Raman spectroscopy is used for the localization and tracking of chemotherapeutic drug, doxorubicin, in the intracellular environment of lung cancer cell line. Results show the potential of the technique to monitor the mechanisms of action and response on a molecular level, with subcellular resolution.Please check this proof carefully. Our staff will not read it in detail after you have returned it.Translation errors between word-processor files and typesetting systems can occur so the whole proof needs to be read. Please pay particular attention to: tabulated material; equations; numerical data; figures and graphics; and references. If you have not already indicated the corresponding author(s) please mark their name(s) with an asterisk. Please e-mail a list of corrections or the PDF with electronic notes attached -do not change the text within the PDF file or send a revised manuscript. Corrections at this stage should be minor and not involve extensive changes. All corrections must be sent at the same time.Please bear in mind that minor layout improvements, e.g. in line breaking, table widths and graphic placement, are routinely applied to the final version.We will publish articles on the web as soon as possible after receiving your corrections; no late corrections will be made. Please ensure that all queries are answered when returning your proof corrections so that publication of your article is not delayed. Vibrational spectroscopy, including Raman spectroscopy, has been widely used over the last few years to explore potential biomedical applications. Indeed, Raman spectroscopy has been demonstrated to be a powerful non-invasive tool in cancer diagnosis and monitoring. In confocal microscopic mode, the technique is also a molecularly specific analytical tool with optical resolution which has potential applications in subcellular analysis of biochemical processes, and therefore as an in vitro screening tool of the efficacy and mode of action of, for example, chemotherapeutic agents. In order to demonstrate and explore the potential in this field, established, model chemotherapeutic agents can be valuable. In study paper, Raman spectroscopy coupled with confocal microscopy were used for the localization and tracking of the commercially available drug, doxorubicin (DOX), in the intracellular environment of the lung cancer cell line, A549. Cytotoxicity assays were employed to establish clinically relevant drug doses for 24 h exposure, and confocal laser scanning fluorescence microscopy was conducted in parallel with Raman spectroscopy profiling to confirm the drug internalisation and localisation. Multivariate statistical analysis, consisting of PCA ( principal components analysis) was used to highlight doxorubicin interaction with cancer cells and spectral variations due to its effects before and after DOX spectral features subtraction from nuclear and nucleolar spectra, were compared to non-exposed control spectra. Results show that Raman micro spectroscopy is not only able to detect doxorubicin inside cells an...
The potential of Raman micro spectroscopy as an in vitro, non-invasive tool for clinical applications has been demonstrated in recent years, specifically for cancer research. To further illustrate its potential as a high content and label free technique, it is important to show its capability to elucidate drug mechanisms of action and cellular resistances. In this study, cytotoxicity assays were employed to establish the toxicity profiles for 24 hr exposure of lung cancer cell lines, A549 and Calu-1, to the commercially available drug, doxorubicin (DOX). Raman spectroscopy, coupled with Confocal Laser Scanning Microscopy and Flow Cytometry, was used to track the DOX mechanism of action, at a subcellular level, and to study the mechanisms of cellular resistance to DOX. Biomarkers related to the drug mechanism of action and cellular resistance to apoptosis, namely reactive oxygen species (ROS) and bcl-2 protein expression, respectively, were also measured and correlated to Raman spectral profiles. Calu-1 cells are shown to exhibit spectroscopic signatures of both direct DNA damage due to intercalation in the nucleus and indirect damage due to oxidative stress in the cytoplasm, whereas the A549 cell line only exhibits signatures of the former mechanism of action. PCA of nucleolar, nuclear and cytoplasmic regions of A549 and Calu-1 with corresponding loadings of PC1 and PC2.
The applications of Raman microspectroscopy have been extended in recent years into the field of clinical medicine, and specifically in cancer research, as a non-invasive diagnostic method in vivo and ex vivo, and the field of pharmaceutical development as a label-free predictive technique for new drug mechanisms of action in vitro. To further illustrate its potential for such applications, it is important to establish its capability to fingerprint drug mechanisms of action and different cellular reactions. In this study, cytotoxicity assays were employed to establish the toxicity profiles for 48 and 72 hours exposure of lung cancer cell lines, A549 and Calu-1, after exposure to Actinomycin D (ACT) and Raman micro-spectroscopy was used to track its mechanism of action at subcellular level and subsequent cellular responses. Multivariate data analysis was used to elucidate the spectroscopic signatures associated with ACT chemical binding and cellular resistances. Results show that the ACT uptake and mechanism of action are similar in the 2 cell lines, while A549 cells exhibits spectral signatures of resistance to apoptosis related to its higher chemoresistance to the anticancer drug ACT. The observations are discussed in comparison to previous studies of the similar anthracyclic chemotherapeutic agent Doxorubicin. A, Preprocessed Raman spectrum of ACT stock solution dissolved in sterile water and mean spectrum with SD of (B) nucleolus, (C) nucleus and (D) cytoplasm of A549 cell lines after 48 hours exposure to the corresponding IC 50 . K E Y W O R D SA549, Actinomycin D, binding signature, Calu-1, Raman micro-spectroscopy | INTRODUCTIONRaman micro-spectroscopy is a non-invasive analytical tool, whose potential in clinical applications to distinguish between normal and cancer cells and monitoring drug mechanisms of action has already been demonstrated [1][2][3][4][5]. Therefore, it can potentially be used in preclinical development for the prediction of chemotherapeutic efficacy and potentially ultimately as a companion diagnostic tool. In order to assure its application as a predictive tool, Raman spectroscopy should provide information about drug mechanisms of action and cellular reactions or resistance. Previous studies were made using Raman microspectroscopy and chemotherapeutic drugs such as Cisplatin [6], Vincristine [6], Erlotinib [7], Panitumumab [8] and Doxorubicin (DOX) [9] showing the potential of this
Raman micro‐spectroscopy is a non‐invasive analytical tool, whose potential in cellular analysis and monitoring drug mechanisms of action has already been demonstrated, and which can potentially be used in pre‐clinical and clinical applications for the prediction of chemotherapeutic efficacy. To further investigate such potential clinical application, it is important to demonstrate its capability to differentiate drug mechanisms of action and cellular resistances. Using the example of Doxorubicin (DOX), in this study, it was used to probe the cellular uptake, signatures of chemical binding and subsequent cellular responses, of the chemotherapeutic drug in two lung cancer cell lines, A549 and Calu‐1. Multivariate statistical analysis was used to elucidate the spectroscopic signatures associated with DOX uptake and subcellular interaction. Biomarkers related to DNA damage and repair, and mechanisms leading to apoptosis were also measured and correlated to Raman spectral profiles. Results confirm the potential of Raman spectroscopic profiling to elucidate both drug kinetics and pharmacodynamics and differentiate cellular drug resistance associated with different subcellular accumulation rates and subsequent cellular response to DNA damage, pointing towards a better understanding of drug resistance for personalised targeted treatment.
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