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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 30, 2005 as doi:10.1096/fj.05-4331fje. |
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Departments of
* Pathology and Laboratory Medicine,
Surgery,
Medicine, and
Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA;
|| Department of Pathology, Kanazawa Medical University, Ishikawa, Japan;
¶ Department of Pathology, South Carolina Cancer Center, University of South Carolina, Columbia, South Carolina, USA; and
# Chemoprevention and Nutritional Carcinogenesis Program, Institute for Cancer Prevention, Valhalla, New York, USA
1 Correspondence: Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 165 Ashley Ave., Suite 309, Charleston, SC 29425, USA. E-mail: kawamori@musc.edu; or zhoud{at}musc.edu
SPECIFIC AIMS
The sphingosine kinase 1 (SK1)/sphingosine 1-phosphate (S1P) pathway has been implicated in cell growth, apoptosis, angiogenesis, and induction of cyclooxygenase-2 (COX-2). Increased expression of COX-2 and production of PGE2 have been implicated in colon carcinogenesis. Therefore, we examined the potential role of SK1 in COX-2 expression during colon carcinogenesis. The expression of SK1 and COX-2 in colon tumors induced by azoxymethane (AOM) in male F344 rats was examined by immunohistochemical and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses. The existence of a potential mechanistic link between SK1 and COX-2 expression in colon carcinogenesis was examined by analysis of COX-2 expression and PGE2 production in HT-29 human colon cancer cells after SK1 down-regulation by small interfering RNA (siRNA) and in RIE-1 rat normal intestinal epithelial cells after SK1 overexpression by gene transfection.
PRINCIPAL FINDINGS
1. The expression of SK1 and COX-2 is up-regulated in colon cancer tissues induced by AOM in male F344 rats
SK1 was positively stained in 21 of 28 (75%) of colon adenocarcinomas including 13 of 17 (76%) well-differentiated, 5 of 6 (83%) moderately differentiated, and 3 of 5 (60%) poorly differentiated adenocarcinomas, whereas no positive SK1 staining was observed in normal colon mucosa. The immunoreactivity of both SK1 and COX-2 exhibited a similar cellular distribution. All COX-2 positive adenocarcinomas showed SK1 positive staining. The increase in SK1 and COX-2 expression in colon adenocarcinoma was confirmed by the analysis of SK1 and COX-2 mRNA expression using real-time RT-PCR (P<0.001 and P<0.01 vs. normal mucosa, respectively).
2. Down-regulation of SK1 using siRNA inhibited COX-2 expression and PGE2 production induced by pro-inflammatory cytokines in HT-29 human colon cancer cells
Stimulation of HT-29 cells with S1P but not with ceramide and sphingosine increased COX-2 expression and PGE2 production
3. Overexpression of SK1 in RIE-1 rat normal intestinal epithelial cells induces COX-2 expression
These in vitro studies provide additional support to our hypothesis that the SK1/S1P pathway may play an important role in colon carcinogenesis in part by regulating the expression of COX-2 and production of PGE2 in colon cancer cells.
CONCLUSIONS AND SIGNIFICANCE
AOM is a well-characterized carcinogen that induces colon cancer in rodents following its intraperitoneal or subcutaneous injection. The AOM-induced rodent colon cancer provides a well-controlled experimental model of colon carcinogenesis, as the induction of colon cancer by AOM in rats mimics the process of human colon carcinogenesis (e.g., via a chronologically progressive manner from aberrant crypt foci, adenoma to adenocarcinoma). The results from this study showed that AOM-induced colon carcinogenesis is associated with an up-regulation of SK1 expression, particularly in a tumor progressive manner. Minimal SK1 immunostaining was observed in normal colon mucosa from either normal rats or adjacent to colon cancer tissue. A low percentage (<22%) of colon adenomas showed weak staining for SK1 (Fig. 1
). In contrast, 75% (21 of 28) of colon adenocarcinomas exhibited intense SK1 immunostaining (Fig. 1)
. The SK1 positive staining was observed primarily in colonic adenocarcinoma cells and localized in cytoplasm, especially in the perinuclear regions. Some stromal cells including macrophages, fibroblasts, and vascular endothelial cells were also stained positive for SK1. The immunostaining of SK1 and COX-2 exhibited a similar cellular distribution. All COX-2 positive colon adenocarcinomas showed SK1 positive staining. The increase in SK1 and COX-2 expression in rat colon adenocarcinoma was confirmed by the analysis of SK1 and COX-2 mRNA expression using real-time RT-PCR (P<0.001 and P<0.01 vs. normal mucosa, respectively). These results demonstrate that SK1 is up-regulated in association with COX-2 during colon carcinogenesis induced by AOM. We hypothesize that SK1 may play an important role in colon carcinogenesis, especially during transformation from adenoma to adenocarcinoma, in part by regulating COX-2 expression.
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To test this hypothesis, we analyzed COX-2 expression and PGE2 production in HT-29 cells after the cells were treated with SK1 siRNA to down-regulate the expression of SK1 or stimulated with various sphingolipids. It was found that down-regulation of SK1 expression by SK1 specific siRNA significantly inhibited TNF
- or IL-1ß-induced COX-2 expression and PGE2 production in HT-29 cells (Fig. 2
). In contrast, down-regulation of SK2 by siRNA had no effect on the induction of PGE2 production. Incubation of HT-29 cells with exogenous S1P stimulated COX-2 expression and PGE2 production, whereas incubation of the cells with ceramide and sphingosine had no effects on PGE2 production in HT-29 cells. These results suggest that SK1, not SK2, mediates the induction of COX-2/PGE2 in HT-29 cells via S1P. This suggestion was further supported by the finding that RIE-1 cells transfected with hSK1 expressed an increased level of COX-2 expression as compared with vector transfected cells.
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Seven adenocarcinomas including 1 (1 of 17) well, 3 (3 of 6) moderately, and 3 (3 of 5) poorly differentiated adenocarcinomas showed SK1 overexpression but no significant COX-2 immunostaining. Since SK1 has been implicated in cell transformation and tumor growth via S1P-mediated cell proliferation, anti-apoptosis and angiogenesis, it has yet to be determined if SK1 can promote colon carcinogenesis via a COX-2 independent mechanism. In addition, the following questions are still unsolved: 1) which S1P receptors are involved in these pathways; and 2) what signal transduction pathways connect these two pathways.
Our results suggest that SK1 may play a key role in colon carcinogenesis. It has the potential to be used as a novel biomarker for colon malignancy and target for the development of mechanisms-based chemopreventive and therapeutic strategies against colon cancer development.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4331fje;
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