gw9662 and Liver-Neoplasms

The purpose of this study was to investigate the effect of FNDC5 expression levels in hepatocellular carcinoma on the phenotypic changes of macrophages in tumor tissues.. In this study, we established an in vitro co-culture system of hepatocellular carcinoma cells and macrophages. Then we performed overexpression or knockdown of FNDC5 gene in hepatocellular carcinoma cells to observe the effect of changes in FNDC5 expression level on the phenotypic changes of THP-1 macrophages. And the conclusions obtained in the in vitro assay were further validated by a subcutaneous tumorigenic nude mice model.. Our findings suggest that elevated FNDC5 expression in hepatocellular carcinoma cells lead to an increased M2 phenotype and decreased M1 phenotype in macrophages. This effect may be achieved by elevating PPARγ levels in macrophages while decreasing NF-κB and NLRP3 levels. These changes could be reversed by using PPARγ inhibitors.. We preliminarily demonstrated that FNDC5 in hepatocellular carcinoma cells promotes the polarization of M2 macrophages by affecting the PPARγ/NF-κB/NLRP3 pathway.

Topics: Anilides; Animals; Carcinogenesis; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Coculture Techniques; Fibronectins; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Nude; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; PPAR gamma; RNA, Small Interfering; Signal Transduction; Survival Analysis; THP-1 Cells; Tumor Burden; Xenograft Model Antitumor Assays

Cannabinoids exert antiproliferative effects in a wide range of tumoral cells, including hepatocellular carcinoma (HCC) cells. In this study, we examined whether the PPARγ-activated pathway contributed to the antitumor effect of two cannabinoids, Δ9-tetrahydrocannabinol (THC) and JWH-015, against HepG2 and HUH-7 HCC cells. Both cannabinoids increased the activity and intracellular level of PPARγ mRNA and protein, which was abolished by the PPARγ inhibitor GW9662. Moreover, genetic ablation with small interfering RNA (siRNA), as well as pharmacological inhibition of PPARγ decreased the cannabinoid-induced cell death and apoptosis. Likewise, GW9662 totally blocked the antitumoral action of cannabinoids in xenograft-induced HCC tumors in mice. In addition, PPARγ knockdown with siRNA caused accumulation of the autophagy markers LC3-II and p62, suggesting that PPARγ is necessary for the autophagy flux promoted by cannabinoids. Interestingly, downregulation of the endoplasmic reticulum stress-related protein tribbles homolog 3 (TRIB3) markedly reduced PPARγ expression and induced p62 accumulation, which was counteracted by overexpression of PPARγ in TRIB3-knocked down cells. Taken together, we demonstrate for the first time that the antiproliferative action of the cannabinoids THC and JWH-015 on HCC, in vitro and in vivo, are modulated by upregulation of PPARγ-dependent pathways.

Topics: Anilides; Antineoplastic Agents; Apoptosis; Cannabinoids; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Hep G2 Cells; Humans; Indoles; Liver Neoplasms; PPAR gamma; RNA Interference; RNA, Small Interfering; Signal Transduction

