Inhibition of cell transformation by sulindac sulfide is confined to specific oncogenic pathways
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are routinely prescribed to reduce swelling and pain in patients suffering from ailments such as arthritis and headache. These compounds are thought to exert their effects by interfering with the cyclooxygenase pathway, thus inhibiting the synthesis of pro-inflammatory prostaglandins [1]. In addition to their prescribed role, NSAIDs have been shown to reduce the risk of developing colorectal cancer (CRC) and adenoma [2], [3], [4]. Of particular interest, sulindac, a specific NSAID, has been shown to have a significant chemopreventive effect in reducing the size and number of adenomatous polyps in patients afflicted with familial adenomatous polyposis (FAP), an autosomal dominant genetic disorder characterized by the development of hundreds of colorectal adenomas during the second to third decade of life [5], [6]. Sulindac is a pro-drug when administered orally and is metabolized to sulindac sulfide and sulindac sulfone by colonic bacteria [7]. Sulindac sulfide is considered the active metabolite and exerts its function by non-selectively inhibiting cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) [7], [8]. COX-2 is overexpressed in human colorectal cancers [9], and its inactivation is associated with decreased carcinogenesis [10]. Although much less active in inhibiting COX activities, sulindac sulfone has also been shown to induce apoptosis and can inhibit mammary carcinogenesis [11], [12].
While its molecular mechanisms remain largely unknown, substantial evidence exists that the antiproliferative and antineoplastic effect of sulindac is not solely dependent on eicosanoid metabolism [13]. Colon cancer cells lacking COX activity are inhibited to the same degree as colon cancer cells with COX activity [14]. Furthermore, the addition of prostaglandins does not rescue COX-producing colon cancer cells from sulindac-associated growth arrest [15]. Other signal transduction pathways have been identified as possible targets of sulindac. Experiments have shown that sulindac sulfide can directly affect Ras-mediated signal transduction by inhibiting Ras/Raf dependent transactivation [16]. In addition, sulindac can inhibit the activation of the nuclear factor-κB pathway (NF-κB) [17]. Finally, studies have identified the peroxisome proliferator-activated receptor δ as a target for sulindac sulfide [18].
The present study examines the effect of sulindac sulfide on cellular transformation caused by several established oncogene products in an attempt to specific signaling pathways targeted by it. The results indicate that sulindac sulfide exclusively inhibits Ha-Ras-mediated transformation of four oncogenes tested, despite the shared targets that exist among the other signal transduction pathways. These observations illustrate the specific nature by which sulindac sulfide exerts its antineoplastic effect and open the possibility of developing novel therapeutic agents that target the sulindac sulfide-resistant pathways of oncogenesis.
Section snippets
Drugs and plasmid constructs
Sulindac sulfide was purchased from Biomol (Plymouth Meeting, PA). The drug was dissolved in dimethyl sulfoxide (DMSO) in stock solutions of 250 μM. Expression plasmids containing Ha-Ras, v-Src, Gα12, and Gα13 were kindly provided by Dr Raul Urrutia (Mayo Clinic, Rochester, MN) [19].
Cell proliferation/cytotoxicity assay
Assays were performed as described by the CellTiter 96® Aqueous One Solution Cell Proliferation Assay Protocol (Promega, Madison, WI). NIH3T3 fibroblasts (obtained from American Type Culture Collection) were
Results
We first determined the optimal concentration of sulindac sulfide with which to conduct the transformation experiments. This was accomplished by incubating NIH3T3 fibroblasts in the presence of increasing concentrations of sulindac sulfide and measuring their proliferative potential by a cell proliferation assay (Fig. 1). As shown, sulindac sulfide did not begin to inhibit cell growth until it reached a concentration of 200 μM. However, in 100-mm tissue culture dishes, treatments with over 100
Discussion
In this study, we investigated whether sulindac sulfide could protect against neoplastic transformation elicited by Ha-Ras, Gα12, Gα13, and v-Src. Strong evidence exists that sulindac sulfide directly inhibits p21Ras activation of Raf and Ha-Ras-induced foci formation [16]. Therefore, we chose to examine those oncoproteins that are linked to eicosanoid metabolism or have been shown to modulate the activity of Ras or its targets. Effectors of Gα12 and Gα13 activate Ras and other monomeric G
Acknowledgements
We would like to thank Dr Raul Urrutia for providing the plasmids used in this study. This work is supported by National Institutes of Health grants DK52230 and CA84197.
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