of prostate cancer cells. We show that MTA1 is expressed in over 90% of prostate cancer tissues, especially metastatic prostate cancer tissue, comparing to non-expression in normal prostate tissue. RT-PCR analysis and Western blot assay showed that MTA1 expression is significantly higher in highly metastatic prostate cancer PC-3M-1E8 cells (1E8) than in poorly metastatic prostate cancer PC-3M-2B4 cells (2B4). Silencing MTA1 expression by siRNA treatment in 1E8 cells increased the cellular malignant characters, including the cellular adhesive ability, decreased the cellular invasive ability and changed the polarity of cellular cytoskeleton. 1E8 cells over-expressing MTA1 had a reduced expression of E-cadherin, while 1E8 cells treated with MTA1 siRNA had a higher expression of E-cadherin. The expression of phosphorylated AKT (p-AKT) or the inhibition of p-AKT by wortmannin treatment (100 nM) significantly altered the function of MTA1 in the regulation of E-cadherin expression. Alterations in E-cadherin expression changed the role of p-AKT in cellular malignant characters. All of these results demonstrate that MTA1 plays an important role in controlling the malignant transformation of prostate cancer cells through the p-AKT/E-cadherin pathway. This study also provides a new mechanistic role for MTA1 in the regulation of prostate cancer metastasis.     

Involvement of HDAC1 in E-cadherin expression in prostate cancer cells; its implication for cell motility and invasion

In this study, we investigate the molecular mechanism by which histone deacetylase (HDAC) inhibitors exert anti-invasiveness effect against prostate cancer cells. We first evaluate the growth inhibition effect of HDAC inhibitors in prostate cancer cells, which is accompanied by induction of p21^WAF1 expression and accumulation of acetylated histones. And we found that the migration and invasion of prostate cancer cells is strongly inhibited by treatment with HDAC inhibitors. In parallel, E-cadherin level is highly up-regulated in HDAC inhibitor-treated prostate cancer cells. And siRNA knockdown of E-cadherin significantly diminishes the anti-invasion effect of HDAC inhibitors, indicating that E-cadherin overexpression is one of possible mechanism for anti-invasion effect of HDAC inhibitors. Furthermore, specific downregulation of HDAC1, but not HDAC2, causes E-cadherin expression and subsequent inhibition of cell motility and invasion. Collectively, our data demonstrate that HDAC1 is a major repressive enzyme for E-cadherin expression as well as HDAC inhibitor-mediated anti-invasiveness.     

The role of calpain in the proteolytic cleavage of E-cadherin in prostate and mammary epithelial cells

The E-cadherin protein mediates Ca(2+)-dependent interepithelial adhesion. Association of E-cadherin with the catenin family of proteins is critical for the maintenance of a functional adhesive complex. We have identified a novel truncated E-cadherin species of 100-kDa (E-cad(100)) in prostate and mammary epithelial cells. E-cad(100) was generated by treatment of cells with ionomycin or TPA. Cell-permeable calpain inhibitors prevented E-cad(100) induction by ionomycin. Immunoblotting for spectrin and mu-calpain confirmed calpain activation in response to ionomycin treatment. Both the mu- and m-isoforms of calpain efficiently generated E-cad(100) in vitro. The E-cad(100) fragment was unable to bind to beta-catenin, gamma-catenin, and p120, suggesting that this cleavage event would disrupt the E-cadherin adhesion complex. Mutational analysis localized the calpain cleavage site to the cytosolic domain upstream of the beta- and gamma-catenin binding motifs of E-cadherin. Because E-cadherin is inactivated in many adenocarcinomas we hypothesized that calpain may play a role in prostate tumorigenesis. A prostate cDNA microarray data base was analyzed for calpain expression in which it was found that m-calpain was up-regulated in localized prostate cancer, and to an even higher degree in metastatic prostate cancer compared with normal prostate tissue. Furthermore, we examined the cleavage of E-cadherin in prostate cancer specimens and found that E-cad(100) accumulated in both localized and metastatic prostate tumors, supporting the cDNA microarray data. These findings demonstrate a novel mechanism by which E-cadherin is functionally inactivated through calpain-mediated proteolysis and suggests that E-cadherin is targeted by calpain during the tumorigenic progression of prostate cancer.     

Does the inhibition of c-myc expression mediate the anti-tumor activity of PPAR's ligands in prostate cancer cell lines?

