Tracking InfraRed signatures of drugs in cancer cells by Fourier Transform microspectroscopy

Abstract

Aimed at developing accurate, reliable and cost-saving analytical techniques for drugs screening we evaluated the potential of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) as a quantitative pre-diagnostic approach for the rapid identification of IR signatures of drugs targeting specific molecular pathways causing Chronic Myeloid Leukemia (CML). To obtain reproducible FTIR absorbance spectra at the necessary spatial resolution we optimized sample preparation and acquisition parameters on a single channel Mercury-Cadmium-Telluride (MCT) detector in the spectral interval of frequencies from 4000 to 800 cm(-1). Single K562 cells were illuminated by Synchrotron Radiation (SR) and a number of ~15 K562 cells spread in monolayer were illuminated by a conventional IR source (Globar), respectively. Combining IR spectral data with the results of complementary biochemical investigations carried out in samples by different analytical methods we identified and cross-validated IR signatures of drugs targeting the oncogenic protein BCR/ABL and its associated abnormal tyrosine kinase activity in K562 cell line. Unsupervised pattern recognition performed by Hierarchical Cluster Analysis (HCA) clustered the spectra of single K562 cells in two distinct groups roughly corresponding to living and to apoptotic cells, respectively. The corresponding IR spectral profiles were assumed to represent drug-resistant and drug-sensitive cells. Significant variations with increasing percentages of apoptotic cells were observed after the treatment of K562 cells with drugs that directly or indirectly target BCR/ABL. In conclusion, we suggest that microFTIR associated with multivariate data analysis may be useful to assess drug compounds in ex vivo cancer cell models and possibly peripheral blast cells from CML patients.

Synchrotron Radiation Spectroscopic Techniques as Tools for the Medicinal Chemist: Microprobe X-Ray Fluorescence Imaging, X-Ray Absorption Spectroscopy, and Infrared Microspectroscopy

“…[92,106] As such, recent attention has been focussed on the use of SR-FTIR microspectroscopy for the study of biomolecular changes that occur following the administration of drugs or potential therapeutic agents in diseased cells. [92,[108][109][110][111][112] Importantly, macromolecules associated with biological samples produce signature IR bands that can be readily discerned from the IR spectra. [94,99,[113][114] Typical spectral features include: amide I ( 1650 cm 1 ) and amide II ( 1549 cm 1 ) bands representative of proteins; symmetric (1080 cm 1 ) and anti-symmetric PO 2  (1232 cm 1 ) bands associated with DNA; C-H stretches of -CH 3 ( as 2955 cm 1 ) and -CH 2 ( as 2850 cm 1 ), the ester band (1740 cm 1 ) and the C-O-P (1080 cm 1 ) band associated with lipids/phospholipids; and C-O stretches (1050 cm 1 ) of carbohydrates.…”

“…The interest in the use of this technique stems from an urgent demand for the development of accurate and cost-saving analytical techniques that can be applied to the screening of new drug candidates. [109] The analysis involves the collection of spectra from large numbers of cells (generally >100 cells per sample) following exposure to the therapeutic agent, and the objective analysis of the resultant data through statistical procedures such as principal component analysis (PCA) and hierarchical cluster analysis. [92] While changes in spectral features can often be visually observed in average spectra, tools such as PCA remove subjectivity and add credence to the evaluation of any biomolecular differences/similarities in the spectra of cell or tissue samples.…”

“…Draux et al [108] reported increases in the molecular vibrations corresponding to characteristic bands of proteins and RNA and it was concluded that these IR changes may be related to the molecular mechanism of the cytostatic drug and the accumulation of the cells in G1 phase. [108] Bellisola, et al [109] cells. [109] For instance, they concluded that significant changes that occurred in the interval 1290-900 cm 1 and in particular between 940-900 cm 1 , were associated with changes in the PO stretching mode in the phosphorylated proteins.…”

“…[108] Bellisola, et al [109] cells. [109] For instance, they concluded that significant changes that occurred in the interval 1290-900 cm 1 and in particular between 940-900 cm 1 , were associated with changes in the PO stretching mode in the phosphorylated proteins. [109] This was significant because the drug was known to cause a rapid decrease of protein tyrosine phosphorylation.…”

“…[109] For instance, they concluded that significant changes that occurred in the interval 1290-900 cm 1 and in particular between 940-900 cm 1 , were associated with changes in the PO stretching mode in the phosphorylated proteins. [109] This was significant because the drug was known to cause a rapid decrease of protein tyrosine phosphorylation. [109] Mantsch and coworkers [125] have also used SR-FTIR microspectroscopy for the detection of apoptosis in K562 cells and the T-lymphoblastic cell line (CEM cells), with the aim of determining its feasibility as an indicator of chemosensitivity of leukaemia cells isolated from individual patients.…”