Available online http://breast-cancer-research.com/content/7/5/R645 ResearchVol 7 No 5 article Open Access Anticancer properties of propofol-docosahexaenoate and propofol-eicosapentaenoate on breast cancer cells Rafat A Siddiqui1,2,3, Mustapha Zerouga1, Min Wu1, Alicia Castillo1, Kevin Harvey1, Gary P Zaloga1,2 and William Stillwell1,3 1Methodist Research Institute, Clarian Health Partners, Indianapolis, IN, USA 2Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA 3Department of Biology, Indiana University-Purdue University, Indianapolis, IN, USA Corresponding author: Rafat A Siddiqui, [email protected] Received: 23 Aug 2004 Revisions requested: 24 Nov 2004 Revisions received: 21 Jan 2005 Accepted: 8 Apr 2005 Published: 7 Jun 2005 Breast Cancer Research 2005, 7:R645-R654 (DOI 10.1186/bcr1036) This article is online at: http://breast-cancer-research.com/content/7/5/R645 © 2005 Siddiqui et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Introduction Epidemiological evidence strongly links fish oil, Methods The conjugates linking an omega-3 fatty acid, either which is rich in docosahexaenoic acid (DHA) and DHA or EPA, with propofol were synthesized and tested for their eicosapentaenoic acid (EPA), with low incidences of several effects on migration, adhesion and apoptosis on MDA-MB-231 types of cancer. The inhibitory effects of omega-3 breast cancer cells. polyunsaturated fatty acids on cancer development and Results At low concentrations (25 µM), DHA, EPA or propofol progression are supported by studies with cultured cells and alone or in combination had minimal effect on cell adhesion to animal models. Propofol (2,6-diisopropylphenol) is the most vitronectin, cell migration against serum and the induction of extensively used general anesthetic–sedative agent employed apoptosis (only 5 to 15% of the cells became apoptotic). In today and is nontoxic to humans at high levels (50 µg/ml). contrast, the propofol-DHA or propofol-EPA conjugates Clinically relevant concentrations of propofol (3 to 8 µg/ml; 20 significantly inhibited cell adhesion (15 to 30%) and migration to 50 µM) have also been reported to have anticancer activities. (about 50%) and induced apoptosis (about 40%) in breast The present study describes the synthesis, purification, cancer cells. characterization and evaluation of two novel anticancer Conclusion These results suggest that the novel propofol-DHA conjugates, propofol-docosahexaenoate (propofol-DHA) and and propofol-EPA conjugates reported here may be useful for propofol-eicosapentaenoate (propofol-EPA). the treatment of breast cancer. Introduction lines are breast cancer cells [7,8,15-17]. Several of these Omega-3 polyunsaturated long-chain fatty acids (ω-3 PUFAs) reports indicate that at low concentrations ω-3 PUFAs (10 to have been documented to inhibit or even prevent cancer. Epi- 100 µM) produce anticancer effects through the induction of demiological evidence strongly links fish oil (rich in docosahex- apoptosis rather than via cytotoxicity. Propofol (2,6 diisopro- aenoic acid (DHA) and eicosapentaenoic acid (EPA)) with low pylphenol) is the most extensively used general anesthetic- incidences of several types of cancer [1-6]. The inhibitory sedative agent employed today [18,19] and is nontoxic to effects of ω-3 PUFAs on cancer development and progression humans at high levels (3 to 8 µg/ml; 20 to 50 µM) [20]. Pro- are supported by studies using cultured cells and animal mod- pofol is a potent antioxidant [21-24] and has been shown to els [7-14]. However, the mechanisms by which ω-3 PUFAs stimulate protein kinase C [25,26], inhibit calcium entry in inhibit cancer remain unclear. Of particular interest are the muscle cells [27] and increase the calcium sensitivity of myo- many reports demonstrating anticancer properties of ω-3 filaments in ventricular myocytes [28]. Propofol is also a potent PUFAs on the growth and survival of various cancer cell lines direct vasodilator and bronchodilator and has recently been cultured in vitro. Included in the growing list of affected cell shown to possess anti-inflammatory and antiseizure BHT = 2,6-di-tert-butyl-4-methylphenol; Chl = chlorambucil; DEVD-AFC = Asp-Glu-Val-Asp-fluoromethylketone-aminotrifluoromethylcoumarin conju- gate; DHA = docosahexaenoic acid; DMEM = Dulbecco's modified Eagle's medium; EPA = eicosapentaenoic acid; GC = gas chromatography; ω- 3 PUFAs = omega-3 polyunsaturated fatty acids; TLC = thin-layer chromatography. R645 Breast Cancer Research Vol 7 No 5 Siddiqui et al. properties. Although the exact signaling systems responsible clohexylcarbodiimide, 2,6-di-tert-butyl-4-methylphenol (BHT), for these effects are unclear, it does indicate that propofol 4-(dimethyl amino)pyridine, hematoxylin, crystal violet and all alters signaling pathways within cells. other reagents were from Sigma Chemical Co. (St Louis, MO). Although most studies concerning the mode of action of pro- Synthesis and purification