[CANCER RESEARCH 62, 1394 –1400, March 1, 2002] Nuclear Magnetic Resonance-visible Lipids Induced by Cationic Lipophilic Chemotherapeutic Agents Are Accompanied by Increased Lipid Droplet Formation and Damaged Mitochondria 1 Edward J. Delikatny, 2 Wendy A. Cooper, Susan Brammah, Nalayini Sathasivam, and Darryl C. Rideout Department of Cancer Medicine, The University of Sydney, New South Wales 2006, Australia [E. J. D., W. A. C., N. S.]; Central Sydney Area Health Service Electron Microscopy Unit, Concord Hospital, Concord, New South Wales 2139, Australia [S. B.]; and Darryl Rideout Structural Bioinformatics, Inc., San Diego, California [D. C. R.] ABSTRACT Proton nuclear magnetic resonance (NMR) spectroscopy, histological lipid staining, and electron microscopy were used to assess the biochemical and structural changes induced by treating the cultured human breast cell line HBL-100 with the cationic lipophilic phosphonium salts p- (triphenylphosphoniummethyl) benzaldehyde chloride (drug A) and [4-(hydrazinocarboxy)-1-butyl] tris-(4-dimethylaminophenyl) phospho- nium chloride (drug B). The major biochemical change detected by 1 H NMR in drug-treated cells was a significant time- and concentration- dependent increase in lipid acyl chain resonances arising from mobile lipids. The amount of NMR-visible lipid strongly correlated with morpho- metric measurements of oil red O-staining lipid detected in the cytoplasm by light microscopy. Ultrastructural investigations revealed substantial damage to mitochondria and the progressive development of lipid droplets accompanied by end-stage autophagic vacuoles, in the form of densely staining myelinoid bodies, after treatment of HBL-100 cells with drug B at the IC 50 . No apparent increase in acid phosphatase activity was observed using electron microscopy, indicating that the accumulation of phospho- lipids in myelinoid bodies may result from substrate inundation of the lysosome, rather than increased lysosomal activity. These results indicate a potential role for lysosomal lipid catabolism in the formation of NMR- visible lipids in models of cytotoxic insult. INTRODUCTION Tetraphenylphosphonium-based CLPS 3 are novel anticancer agents with potential utility in the treatment of neoplasia. TPP is the parent compound of a series of CLPS, including drug A and the cationic acylhydrazine drug B, which have been shown to selectively inhibit the growth of cell lines derived from a wide variety of carcinomas (breast, colon, pancreas, bladder, and hypopharynx) relative to un- transformed cell lines in vitro [CV-1 monkey kidney epithelial cells; IEC-18 rat ileal epithelial cells (1, 2)]. These drugs are thought to accumulate intracellularly as a function of membrane potential be- cause collapsing the membrane potential with either valinomycin or high extracellular potassium concentrations reduces both the accumu- lation and the cytotoxicity of these agents (1, 2). The high negative plasma membrane potentials characteristic of neoplastic cells are believed to account for the selective accumulation and toxicity of CLPS and other cationic lipophilic compounds against malignant cells (3–5). Studies of various cationic lipophilic compounds suggest that mi- tochondrial toxicity provides the basis for the cytotoxicity of these compounds. Inhibition of mitochondrial respiration has been demon- strated in studies of TPP (2) and other lipophilic cationic compounds, such as MKT-077 (5, 6) and rhodamine 123 (4). Loss of ATP and phosphocreatine and decreases in intracellular pH have also been shown to accompany treatment with cationic lipophilic compounds (7, 8). It is thought that CLPS such as drugs A, B, and TPP as well as rhodamine 123 all have a similar mechanism of action because sen- sitivity profiles determined with a fixed set of cultured cells are closely similar for these agents (2). Although loss of respiratory control has been suggested as the main mechanism of toxicity of lipophilic cationic agents, this may not be the sole cause for cell death. Damage to the mitochondria can cause changes in mitochondrial permeability and the release of apoptotic factors that would ultimately result in cell death (9, 10). Indeed, the full range of biochemical effects these drugs have on cancer cells has not been fully elucidated, and, consequently, it is currently not possible to unequivocally iden- tify the final mediators of CLPS-induced cell death. NMR studies of human breast cancer cells treated with TPP have revealed increases in NMR-visible or mobile lipids that are not readily explained by mitochondrial respiratory enzyme inhibition (11–13). The drug-induced 1 H NMR spectral changes were independent of cellular phenotype because similar concentration-dependent increases in lipid resonances were demonstrated in both the highly tumourigenic DU4475 breast carcinoma and the transformed HBL-100 human breast cell lines. However, the early and persistent detection of these lipid signals in response to TPP suggests that these alterations in lipid pools may reflect some of the primary cellular responses prior to or leading to cell death. Consequently, the aim of the current studies was to use histochemical staining and electron microscopy to identify subcellular alterations that give rise to the 1 H NMR-visible lipids. These observations shed light on the ongoing controversy over the origins of NMR-visible lipid signals by demonstrating cytoplasmic inclusions of neutral lipids accompanied by evidence of lysosomal activity in this model system of cytotoxic insult. MATERIALS AND METHODS Cell Culture. Human HBL-100 breast cells were grown as described previously (11, 13) in RPMI-1640 supplemented with 10% (v/v) fetal bovine serum (batch number 81012053; Trace Biosciences), 2 mML-glutamine, gen- tamicin (0.1% v/v; 40,000 units/ml), and 250 units/liter human insulin and buffered with 26.2 mM sodium bicarbonate using standard culture conditions of 37°C and 5% CO 2 in air. HBL-100 cells grew as an adherent monolayer with a doubling time of approximately 40 h. In all experiments, HBL-100 cells were inoculated at a concentration of 2.5  10 5 cells/ml and treated with drug A or B (or equal volumes of PBS for controls) 5 h after seeding. Cells were treated at concentrations of drug A (300 or 10 M) or drug B (10 and 2 M) that represents the IC 50 and IC 10 for each drug, respectively (11). Cells were harvested and examined using various techniques after 24, 48, or 72 h of drug exposure. Drugs. Two CLPS were used in this study, a cationic aldehyde, drug A (M r = 417) and a cationic acylhydrazine, drug B (M r = 542). Before each experiment, fresh stock at concentrations of 10 mM were prepared in PBS. Received 7/27/01; accepted 1/3/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Australian National Health and Medical Research Council Grant 940630, the Leo and Jenny Leukaemia and Cancer Foundation, and NIH Grant R21 CA79718. This article is dedicated to the memory of Josephine Vandeleur. 2 To whom requests for reprints should be addressed. Present address: Department of Radiology, University of Pennsylvania Medical Center, Room B1, Stellar-Chance Build- ing, 422 Curie Boulevard, Philadelphia, PA 19104-6140. Phone: (215) 898-1805; Fax: (215) 573-2113; E-mail: delikatn@oasis.rad.upenn.edu. 3 The abbreviations used are: CLPS, cationic lipophilic phosphonium salts; drug A, p-(triphenylphosphoniummethyl) benzaldehyde chloride; drug B, [4-(hydrazinocarboxy)- 1-butyl] tris-(4-dimethylaminophenyl) phosphonium chloride; NMR, nuclear magnetic resonance; PABA, p-aminobenzoic acid; TPP, tetraphenylphosphonium chloride. 1394 Research. on October 2, 2015. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from