Régis Gasper

Protein secondary structure content in solution, films and tissues: redundancy and complementarity of the information content in circular dichroism, transmission and ATR FTIR spectra.

The paper presents a simple and robust method to determine protein secondary structure from circular dichroism, transmission and attenuated total reflection (ATR) Fourier transform infrared spectra. It is found that the different spectroscopic methods bring valuable but roughly identical information on the secondary structure of proteins. ATR and transmission FTIR spectra display distinct differences, yet the secondary structure can be predicted from their spectra with roughly the same success. It is also found that one wavenumber or wavelength includes the large majority of the information correlated with secondary structure content and no more than 3 significant independent wavenumbers/wavelengths could be found for any of the spectroscopic data. This finding indicates that more complex linear combinations of the absorbance or ellipticities will not further improve secondary structure predictions. Furthermore, the information content in CD, transmission and ATR FTIR spectra is largely redundant. If combining CD and FTIR results in some improvement of structure prediction quality, the improvement is too modest to prompt spectroscopists to collect different spectroscopic data for structure prediction purposes. On the other hand, the data collected show that the quality of the FTIR spectrometers is such that biosensors or imaging methods sampling from 10(-9) to 10(-15) g yield spectra of sufficient quality to analyze protein secondary structure. These new techniques open the way to a new area of research, both in protein conformational response to ligand and imaging at sub-cellular scales.

FTIR spectral signature of the effect of cardiotonic steroids with antitumoral properties on a prostate cancer cell line

We show in the present work that the infrared (IR) spectrum of human PC-3 prostate cancer cells exposed to anticancer drugs could offer a unique opportunity to get a fingerprint of all the major biochemical components (DNA, RNA, proteins, lipids, etc.) present in the cells and to identify with high sensitivity the signature of the metabolic changes induced by anticancer drugs. We investigated here the FTIR-related signatures of the effect of 4 structurally-related cardiotonic steroids (CS), i.e. ouabain, 19-hydroxy-2″-oxovoruscharin, hellebrin and 19-hydroxy-hellebrin on PC-3 cancer cells incubated between 0 and 36 h in the absence (control) or the presence of the CS. For each molecule a single spectral signature described the largest part of the time dependent modifications with a possible very minor second component. The spectral signatures characterizing the effects of each of the four CS were unique but very similar when compared to the signature of the effect of an intercalating anticancer drug, i.e. doxorubicin, selected as a positive reference compound in our study, suggesting a fully distinct set of cellular perturbations. The current study thus illustrates that Fourier Transform Infrared (FTIR) analyses can be used to identify, among the perturbations induced on a given cancer cell line, the features common to a group of anticancer compounds as well as features specific to every single drug.

Effects of the confluence rate on the FTIR spectrum of PC-3 prostate cancer cells in culture

Recently, the possibility of using IR spectroscopy to fingerprint the mode of action of potent antitumor drugs on cancer cells at sub-lethal concentrations has been demonstrated by comparing spectra recorded from untreated or drug-treated cells. The present study investigates the potential interference of the cell culture confluence rate on cell FTIR signature. Significant spectral differences were observed on cells harvested at different confluence rates. Yet, these differences were weak when compared to those induced by sub-lethal ouabain concentrations, used as a model of cardenolide drug. Furthermore, principal component analysis reveals that the impact of the confluence rate, above 50% coverage, on the FTIR spectra is essentially unique and orthogonal to the one induced by the drug model.

Ouabain-induced modifications of prostate cancer cell lipidome investigated with mass spectrometry and FTIR spectroscopy

Fourier transform infrared (FTIR) spectroscopy was used to investigate modifications of prostate cancer PC-3 cell lipidome after exposure to sub-lethal concentrations of ouabain. FTIR spectroscopy offered an overview of the lipid classes present in the whole sample. The method is simple, label free and some features can be detected on entire cells. We compared the achievements of FTIR spectroscopy with data obtained by mass spectrometry (MS) on the same samples. It appears that FTIR spectroscopy could identify content variations in some lipid classes, e.g., these containing choline head groups such as phosphatidylcholine and sphingomyelin. MS analysis could confirm this result as indicated by principal component analysis and 2D heterocorrelation maps. FTIR spectra were also able to report changes in ester/choline/phosphate ratios characterizing lipid changes induced by ouabain. Furthermore, quantization of major lipid classes (PC, PE, PG, SM) could be obtained by curve fitting of the FTIR spectra. Yet, FTIR failed to resolve lipid classes for which the polar heads do not display specific IR features such as phosphatidylglycerol and cardiolipin.

Time Dependence of Cellular Chemical Changes Induced in Prostate PC-3 Cancer Cells by Two Structurally Related Cardenolides Monitored by Fourier Transform Infrared (FT-IR) Spectroscopy

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 two-dimensional 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−1 for ouabain, and 1400 → 1655 → 1100 → 1250 → 1020 cm−1” 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.

FTIR spectroscopy reveals the concentration dependence of cellular modifications induced by anticancer drugs

Large-scale screening to determine the mechanisms of anti-cancer actions of chemical libraries still presents tech- nical challenges that are beyond the capabilities of conventional methods used in cellular or molecular biology. We recently demonstrated in a proof-of-concept study that infrared (IR) spectrum of cells exposed to anticancer drugs could be used to clas- sify their mechanisms of actions. This study highlighted the fact that molecules inducing unique metabolic modifications could be selected for further pharmacological improvements. We show in this paper that drug concentration is an important parameter to be taken into account when analyzing mechanisms of anti-cancer actions by means of FTIR. The data indeed demonstrated that distinct spectral modifications occur in human PC-3 prostate cancer cells when exposed to ouabain at 10 × IC50 versus 1 × IC50. Longer incubation times at 1 × IC50 never resulted in spectral modifications fitting with those observed at 10 × IC50.

Monitoring of metabolism perturbation in prostate PC-3 cancer cells by sub-lethal concentrations of methotrexate

Infrared imaging was used to investigate the effect of sub-lethal concentration of methotrexate on prostate PC-3 cancer cells. Cells were exposed for 0, 24 and 48 h to 9 nM of methotrexate, the concentration that inhibits 50% of cell growth after 72 h. Sub-lethal concentrations of methotrexate induced a reproducible signature that can be measured by infrared spectroscopy. Student t-test revealed that the process occurs in at least two steps, the first one affecting the nucleic acid region of the spectrum and the second one, the protein region. Imaging of individual cells demonstrated that the recorded differences are not due to a specific subpopulation of cells but homogenously affects all the cells. Near perfect identification of treated cells could be obtained using a supervised classification.

Data processing in FTIR imaging of cells: Towards protein secondary structure imaging

Focal plane arrays provide a rapid method for infrared imaging of cells and tissues. Beside statistical analyses designed to classify image features, insight into the structure of the molecules present in the sample can be obtained. The paper presents a simple way to retrieve complete information on protein secondary structure from the thousands of spectra obtained by imaging. Human PC-3 prostate cancer cells were shown here to present cell-to-cell variations in protein secondary structure which might be related to the cell cycle. Intracellular variations are small but intercellular variations can be quite significant. This observation could yield unanticipated insight in cell population analysis. Furthermore, the results demonstrate the robustness of secondary structure determination on individual pixels at a sub-cellular scale.

Classification of drug modes of action on cancer cells by FTIR fingerprinting

IR spectrum of a cell provide an accurate fingerprint of cell metabolism at the moment of the measure. This contribution explores the feasibility and the limits of using IR spectroscopy for the selection of new drugs based purely on their mode of action.