Growth compensatory role of sulindac sulfide-induced thrombospondin-1 linked with ERK1/2 and RhoA GTPase signaling pathways
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are used to relieve pain and inflammation but have also received considerable attention because of their protective effects against human cancer (Rao et al., 1995, Reddy et al., 1996). NSAIDs inhibit enzymatic activity of cyclooxygenases (COX) whose products prostaglandins are known to inhibit apoptosis, stimulate tumor growth, and enhance angiogenesis, tumor cell invasion and metastasis in many cancer models (Connolly et al., 2002, Yoshida et al., 2003). NSAIDs, by inhibiting COX activity, enhance apoptosis and exert anti-metastatic and anti-angiogenesis effects, thereby inhibiting tumor growth. However, some lines of evidences suggested that NSAIDs modulate tumor growth by cycloooxygenase-independent signaling pathways (Zhang et al., 1999, Tegeder et al., 2001).
In terms of COX-independent pathway, evidence has been presented that NSAIDs suppress tumor cell invasion which was mediated by anti-metastatic factor thrombospondin-1 (TSP-1) via early growth response gene product-1 (EGR-1) (Moon et al., 2005). TSP-1 is a high-molecular-weight, multifunctional glycoprotein, which is synthesized and secreted by various cell types such as fibroblasts, smooth muscle cells, monocytes, macrophages, osteoblasts, and neoplastic cells (Adams, 2001, Adams and Lawler, 2004). However, the role of TSP-1 in tumor progression is very complex and controversial. Although the reduced expression of TSP-1 is known to correlate with a poor prognosis in cancer patients as well as animal tumor models (Yamaguchi et al., 2002), released TSP-1 is also capable of fostering the metastatic spread, angiogenesis as well as tumor cell survival in some progressive tumors such as invasive cervical cancer, pancreatic and ductal carcinoma, and breast carcinomas (Clezardin et al., 1993, Roberts, 1996). In addition to the effects on the tumor cell growth and metastasis, TSP-1 can promote the wound healing process in response to the external stresses as well (DiPietro et al., 1996, Anilkumar et al., 2002).
Generally speaking on the mitogenic signals, ERK1/2 MAP kinase signals function as the cytoprotective roles and maintain the cellular homeostasis by enhancing the cellular growth and proliferation after diverse injuries (Kohno and Pouyssegur, 2003, Sun and Sinicrope, 2005). However, whereas ERK1/2 signals can promote the recovery of the injured tissue, sometimes chronic stimulation of mitogenic signals can provide the tumor cells with the growth advantage over the normal tissues. Recently, we reported that the human intestinal epithelial cells can develop compensatory protection mechanisms after NSAID exposure (Moon et al., 2007). Mitogenic signals associated with ERK1/2 MAP kinase and early growth response gene 1 (EGR-1) during gastrointestinal injury mediated the epithelium recovery processes after NSAID exposure. Epithelial toxic stresses have been known to stimulate the compensatory signals of wound healing and tissue reconstitution including growth factor-activated ras-associated MAP kinase pathway and other small GTP-binding protein family signals as well (Guo et al., 2003, Tarnawski, 2005). Rho small GTPases such as RhoA, CDC42, and Rac1 GTPas are a multimember family of RAS small GTPase, which are also mitogenic and tumorigeneic when over-expressed in the intestinal epithelium.
Based on the hypothesis that TSP-1 can mediate the cellular survival after NSAID treatment, we now evaluated the cellular growth in terms of TSP-1 production and its induction signaling pathways. Induction of TSP-1 by sulindac sulfide was mediated by mitogenic signals and the TSP-1 had growth modulating function in sulindac sulfide-treated HCT-116 cells. These findings provide the insight into the possible compensatory mechanism under the cytotoxic condition by chemopreventive NSAID intake. Although there are chances that NSIAD-treated tumor cells can survive the chemopreventive action, the growth-compensatory actions of TSP-1 may contribute to the reconstitution after NSAID-mediated cytotoxic insults.
Section snippets
Cell culture conditions and reagents
HCT-116 colonic adenocarcinoma cells were purchased from American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 (Welgene bioscience, Daeu, Korea) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Sigma Chemical Company, St. Louis, MO), 50 unit/ml penicillin (Sigma Chemical Company), and 50 µg/ml streptomycin (Sigma). During incubation with chemicals, cells were cultured in serum-free RPMI 1640 media. All the chemicals were purchased from Sigma.
Cellular viability assay
Induction of human thrombospondin-1 gene expression and its effects on the cellular proliferation in presence of NSAIDs
HCT-116 cells were selected to investigate the effect of NSAIDs because they serve as a well established cell line model for the study of chemopreventive effects on colon cancer in cyclooxygenase (COX)-2-independent way (Baek et al., 2002, Maier et al., 2005). We first needed to determine whether COX inhibitors would increase the expression of thrombospondin-1 in HCT-1116 cells as observed in other cancer cell line according to our previous report (Moon et al., 2005). HCT-116 cells were treated
Discussion
This study investigated the influence of sulindac sulfide on the induction of TSP-1 in the colonic HCT-116 cells and demonstrated that the enhanced TSP-1 production was associated with survival in sulindac sulfide-treated cells. The TSP-1 gene induction mechanism was linked with ERK1/2 and RhoA GTPase signaling pathways (Fig. 5). TSP-1 has been generally associated with both the tumor progression and the tumor suppression. Many different and sometimes opposite functions of TSP-1 have been
Acknowledgment
This work was supported by Medical Research Institute Grant (2007-11), Pusan National University.
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Yuseok Moon and Jeung Il Kim contributed equally to this study.