Determination of cellular changes in response to anticancer agents using live-cell FTIR spectrosocpy

Abstract

The survival rate of cancer has improved in the past few years with the development of many targeted therapies, yet many cancer types acquired resistance to these anticancer drugs and hence lead to the relapse of cancer cells. Current pre-clinical approaches employed in drug discovery of novel anticancer compounds have not met the increasing demand and are associated with an excessive cost and a high attrition rate. Consequently, a screening approach, that is cost-effective, reliable and thoroughly provides the critically important mechanistic information on the drug-cell interaction, is urgently needed in the pre-clinical selection of new drug candidates. This thesis aims to develop a new screening approach based on live-cell FTIR spectroscopy for drug screening that has a low running cost and can discriminate anticancer drugs according to their biochemical pathways or modes of actions (MoAs). In the first result chapter (Chapter 3), we developed the measurement approach and demonstrated that multi-reflection ATR-FTIR spectroscopy could be used to obtain high-quality spectra of live cells in their aqueous culture medium, and the results were validated with a standard orthogonal method, MTT assay. Three cancer cell lines, namely HeLa, PC-3 and Caco-2 with different degree of resistance to doxorubicin (a known DNA targeting drug) were investigated. The first spectrum after adding doxorubicin was used as a background, which is ratioed to the subsequent spectra to obtain the difference spectra.The difference spectra were analysed and showed significant spectral changes in the IR absorbance bands that are associated with phosphates backbone of nucleic acids. These cellular changes provided evidence of the DNA synthesis inhibition and disintegration, which could be related to the DNA intercalating effect of doxorubicin. In Chapter 4, we applied this approach to study the cellular changes of MDA-MB-231 (a triple-negative breast cancer cell line) to four well-known anticancer drugs from different classes; tamoxifen and toremifene (Selective Estrogen Receptor Modulators, SERMs), imatinib (Tyrosine Kinase Inhibitor) and doxorubicin (DNA-intercalating agent). Principal component analysis (PCA) of the difference spectra has shown that drugs with different modes of action are well-separated, while the drugs with the same mode of action are clustered together 3 on the score plot. The results have also shown that when cells are treated at IC50, the separation appears to be the clearest at 2 hours for imatinib and tamoxifen/toremifene and 6 hours for doxorubicin. However, at the 50% of IC50, the separation appears to be the best at a longer incubation time (i.e. 24 hours) for all the four drugs. In Chapter 5, we attempted to compare the results obtained with the triple-negative MDA-MB-231 cells to a triple-positive breast cancer cells (MCF7). The results demonstrated that drugs are discriminated according to their modes of action, showing that the method is applicable for different cell lines. Tamoxifen and toremifene induced similar spectral changes in the cellular compositions of MCF7 cells and clustered in the same PC score plots, while doxorubicin-treated MCF7 cells highlighted different spectral changes and clustered separately. The comparison between the two cell lines indicated that SERMs induced different spectral changes that are attributed to the different modes of actions of these drugs in both cell lines. On the other hand, doxorubicin-treated MDA-MB-231 and MCF7 cells have shown relatively similar spectral changes in the DNA region, which indicates the DNA intercalation effect of doxorubicin in both cell lines. In conclusion, the work presented in this thesis has successfully shown that live cell FTIR spectroscopy combined with PCA can distinguish and group anticancer drugs based on the induced spectral changes in live cell