To investigate whether the apoptotic activities of 8-bromo-7-methoxychrysin (BrMC) involve reactive oxygen species (ROS) generation and c-Jun N-terminal kinase (JNK) activation in human hepatocellular carcinoma cells (HCC).. HepG2, Bel-7402 and L-02 cell lines were cultured in vitro and the apoptotic effects of BrMC were evaluated by flow cytometry (FCM) after propidium iodide (PI) staining, caspase-3 activity using enzyme-linked immunosorbent assay (ELISA), and DNA agarose gel electrophoresis. ROS production was evaluated by FCM after dichlorodihydrofluorescein diacetate (DCHF-DA) probe labeling. The phosphorylation level of JNK and c-Jun protein was analyzed by Western blotting.. FCM after PI staining showed a dose-dependent increase in the percentage of the sub-G1 cell population (P < 0.05), reaching 39.0% +/- 2.8% of HepG2 cells after 48 h of treatment with BrMC at 10 micromol/L. The potency of BrMC to HepG2 and Bel-7402 (32.1% +/- 2.6%) cells was found to be more effective than the lead compound, chrysin (16.2% +/- 1.6% for HepG2 cells and 11.0% +/- 1.3% for Bel-7402 cell) at 40 micromol/L and similar to 5-fluorouracil (33.0% +/- 2.1% for HepG2 cells and 29.3% +/- 2.3% for Bel-7402 cells) at 10 micromol/L. BrMC had little effect on human embryo liver L-02 cells, with the percentage of sub-G1 cell population 5.4% +/- 1.8%. Treatment of HepG2 cells with BrMC for 48 h also increased the levels of active caspase-3, in a concentration-dependent manner. z-DEVD-fmk, a caspase-3-specific inhibitor, prevented the activation of caspase-3. Treatment with BrMC at 10 micromol/L for 48 h resulted in the formation of a DNA ladder. Treatment of cells with BrMC (10 micromol/L) increased mean fluorescence intensity of DCHF-DA in HepG2 cells from 7.2 +/- 1.12 at 0 h to 79.8 +/- 3.9 at 3 h and 89.7 +/- 4.7 at 6 h. BrMC did not affect ROS generation in L-02 cells. BrMC treatment failed to induce cell death and caspase-3 activation in HepG2 cells pretreated with N-acetylcysteine (10 mmol/L). In addition, in HepG2 cells treated with BrMC (2.5, 5.0, 10.0 micromol/L) for 12 h, JNK activation was observed. Peak JNK activation occurred at 12 h post-treatment and this activation persisted for up to 24 h. The expression of phosphorylated JNK and c-Jun protein after 12 h with BrMC-treated cells was inhibited by N-acetylcysteine and SP600125 pre-treatment, but GW9662 had no effect. SP600125 substantially reduced BrMC-induced cell death and caspase-3 activation of HepG2 cells. N-acetylcysteine and GW9662 also attenuated induction of cell death and caspase-3 activation in HepG2 cells treated with BrMC.. BrMC induces apoptosis of HCC cells by ROS generation and sustained JNK activation.

Topics: Acetylcysteine; Anilides; Antineoplastic Agents; Antioxidants; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Enzyme Activation; Flavonoids; Hep G2 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Reactive Oxygen Species

Peroxisome proliferator-activated receptor (PPAR) agonists such as clofibrate are known to affect liver fatty acid binding protein (L-FABP) levels, which in turn influence hepatocellular oxidant status. The mechanism of clofibrate's modulation of L-FABP levels is not clear. In this study we used clofibrate (PPARα agonist), MK886 (PPARα antagonist), and GW9662 (PPARγ antagonist) in determining the regulating mechanism of L-FABP expression and its antioxidant activity in CRL-1548 hepatoma cells. Antioxidant activity was assessed by determining intracellular reactive oxygen species (ROS) using dichlorofluorescein (DCF) fluorescence. The effect of clofibrate on cytosolic activity of the intracellular antioxidant enzymes was also assessed. RT-PCR and mRNA stability assay showed that clofibrate treatment enhanced L-FABP mRNA stability, which resulted in increased L-FABP levels. A nuclear run-off assay and RT-PCR measurements of L-FABP mRNA revealed that clofibrate increased the L-FABP gene transcription rate. The increased L-FABP was associated with reduced cytosolic ROS. Levels of superoxide dismutase, glutathione peroxidase, and catalase were not affected by clofibrate treatment. L-FABP siRNA knockdown studies showed that a reduction in L-FABP expression was associated with increased DCF fluorescence. We conclude that clofibrate enhanced L-FABP gene transcription and mRNA stability, thus affecting L-FABP expression and ultimately cellular antioxidant activity.

