Human Peritoneal Mesothelial Cell Line Research Articles

Destruction of gastric cancer cells to mesothelial cells by apoptosis in the early peritoneal metastasis

This study examined the mechanism by which the gastric cancer cells lead to early peritoneal metastasis. HMrSV5 cells, a human peritoneal mesothelial cell line, were co-incubated with the supernatants of gastric cancer cells. Morphological changes of HMrSV5 cells were observed. The cell damage was quantitatively determined by MTT assay. The apoptosis of HMrSV5 cells was observed under transmission electron microscope. Acridine orange/ethidium bromide-stained condensed nuclei was detected by fluorescent microscopy and flow cytometry. The expressions of Bcl-2 and Bax was immunochemically evaluated. The results showed that conspicuous morphological changes of apoptosis were observed in HMrSV5 cells 24 h after treatment with the supernatants of gastric cancer cells. The supernatants could induce apoptosis of HMrSV5 cells in a time-dependent manner. The supernatants could up-regulate the expression of Bax and suppress that of Bcl-2 in HMrSV5 cells. These findings demonstrated that gastric cancer cells can induce the apoptosis of HPMCs through supernatants in the early peritoneal metastasis. The abnormal expressions of Bcl-2 and Bax may contribute to the apoptosis. Anti-apoptosis drugs promise to be adjuvant chemotherapeutic agents in the treatment of peritoneal metastasis of gastric cancer.

A neurokinin‐1 receptor antagonist that reduces intraabdominal adhesion formation increases peritoneal matrix metalloproteinase activity

Adhesions remain a significant complication of abdominal surgery. There is a growing body of evidence suggesting that remodeling of peritoneal extracellular matrix by matrix metalloproteinases (MMPs) is involved in adhesion formation. We have shown that administration of a specific neurokinin-1 receptor (NK-1R) antagonist (CJ-12,255, Pfizer) to rats within 5 hours of surgery reduces intraabdominal adhesion formation. Because substance P (SP), the primary NK-1R ligand, is known to augment tissue fibrosis, the aim of this study was to determine the effects of NK-1R antagonist administration on peritoneal MMP expression and activity 24 hours after surgery in a rat adhesion model. Following laparotomy, four ischemic buttons were created on the peritoneum of rats that received either an intraperitoneal NK-1R antagonist or a vehicle at surgery. Adhesion formation was assessed 7 days later. Peritoneal fluid and tissue were collected at 24 hours to assess total MMP activity, as well as MMP-2, MMP-8, and MMP-9 activity. Specific MMP and tissue inhibitors of MMP mRNAs were measured, and the effects of SP on MMP-3 expression were determined in Met-5A cells, a human peritoneal mesothelial cell line. NK-1R antagonist administration reduced adhesion formation by 47% (p<0.05) at 7 days and significantly increased the total MMP activity in peritoneal fluid at 24 hours. There was an accompanying increase (p<0.05) in MMP-8 and MMP-9 mRNA expression and activity in peritoneal tissue and fluid, respectively. MMP-3 mRNA was also increased in the 24-hour peritoneal tissue, and exposure of Met-5A cells to SP reduced MMP-3 expression and activity. These data support a role for MMPs, specifically MMP-3, MMP-8, and MMP-9, in intraabdominal adhesion formation and suggest that the NK-1R antagonist may reduce adhesions, in part, by increasing MMP activity in the peritoneum by 24 hours after surgery.

CD44 and β1 Integrin Mediate Ovarian Carcinoma Cell Adhesion to Peritoneal Mesothelial Cells

Epithelial cancer of the ovary spreads by implantation of tumor cells onto the mesothelial cells lining the peritoneal cavity. The aim of this study was to identify the adhesion molecules involved in the interaction of ovarian carcinoma cells with mesothelial cells. The human ovarian carcinoma cell lines SKOV3 and NIH:OVCAR5 as well as LP9 cells, a human peritoneal mesothelial cell line, were analyzed by flow cytometry for the expression of CD44 and the beta1 integrin subunit. An in vitro adhesion assay was developed whereby LP9 cells were grown as confluent monolayers, and radiolabeled ovarian carcinoma cells were monitored for their ability to adhere to the mesothelial monolayer in the presence of potential inhibitors. Each cell line was evaluated for the presence of a pericellular matrix by a particle exclusion assay. A monoclonal antibody (MAb) against the beta1 integrin subunit significantly reduced the adhesion of SKOV3 cells to LP9 cells, whereas NIH:OVCAR5 adhesion to LP9 cells was significantly inhibited by a CD44 MAb. The LP9 cells produced both hyaluronic acid (a ligand for CD44) as well as several extracellular matrix molecules (ligands for the beta1 integrin heterodimers). These results suggest that both CD44 and the beta1 integrin heterodimers may play a role in mediating the adhesion of ovarian carcinoma cells to mesothelial cells.

PAI-1 secretion and matrix deposition in human peritoneal mesothelial cell cultures: Transcriptional regulation by TGF-β1

Plasminogen activator inhibitor-1 (PAI-1), the main inhibitor of plasminogen activators in plasma and in peritoneum, impairs plasmin formation that is essential for the repair processes of the mesothelium damaged by peritoneal dialysis fluids and peritonitis. The fibrogenetic cytokine transforming growth factor-beta (TGF-beta) displays variable effects on extracellular matrix remodeling enzymes and their inhibitors depending on tissues and cell lines. We previously found an unexpected stimulating effect of TGF-beta 1 on matrix metalloproteinase-9 in peritoneal mesothelial cells. In this study, we analyzed the effects of TGF-beta 1 on PAI-1 production and deposition in extracellular matrix. We used primary cultured mesothelial cells and a recently established human peritoneal mesothelial cell line (HMrSV5). Cell-associated and secreted plasminogen activators and their inhibitors were detected and characterized by substrate gel zymography. PAI-1 was identified by reverse zymography and by Western blotting, and total PAI-1 was measured by ELISA. Secreted and cell-associated PA activity was measured by its ability to activate plasminogen into plasmin, that is, by the release of paranitroaniline from the plasmin synthetic substrate S-2251. PAI-1 mRNA accumulation was assessed by Northern blot. In vitro nuclear run-on assays were carried out to determine whether TGF-beta 1 had transcriptional effects on PAI-1 expression. Finally, the subcellular distribution of PAI-1 was analyzed by immunofluorescence and by immunogold silver staining. TGF-beta 1 increased PAI-1 antigen in the conditioned media of HMrSV5 cells, in a time- and concentration-dependent manner. This induced a dramatic decrease of free tPA in the cell medium and of membrane-bound uPA, and a parallel increase of high molecular weight PA-PAI complexes. Consequently, secreted and cell-associated plasminogen activator activities were considerably reduced. In primary cultured peritoneal mesothelial cells, TGF-beta 1 also induced PAI-1 secretion and the shift of tPA toward high molecular weight complexes. TGF-beta 1 increased PAI-1 mRNA in a time- and concentration-dependent manner. This effect was at least in part transcriptional since an approximately threefold increase in the rate of PAI-1 gene transcription was observed in nuclei sampled after a four-hour cell exposure to 5 ng/ml TGF-beta 1. Finally, TGF-beta 1 substantially increased the amount of intracellular and matrix-associated PAI-1. These results suggest that excessive TGF-beta 1 stimulated PAI-1 could prevent appropriate peritoneal healing by impairing the degradation of fibrin and of unorganized matrix components, and by interfering with cell migration.