hymecromone and Carcinoma--Squamous-Cell

Although cells with tumorigenic/stem cell-like properties have been identified in many cancers, including oral squamous cell carcinoma (OSCC), their isolation and characterisation is still at early stages. The aim of this study is to characterise the electrophysiological properties of OSCC cells with different tumorigenic properties in order to establish if a correlation exists between tumorigenicity and cellular electrical characteristics.. Rapid adherence to collagen IV was used as a non-invasive, functional method to isolate subsets of cells with different tumorigenic abilities from one oral dysplastic and three OSCC-derived cell lines. The cell subsets identified and isolated using this method were further investigated using dielectrophoresis, a label-free method to determine their electrophysiological parameters. Cell membrane morphology was investigated using scanning electron microscopy (SEM) and modulated by use of 4-methylumbelliferone (4-MU).. Rapid adherent cells (RAC) to collagen IV, enriched for increased tumorigenic ability, had significantly higher effective membrane capacitance than middle (MAC) and late (LAC) adherent cells. SEM showed that, in contrast to MAC and LAC, RAC displayed a rough surface, extremely rich in cellular protrusions. Treatment with 4-MU dramatically altered RAC membrane morphology by causing loss of filopodia, and significantly decreased their membrane capacitance, indicating that the highest membrane capacitance found in RAC was due to their cell membrane morphology.. This is the first study showing that OSCC cells with higher tumour formation ability exhibit higher effective membrane capacitance than cells that are less tumorigenic. OSSC cells with different tumorigenic ability possessed different electrophysiological properties mostly due to their differences in the cell membrane morphology. These results suggest that dielectrophoresis could potentially used in the future for reliable, label-free isolation of putative tumorigenic cells.

Topics: Animals; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line, Tumor; Cell Membrane; Electric Capacitance; Humans; Hymecromone; Mice, Inbred NOD; Mice, SCID; Microscopy, Electron, Scanning; Mouth Neoplasms; Neoplastic Stem Cells; Specific Pathogen-Free Organisms

Oesophageal cancer is a highly aggressive tumour entity with at present poor prognosis. Therefore, novel treatment options are urgently needed. Hyaluronan (HA) is a polysaccharide present in the matrix of human oesophageal squamous cell carcinoma (ESCC). Importantly, in vitro ESCC cells critically depend on HA synthesis to maintain the proliferative phenotype. The aim of the present study is (1) to study HA-synthase (HAS) expression and regulation in human ESCC, and (2) to translate the in vitro results into a mouse xenograft model of human ESCC to study the effects of systemic versus tumour targeted HAS inhibition on proliferation and distribution of tumour-bound and stromal hyaluronan.. mRNA expression was investigated in human ESCC biopsies by semiquantitative real-time RT PCR. Furthermore, human ESCC were xenografted into NMRI nu/nu mice. The effects on tumour progression and morphology of 4-methylumbelliferone (4-MU), an inhibitor of HA-synthesis, and of lentiviral knock down of HA-synthase 3 (HAS3), the main HAS isoform in the human ESCC tissues and the human ESCC cell line used in this study, were determined. Tumour progression was monitored by calliper measurements and by flat-panel detector volume computed tomography (fpVCT). HA content, cellular composition and proliferation (Ki67) were determined histologically.. mRNA of HAS isoform 3 (HAS3) was upregulated in human ESCC biopsies and HAS3 mRNA was positively correlated to expression of the epidermal growth factor (EGF) receptor. EGF was also proven to be a strong inductor of HAS3 mRNA expression in vitro. During the course of seven weeks, 4-MU inhibited progression of xenograft tumours. Interestingly, remodelling of the tumour into a more differentiated phenotype and inhibition of cell proliferation were observed. Lentiviral knockdown of HAS3 in human ESCC cells prior to xenografting mimicked all effects of 4-MU treatment suggesting that hyaluronan produced by ESCC is accountable for major changes in tumour environment in vivo.. Systemic inhibition of HA-synthesis and knockdown of tumour cell HAS3 cause decreased ESCC progression accompanied by tumour stroma remodelling and may therefore be used in novel approaches to ESCC therapy.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Line, Tumor; Cell Proliferation; Disease Progression; ErbB Receptors; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Glucuronosyltransferase; Humans; Hyaluronan Synthases; Hyaluronic Acid; Hymecromone; Immunohistochemistry; Mice; Mice, Inbred Strains; Mice, Nude; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; Xenograft Model Antitumor Assays

Hyaluronan (HA) is a polysaccharide component in the parenchyma and stroma of human esophageal squamous cell carcinoma (ESCC). Clinically, esophageal cancer represents a highly aggressive tumor type with poor prognosis resulting in a 5-year survival rate of 5%. The aim of the present study was the detailed analysis of the role of HA synthesis for ESCC phenotype in vitro using the ESCC cell line OSC1. In OSC1 cells, pericellular HA-matrix surrounding extended actin-dependent filopodia was detected. The small molecule inhibitor of HA synthesis, 4-methylumbelliferone (4-MU, 0.3 mm) caused loss of these filopodia and focal adhesions and inhibited proliferation and migration. In search of the underlying mechanism cleavage of focal adhesion kinase (FAK) was detected by immunoblotting. In addition, displacing HA by an HA-binding peptide (Pep-1, 500 mug/ml) and digestion of pericellular HA by hyaluronidase resulted in cleavage of focal adhesions. Furthermore, real-time reverse transcription PCR revealed that HA synthase 3 (HAS3) > HAS2 are the predominant HA-synthases in OSC1. Lentiviral transduction with shHAS3, and to a lesser extent with shHAS2, reduced intact FAK protein and filopodia as well as proliferation and migration. Furthermore, down-regulation by lentiviral shRNA of RHAMM (receptor of HA-mediated motility) but not CD44 induced loss of filopodia and caused FAK cleavage. In contrast, knockdown of both HA receptors inhibited proliferation and migration of OSC1. In conclusion, HA synthesis and, in turn, RHAMM and CD44 signaling promoted an activated phenotype of OSC1. Because RHAMM appears to support both filopodia, FAK, and the proliferative and migratory phenotype, it may be promising to explore RHAMM as a potential therapeutic target in esophageal cancer.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Esophageal Neoplasms; Extracellular Matrix Proteins; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Gene Knockdown Techniques; Glucuronosyltransferase; Humans; Hyaluronan Receptors; Hyaluronan Synthases; Hyaluronic Acid; Hymecromone; Molecular Sequence Data; Phenotype; Pseudopodia