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands seem to induce anticancer effects on prostate cancer cells, but the mechanism is not clear. The effect of PPARgamma ligands omega-6 fatty acids and ciglitazone (2-15 microM)--on proliferation, and apoptosis of LNCaP, PC-3, DU145, CA-K and BPH-K cells was studied. PPARgamma ligands led to: (1) reduction of proliferation (20-50%) of all the studied cell lines, (2) stimulation of differentiation of prostate cancer cells through an increased expression (1.5-3-fold: LNCaP, DU145, BPH-K) or reexpression (PC-3, CA-K) of E-cadherin with parallel inhibition of N-cadherin expression (PC-3, CA-K) and (3) down-regulation (1-2-fold) of beta-catenin and c-myc expression. The selective PPARgamma antagonist GW9662 abolished the effect of those ligands on prostate cancer cells. These results suggest that inhibition of beta-catenin and in effect c-myc expression through activation of PPARgamma may help prostate cancer cells to restore several characteristics of normal prostate cells phenotype.     

Antiproliferative effect of conjugated linoleic acid in caco-2 cells: involvement of PPARgamma and APC/beta-catenin pathways

Conjugated linoleic acid (CLA), a naturally occurring substance in food sources, occurs as mixtures of positional and geometrical isomers of octadecadienoate (18:2), and may inhibit colon tumorigenesis. It has been hypothesized that CLA can modulate cell proliferation and differentiation through the activation of peroxisome proliferator-activated receptors (PPARs), among which PPARgamma is involved in growth inhibition of transformed cells. The aim of the present study was to investigate whether the antiproliferative effects of CLA are mediated by its interaction with PPARgamma and APC/beta-catenin signalling pathway in human colon cancer cells. In CLA-treated caco-2 cells we found a remarkable increase in the expression of PPARgamma, which translocated into the nucleus, while PPARalpha and beta/delta protein levels were not affected. GW259662, a well known PPARgamma antagonist, blocked the increase in PPARgamma protein rate and abrogated some biological effects of CLA, as it restored the proliferative capability of the cells and ERK1/2 phosphorylation level. We demonstrated that CLA treatment determined the down-regulation of APC and c-myc proteins, but in this case the administration of the antagonist was not able to revert CLA effects. Furthermore, CLA induced a reorganization of E-cadherin and beta-catenin, as well as a redistribution of actin and tubulin filaments. Our data suggest that CLA may regulate PPARgamma expression by selectively acting as an agonist; however, the discrepancies in PPARgamma antagonist efficacy suggest the involvement of other pathways, independent of PPARgamma, in CLA antiproliferative activity.     

Upregulation of SATB1 Is Associated with Prostate Cancer Aggressiveness and Disease Progression

Disease aggressiveness remains a critical factor to the progression of prostate cancer. Transformation of epithelial cells to mesenchymal lineage, associated with the loss of E-cadherin, offers significant invasive potential and migration capability. Recently, Special AT-rich binding protein (SATB1) has been linked to tumor progression. SATB1 is a cell-type restricted nuclear protein, which functions as a tissue-specific organizer of DNA sequences during cellular differentiation. Our results demonstrate that SATB1 plays significant role in prostate tumor invasion and migration and its nuclear localization correlates with disease aggressiveness. Clinical specimen analysis showed that SATB1 was predominantly expressed in the nucleus of high-grade tumors compared to low-grade tumor and benign tissue. A progressive increase in the nuclear levels of SATB1 was observed in cancer tissues compared to benign specimens. Similarly, SATB1 protein levels were higher in a number of prostate cancer cells viz. HPV-CA-10, DU145, DUPro, PC-3, PC-3M, LNCaP and C4-2B, compared to non-tumorigenic PZ-HPV-7 cells. Nuclear expression of SATB1 was higher in biologically aggressive subclones of prostate cancer cells with their respective parental cell lines. Furthermore, ectopic SATB1 transfection conferred increased cell motility and invasiveness in immortalized human prostate epithelial PZ-HPV-7 cells which correlated with the loss of E-cadherin expression. Consequently, knockdown of SATB1 in highly aggressive human prostate cancer PC-3M cells inhibited invasiveness and tumor growth in vivo along with increase in E-cadherin protein expression. Our findings demonstrate that SATB1 has ability to promote prostate cancer aggressiveness through epithelial-mesenchymal transition.     

