TIME-RELATED VARIATIONS IN 1H-NMR-VISIBLE
METABOLITES DURING RADIATION-INDUCED APOPTOSIS
IN MG-63 HUMAN OSTEOSARCOMA SPHEROIDS
Maria Teresa Santini1,2, Rocco Romano2,3, Gabriella Rainaldi1,2,
Antonella Ferrante1, Paola Indovina1, Andrea Motta4 and Pietro Luigi Indovina2,5
1Dipartimento di Ematologia, Oncologia e Medicina Molecolare, ISS, Roma
2CNR-INFM, Unità di Napoli, Napoli
3Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Salerno
4Consiglio Nazionale delle Ricerche, Istituto di Chimica Biomolecolare, Pozzuoli
5Dipartimento di Scienze Fisiche, Università di Napoli “Federico II”, Napoli
High resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy can be a useful
tool in examining, non-invasively, various aspects of cell behavior, including apoptosis in
different cell types (1,2). Specifically, it has been utilized to demonstrate that the spectral
peaks resonating at about 0.9 and 1.3 ppm arising from methyl and methylene groups of fatty
acyl chains of lipids, respectively, are related to cell death processes both in vitro and in vivo
(3,4). In fact, these studies show that an
accumulation of CH2 and CH3 mobile lipids,
particularly of the CH2 groups, occurs during apoptosis while necrosis does not result in such
an accumulation. In addition, 1H-NMR has also been employed to characterize the
secondary metabolites associated with neutrophil apoptosis (5).
It is well-established that besides metabolic changes, cell cycle delay, and damage to DNA
and the cell membrane, ionizing radiation can also induce cell death through apoptosis (6,7).
However, apoptosis induced by ionizing radiation has never been studied, at least to our
knowledge, by 1H-NMR spectroscopy. This is especially true in spheroids, a cell model of
great complexity that resembles in vivo tumors much more closely than monolayer cultures
(8,9). Consequently, no detailed analysis of the 1H-NMR spectroscopic changes that take
place during radiation-induced apoptosis exist, nor do thorough investigations into the
temporal dynamics of these eventual variations.
With this in mind, it was the purpose of the present study to use 1H-NMR to examine the
metabolic changes that occur in MG-63 osteosarcoma three-dimensional tumor spheroids
undergoing apoptosis. In fact, small (about 50 - 80 µm in diameter) spheroids with no
hypoxic center were exposed to 5 Gy of ionizing radiation and apoptosis verified by nuclear
dye Hoechst 33258 staining of paraffin-embedded spheroid sections and also by caspase-3
activation. The one-dimensional 1H-NMR spectra of control and exposed MG-63 spheroids
were collected at 24, 48 and 72 h after irradiation and compared using an algorithm
developed by our group (10). The two-dimensional spectra were also examined. The
results demonstrate that numerous metabolic changes take place in spheroids exposed to 5
Gy. In particular, significant increases in both CH2 and CH3 mobile lipids take place,
becoming more pronounced as a function of time and in relation to the rise in the percentage
of apoptosis. Other metabolites also appear to follow precise temporal dynamics. Of
particular interest are the changes in taurine and reduced glutathione (GSH). These data
show that 1H-NMR can be extremely useful in examining the temporal dynamics of
radiation-induced apoptosis in spheroids. The importance of a better understanding of the
time-related variations that occur during apoptosis not only in CH2 and CH3 lipids, but also
in other important metabolites can serve in comprehending more fully the changes that take
place in vivo and thus in evaluating more accurately the outcome of radiotherapy protocols.
References
1.
J. L. Evelhoch, R. J. Gillies, G. S. Karczmar, J. A. Koutcher, R. J. Maxwell, O.
Nalcioglu, N. Raghunand, S. M. Ronen, B. D. Ross and H. M. Swartz, Applications of
magnetic resonance in model systems: cancer therapeutics. Neoplasia 2, 152-165 (2000).
2.
J. M. Hakumäki and R. A. Kauppinen, 1H-NMR visible lipids in the life and death of
cells. Trends Biochem. Sci. 25, 357-362 (2000).
3.
J. M. Hakumäki and K. A. Brindle, Techniques: Visualizing apoptosis using nuclear
magnetic resonance. Trends Pharmacol. Sci. 24, 146-149 (2003).
4.
F. G. Blankenberg, R. W. Storrs, L. Naumovski, T. Goralski and D. Spielman,
Detection of apoptotic cell death by proton nuclear magnetic resonance spectroscopy. Blood
87, 1951-1956 (1996).
5.
A. V. W. Nunn, M. L. Barnard, K. Bhakoo, J. Murray, E. J. Chilvers and J. D. Bell,
Characterisation of secondary metabolites associated with neutrophil apoptosis. FEBS Lett.
392, 295-298 (1996).
6.
D. Watters, Molecular mechanisms of ionizing radiation-induced apoptosis. Immunol.
Cell Biol. 77, 263-271 (1999).
7.
N. J. Stapper, M. Stuschke, A. Sak and G. Stuben, Radiation-induced apoptosis in
human sarcoma and glioma cell lines. Int. J. Cancer 62, 58-62 (1995).
8.
M. T. Santini and G. Rainaldi, Three-dimensional spheroid model in tumor biology.
Pathobiol. 67, 148-157 (1999).
9.
M. T. Santini, G. Rainaldi and P. L. Indovina, Apoptosis, cell adhesion and the
extracellular matrix in the three-dimensional growth of multicellular tumor spheroids. Crit.
Rev. Oncol. Hematol. 36, 75-87 (2000).
10.
R. Romano, M. T. Santini and P. L. Indovina, A time-domain algorithm for NMR
spectral normalization. J. Magn. Reson. 146, 89-99 (2000).