Intracellular investigation on the differential effects of 4 polyphenols on MCF-7 breast cancer cells by Raman imaging.

The past decades have seen significant interest in the study of polyphenolic compounds as potential therapeutic agents in medicine because they display a vast array of cellular effects beneficial to treat or manage a plethora of chronic diseases including inflammatory diseases, cardiovascular abnormalities and several types of cancer. These compounds act at different stages of carcinogenesis but deciphering their mode of action is a complex task. Live MCF-7 breast cancer cells were investigated using Raman imaging to evaluate the perturbations induced after incubating cells with four different polyphenols: EGCG, gallic acid, resveratrol and tannic acid. First, clear spectral changes could be observed between the spectra of the cytoplasm and the nucleus of live MCF-7 cancer cells demonstrating a difference in their respective global chemical composition. The treatments induced significant modifications in the cells but no clear common pattern of modifications from the 4 drugs could be observed in the cell spectra in the 1800-600 cm-1 region. The high spatial resolution of Raman confocal microscopy enabled both the nucleus and cytoplasm to be independently targeted to study the impact of the polyphenols on the cell line. Positive spectral variations at 2851 cm-1 and 2920 cm-1 as well as in the 1460-1420 cm-1 and 1660-1650 cm-1 spectral regions inside cell cytoplasm reflected an increase of the lipid content after exposure to polyphenols. Lipid accumulation appears to be an early biomarker of drug-induced cell stress and subsequent apoptosis. Interestingly an increase of cytochrome c into the cytosol was also induced by EGCG. These multiple events are possibly associated with cell apoptosis. In conclusion, Raman micro-spectroscopy provides a complementary spectroscopic method to realize biological investigations on live cancer cells and to evaluate the effects of polyphenols at the subcellular level.

HTS-FTIR spectroscopy allows the classification of polyphenols according to their differential effects on the MDA-MB-231 breast cancer cell line.

Breast cancer is a major public health issue among women in the world. Meanwhile new anticancer treatments struggle more and more to be accepted in the pharmaceutical market and research costs still increase. There is therefore a need to find new treatments and new screening methods to test them more quickly and efficiently. Among natural compounds, an increasing interest has been given to polyphenols as they can take action at the different stages of carcinogenesis, from tumour initiation to metastasis formation, by disturbing multiple cellular signalling pathways. They constitute one of the largest groups of plant metabolites and more than 8000 compounds have already been identified based on their chemical structure. Traditionally in pharmacology, new anticancer drugs are first evaluated for their potential to inhibit the proliferation of cancer cell lines. Numerous potential drugs are discarded at this stage even though they could show interesting modes of action. In turn, there is an increasing demand for more systemic approaches in order to obtain a global and accurate insight into the biochemical processes mediated by drugs. Recently, FTIR spectroscopy was demonstrated to be an innovative tool to obtain a unique fingerprint of the effects of anticancer drugs on cells in culture. While this spectral technique appears to have a definite potential to sort drugs according to their spectral fingerprints, characteristic of the metabolic modifications induced, the present challenge remains to evaluate the drug-induced spectral changes in cancer cells on a larger scale. This article presents the results obtained for a 24 h-exposure of the breast cancer cell line MDA-MB-231 to 15 compounds belonging to different classes of polyphenols using FTIR spectroscopy connected to a high throughput screening extension. Through unsupervised and supervised statistical analyses (PCA, MANOVA, Student's t-tests and HCA), a distinction between polyphenol treatments and controls could be well established.

High throughput absorbance spectra of cancerous cells: a microscopic investigation of spectral artifacts.

FTIR spectroscopy was recently demonstrated to be a useful tool to obtain a unique fingerprint of the effects of several anticancer drugs on cells in culture. While FTIR spectroscopy appears to have a definite potential to sort anticancer drugs on the basis of the metabolic modifications they induced, the present challenge is to evaluate the drug-induced spectral changes in cancer cells on a larger scale. The coupling of FTIR spectroscopy with a high throughput screening extension could become a useful method to generate drug classifications based on their "modes of action". Practically, the robustness of this approach is jeopardized by the variability that can appear from one cell smear to the next. When a few cells are scattered on the support, strong scattering effects are observed and locally dense cell aggregates could result in non-linearity of the signal. A microscopic study using infrared imaging demonstrates that the mean HTS (96-well High Throughput Spectroscopy) spectra recorded on 96 well ZnSe plates are the averages of contributions characterized by a wide absorbance distribution and by Mie scattering effects which significantly vary from point to point. Spectrum quality is at its best at the highest cell concentrations, i.e. from 300 000 to 400 000 cells per well, which present the best S/N and a relatively smaller Mie scattering effect. When the breast cancer cell line MDA-MB-231 was treated with four different polyphenols, spectra showed quite similar variations with respect to control spectra, with more intense variations for the quercetin and EGCG compared to resveratrol and capsaicin. Correction of the spectra with the RMieS algorithm improved their clustering according to the polyphenolic treatment.