Jinyi Shao

Prostaglandin E2 Increases Growth and Motility of Colorectal Carcinoma Cells

Chronic use of nonsteroidal anti-inflammatory drugs results in a significant reduction of risk and mortality from colorectal cancer in humans. All of the mechanism(s) by which nonsteroidal anti-inflammatory drugs exert their protective effects are not completely understood, but they are known to inhibit cyclooxygenase activity. The cyclooxygenase enzymes catalyze a key reaction in the conversion of arachidonic acid to prostaglandins, such as prostaglandin E2 (PGE2). Here we demonstrate that PGE2 treatment of LS-174 human colorectal carcinoma cells leads to increased motility and changes in cell shape. The prostaglandin EP4 receptor signaling pathway appears to play a role in transducing signals which regulate these effects. PGE2 treatment results in an activation of phosphatidylinositol 3-kinase/protein kinase B pathway that is required for the PGE2-induced changes in carcinoma cell motility and colony morphology. Our results suggest that PGE2 might enhance the invasive potential of colorectal carcinoma cells via activation of major intracellular signal transduction pathways not previously reported to be regulated by prostaglandins.

Regulation of Constitutive Cyclooxygenase-2 Expression in Colon Carcinoma Cells

Cyclooxygenase-2 (COX-2) is not normally expressed in the human large intestine, but its levels are increased in the majority of human colorectal carcinomas. Here we investigate the regulation of constitutive COX-2 expression and prostaglandin production in human colorectal carcinoma cells. Both COX-2 mRNA and protein were expressed in well differentiated HCA-7, Moser, LS-174, and HT-29 cells, albeit at different levels. COX-2 expression was not detected in several poorly differentiated colon cancer cell lines including DLD-1. Transcriptional regulation played a key role for the expression of COX-2 in human colon carcinoma cells, and both the nuclear factor for interleukin-6 regulatory element and the cAMP-response element were responsible for regulation ofCOX-2 transcription. COX-2 mRNA was more stable in HCA-7 cells than in the other cell lines tested. Both transcriptional and post-transcriptional regulation of COX-2 involved the MAP kinase pathway. Modulation of the Akt/protein kinase B or Rho B signaling pathways altered the levels of COX-2 expression. Furthermore, COX-2 protein is degraded through ubiquitin proteolysis, and its half-life was ∼3.5–8 h. HCA-7 cells produced significant quantities of prostaglandin E2 and other prostaglandins. Moser and LS-174 cells also generated prostaglandins, but levels were significantly lower than that observed in HCA-7 cells.

A selective cyclooxygenase 2 inhibitor suppresses the growth of H-ras-transformed rat intestinal epithelial cells

BACKGROUND & AIMS Constitutive expression of cyclooxygenase 2 (COX-2) has been found in 85% of colorectal cancers. Ras mutations are found in 50% of colorectal adenocarcinomas. The aim of this study was to determine the role of COX-2 in ras-induced transformation in rat intestinal epithelial (RIE) cells. METHODS Cell growth was determined by cell counts. The expression of COX-2 was examined by Northern and Western analyses. For tumorigenicity assays, cells were inoculated into dorsal subcutaneous tissue of athymic nude mice. DNA-fragmentation assays were performed to detect apoptosis. RESULTS The expression of COX-2 was increased in RIE-Ras cells at both messenger RNA (9-fold) and protein (12-fold) levels. Prostaglandin I2 levels were elevated 2.15-fold in RIE-Ras cells. Serum deprivation further increased COX-2 expression 3.8-fold in RIE-Ras cells. Treatment with a selective COX-2 antagonist (SC58125) inhibited the growth of RIE-Ras cells through inhibition of cell proliferation and by induction of apoptosis. SC-58125 treatment reduced the colony formation in Matrigel by 83.0%. Intraperitoneal administration of SC-58125 suppressed RIE-Ras tumor growth in nude mice by 60.3% in 4 weeks. SC-58125 treatment also induced apoptosis in RIE-Ras cells as indicated by increased DNA fragmentation. CONCLUSIONS Overexpression of COX-2 may contribute to tumorigenicity of ras-transformed intestinal epithelial cells. Selective inhibition of COX-2 activity inhibits growth of ras-transformed intestinal epithelial cells and induces apoptosis.