of propofol-DHA and pofol have concentrated on its action as an anesthetic, there propofol-EPA are a few reports indicating that this compound may also affect Although only propofol-DHA synthesis is described here, the cellular processes related to cancer. Clinically relevant con- procedure is analogous for the propofol-EPA conjugate. Syn- centrations of propofol (3 to 8 µg/ml) were reported to thesis was performed in two steps. First, docosahexaenoic decrease the metastatic potential of human cancer cells, acid anhydride (DHA-anhydride) was synthesized, followed by including HeLa, HT1080, HOS and RPMI-7951 cells [29]. In its esterification to propofol. Synthesis of the conjugate was addition, continuous infusion of propofol inhibited pulmonary performed under reduced light and under nitrogen to minimize metastasis of murine osteosarcoma (LM8) cells in mice auto-oxidation. In brief, DHA or EPA (0.300 mmol), a coupling through the modulation of Rho A [29]. In HL-60 human promy- reagent, N,N-dicyclohexylcarbodiimide (0.45 mmol), and an elocytic leukemia cells, propofol was shown to inhibit growth antioxidant, BHT (1.5 µM), were dissolved in 5 ml of chloro- and induce the formation of apoptotic bodies, increase DNA form and the reaction was stirred for 60 min at room tempera- fragmentation and laddering, activate caspase-3, caspase-6, ture (23–25°C). To this mixture, propofol (0.29 mmol) and 4- caspase-8 and caspase-9, and induce the cytosolic release of (dimethyl amino)pyridine (0.152 mmol) were added. The mix- cytochrome c [30]. The conclusion from these studies was ture was stirred for a period of 12 hours; the suspension was that propofol induces apoptosis through both a cell-surface then filtered, washed with light petroleum (38.3–53.2°C) and death receptor (extrinsic) and the mitochondrial (intrinsic) subjected to purification on analytical thin-layer plates (silica pathway. These studies suggest that propofol possesses anti- gel, 60 A, 0.2 mm thickness; Alltech Associates, Inc., Deer- cancer properties in addition to its sedative effects. field, IL). The plates were developed in a solvent mixture of petroleum ether and ethyl acetate (92:8, v/v) and the products The omega-3 PUFAs DHA and EPA are natural nontoxic food were revealed with iodine vapor. The spots were compared substances that have interesting anticancer properties. Propo- with authentic DHA, propofol, BHT, pyridine and N,N-dicy- fol is a widely employed nontoxic anesthetic that also has anti- clohexylcarbodiimide and the spot corresponding to the new cancer properties. Although several preparations of propofol compound was scraped, suspended in chloroform/methanol with lipid mixtures are available for anesthetic usage (Astra- (20:80, v/v), passed through a glass filter and stored at -70°C. Zeneca, Wilmington, DE), to our knowledge propofol has not The propofol-DHA and propofol-EPA conjugates were charac- previously been covalently conjugated with fatty acids. The terized as described below. purpose of this study was to synthesize and investigate novel compounds composed of DHA or EPA conjugated to propo- Characterization of propofol-DHA fol. The conjugates were tested for their ability to inhibit breast Characterization of the propofol-DHA conjugate was per- cancer cell migration, alter adhesion to the matrix protein vit- formed by a combination of techniques. First, the presence of ronectin and induce apoptosis. The results indicate that these propofol in the conjugate was assessed by UV spectroscopy novel conjugates might represent a new class of anticancer with an SLM Aminco 3000 spectrophotometer. The conjugate agents. was dissolved in ethanol and the absorption spectra was measured from 200 to 600 nm. Next, the new compound was Materials and methods hydrolyzed and methylated in 3 M methanolic HCl [31]. The Materials product of the reaction was extracted with hexane/water (2:1, MDA-MB-231 breast cancer cells were purchased from the v/v) and the organic phase was analyzed with a Shimatzu 17A American Type Culture Collection (ATCC; Manassas, VA). gas chromatograph with a 0.25 mm × 30 m Stabilwax capillary The Vybrant Apoptosis assay kit was from Molecular Probes column (Resteck, Belfont, PA). The temperature ramp was (Eugene, OR), and DMEM, penicillin, streptomycin and 180 to 240°C at 3°C/min (hold 3 min), followed by 240 to glutamine were from Invitrogen Corporation (Grand Island, 245°C at 1°C/min. To determine the presence of a possible NY). Fetal bovine serum was from BioWhittaker (Walkersville, ester group formed in the new product, an infrared spectrum MD). Transwell chemotaxis chamber plates were from Corning was taken on a Perkin Elmer/2000FT-IR spectrometer. A thin Incorporated (Corning, NY). Cytomatrix Human Vitronectin- film of the product was made by evaporation from chloroform. Coated Strips were from Chemicon International, Inc. (Temec- Finally, the molecular mass of the product was determined by ula, CA).