SynchrotronFTIR analysis of drug treated ovarian A2780 cells: an ability to differentiate cell response to different drugs?

Abstract

Recently a new di-gold(I) organometallic complex [1,3-(Ph(3)PAu)(2)-C(6)H(4)] (KF0101) has been synthesised and found to exhibit cytotoxic activity in vitro. Subsequently it has been demonstrated that KF0101 shows little or no cross-resistance against a number of the cisplatin resistant ovarian cancer cell lines in vitro suggesting a different mode of action for the drug. In this study, syncrotron radiation infrared microspectroscopy (SR-IRMS) has been used on drug treated single A2780 cells in order to determine if this different mode of action can be identified spectroscopically. The aim of the study was to establish: (i) if single cell SR-IRMS could be used to give insight into the cellular response on treatment with different cytotoxic agents relative to non-treated cells (control) and (ii) that if the cytotoxic drugs elicit a different biochemical response these responses could be distinguished from each other. The most striking features obtained after Principal Components Analysis (PCA) of Resonant Mie Scattering (RMieS) corrected single cell spectra of drug treated ovarian A2780 cells are: (i) The spectra obtained for the control are quite heterogeneous and several hundred spectra are required to adequately define the nature of the control; (ii) after drug treatment at the IC50 level for 24 h with cisplatin, KF0101, methotrexate, paclitaxel or 5-fluorouracil the cell spectra, as represented on a PCA scores plot, generally concentrate in certain well defined areas of the control, there are however a small number of spectra that fall outside of the area defined by the control; and (iii) a differentiation between cell spectra obtained on treatment with different drugs is observed which fits well with different in vitro cell culture behaviour and a flow cytometry cell cycle analysis of the control and drug treated cells. Inspection of the loading plots shows that PC1 is essentially the same for all plots and reflects changes in cell biochemistry related to the cell cycle. PC2, however, on comparison of the control versus cisplatin or cisplatin versus KF0101 is indicative of differences induced by drug treatment and has been termed as cell cycle-plus behaviour. These data are shown to be consistent with that obtained using bench-top IRMS by averaging a number of single cell spectra and carrying out a PCA, but SR-IRMS offers more insight into how the drug is affecting the cell population. More importantly, this approach enables the influence of the cell cycle on both the control and drug treated samples to be taken into consideration when evaluating the drug-cell interaction.

Cellular injury evidenced by impedance technology and infrared microspectroscopy

“…Many publications focus on the cytotoxic drugs and plant extracts or compounds affecting FTIR spectra of treated cells [11,13,14,25,26] while little research had been done on cytostatic responses [15].…”

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

“…[92] There have since been significant developments towards disease diagnosis, improvements in the understanding of disease progression and validations of hypotheses regarding diseases such as prostate, [93] skin, [94] colon, [95] cervical, [94,96] and breast cancers, [97] melanoma, [98] leukemia, [99] transmissible spongiform encephalopathies/prion diseases, [100] Alzheimers disease, [101][102] diabetes, [103] malaria, [104] ophthalmic disorders, [105] and arthritic disorders. [94,99] The usefulness of SR-FTIR microspectroscopy derives from the fact that the IR source is 10-1000 times more brilliant than the conventional globar source.…”

“…[106][107] This equates to superior signal to noise ratios in the resultant spectra, improving acquisition times and spatial resolution, both of which are important for analysing biological samples. [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.…”

“…[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.…”

“…[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. [118] SR-FTIR can be used for mapping individual cells and tissues; [119][120] however, this is diffraction limited and noise becomes apparent at approximately 1000 cm 1 when an aperture of 5 µm 2 or smaller is used.…”