fh535 and Liver-Neoplasms

Autophagy targets cellular components for lysosomal-dependent degradation in which the products of degradation may be recycled for protein synthesis and utilized for energy production. Autophagy also plays a critical role in cell homeostasis and the regulation of many physiological and pathological processes and prompts this investigation of new agents to effect abnormal autophagy in hepatocellular carcinoma (HCC). 2,5-Dichloro-N-(2-methyl-4-nitrophenyl) benzenesulfonamide (FH535) is a synthetic inhibitor of the Wnt/β-catenin pathway that exhibits anti-proliferative and anti-angiogenic effects on different types of cancer cells. The combination of FH535 with sorafenib promotes a synergistic inhibition of HCC and liver cancer stem cell proliferation, mediated in part by the simultaneous disruption of mitochondrial respiration and glycolysis. We demonstrated that FH535 decreased HCC tumor progression in a mouse xenograft model. For the first time, we showed the inhibitory effect of an FH535 derivative, FH535-N, alone and in combination with sorafenib on HCC cell proliferation. Our study revealed the contributing effect of Wnt/β-catenin pathway inhibition by FH535 and its derivative (FH535-N) through disruption of the autophagic flux in HCC cells.

Topics: Animals; Antineoplastic Agents; Autophagy; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Female; Gene Knockdown Techniques; Humans; Liver Neoplasms; Liver Neoplasms, Experimental; Mice; Mice, Nude; Sorafenib; Sulfonamides; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

Treatment of advanced hepatocellular carcinoma (HCC) remains a challenge due to the high tumor heterogeneity. In the present study, we aim to evaluate the impact of the β-catenin inhibitor, FH535, alone or in combination with the Ras/Raf/MAPK inhibitor Sorafenib, on the bioenergetics profiles of the HCC cell lines Huh7 and PLC/PRF/5. Single low-dose treatments with FH535 or Sorafenib promoted different effects on mitochondrial respiration and glycolysis in a cell type specific manner. However, the combination of these drugs significantly reduced both mitochondrial respiration and glycolytic rates regardless of the HCC cells. The significant changes in mitochondrial respiration observed in cells treated with the Sorafenib-FH535 combination may correspond to differential targeting of ETC complexes and changes in substrate utilization mediated by each drug. Moreover, the bioenergetics changes and the loss of mitochondrial membrane potential that were evidenced by treatment of HCC cells with the combination of FH535 and Sorafenib, preceded the induction of cell apoptosis. Overall, our results demonstrated that Sorafenib-FH535 drug combination induce the disruption of the bioenergetics of HCC by the simultaneous targeting of mitochondrial respiration and glycolytic flux that leads the synergistic effect on inhibition of cell proliferation. These findings support the therapeutic potential of combinatory FH535-Sorafenib treatment of the HCC heterogeneity by the simultaneous targeting of different molecular pathways.

Topics: Apoptosis; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Energy Metabolism; Humans; Liver Neoplasms; Mitochondria; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Signal Transduction; Sorafenib; Sulfonamides

The Wnt signaling pathway is activated in hepatocellular carcinoma (HCC). This study investigated the effects of FH535, an inhibitor of the Wnt signaling pathway, on the proliferation and motility of HCC cells. HLF cells and PLC/PRF/5 cells, HCC cells, were subjected to 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay with the addition of FH535. RNA was isolated from the cells and subjected to real-time quantitative PCR. Hematoxylin and eosin (H&E) staining was performed to analyze apoptosis. A scratch assay was performed to analyze cell motility. Cell proliferation significantly decreased (P<0.05). The expression levels of cyclin D1 significantly decreased in both cell lines (P<0.05). Pyknotic nuclei were observed in the cells cultured with FH535 (50 µM). In the scratch assay, the distance between the growing edges of cells and the scratched line significantly decreased with the addition of FH535 at 50 µM (P<0.05). The expression levels of matrix metalloproteinase 9 significantly decreased at 50 µM (P<0.05). FH535 suppressed the proliferation of HCC cells by downregulating the expression of cyclin D1 and by inducing apoptosis. Further, it suppressed cell motility by downregulating the expression of matrix metalloproteinase.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Matrix Metalloproteinase 9; Sulfonamides; Wnt Signaling Pathway