Induction of cyclooxygenase-2 by activated Ha-ras oncogene in Rat-1 fibroblasts and the role of mitogen-activated protein kinase pathway.

Elevated cyclooxygenase-2 (COX-2) expression and activity have been observed in several different transformed cell types that express mutated ras genes. To investigate the mechanism of increased COX-2 expression following Ras-mediated transformation, Rat-1:iRas cell line was transfected with an Ha-Ras Val-12 cDNA expression vector that is under the transcriptional control of the lac operon and is inducible with isopropyl-1-thio-β-d-galactopyranoside (IPTG). IPTG treatment caused parallel increases in the levels of Ha-Ras and COX-2 proteins in Rat-1:iRas cells. The increased expression of COX-2 was accompanied by increased prostaglandin E2 production. Selective inhibition of COX-2 activity suppressed the production of prostaglandin E2 by >90% but did not alter the progress of the morphological transformation. The level of COX-2 mRNA was up-regulated by activated Ha-Ras. Induction of Ras increased the transcription of COX-2 by 44.3 ± 10.1% and increased the half-life of COX-2 mRNA by ∼3.5-fold. A specific mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) inhibitor (PD 98059) caused a delay in both the activation of ERK1/2 and the induction of COX-2 in IPTG-induced Rat-1:iRas cells. Inhibition of ERK activity by PD 98059 also suppressed the induction of COX-2 by epidermal growth factor in intestinal epithelial cells and significantly reduced the expression of COX-2 in Ha-Ras-transformed rat intestinal epithelial cells. ERK activity appears to be required for induction of COX-2 by Ras.

Akt/PKB Activity Is Required for Ha-Ras-mediated Transformation of Intestinal Epithelial Cells

Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) is thought to serve as an oncogenic signaling pathway which can be activated by Ras. The role of PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells is currently not clear. Here we demonstrate that inducible expression of oncogenic Ha-Ras results in activation of PKB/Akt in rat intestinal epithelial cells (RIE-iHa-Ras), which was blocked by treatment with inhibitors of PI3K activity. The PI3K inhibitor, LY-294002, partially reversed the morphological transformation induced by Ha-Ras and resulted in a modest stimulation of apoptosis. The most pronounced phenotypic alteration following inhibition of PI3K was induction of G1phase cell cycle arrest. LY-294002 blocked the Ha-Ras-induced expression of cyclin D1, cyclin-dependent kinase (CDK) 2, and increased the levels of p27 kip . Both LY-294002 and wortmannin significantly reduced anchorage-independent growth of RIE-iHa-Ras cells. Forced expression of both the constitutively active forms of Raf (ΔRaf-22W or Raf BXB) and Akt (Akt-myr) resulted in transformation of RIE cells that was not achieved by transfection with either the Raf mutant construct or Akt-myr alone. These findings delineate an important role for PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells.

Oncogenic Ras-mediated Cell Growth Arrest and Apoptosis are Associated with Increased Ubiquitin-dependent Cyclin D1 Degradation

The cellular responses to activated Ras vary depending on cell type. Normal cells are often induced into pathways that lead to cell growth arrest, senescence, and/or apoptosis in response to activated Ras expression. These are important protective anti-tumorigenic responses that restrict the propagation of cells bearing activated oncogenes. Here we show that induction of Ha-RasVal-12 in Rat-1 fibroblasts resulted in G1 growth arrest and apoptosis with loss of viable cells that is accompanied by a marked decrease in cyclin D1 levels via increased ubiquitin-proteasome-dependent cyclin D1 turnover. This is in contrast with a rat intestinal epithelial cell line in which induction of Ha-RasVal-12 results in transformation associated with sustained proliferation and increased levels of cyclin D1, that is not accompanied by anoikis or apoptosis. Expression of the cyclin D1 mutant (T286A) that contains an alanine for threonine 286 substitution and is resistant to ubiquitin-proteasome degradation in the Ha-RasVal-12 expressing Rat-1 cells resulted in a sustained transformed phenotype with no accumulation of cells in G1. Inhibition of mitogen-activated protein kinase (MEK1/2) pathway partially reversed the Ras-mediated decrease in cyclin D1. Induction of Ha-RasVal-12 resulted in activation of Akt kinase and inactivation of glycogen-synthase-3β kinase that are associated with reduction of cyclin D1 protein. These results suggest that Ras-mediated cyclin D1 degradation in Rat-1 cells appears to be partially dependent on activation of mitogen-activated protein kinase pathway and independent of glycogen-synthase-3β kinase pathway.

