Peroxisome proliferator-activated receptor gamma ligand-induced growth inhibition of human hepatocellular carcinoma

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been implicated in the growth inhibition and differentiation of certain human cancers with diverse tissue origin. In this study, expression of PPARgamma in human hepatocellular carcinoma (HCC) and the effect of PPARgamma ligands on HCC cells were investigated in vitro using Hep G2, HuH-7, KYN-1 and KYN-2 cell lines. All cell lines were found to express functionally active PPARgamma and a marked growth inhibition was induced by thiazolidinedione ligands troglitazone, and pioglitazone as well as with its natural ligand 15-deoxy-Delta(12,14)-prostaglandin J(2). The growth inhibitory effect was associated with a dose-dependent inhibition of DNA synthesis, cell cycle progression and alpha fetoprotein expression.     

The high affinity peroxisome proliferator-activated receptor-gamma agonist RS5444 inhibits both initiation and progression of colon tumors in azoxymethane-treated mice

We evaluated RS5444, a thiazolidinedione high affinity PPARgamma agonist, for the ability to inhibit colon carcinogenesis in azoxymethane (AOM)-treated mice. In our initial experiment, mice were treated with RS5444 during AOM treatment, and the drug was withdrawn 12 weeks after the last injection of AOM. RS5444 significantly inhibited aberrant crypt focus formation under these circumstances. Furthermore, exposure to RS5444 during the course of AOM treatment effectively blocked colon tumor formation after withdrawal of the agonist. PPARgamma expression and nuclear localization were reduced in adenomas. RS5444 did not inhibit DNA synthesis in tumor cells, suggesting that PPARgamma activity was impaired in adenomas. To test this hypothesis, pre-existing adenomas were treated with RS5444 for 16 weeks. We observed a slight, albeit not statistically significant, reduction in tumor incidence in RS5444-treated mice. However, histological examination revealed that tumors from RS5444-treated mice were of significantly lower grade, as evaluated by the extent of dysplasia. Furthermore, carcinoma in situ was observed in about one-third of control tumors, but was never observed in RS5444-treated tumors. We conclude that RS5444 inhibits both initiation and progression of colon tumors in the AOM model of sporadic colon carcinogenesis.     

Hepatitis C virus core protein induces spontaneous and persistent activation of peroxisome proliferator-activated receptor alpha in transgenic mice: implications for HCV-associated hepatocarcinogenesis

Persistent infection of hepatitis C virus (HCV) can lead to a high risk for hepatocellular carcinoma (HCC). HCV core protein plays important roles in HCV-related hepatocarcinogenesis, because mice carrying the core protein exhibit multicentric HCCs without hepatic inflammation and fibrosis. However, the precise mechanism of hepatocarcinogenesis in these transgenic mice remains unclear. To evaluate whether the core protein modulates hepatocyte proliferation and apoptosis in vivo, we examined these parameters in 9- and 22-month-old transgenic mice. Although the numbers of apoptotic hepatocytes and hepatic caspase 3 activities were similar between transgenic and nontransgenic mice, the numbers of proliferating hepatocytes and the levels of numerous proteins such as cyclin D1, cyclin-dependent kinase 4 and c-Myc, were markedly increased in an age-dependent manner in the transgenic mice. This increase was correlated with the activation of peroxisome proliferator-activated receptor alpha (PPARalpha). In these transgenic mice, spontaneous and persistent PPARalpha activation occurred heterogeneously, which was different from that observed in mice treated with clofibrate, a potent peroxisome proliferator. We further demonstrated that stabilization of PPARalpha through a possible interaction with HCV core protein and an increase in nonesterified fatty acids, which may serve as endogenous PPARalpha ligands, in hepatocyte nuclei contributed to the core protein-specific PPARalpha activation. In conclusion, these results offer the first suggestion that HCV core protein induces spontaneous, persistent, age-dependent and heterogeneous activation of PPARalpha in transgenic mice, which may contribute to the age-dependent and multicentric hepatocarcinogenesis mediated by the core protein.     

Ligands for peroxisome proliferator-activated receptor gamma inhibit growth of pancreatic cancers both in vitro and in vivo.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed largely in adipose tissues and plays an important role in adipocyte differentiation. Several studies have recently shown that ligands of PPARgamma could lead to growth inhibition in some malignancies. In our study, we focused on pancreatic cancers, because the prognosis of advanced pancreatic cancer has not significantly improved due to its resistance to various chemotherapeutic regimens, so that a novel strategy should be required. We show here that PPARgamma is expressed in 5 pancreatic cancer cell lines detected in both mRNA and protein level as well as in human primary and metastatic pancreatic carcinomas examined by immunohistochemical studies. A specific ligand of PPARgamma, troglitazone, led to G1 accumulation with the increase in p27(Kip1), but not p21(Waf1/Cip1) and inhibited cellular proliferation in a pancreatic cancer cell line, Panc-1. The overexpression of PPARgamma in a pancreatic cancer cell line, KMP-3, caused lipid accumulation, which suggested cell growth in some cancers might be inhibited, at least in part, through terminal differentiation in the adipogenic lineage. In addition, implanted Panc-1 tumors in nude mice showed significant inhibition of tumor growth, when treated with pioglitazone, another specific ligand of PPARgamma. Our results suggest that ligands of PPARgamma may be a novel therapeutic agent for the treatment of pancreatic carcinomas.     

