Transcriptomic Profiling Predicts Multiple Pathways and Molecules Associated With the Metastatic Phenotype of Oral Cancer Cells.

BACKGROUND/AIM: Metastasis to cervical lymph nodes of oral squamous cell carcinoma (OSCC) leads to a poor prognosis. The present study aimed at investigating the pathways and molecules associated with OSCC metastasis. MATERIALS AND METHODS: The transcriptome between HSC-3 cells and their highly metastatic subline, HSC-3-M3 cells, was examined using gene expression microarray. Gene enrichment analyses and Ingenuity Pathway Analysis were performed. Kaplan-Meier plot analysis using a publicly available dataset was conducted to assess whether candidate molecules are prognosticators. RESULTS: A total of 1,018 genes were differentially expressed, and the inflammatory pathway and NF-kB were predicted to be activated in HSC-3-M3 cells. CSF2 was suggested to be an indicator of poor prognosis in head and neck cancers. CONCLUSION: Inflammation and NF-kB may be involved in the metastasis of OSCC, and CSF2 is a promising diagnostic and therapeutic molecule. Moreover, HSC-3-M3 cells are a useful cell line model for studying OSCC progression.

Effect of prostaglandin E2 on urokinase-type plasminogen activator production by human lung fibroblasts.

Human lung fibroblasts are components of stromal tissue and produce various proteins as occasion demands, such as extracellular matrix (ECM) components and proteases. Pulmonary tumour cells produce high levels of prostaglandin E(2) (PGE(2)), which regulates tumour growth and metastasis. Urokinase-type plasminogen activator (uPA) is essential in the degradation of peritumour ECM. Furthermore, uPA is an important protease believed responsible for several tumour characteristics through its activation of certain proteases and growth factors. We hypothesized that the PGE(2) overexpression from tumour cells would have some effect on uPA expression in lung fibroblasts. In this study, the influence of PGE(2) on uPA expression in human lung fibroblasts was investigated using two lines of such fibroblasts. Although the cell surface uPA level was comparable to that of PGE(2) untreated cells, the expression of uPA mRNA and production was increased by the addition of PGE(2) in both lines of fibroblasts. These fibroblasts expressed both the EP(2) and EP(4) PGE(2) receptor mRNAs. Pretreatment with EP(2) and/or EP(4) receptor antagonists reduced the intercellular and cell surface uPA expression of the human lung fibroblasts. These results indicated that there is a relationship between the PGE(2) system and uPA production in human lung fibroblasts operating through EP(2) and/or EP(4) receptor signalling. uPA induced by PGE(2) from stromal fibroblasts surrounding lung tumour thus appears to play an important role through these EP receptors. Inhibition of EPs in tumour tissue might be a useful strategy for anti-metastasis therapy.

The effects of cyclooxygenase2-prostaglandinE2 pathway on Helicobacter pylori-induced urokinase-type plasminogen activator system in the gastric cancer cells.

BACKGROUND: Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in the destruction of the extracellular matrix and basement membrane. The induction of uPA and uPAR in the gastric cancer cells with H. pylori has been demonstrated previously. The involvement of COX-2-PGE2 pathway in the uPA system (uPA and uPAR) expression is unclear. METHODS: Gastric cancer cells (MKN45) were co-cultured with H. pylori standard strain (NCTC11637). The specific inductions of uPA and uPAR mRNA were examined by reverse transcription-polymerase chain reaction amplification. The secreted uPA antigen was measured by ELISA. To evaluate the involvement of COX-2 and PGE2 pathway in H. pylori-induced uPA and uPAR expressions, we examined the effects of COX-2 inhibitor and PGE2 receptor antagonist on H. pylori-induced uPA and uPAR expression in the gastric cancer cells. RESULTS: The expressions of both uPA and uPAR mRNAs in the gastric cancer cells increased obviously (12-fold and 3-fold, respectively) with H. pylori stimulation. The amount of uPA antigen into the culture medium increased dramatically with H. pylori stimulation. The COX-2 expression level in the gastric cancer cells increased remarkably with H. pylori stimulation. H. pylori-induced uPA and uPAR expression levels were suppressed with COX2 inhibitor treatment. The amount of PGE2 antigen into the culture medium increased dramatically 24 hours after H. pylori stimulation. The gastric cancer cells expressed EP2 and EP4 subtypes. EP2 receptor antagonist suppressed the H. pylori-induced uPA and uPAR expressions in the gastric cancer cells. CONCLUSIONS: Our results indicated that COX2-PGE2 pathway may be involved in H. pylori-associated uPA and uPAR induction, and that COX-2 inhibitor or EP2 receptor antagonist may inhibit angiogenesis and tumor invasion via suppression of the uPA system.

