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.