Inhibitory effect of meloxicam, a selective cyclooxygenase-2 inhibitor, and ciglitazone, a peroxisome proliferator-activated receptor gamma ligand, on the growth of human ovarian cancers

BACKGROUND: It was recently reported that high expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and low expression of cyclooxygenase-2 (COX-2) might be involved in the inhibition of ovarian tumor progression and confirmed that PPARgamma activation could suppress COX-2 expression via the nuclear factor-kappaB pathway in ovarian cancer cells. METHODS: The current study investigated whether meloxicam, a selective COX-2 inhibitor, and ciglitazone, a ligand for PPARgamma, inhibit the growth of human ovarian cancer cell lines and aimed to elucidate the molecular mechanism of their antitumor effect. Tumor growth and survival were examined in female nu/nu mice xenografted with subcutaneous OVCAR-3 tumors or with intraperitoneal DISS tumors and treated with meloxicam (162 ppm in diet, every day) or ciglitazone (15 mg/kg intraperitoneally once a week). RESULTS: Both meloxicam and ciglitazone treatments significantly suppressed the growth of OVCAR-3 tumors xenotransplanted subcutaneously and significantly prolonged the survival of mice with malignant ascites derived from DISS cells as compared with controls. Meloxicam treatment decreased COX-2 expression in tumors by 2.5-fold compared with that observed in untreated tumors. Although ciglitazone treatment did not alter COX-2 expression in tumors, it reduced the expression of microsomal prostaglandin (PG) E synthase, which converts COX-derived PGH(2) to PGE(2). Both meloxicam and ciglitazone decreased PGE(2) levels in serum as well as in ascites. Reduced microvessel density and induced apoptosis were found in solid OVCAR-3 tumors treated with either meloxicam or ciglitazone. CONCLUSIONS: These results indicate that both meloxicam and ciglitazone produce antitumor effects against ovarian cancer in conjunction with reduced angiogenesis and induction of apoptosis.     

High-dose methotrexate in combination with interferons in the treatment of malignant pleural mesothelioma.

Twenty six patients with pleural mesothelioma of UICC stage I-IV excluding M1 disease (46% of whom had stage I disease and 38% stage III disease) were treated intravenously with high dose MTX (3 g) and calcium folinate rescue three times at intervals of 2 weeks and three times at intervals of 3 weeks. Natural interferon (IFN)-alpha (3 MIU days 2-10) and recombinant IFN-gamma1b (50 microg m(-2) on days 2, 6 and 10) were injected subcutaneously after each MTX dose. At the end of MTX treatment the IFNs were continued as maintenance therapy until disease progression. Seven partial responses were observed among 24 patients evaluable for response (response rate 29%, 95% confidence interval 13-51%). Median duration of response was 10 months (range 3-24 months). Median survival was 17 months and 1-year and 2-year survival rates 62% and 31% respectively. The toxicity of the chemo-immunotherapy was acceptable. Treatment was stopped in one patient who developed grade IV neurological toxicity. MTX dose reductions were rare (two patients with grade 1-2 renal toxicity). The combination of high dose MTX and IFN-alpha and IFN-gamma is active against malignant pleural mesothelioma and well-tolerated. The survival rates are encouraging.     

Molecular classification of selective oestrogen receptor modulators on the basis of gene expression profiles of breast cancer cells expressing oestrogen receptor alpha

The purpose of this study was to classify selective oestrogen receptor modulators based on gene expression profiles produced in breast cancer cells expressing either wtERalpha or mutant(351)ERalpha. In total, 54 microarray experiments were carried out by using a commercially available Atlas cDNA Expression Arrays (Clontech), containing 588 cancer-related genes. Nine sets of data were generated for each cell line following 24 h of treatment: expression data were obtained for cells treated with vehicle EtOH (Control); with 10(-9) or 10(-8) M oestradiol; with 10(-6) M 4-hydroxytamoxifen; with 10(-6) M raloxifene; with 10(-6) M idoxifene, with 10(-6) M EM 652, with 10(-6) M GW 7604; with 5 x 10(-5) M resveratrol and with 10(-6) M ICI 182,780. We developed a new algorithm 'Expression Signatures' to classify compounds on the basis of differential gene expression profiles. We created dendrograms for each cell line, in which branches represent relationships between compounds. Additionally, clustering analysis was performed using different subsets of genes to assess the robustness of the analysis. In general, only small differences between gene expression profiles treated with compounds were observed with correlation coefficients ranged from 0.83 to 0.98. This observation may be explained by the use of the same cell context for treatments with compounds that essentially belong to the same class of drugs with oestrogen receptors related mechanisms. The most surprising observation was that ICI 182,780 clustered together with oestrodiol and raloxifene for cells expressing wtERalpha and clustered together with EM 652 for cells expressing mutant(351)ERalpha. These data provide a rationale for a more precise and elaborate study in which custom made oligonucleotide arrays can be used with comprehensive sets of genes known to have consensus and putative oestrogen response elements in their promoter regions.     