Involvement of cyclooxygenase-2--prostaglandin E2 pathway in interleukin-8 production in gastric cancer cells.

Prostaglandin E(2) (PGE(2)) is thought to play an important role in both inflammatory and anti-inflammatory effects. The effect of PGE(2) on the proinflammatory chemokine interleukin-8 (IL-8) in the gastric epithelial cells has not been defined yet. A gastric cancer cell line (MKN45) and primary gastric fibroblasts were cocultured with Helicobacter pylori standard strain (NCTC11637). The expressions of IL-8 and cyclooxygenase 2 (COX-2) mRNA were examined by reverse transcription polymerase chain reaction (RT-PCR) amplification. The amount of IL-8 antigen secreted by the MKN45 cells and gastric fibroblasts was measured by enzyme-linked immunosorbent assay (ELISA). We examined the effects of H pylori stimulation on IL-8 and COX-2 expression levels and the effects of COX-2 inhibitor on H pylori-induced IL-8 production in the MKN45 cells and gastric fibroblasts. Furthermore, we examined the expressions of subtypes of PGE(2) receptors, the effects of arachidonic acid and PGE(2) on IL-8 production, and the effects of PGE(2) on the total cellular cyclic adenosine monophosphate (cAMP) in MKN45 cells. MKN45 cells and gastric fibroblasts expressed IL-8 and COX-2 mRNA under stimulation with H pylori. The MKN45 cells produced IL-8 and PGE(2) antigen into the culture medium with H pylori stimulation, and the production level of IL-8 and PGE(2) antigen decreased significantly with COX-2 inhibitor pretreatment (concentration: 50 muM). On the other hand, the gastric fibroblasts strongly produced IL-8 antigen even in the unstimulated condition, and the amount of IL-8 antigen was not affected by H pylori stimulation and/or COX-2 inhibitor pretreatment. The MKN45 cells expressed IL-8 mRNA and released IL-8 antigen slightly, and the expression level of IL-8 mRNA and the amount of IL-8 antigen increased significantly with PGE(2) treatment in a dose-dependent manner. PGE(2)-induced IL-8 production was inhibited by pretreatment with EP2 and EP4 antagonists. The MKN45 cells expressed EP2 and EP4 subtypes of PGE(2) receptors, and these expression levels were not affected by H pylori stimulation or PGE(2) treatment. The amount of IL-8 antigen increased slightly, but not significantly, with arachidonic acid treatment. PGE(2) treatment for 15 minutes increased the total cellular cAMP in the MKN45 cells. These results suggest that the COX-2-PGE(2) pathway may be involved in IL-8 production in gastric epithelial cells.

Expressions of urokinase-type plasminogen activator, its receptor and plasminogen activator inhibitor-1 in gastric cancer cells and effects of Helicobacter pylori.

OBJECTIVE: Destruction of the extracellular matrix is essential for tumor invasion and metastasis. The relationship between Helicobacter pylori (H. pylori) infection and destruction of the extracellular matrix is not yet clear. Urokinase-type plasminogen activator (uPA) plays an important role in the destruction of the extracellular matrix and basement membrane. Urokinase-type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1(PAI-1) appear to be associated with these processes. To clarify the role of H. pylori infection in the processes of destruction of the extracellular matrix and basement membrane in cancerous tissue, the effect of H. pylori on the expressions of uPA, uPAR and PAI-1 in cancer cells was investigated. MATERIALS AND METHODS: Gastric cancer cell lines (MKN45, KATO-III) were co-cultured with H. pylori standard strain (NCTC11637), cagA-negative strain and clinical isolated strain. The specific inductions of uPA, uPAR and PAI-1 mRNA were examined by reverse transcription-polymerase chain reaction (RT-PCR) amplification. The secreted uPA antigen was measured by enzyme-linked immunosorbent assay (ELISA). To evaluate the role of transcription factor NF-kappaB in uPA and uPAR gene transcription with H. pylori stimulation, the effect of NF-kappaB inhibitor MG132 on H. pylori-induced uPA and uPAR mRNA expression was examined. RESULTS: The expressions of both uPA and uPAR mRNAs in the gastric cancer cell lines (MKN45 and KATO- III) were increased markedly (uPA mRNA; MKN45: 12-fold, KATO-III: 5-fold) (uPAR mRNA; MKN45: 3-fold, KATO-III: 3-fold) with H. pylori NCTC11637 strain stimulation, whereas the expression levels of uPA and uPAR mRNA did not increase with cagA-negative strain stimulation. These cancer cell lines slightly secreted uPA antigen into the culture medium, and the amount of uPA antigen increased dramatically by stimulation with H. pylori NCTC11637 and cagA-positive clinical isolated strains. These gastric cancer cell lines also slightly secreted PAI-1 antigen into the culture medium, and the amount of PAI-1 antigen was not affected by H. pylori NCTC11637 stimulation. H. pylori-induced uPA and uPAR mRNA expressions were strongly down-regulated by pretreatment with MG132 in both cell lines. CONCLUSIONS: The results of this study indicated the possibility that cagA-positive H. pylori may play an important role not only in tissue remodeling, angiogenesis and wound healing but also in the process of degradation of the extracellular matrix breakdown, tumor invasion and metastasis by inducing uPA and uPAR complex in the gastric cancer cells.