Recurrence and metastasis are the two leading causes of poor prognosis of hepatocellular carcinoma (HCC) patients. Cyclooxygenase (COX)-2 is overexpressed in many types of cancers including HCC and promotes its metastasis. Meloxicam is a selective COX-2 inhibitor that has been reported to exert an anti-proliferation and invasion/migration response in various tumors. In this study, we examined the role of meloxicam on HCC cell proliferation and migration and explored the molecular mechanisms underlying this effect. We found that meloxicam inhibited HCC cell proliferation and had a cell cycle arrest effect in human HCC cells. Furthermore, meloxicam suppressed the ability of HCC cells expressing higher levels of COX-2 and prostaglandin E2 (PGE2) to migration via potentiating expression of E-cadherin and alleviating expression of matrix metalloproteinase (MMP)-2 and -9. COX-2/PGE2 has been considered to activate the β-catenin signaling pathway which promotes cancer cell migration. We found that treatment with PGE2 significantly enhanced nuclear accumulation of β-catenin and the activation of GSK3β which could be reversed by meloxicam in HCC cells. We also observed that HCC cell migration and upregulation of the level of MMP-2/9 and downregulation of E-cadherin induced by PGE2 were suppressed by FH535, an inhibitor of β-catenin. Taken together, these findings provide a new treatment strategy against HCC proliferation and migration.

Topics: Antigens, CD; beta Catenin; Cadherins; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Dinoprostone; Enzyme Activation; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Meloxicam; Neoplasm Invasiveness; RNA Interference; RNA, Small Interfering; Signal Transduction; Sulfonamides; Thiazines; Thiazoles

The aim of this study is to find synergistic effect using FH535 and sorafenib by targeting the RAS/RAF/MAPK and WNT/β-catenin pathways.. 3H-Thymidine incorporation assays were performed to address Huh7 and liver cancer stem cell (LCSC) inhibition using FH535 and sorafenib, alone and in combination. Calcusyn analysis was used to calculate the combination index (CI). A western blot assay was performed to check for potential targets.. FH535 and sorafenib caused inhibition of Huh7 and LCSC. Combination therapy was significantly better than monotherapy in inhibition of HuH7. Combination with sorafenib and FH535 was found to be synergistic in inhibition of LCSC with a CI of less than 1. The western blot assay demonstrated enhanced cleaved poly (ADP-ribose) polymerase (PARP) and inhibition of cyclin D1, B-cell lymphoma 2 (Bcl2), survivin and cellular myelocytomatosis oncogene (c-MYC).. FH535 and sorafenib combination produced synergistic effect on inhibition of HCC and LCSC. Our study demonstrated that FH535 can induce apoptosis in these two different hepatocellular carcinoma (HCC) cell lines.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; Drug Synergism; Female; Humans; Immunophenotyping; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Mice; Mitogen-Activated Protein Kinases; Neoplastic Stem Cells; Niacinamide; Phenotype; Phenylurea Compounds; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins p21(ras); raf Kinases; Signal Transduction; Sorafenib; Sulfonamides; Survivin; Wnt Signaling Pathway

Activation of the Wnt/β-catenin pathway has been observed in at least 1/3 of hepatocellular carcinomas (HCC), and a significant number of these have mutations in the β-catenin gene. Therefore, effective inhibition of this pathway could provide a novel method to treat HCC. The purposed of this study was to determine whether FH535, which was previously shown to block the β-catenin pathway, could inhibit β-catenin activation of target genes and inhibit proliferation of Liver Cancer Stem Cells (LCSC) and HCC cell lines. Using β-catenin responsive reporter genes, our data indicates that FH535 can inhibit target gene activation by endogenous and exogenously expressed β-catenin, including the constitutively active form of β-catenin that contains a Serine37Alanine mutation. Our data also indicate that proliferation of LCSC and HCC lines is inhibited by FH535 in a dose-dependent manner, and that this correlates with a decrease in the percentage of cells in S phase. Finally, we also show that expression of two well-characterized targets of β-catenin, Cyclin D1 and Survivin, is reduced by FH535. Taken together, this data indicates that FH535 has potential therapeutic value in treatment of liver cancer. Importantly, these results suggest that this therapy may be effective at several levels by targeting both HCC and LCSC.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Screening Assays, Antitumor; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Molecular Targeted Therapy; Neoplastic Stem Cells; Sulfonamides; Survivin; Transcriptional Activation; Wnt Signaling Pathway