NF-κB is required for H- ras oncogene induced abnormal cell proliferation and tumorigenesis

Oncogenic mutations in ras lead to constitutive activation of downstream signaling pathways that modulate the activities of transcription factors. In turn, these factors control the expression of a subset of genes responsible for neoplastic cell transformation. Recent studies suggest that transcription factor NF-κB contributes to cell transformation by inhibiting the cell death signal activated by oncogenic Ras. In this study, inhibition of NF-κB activity by forced expression of a super-repressor form of IκBα, the major inhibitor of NF-κB, markedly decreased the growth rate, saturation density and tumorigenicity of oncogenic H-Ras transformed rat embryo fibroblasts. Such clonally isolated cells overexpressing IκBα super-repressor not only were viable but also exhibited no sign of spontaneous apoptosis. Inhibition of NF-κB in these cells was functionally demonstrated by both the loss of cytokine induced DNA binding activity and a profoundly increased sensitivity to cell death in response to TNF-α treatment. In contrast, inhibition of NF-κB activity in non-transformed fibroblasts had minimal effect on growth, but rendered the cells resistant to a subsequent transformation by H-ras oncogene. Similar results were also obtained with rat intestinal epithelial cells harboring an inducible ras oncogene. Taken together, these findings suggest that NF-κB activity is essential for abnormal cell proliferation and tumorigenicity activated by the ras oncogene and highlight an alternative functional role for NF-κB in oncogenic Ras-mediated cell transformation that is distinct from its anti-apoptotic activity.

Cyclooxygenase-2 alters transforming growth factor-β1 response during intestinal tumorigenesis

BACKGROUND Recent investigation suggests that cyclooxygenase-2 plays an important role in colorectal carcinogenesis. Transforming growth factor-beta1 (TGF-beta 1) is one of the most potent stimulators of cyclooxygenase-2 expression. A key step in intestinal tumorigenesis involves alteration of the normal cellular response to TGF-beta 1. We have hypothesized that overexpression of cyclooxygenase-2 alters intestinal epithelial response to TGF-beta 1. METHODS RIE-1 cells were stably transfected with rat cyclooxygenase-2 complementary DNA in either the sense (RIE-S) or antisense (RIE-AS) orientation. Tumor cell invasion was assessed with a modified Boyden collagen type I invasion assay in the presence of TGF-beta 1, antibody to urokinase plasminogen activator (uPA), or the selective cyclooxygenase-2 inhibitor SC-58125. Expression of uPA, uPA receptor, and plasminogen activator inhibitor-1 were determined by Western blot and enzyme-linked immunosorbent assay. RESULTS RIE-1 and RIE-AS did not invade although RIE-S cells were minimally invasive at baseline. TGF-beta 1 had no effect on RIE-1 or RIE-AS invasion; however, TGF-beta 1 significantly upregulated RIE-S cell invasion. All 3 RIE cell lines produce minimal uPA under basal conditions. TGF-beta 1 upregulated uPA production only in the RIE-S cells. Both antibody to uPA and SC-58125 reversed TGF-beta-mediated RIE-S cell invasion. SC-58125 inhibited TGF-beta-mediated RIE-S uPA production. CONCLUSIONS These results demonstrate that overexpression of cyclooxygenase-2 alters intestinal epithelial response to TGF-beta 1, which may be a mechanism by which cyclooxygenase-2 promotes colon carcinogenesis.

Transforming growth factor β (TGF-β) promotes invasiveness of ras-transformed rat intestinal epithelial (RIE) cells

Methods: An inducible form of H-Ras (V12) was transfected into the RIE cells (RIE:iRas) and was inducible by treatment with isopropyl-1thio-b-D-galactopyranoside (IPTG). Separately, The RIE:iRas cells were also transfected with a dominant negative form of the TbRII (DNIIR). The effect of TGF-b1 on cell invasiveness was examined in a Boyden chamber with type I collagen-coated transwell inserts. Cells were seeded on the inserts after the treatment with IPTG for 0168 h and stimulated with 5 ng/ml TGF-b1 for 24 h. TbRII expression was assessed by immunoblotting and immunofluorescence.