Human PC-3 prostate cancer cells were incubated in the presence of two cardenolides, i.e., ouabain and 19-hydroxy-2′′-oxovoruscharin. Their effects were monitored by infrared spectroscopy of the cells after different exposure times to the cardenolides. Analysis of changes in absorbance intensities indicated that, for both compounds, the absorbance at one wavenumber with a minor contribution of a second wavenumber is sufficient to build a linear model accurate enough to assign more than 97% of the spectra to their correct time slot. Student t-tests and twodimensional correlation analysis (2D-COS) indicated that both drugs have very similar effects on PC-3 cells. However, asynchronous 2D maps revealed significant differences and allowed the sequence of the spectral changes to be determined: 1395 → 1695 cm<sup>–1</sup> for ouabain, and 1400 → 1655 → 1100 → 1250 → 1020 cm<sup>–1</sup> for 19-hydroxy-2′′-oxovoruscharin. 2D correlation map subtraction allowed the identification of very specific differences in the impact of both compounds on PC-3 cells, in particular the ability of 19-hydroxy-2′′-oxovoruscharin to affect nucleic acid of PC-3 cells.
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