Preservation of the characteristics of the cultured human type II alveolar epithelial cells.

The human type II alveolar epithelial cells lost their specific characteristics during cultivation. We examined the ultrastructural and biochemical nature of the human type II cells cultured by two culture systems. To make a physiological alveoli model, the epithelial cells were seeded onto the cell culture insert and allowed contact with the air directly. The cells exposed to the air expressed polarity and immature lamellar bodies in their cytoplasm. Separately, the alveolar epithelial cells were cultured as spheroids to construct the three-dimensional condition. These cells expressed mature morphological characteristics as epithelial cells and lamellar bodies. The expression of the surfactant apoprotein-A (SP-A) and -C (SP-C) mRNA was compared in the cells cultured as a monolayer, the air exposed and the spheroids. SP-A mRNA was detected in all the cultured epithelial cells, but SP-C mRNA, a specific protein for the type II cells, was expressed only in the cells forming spheroids. The expression of uPA, one of the fibrinolytic enzymes, its receptor (uPAR) and its inhibitor-1 (PAI-1) were also examined. The epithelial cells exposed to the air and formed spheroids expressed a larger amount of uPA mRNA than the monolayer, although the amount of uPAR mRNA were comparable in these cells. The amount of PAI-1 mRNA significantly increased when the epithelial cells were exposed to the air. These results indicate that the type II alveolar epithelial cells induced and preserved their specific characteristics by taking the physiological three-dimensional structure, and these characteristics were partially restored by exposure to the air. Those findings suggest that the alveolar epithelial cells should be cultivated in three-dimensional form with contact to the air to regenerate an appropriate alveolar tissue.

Expression of urokinase-type plasminogen activator and its receptor in gastric fibroblasts and effects of nonsteroidal antiinflammatory drugs and prostaglandin.

In acute inflammatory condition, little is known about the expression of the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in the gastric fibroblasts. To clarify the role of human gastric fibroblasts in acute inflammatory conditions such as gastric ulcer, the effects of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha on the expression of uPA and uPAR, which were suggested to be associated with tissue remodeling, in gastric fibroblasts were investigated. The expression level of uPA mRNA and the amount of uPA antigen increased significantly on treatment with each concentration of IL-1beta (1 and 10 ng/ml) and 10 ng/ml TNF-alpha. On the other hand, the amounts of uPA antigen on cell surfaces were not affected significantly by IL-1beta and TNF-alpha stimulation. The expression level of uPAR mRNA increased in a dose-dependent manner on IL-1beta stimulation. The effect of indomethacin on uPA and uPAR expression in these cells was also examined. When gastric fibroblasts were treated with 50 microM indomethacin, the expression level of uPA mRNA decreased significantly, and the amount of uPA antigen in the culture medium and on cell surfaces decreased significantly with indomethacin in a dose-dependent manner. The increased uPAR mRNA expression caused by IL-1beta was reduced to the basal level by treatment with 50 microM indomethacin. Furthermore, we investigated the role of prostaglandin E2 (PGE2), which is suggested to play major roles in acute inflammation of the stomatch, on uPA and uPAR expression in gastric fibroblasts. The expression level of uPAR mRNA and the amount of uPA antigen on cell surfaces increased in a dose-dependent manner on treatment with PGE2 (10 and 50 ng/ml). These results suggest that uPA and uPAR expression in gastric fibroblasts is involved in the regulating system of PGE2 and that NSAIDs may delay healing of gastric mucosal injury in part through suppressing uPA production via inhibition of endogenous PG production.