An in vitro study of the interaction of the chemotherapeutic drug Actinomycin D with lung cancer cell lines using Raman micro‐spectroscopy

Abstract

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

Doxorubicin kinetics and effects on lung cancer cell lines using in vitro Raman micro‐spectroscopy: binding signatures, drug resistance and DNA repair

“…In both nucleolus and nucleus of Calu-1 cells, features at 1430-1450 cm À1 are also observed, corresponding to guanine and cytosine (absent in control cells) and are related to DOX-DNA intercalation inducing damage and/or conformational changes [15]. The same features have been observed after 24 hrs of DOX interaction in A549 cells [20] confirming the early stage DOX-DNA and RNA binding for both cell lines [38]. The decrease in the bands at 785 and 813 cm À1 , corresponding respectively to DNA backbone O-P-O and RNA O-P-O phosphodiester bond stretching, confirm an early DOX effect in Calu-1 cells consistent with its main mechanism of action.…”

“…The present study expands the previous work exploring the use of Raman micro-spectroscopy to monitor DOX effects and cellular responses in vitro [17,20] to a comparison of the uptake kinetics and responses of two different histological subtype cell lines, A549 and Calu-1. In order to extract biological information contained in Raman spectra, multivariate data analysis consising of Principal Component Analysis (PCA), Partial Last Square Regression (PLSR) and Independent Component Analysis (ICA) is used.…”

“…In both cell lines, for the nucleolar regions (Figure 5A), a notable decrease in features at 728 cm À1 (adenine) 785 cm À1 (cytosine, thymine and DNA backbone O-P-O), 813 cm À1 (RNA O-P-O stretching), 1095 cm À1 (DNA PO 2 À symmetric stretching) and 1376 cm À1 (thymine), are consistent with a decrease in nucleic acid contributions due to the DOX mechanism of action by nucleolar interaction, inducing DNA replication blockage, and decrease of RNA features by inhibiting RNA synthesis at the level of rRNA transcription, inhibiting ribosome biogenesis, and, as a consequence, alteration of nucleolar structure [42], size, shape and fragmentation [17,20,29], inducing nucleolar stress dependent apoptosis [43,44].…”

“…Raman micro-spectrosopy has previously demonstrated that A549 and Calu-1 present different sensitivities to DOX treatment after 24 hrs exposureas a result of different relative contributions of the different mechanisms of action involved [20]. In order to further investigate the subcellular differences at early and late stages of exposure, Raman profiles for both cell lines were compared after 2, 6, 12, 24, 48 and 72 hrs exposure to DOX and spectra were taken from the three subcellular compartments, nucleolus, nucleus and cytoplasm (the example of original and pre-processed data of Calu-1 24 hrs exposure is shown in Figure S1).…”

“…DOX is an anthracycline widely used in chemotherapy for the treatment of various human cancers and aggressive tumors [15,16], despite its serious side effects, principally cardiotoxicity, and a not fully understood mechanism of action [17][18][19]. In previous in vitro reports, different relative contributions of subcellular processes have been observed, depending on cell line, [20] including ROS production, intercalation between nucleic base pairs and Topoisomerase II inhibition, blocking of DNA replication [17,21] and induction of DNA double stand breaks (DSB) by nucleosome turnover increase, leading to cellular apoptosis [22][23][24]. DSB induce a DNA damage response (DDR) which can be visualised locally as an accumulation of repair proteins, known as nuclear foci [25] and chromatin modification acting as an adaptor attracting and localizing retention of DNA repair proteins.…”