Combination therapy with PPARgamma and PPARalpha agonists increases glucose-stimulated insulin secretion in db/db mice

Although peroxisome proliferator-activated receptor (PPAR)gamma agonists ameliorate insulin resistance, they sometimes cause body weight gain, and the effect of PPAR agonists on insulin secretion is unclear. We evaluated the effects of combination therapy with a PPARgamma agonist, pioglitazone, and a PPARalpha agonist, bezafibrate, and a dual agonist, KRP-297, for 4 wk in male C57BL/6J mice and db/db mice, and we investigated glucose-stimulated insulin secretion (GSIS) by in situ pancreatic perfusion. Body weight gain in db/db mice was less with KRP-297 treatment than with pioglitazone or pioglitazone + bezafibrate treatment. Plasma glucose, insulin, triglyceride, and nonesterified fatty acid levels were elevated in untreated db/db mice compared with untreated C57BL/6J mice, and these parameters were significantly ameliorated in the PPARgamma agonist-treated groups. Also, PPARgamma agonists ameliorated the diminished GSIS and insulin content, and they preserved insulin and GLUT2 staining in db/db mice. GSIS was further increased by PPARgamma and -alpha agonists. We conclude that combination therapy with PPARgamma and PPARalpha agonists may be more useful with respect to body weight and pancreatic GSIS in type 2 diabetes with obesity.     

SIK1作为miR-93新的靶基因抑制前列腺癌细胞增殖、侵袭和迁移

该文探讨了SIK1作为miR-93新的靶基因对前列腺癌细胞增殖、侵袭和迁移的抑制作用.采用重组质粒pcDNA3.1-SIK1上调前列腺癌细胞中SIK1的表达后,利用CCK8和克隆形成实验检测细胞增殖;利用细胞划痕和Transwell实验检测细胞侵袭和迁移;利用West-ern blot检测E-cadherin和Vime...     

Activation of PPARgamma reverses a defect of surfactant synthesis in mice lacking two types of fatty acid binding protein

Lung surfactant is a lipid-protein-film covering the inner alveolar surface. We have previously shown that double knock-out (d-ko) mice lacking both the epidermal-type (E-) and the heart-type (H-) fatty acid binding protein (FABP) exhibit a defect of surfactant synthesis in alveolar type II cells that can be corrected by feeding pioglitazone, a drug that activates peroxisome proliferator-activated receptor gamma (PPARgamma). Here, we demonstrate first that healthy surfactant at collapse pressure produces protrusions composed of bilayers but not folds, second that the d-ko effect profoundly perturbs lipid/hydrophobic protein composition, pressure-area isotherm, and structural organisation of the surfactant at nanoscale, parameters that are critical for the normal breathing cycle. In support of these data in vivo measurements of lung function reveal that maximum compliance in d-ko vs. wild-type mice is significantly reduced. Further, we show that the biophysical phenotype can be corrected substantially with pioglitazone. Finally, we show that d-ko alveolar cells up-regulate liver-type (L-) FABP, a member of the FABP family that we have previously shown to interact with PPARgamma. Taken together, these data suggest that PPARgamma agonists could be a tool to repair surfactant damage caused by dysfunctional alveolar lipid metabolism, and provide in vivo support for L-FABP aided signaling.     

Peroxisome proliferator-activated receptor gamma agonists promote TRAIL-induced apoptosis by reducing survivin levels via cyclin D3 repression and cell cycle arrest

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy that preferentially induces apoptosis in cancer cells. However, many neoplasms are resistant to TRAIL by mechanisms that are poorly understood. Here we demonstrate that human breast cancer cells, but not normal mammary epithelial cells, are dramatically sensitized to TRAIL-induced apoptosis and caspase activation by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists of the thiazolidinedione (TZD) class. Although TZDs do not significantly alter the expression of components of the TRAIL signaling pathway, they profoundly reduce protein levels of cyclin D3, but not other D-type cyclins, by decreasing cyclin D3 mRNA levels and by inducing its proteasomal degradation. Importantly, both TRAIL sensitization and reduction in cyclin D3 protein levels induced by TZDs are likely PPARgamma-independent because a dominant negative mutant of PPARgamma did not antagonize these effects of TZDs, nor were they affected by the expression levels of PPARgamma. TZDs also inhibit G(1) to S cell cycle progression. Furthermore, silencing cyclin D3 by RNA interference inhibits S phase entry and sensitizes breast cancer cells to TRAIL, indicating a key role for cyclin D3 repression in these events. G(1) cell cycle arrest sensitizes breast cancer cells to TRAIL at least in part by reducing levels of the anti-apoptotic protein survivin: ectopic expression of survivin partially suppresses apoptosis induced by TRAIL and TZDs. We also demonstrate for the first time that TZDs promote TRAIL-induced apoptosis of breast cancer in vivo, suggesting that this combination may be an effective therapy for cancer.     

