Synchrotron based Fourier‐transform infrared microspectroscopy as sensitive technique for the detection of early apoptosis in U‐87 MG cells

The aim of this study is to show that SR‐FTIRM can be successfully applied for the detection of early apoptotic processes in the human glioma cells (U‐87 MG). The apoptosis was induced by photodynamic action after irradiation of either photosensitizer hypericin (Hyp) or a complex of Hyp with low‐density lipoproteins (Hyp/LDL) incorporated into the cells. The differences between infrared (IR) spectra of non‐treated and photodynamically treated U‐87 MG cells are mainly manifested in position of Amide I vibrational band, sensitive to changes in protein secondary structure. The conformational transition α ‐helix to β ‐sheet results in the shift of Amide I vibration to lower frequency, and can be explained as a consequence of the processes leading to apoptosis. It is also shown that the sensitivity of SR‐FTIRM for the detection of early apoptotic signs is comparable, or even higher, than that of classic technique usually used for the detection of early apoptosis, flow‐cytometry study of cell staining by Annexin V. Moreover, we have demonstrated by both methods, SR‐FTIRM and flow‐cytometry, that Hyp/LDL complex loaded U‐87 MG cells 24 hours after photodynamic action are mostly late apoptotic/necrotic. In contrast, U‐87 MG cells loaded with Hyp alone display apoptosis as prevailing mode of cell death at 4 hours and 24 hours after photodynamic action. (© 2010 by Astro Ltd., Published exclusively by WILEY‐VCH Verlag GmbH & Co. KGaA)