Apoptotic and Necrotic Cell Death in Leukemic HL60 Cells: a Metabolomics Study Using High Resolution Nuclear Magnetic Resonance (1H-NMR) Spectroscopy

Apoptosis and necrosis represent two diverse mechanisms by which cells die. Apoptosis is triggered by different stimuli and plays a key role in the cell deletion that occurs during embryogenesis, physiological tissue turnover in adults, tissue atrophy and tumor regression (1). It is characterized by cell shrinkage, membrane blebbing, deep condensation and marginalization of nuclear chromatin, formation of apoptotic bodies and fragmentation of DNA at internucleosomal linker sites giving rise to multiples of 180-200 base pairs. Apoptosis can occur after exposure to many anticancer treatments including ionizing radiation and chemotherapeutic drugs (2-3). In particular, ionizing radiation is widely used in the treatment of cancer, and apoptosis has been widely described to be the major mechanism of radiation-induced cell death. As far as chemotherapeutic cancer treatments are concerned, it is well documented that many of these kill cells in vitro and in vivo through the activation of the apoptotic process (4). In particular, the anthracycline doxorubicin (Dox), which is one the best known and most widely used antitumor agents, induces apoptosis in tumor cells. Necrosis is characterized by swelling of cells, destruction of organelles, disruption of membranes, nuclear chromatin lysis without condensation and DNA degradation into a continuous spectrum of sizes of base pairs. Necrotic cell death may occur in response to strong insults such as ischemia, toxins, hyperthermia and direct cell trauma. High resolution nuclear magnetic resonance (1H-NMR) spectroscopy is a versatile, dynamic, non-invasive and non-destructive technique extremely useful in obtaining metabolic, bioenergetic and structural information of cells and tissues both in vivo and in vitro. A further advantage of 1H-NMR is the identification and quantification of a large number of cellular metabolites from a single spectrum. Therefore, it has been widely used to characterize cancer and normal human tissues, to monitor tumor progression or the response to therapeutic treatments and to identify signals associated with apoptosis (5). Especially important signals that have been closely associated to cell death processes are those originating from methyl (CH3 at 0.9 ppm) and methylene (CH2 at 1.3 ppm) lipid acyl chains (3, 6-7). The purpose of the present study was to identify a specific 1H-NMR spectral profile characteristic of apoptosis and to determine if this profile is independent of the type of apoptotic stimulus used. Consequently, the HL60 promyelocytic leukemic cell line was exposed to both a physical (i.e., ionizing radiation) or a chemical (i.e., Dox) treatment. In addition, another goal of this paper was to establish if 1H-NMR can also be used to distinguish between apoptosis and necrosis. Necrosis was induced in the same cells by heat treatment. Apoptosis or necrosis were confirmed by fluorescence microscopy utilizing the chromatin stain Hoechst, agarose gel electrophoresis of DNA and determination of caspase-3 enzymatic activity. The 1H-NMR data reveal that spectra from both irradiated and Dox-treated HL60 cells have the same general appearance and that several important metabolites have very similar quantitative variations. Important variations were found in the signals resonating between 3.41 and 3.25 ppm (taurine; Tau), at about 3.22 ppm (glycerophosphocholine; GPC), at about 3.2 ppm (choline-containing metabolites including choline and phosphocholine, Cho), at about 3.03 ppm (creatine and phosphocreatine; Cr + PCr), at about 2.56 ppm (reduced glutathione; GSH), between 2.34 and 2.04 ppm (_-_ –glutamine and glutamate; Glu), at about 1.48 ppm (alanine; Ala), at about 1.33 ppm (lactate; Lac) and between 0.90-1.30 ppm (CH3 and CH2 groups arising from methyl and methylene groups of fatty acyl chains of lipids, respectively; CH3 lipids and CH2 lipids). In irradiated HL60 cells, significant increases were seen in CH3 and CH2 lipids, Lac and GPC, while significant decreases were observed in Glu, GSH, Cr + PCr, Cho and Tau. No significant variations were seen in Ala. In HL60 cells treated with Dox, significant increases were observed only in CH3 and CH2 lipids, while significant decreases were seen in Glu, GSH, Cho and Tau. No significant variations were found in Lac, Ala, Cr +PCr and GPC. As far as heat-treated HL60 cells are concerned, significant increases were observed in CH3 lipids, Lac, Ala, Glu, Cr + PCr, Cho, GPC and Tau, while a significant decrease was observed in GSH. CH2 lipids did not vary significantly. The data presented suggest that variations in certain important 1H-NMR metabolites are specific to apoptosis independently of the physical or chemical nature of the stimulus used to induce this mode of cell death, while cells dying from necrosis are characterized by a completely diverse behavior of the same metabolites. A deeper understanding of the mechanisms of these two types of cell death may be useful in a clinical setting where it is pivotal to distinguish apoptosis from necrosis in monitoring the outcome of radio and chemotherapeutic protocols. References 1. A.H. Wyllie, Apoptosis: an overview. Br. Med. Bull. 53, 451-465 (1997). 2. K.D. Held, Radiation-induced apoptosis and its relationship to loss of clonogenic survival. Apoptosis 2, 265-282 (1997). 3. T. Bezabeh, M.R.A. Mowart, L. Jarolim, A.H. Greenberg and I.C.P. Smith, Detection of drug-induced apoptosis and necrosis in human carcinoma cells using 1H NMR spectroscopy. Cell Death Differ. 8, 219-224 (2001). 4. R. Kim, K. Tanabe, Y. Uchida, M. Emi, H. Inoue and T. Toge, Current status of the molecular mechanisms of anticancer drug-induced apoptosis. The contribution of molecular-level analysis to cancer chemotherapy. Cancer Chemother. Pharmacol. 50, 343-352 (2002). 5. J.M. Hakumaki and K.M. Brindle, Techiques: visualizing apoptosis using nuclear magnetic resonance. Trends Pharmacol. Sci. 24, 146-149 (2003). 6. J.M. Hakumaki and R.A. Kauppinen, 1H-NMR visible lipids in the life and death of cells. Trends Biochem. Sci. 25, 357-362 (2000). 7. C.M. Shih, W.C. Ko, L.Y. Yang, C.J. Lin, J.S. Wu, T.Y. Lo, S.H. Wang and C.T. Chen, Detection of apoptosis and necrosis in normal human lung cells using 1H NMR spectroscopy. Ann. N.Y. Acad. Sci. 1042, 488-496 (2005).