Probing the action of a novel anti-leukaemic drug therapy at the single cell level using modern vibrational spectroscopy techniques

Abstract

Acute myeloid leukaemia (AML) is a life threatening cancer for which there is an urgent clinical need for novel therapeutic approaches. A redeployed drug combination of bezafibrate and medroxyprogesterone acetate (BaP) has shown anti-leukaemic activity in vitro and in vivo. Elucidation of the BaP mechanism of action is required in order to understand how to maximise the clinical benefit. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Synchrotron radiation FTIR (S-FTIR) and Raman microspectroscopy are powerful complementary techniques which were employed to probe the biochemical composition of two AML cell lines in the presence and absence of BaP. Analysis was performed on single living cells along with dehydrated and fixed cells to provide a large and detailed data set. A consideration of the main spectral differences in conjunction with multivariate statistical analysis reveals a significant change to the cellular lipid composition with drug treatment; furthermore, this response is not caused by cell apoptosis. No change to the DNA of either cell line was observed suggesting this combination therapy primarily targets lipid biosynthesis or effects bioactive lipids that activate specific signalling pathways.

Challenges in application of Raman spectroscopy to biology and materials

“…47 Further improvements in the instrumentation have enabled studying of various problems concerning the structure and interaction in nucleic acids, nucleic acid hydration, interaction with metal ions, DNA melting, DNA damage etc. [48][49][50][51] Raman spectra of nucleic acids contain a large number of bands originating from various moieties like sugar, phosphate, bases etc. which provide very useful information about backbone conformation, base pairing etc.…”

Towards identifying the mode of action of drugs using live-cell FTIR spectroscopy

“…3 The application of FTIR spectroscopy for drug-cells interaction analysis has attracted much attention, 4,5 with number of studies focusing on dry or fixed cells. [5][6][7][8][9][10] The method has shown great promises in characterising the mode of action of drugs based on the changes to the chemical composition of the drug-treated cells at specific time point. 11 However, the development in the FTIR measurement of live cells has allowed measurement of living cells at different time points during treatment.…”

“…[23][24][25][26] Infrared synchrotrons with brightness of several orders of magnitude higher than an ordinary Globar source have been used to partially overcome this problem. Using synchrotron sources, the measurement of infrared spectra of living cells within a reasonable time frame has been successfully demonstrated, 27,28 including the characterisation of normal cells from carcinoma, 29 response of cells to mechanical stress, 30 apoptotic pathways, 31 chemical stress, 12 optical stimulation, 32 protein aggregation, 17,33 drug actions, 10 toxicity in living algae 34 and imaging with subcellular spatial resolution using ZnS hemispheres. 35 However, access to synchrotron facilities are limited and therefore measurements of living cells that can be carried out in an ordinary laboratory remains highly desirable.…”

Live single cell analysis using synchrotron FTIR microspectroscopy: development of a simple dynamic flow system for prolonged sample viability

“…35 The lack of significant changes in the lipid bands is surprising, as changes to the membrane structure were expected with increasing temperature, as well as potentially apoptosis signatures as the cells began to die. 2,56,57 It is possible that changes to the cells were more gradual than anticipated, and longer exposure to higher temperatures before measurements would have led to some of these changes being observed. Figure 5B) shows a much wider range of differences in the mean spectra, particularly between the spectrum at 60 °C, shown in red, and those at the remaining temperatures.…”