Pioglitazone induces apoptosis of macrophages in human adipose tissue

Metabolic syndrome and type 2 diabetes mellitus are associated with an increased number of macrophage cells that infiltrate white adipose tissue (WAT). Previously, we demonstrated that the treatment of subjects with impaired glucose tolerance (IGT) with the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist pioglitazone resulted in a decrease in macrophage number in adipose tissue. Here, adipose tissue samples from IGT subjects treated with pioglitazone were examined for apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. TUNEL-positive cells were identified, and there was a significant 42% increase in TUNEL-positive cells following pioglitazone treatment. Overlay experiments with anti-CD68 antibody demonstrated that most of the TUNEL-positive cells were macrophages. To determine whether macrophage apoptosis was a direct or indirect effect of pioglitazone treatment, human THP1 cells were treated with pioglitazone in vitro, demonstrating increased TUNEL staining in a dose- and time-dependent manner. Furthermore, the appearance of the active proteolytic subunits of caspase-3 and caspase-9 were detected in cell lysate from THP1 cells and also increased in a dose- and time-dependent manner following pioglitazone treatment. Pretreatment with a PPARgamma inhibitor, GW9662, prevented pioglitazone induction of the apoptotic pathway in THP1 cells. Differentiated human adipocytes did not show any significant increase in apoptosis after treatment in vitro with piolgitazone. These findings indicate that PPARgamma has distinct functions in different cell types in WAT, such that pioglitazone reduces macrophage infiltration by inducing apoptotic cell death specifically in macrophages through PPARgamma activation.     

Rosiglitazone promotes tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by reactive oxygen species-mediated up-regulation of death receptor 5 and down-regulation of c-FLIP

Death receptor 5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we show that rosiglitazone sensitizes human renal cancer cells to TRAIL-mediated apoptosis, but not normal human mesangial cells. Furthermore, because rosiglitazone-enhanced TRAIL-mediated apoptosis is induced in various types of cancer cells but is not interrupted by Bcl-2 overexpression, this combinatory treatment may provide an attractive strategy for cancer treatment. We found that treatment with rosiglitazone significantly induces DR5 expression at both its mRNA and its protein levels, accompanying the generation of reactive oxygen species (ROS). Both treatment with DR5/Fc chimeric protein and silencing of DR5 expression using small interfering RNAs attenuated rosiglitazone plus TRAIL-induced apoptosis, showing the critical role of DR5 in this cell death. Pretreatment with GSH significantly inhibited rosiglitazone-induced DR5 up-regulation and the cell death induced by the combined treatment with rosiglitazone and TRAIL, suggesting that ROS mediate rosiglitazone-induced DR5 up-regulation, contributing to TRAIL-mediated apoptosis. However, both DR5 up-regulation and sensitization of TRAIL-mediated apoptosis induced by rosiglitazone are likely PPARgamma-independent, because a dominant-negative mutant of PPARgamma and a potent PPARgamma inhibitor, GW9662, failed to block DR5 induction and apoptosis. Interestingly, we also found that rosiglitazone treatment induced down-regulation of cellular FLICE-inhibitory protein (c-FLIPs), and ectopic expression of c-FLIPs attenuated rosiglitazone plus TRAIL-mediated apoptosis, demonstrating the involvement of c-FLIPs in this apoptosis. Taken together, the results of this study demonstrate that rosiglitazone enhances TRAIL-induced apoptosis in various cancer cells by ROS-mediated DR5 up-regulation and down-regulation of c-FLIPs.     

Protection by pioglitazone in the MPTP model of Parkinson's disease correlates with I kappa B alpha induction and block of NF kappa B and iNOS activation

