latrunculin-a and Carcinoma--Hepatocellular

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D1 encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E2 synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC.

Topics: Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Cyclin A; Cyclin D; Cyclooxygenase 2; Cytoskeleton; Dinoprostone; ELAV Proteins; Hep G2 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Liver Neoplasms; Nonmuscle Myosin Type IIA; Polyribosomes; Protein Transport; Ribonucleoproteins; RNA, Messenger; Thiazolidines

Recent research has indicated a new mode of intercellular communication facilitated by the movement of RNA between cells. There is evidence that RNA can transfer between cells in a multitude of ways, including in complex with proteins or lipids or in vesicles, including apoptotic bodies and exosomes. However, there remains little understanding of the function of nucleic acid transfer between human cells. In this article, we report that human macrophages transfer microRNAs (miRNAs) to hepato-carcinoma cells (HCCs) in a manner that required intercellular contact and involved gap junctions. Two specific miRNAs transferred efficiently between these cells--miR-142 and miR-223--and both were endogenously expressed in macrophages and not in HCCs. Transfer of these miRNAs influenced posttranscriptional regulation of proteins in HCCs, including decreased expression of reporter proteins and endogenously expressed stathmin-1 and insulin-like growth factor-1 receptor. Importantly, transfer of miRNAs from macrophages functionally inhibited proliferation of these cancerous cells. Thus, these data led us to propose that intercellular transfer of miRNA from immune cells could serve as a new defense against unwanted cell proliferation or tumor growth.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Cell Communication; Cell Division; Cell Line, Transformed; Cell Line, Tumor; Cell-Derived Microparticles; Coculture Techniques; Cytoskeleton; Exosomes; Gene Expression Regulation, Neoplastic; Genes, Reporter; Hep G2 Cells; Humans; Leukemia, Monocytic, Acute; Liver Neoplasms; Macrophages; Mastocytoma; MicroRNAs; Receptor, IGF Type 1; Recombinant Fusion Proteins; RNA, Small Interfering; Stathmin; Thiazolidines; Transfection

Des-gamma-carboxy prothrombin (DCP) has been well established as a hepatocellular carcinoma (HCC) tumor marker. However, the precise mechanism by which HCC cells produce DCP remains unknown. Importantly, DCP is not specific for HCC. For example, vitamin K-deficiency or ingestion of a vitamin K antagonist (warfarin) also leads to DCP production. In addition, supplementary administration of vitamin K2 analogues to HCC patients has led to reduce serum DCP levels. From these observations, we hypothesize that DCP might be produced from HCC cells with functional impairment of vitamin K uptake. Because, as previously reported, the down-regulation of E-cadherin or high serum DCP in HCC patients is associated with a high risk of vascular invasion, intra-hepatic metastasis and tumor recurrence, we examined if HCC cells might produce DCP by epithelial to fibroblastoid conversion (EFC) in vitro. HepG2 cells were induced EFC by tumor promoter, 12-O-tetracanoylphorbol-13-acetate (TPA). DCP production was observed in HepG2 cells that had lost E-cadherin expression in a TPA-dose-dependent manner. The DCP production was inhibited by introducing additional vitamin K2 into the treated cells. In addition, LDL uptake as a surrogate of vitamin K uptake was significantly impaired in TPA-treated HepG2 cells. The cells with impairment of LDL uptake produced DCP. Fat soluble vitamins are taken up into cells through clathrin-mediated endocytosis, in which the dynamic polymerization of F-actin plays a crucial role. We found that HepG2 cells with F-actin rearrangement produced DCP. In addition, latrunculin A, an actin depolymerizer, induced naïve HepG2 cells to produce DCP, confirming that impairment of F-actin polymerization is a key mechanism of DCP production. We showed in vitro that cytoskeletal filament change by EFC is crucial for DCP production in HepG2 cells.

Topics: Actins; Biomarkers; Biomarkers, Tumor; Bridged Bicyclo Compounds, Heterocyclic; Cadherins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Transdifferentiation; Clathrin; Cytoskeleton; Dose-Response Relationship, Drug; Endocytosis; Epithelial Cells; Fibroblasts; Humans; Lipoproteins, LDL; Liver Neoplasms; Protein Precursors; Prothrombin; Tetradecanoylphorbol Acetate; Thiazolidines; Vitamin K 2

Tumor necrosis factor-alpha (TNF-alpha) is an important component of the early signaling pathways leading to liver regeneration and proliferation, but it is also responsible for several hepatotoxic effects. We have investigated the effect of TNF-alpha on thapsigargin (TG)-induced store-mediated Ca2+ entry (SMCE) in the human hepatocellular carcinoma cell line HepG2. In these cells, short-term (10 min) exposure to TNF-alpha slightly increased SMCE. In contrast, long-term (12 h) exposure to TNF-alpha significantly reduced SMCE. This effect was reversed by coincubation with atrial natriuretic peptide (ANP), which itself had no effect on SMCE. Cytochalasin D and latrunculin A, inhibitors of actin polymerization, abolished SMCE. Long-term exposure of HepG2 cells to TNF-alpha abolished TG-induced actin polymerization and membrane association of Ras proteins. When TNF-alpha was added in combination with ANP, these effects were reduced. These findings suggest that in HepG2 cells, TNF-alpha inhibits SMCE by affecting reorganization of the actin cytoskeleton, probably by interfering with the activation of Ras proteins, and that ANP protects against these inhibitory effects of TNF-alpha.

Topics: Actins; Atrial Natriuretic Factor; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Carcinogens; Carcinoma, Hepatocellular; Cell Compartmentation; Cytochalasin D; Cytoskeleton; Humans; Liver Neoplasms; Nucleic Acid Synthesis Inhibitors; Polymers; ras Proteins; Thapsigargin; Thiazoles; Thiazolidines; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Adenomatous polyposis coli (APC) is mutated in most colorectal cancers. APC downregulates nuclear beta-catenin, which is thought to be critical for its tumour suppressor function. However, APC may have additional and separate functions at the cell periphery. Here, we examine polarized MDCK and WIF-B hepatoma cells and find that APC is associated with their lateral plasma membranes. This depends on the actin cytoskeleton but not on microtubules, and drug wash-out experiments suggest that APC is delivered continuously to the plasma membrane by a dynamic actin-dependent process. In polarized MDCK cells, APC also clusters at microtubule tips in their basal-most regions. Microtubule depolymerization causes APC to relocalize from these tips to the plasma membrane, indicating two distinct peripheral APC pools that are in equilibrium with each other in these cells. Truncations of APC such as those found in APC mutant cancer cells can neither associate with the plasma membrane nor with microtubule tips. The ability of APC to reach the cell periphery may thus contribute to its tumour suppressor function in the intestinal epithelium.

Topics: Actins; Adenomatous Polyposis Coli Protein; Animals; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Cell Line; Cell Membrane; Cell Polarity; Cytoskeleton; Dogs; Epithelial Cells; Green Fluorescent Proteins; Humans; Kidney; Luminescent Proteins; Macromolecular Substances; Microtubules; Mutagenesis, Site-Directed; Nocodazole; Protein Binding; Protein Transport; Rats; Recombinant Fusion Proteins; Thiazoles; Thiazolidines; Transfection