Topics: Anilides; Animals; Carcinoma, Hepatocellular; Catalase; Cell Line, Tumor; Clofibrate; Fatty Acid-Binding Proteins; Fluoresceins; Gene Expression Regulation, Neoplastic; Glutathione Peroxidase; Hepatocytes; Humans; Hydrogen Peroxide; Indoles; Liver Neoplasms; Oxidative Stress; PPAR alpha; Rats; RNA Stability; RNA, Messenger; Superoxide Dismutase

It has recently been shown that cannabinoids induce growth inhibition and apoptosis in different tumour cell lines. In the current study, the effects of WIN 55,212-2 (WIN), a synthetic and potent cannabinoid receptor agonist, are investigated in hepatoma HepG2 cells and a possible signal transduction pathway is proposed. In these cells, WIN induces a clear apoptotic effect which was accompanied by up-regulation of the death-signalling factors Bax, Bcl-X(S), t-Bid and down-regulation of the survival factors survivin, phospho-AKT, Hsp72 and Bcl-2. Moreover, WIN-induced apoptosis is associated with JNK/p38 MAPK pathway activation and mitochondrial depolarisation demonstrated by a cytofluorimetric assay. The results also show that in HepG2 cells WIN markedly increases the level of the transcription factor PPARgamma in a dose- and time-dependent manner. The addition of the PPARgamma antagonists GW9662 and T0070907 significantly reduces the effects of the drug on both cell viability and the levels of survivin, phospho-AKT and phospho-BAD, suggesting that PPARgamma plays a key role in WIN-induced apoptosis. Altogether, the results seem to indicate a potential therapeutic role of WIN in hepatic cancer treatment.

Topics: Anilides; Apoptosis; Apoptosis Regulatory Proteins; Benzamides; Benzoxazines; Cannabinoids; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Humans; Liver Neoplasms; Membrane Microdomains; Morpholines; Naphthalenes; PPAR gamma; Protein Kinases; Pyridines; Receptor, Cannabinoid, CB2; Signal Transduction

Increased expression of vimentin in carcinomas correlates with parameters of malignant potential such as tumor grade and tumor metastasis. Peroxisome proliferator-activated receptor gamma (PPARgamma) has been intensively evaluated as a potential target for the inhibition of cell growth and metastasis in cancer cells. In the present study, we examined whether PPARgamma is a possible target molecule for the prevention of cell growth and invasion by treatment with agonists (troglitazone, rosiglitazone) and antagonists (T0070907, GW9662) in four different hepatocellular carcinoma (HCC) cell lines. We also evaluated the effects of the PPARgamma agonists and antagonists on tumor cell migration and invasion. The expression level of PPARgamma protein was higher in the sarcomatoid SH-J1 and poorly differentiated HLE cell lines than that in the well-differentiated HCC cell lines (HepG2 and Huh-7). Expression of vimentin was high in the SH-J1 HCC cell line and minimally detected in the HLE cell line. Treatment with low doses of the PPARgamma antagonists inhibited cell growth and colony formation of all four of the HCC cell lines. Vimentin in the high-grade HCC cells was cleaved by the treatment with the PPARgamma antagonists. Furthermore, treatment with the PPARgamma antagonists also strongly inhibited migration and invasion of the SH-J1 and HLE cells. However, treatment with low doses of the agonists had no effect on vimentin expression, migration, and invasion of the high-grade HCC cells but cell growth was inhibited by treatment with high concentrations of the agonists. Our results indicate that treatment with a PPARgamma antagonist may prevent cell growth and invasion of high-grade HCC cells. Our findings also suggest that PPARgamma antagonists inhibit cell growth and invasion through vimentin disarrangement in high-grade HCC.

Topics: Anilides; Benzamides; Blotting, Western; Carcinoma, Hepatocellular; Cell Adhesion; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Chromans; Colony-Forming Units Assay; Humans; Hypoglycemic Agents; Immunoenzyme Techniques; Ligands; Liver Neoplasms; Neoplasm Invasiveness; PPAR gamma; Pyridines; Rosiglitazone; Thiazolidinediones; Troglitazone; Vimentin

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.

Topics: Anilides; Anoikis; Benzamides; Carcinoma, Hepatocellular; Cell Adhesion; Cell Growth Processes; Cell Line, Tumor; Cell Shape; Chromans; Extracellular Matrix; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin beta1; Liver Neoplasms; PPAR gamma; Protein-Tyrosine Kinases; Pyridines; Rosiglitazone; Thiazolidinediones; Troglitazone