Inflammation has been implicated in the pathogenesis of Parkinson's disease (PD). In the chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD, inducible NO synthase (iNOS) derived nitric oxide (NO) is an important mediator of dopaminergic cell death. Ligands of the peroxisome proliferator-activated receptor (PPAR) exert anti-inflammatory effects. We here investigated whether pioglitazone, a PPARgamma agonist, protected mice from MPTP-induced dopaminergic cell loss, glial activation, and loss of catecholamines in the striatum. As shown by western blot, PPARgamma was expressed in the striatum and the substantia nigra of vehicle- and MPTP-treated mice. Oral administration of 20 mg/(kg day) of pioglitazone protected tyrosine hydroxylase (TH)-positive substantia nigra neurons from death induced by 5 x 30 mg/kg MPTP. However, the decrease of dopamine in the striatum was only partially prevented. In mice treated with pioglitazone, there were a reduced activation of microglia, reduced induction of iNOS-positive cells and less glial fibrillary acidic protein positive cells in both striatum and substantia nigra pars compacta. In addition, treatment with pioglitazone almost completely blocked staining of TH-positive neurons for nitrotyrosine, a marker of NO-mediated cell damage. Because an increase in inhibitory protein-kappa-Balpha (IkappaBalpha) expression and inhibition of translocation of the nuclear factor kappaB (NFkappaB) subunit p65 to the nucleus in dopaminergic neurons, glial cells and astrocytes correlated with the protective effects of pioglitazone, our results suggest that pioglitazone sequentially acts through PPARgamma activation, IkappaBalpha induction, block of NFkappaB activation, iNOS induction and NO-mediated toxicity. In conclusion, treatment with pioglitazone may offer a treatment opportunity in PD to slow the progression of disease that is mediated by inflammation.     

Pioglitazone induces plasma platelet activating factor-acetylhydrolase and inhibits platelet activating factor-mediated cytoskeletal reorganization in macrophage

Platelet activating factor (PAF) is a key molecule for inflammation. To examine a role of peroxisome proliferator-activated receptor gamma (PPARgamma) in inflammatory reactions of atherosclerosis, we investigated the effects of 15-deoxy-(Delta12,14)-Prostaglandin J2 (15d-PGJ2) and pioglitazone, PPARgamma ligands, on plasma PAF-acetylhydrolase (PAF-AH) expression in THP-1 macrophages. PAF-AH mRNA and protein were up-regulated by the PPARgamma ligands. Prostaglandin F2alpha (PGF2alpha), a PARgamma inhibitor, abrogated the up-regulation of PAF-AH mRNA by pioglitazone, suggesting that PPARgamma activation is involved in the induction of PAF-AH by pioglitazone. As PAF promotes the cell motility with cytoskeletal reorganization, we investigated the effect of pioglitazone on PAF-mediated morphological changes in THP-1 macrophages. In the absence of pioglitazone, PAF promoted the elongation of actin cytoskeleton, which was inhibited by pretreatment with pioglitazone. In contrast, pioglitazone was not able to inhibit the morphological changes induced by C-PAF, a non-hydrolyzable PAF agonist. Thus, it is suggested that PAF-induced morphological changes could be inhibited by pioglitazone through PAF-AH, which rapidly hydrolyzed PAF. These data propose that PPARgamma/PAF-AH pathway is a clinical target for the prevention against atherosclerosis.     

Analysis of the genomic response of human prostate cancer cells to histone deacetylase inhibitors

Histone deacetylases (HDACs) have emerged as important targets for cancer treatment. HDAC-inhibitors (HDACis) are well tolerated in patients and have been approved for the treatment of patients with cutaneous T-cell lymphoma (CTCL). To improve the clinical benefit of HDACis in solid tumors, combination strategies with HDACis could be employed. In this study, we applied Analysis of Functional Annotation (AFA) to provide a comprehensive list of genes and pathways affected upon HDACi-treatment in prostate cancer cells. This approach provides an unbiased and objective approach to high throughput data mining. By performing AFA on gene expression data from prostate cancer cell lines DU-145 (an HDACi-sensitive cell line) and PC3 (a relatively HDACi-resistant cell line) treated with HDACis valproic acid or vorinostat, we identified biological processes that are affected by HDACis and are therefore potential treatment targets for combination therapy. Our analysis revealed that HDAC-inhibition resulted among others in upregulation of major histocompatibility complex (MHC) genes and deregulation of the mitotic spindle checkpoint by downregulation of genes involved in mitosis. These findings were confirmed by AFA on publicly available data sets from HDACi-treated prostate cancer cells. In total, we analyzed 375 microarrays with HDACi treated and non-treated (control) prostate cancer cells. All results from this extensive analysis are provided as an online research source (available at the journal’s website and at http://luigimarchionni.org/HDACIs.html). By publishing this data, we aim to enhance our understanding of the cellular changes after HDAC-inhibition, and to identify novel potential combination strategies with HDACis for the treatment of prostate cancer patients.