A new class of small molecule estrogen receptor-alpha antagonists that overcome anti-estrogen resistance

Previous studies indicate that BRCA1 protein binds to estrogen receptor-alpha (ER) and inhibits its activity. Here, we found that BRCA1 over-expression not only inhibits ER activity in anti-estrogen-resistant LCC9 cells but also partially restores their sensitivity to Tamoxifen. To simulate the mechanism of BRCA1 inhibition of ER in the setting of Tamoxifen resistance, we created a three-dimensional model of a BRCA1-binding cavity within the ER/Tamoxifen complex; and we screened a pharmacophore database to identify small molecules that could fit into this cavity. Among the top 40 “hits”, six exhibited potent ER inhibitory activity in anti-estrogen-sensitive MCF-7 cells and four of the six exhibited similar activity (IC₅₀ ≤ 1.0 μM) in LCC9 cells. We validated the model by mutation analysis. Two representative compounds (4631-P/1 and 35466-L/1) inhibited ER-dependent cell proliferation in Tamoxifen-resistant cells (LCC9 and LCC2) and partially restored sensitivity to Tamoxifen. The compounds also disrupted the association of BRCA1 with ER. In electrophoretic mobility shift assays, the compounds caused dissociation of ER from a model estrogen response element. Finally, a modified form of compound 35446 (hydrochloride salt) inhibited growth of LCC9 tumor xenografts at non-toxic concentrations. These results identify a novel group of small molecules that can overcome Tamoxifen resistance.     

Discrete functions of GSK3α and GSK3β isoforms in prostate tumor growth and micrometastasis

Isoform specific function of glycogen synthase kinase-3 (GSK3) in cancer is not well defined. We report that silencing of GSK3α, but not GSK3β expression inhibited proliferation, survival and colony formation by the PC3, DU145 and LNCaP prostate cancer cells, and the growth of PC3 tumor xenografts in athymic nude mice. Silencing of GSK3α, but not GSK3β resulted in reduced proliferation and enhanced apoptosis in tumor xenografts. ShRNA-mediated knockdown of GSK3α and GSK3β equally inhibited the ability of prostate cancer cells to migrate and invade the endothelial-barrier in vitro, and PC3 cell micrometastasis to lungs in vivo. Mechanistically, whereas silencing GSK3α resulted in increased expression of pro-apoptotic markers cleaved caspase-3 and cleaved caspase-9 in LNCaP, PC3 and DU145 cells, silencing GSK3β resulted in the inhibition of cell scattering, establishment of cell-cell contacts, increased expression and membrane localization of β-catenin, and reduced expression of epithelial to mesenchymal transition (EMT) markers such as Snail and MMP-9. This indicated the specific role of GSK3β in EMT, acquisition of motility and invasive potential. Overall, our data demonstrated the isoform specific role of GSK3α and GSK3β in prostate cancer cells in vitro, and tumor growth and micrometastasis in vivo, via distinct molecular and cellular mechanisms.     

Enhanced growth inhibition by combination differentiation therapy with ligands of peroxisome proliferator-activated receptor-gamma and inhibitors of histone deacetylase in adenocarcinoma of the lung.

PURPOSE: Histone deacetylase (HDAC) inhibitors and ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma) have been shown previously to induce growth arrest and differentiation in a variety of cancer cell lines. The purpose of this study was to determine whether HDAC inhibitors function similarly in non-small cell lung cancer (NSCLC) and whether combination treatment with HDAC inhibitors and PPARgamma ligands is more efficacious than either agent alone. EXPERIMENTAL DESIGN AND RESULTS: Nanomolar concentrations of trichostatin A induced growth arrest in five of seven NSCLC cell lines, whereas sodium phenylbutyrate (PB) was markedly less potent. In adenocarcinomas, trichostatin A up-regulated general differentiation markers (gelsolin, Mad, and p21/WAF1) and down-regulated markers of the type II pneumocyte progenitor cell lineage (MUC1 and SP-A), indicative of a more mature phenotype. PB had a similar effect. Simultaneous treatment with a PPARgamma ligand and PB enhanced the growth inhibition in adenocarcinomas but not in nonadenocarcinomas. Growth arrest was accompanied by markedly decreased cyclin D1 expression but not enhanced differentiation. CONCLUSIONS: The present study demonstrates potent growth-inhibitory and differentiation-inducing activity of HDAC inhibitors in NSCLC and suggests that combination differentiation therapy should be explored further for the treatment of lung adenocarcinomas.