Keywords: Confocal Raman Microscopy, living cell, AFM, cancer, Paclitaxel .... acid, protein, covalent bound. Raman shifts. Cell. Tryptophane. 758 cm-1. Protein.
Raman confocal microscopy and AFM combined studies of cancerous cells treated with Paclitaxel L. Derelya, P-Y Collart Dutilleula, Sylvain Michotte de Welleb, V. Szabob, C. Gergelyb, F.J.G. Cuisiniera* a
LBN, UFR Odontologie, Université Montpellier 1, 34193 Montpellier, France, 0033 4 6710 7431 b Groupe d’Etude des Semi-conducteurs, UMR 5650, CNRS-Université Montpellier 2, 34095 Montpellier, France ABSTRACT
Paclitaxel interferes with the normal function of microtubule breakdown, induces apoptosis in cancer cells and sequesters free tubulin. As this drug acts also on other cell mechanisms it is important to monitor its accumulation in the cell compartments. The intracellular spreading of the drug was followed using a WITEC 300R confocal Raman microscope equipped with a CCD camera. Hence Atomic force microscopy (an MFP3D- Asylum Research AFM) in imaging and force mode was used to determine the morphological and mechanical modifications induced on living cells. These studies were performed on living epithelial MCF-7 breast cancer cells. Paclitaxel was added to cell culture media for 3, 6 and 9 hours. Among the specific paclitaxel Raman bands we selected the one at 1670 cm-1 because it is not superposed by the spectrum of the cells. Confocal Raman images are formed by monitoring this band, the NH2 and the PO4 band. Paclitaxel slightly accumulates in the nucleus forming patches. The drug is also concentrated in the vicinity of the cell membrane and in an area close to the nucleus where proteins accumulate. Our AFM images reveal that the treated cancerous MCF-7 cells keep the same size as the non treated ones, but their shape becomes more oval. Cell’s elasticity is also modified: a difference of 2 kPa in the Young Modulus characterizes the treated MCF-7 mammary cancerous cell. Our observations demonstrate that paclitaxel acts not only on microtubules but accumulates also in other cell compartments (nucleus) where microtubules are absent. Keywords: Confocal Raman Microscopy, living cell, AFM, cancer, Paclitaxel
1. INTRODUCTION Paclitaxel (taxol), a natural product extracted from the bark of Pacific yew, Taxus brevifolia, can promote tubulin polymerization and inhibit microtubules disassembly, causing cell death by disrupting the microtubule dynamics required for cell division and vital interphase process1. This agent also induces apoptosis by binding and blocking the function of the apoptosis inhibitor protein Bcl-2 (B-cell Leukemia 2). Many reports have shown that paclitaxel induces various cancer cell death through apoptosis2, which is characterized by the cellular rounding-up, cytoplasmic contraction, plasma membrane blebbing, chromatin condensation, DNA fragmentation and many biochemical characteristics, including the activation of death receptor pathway, mitochondrial pathway, and or cysteine aspartic-specific proteases (caspases) cascades3,4. The activation of caspases is commonly thought to be one of the earliest points in the no-return pathway of apoptosis. Caspases are broadly categorized into upstream initiator caspases and downstream effector caspases5. The initiator caspases, such as caspase-8 (death receptor apoptosis pathway) and caspase-9 (mitochondrial apoptosis pathway), typically have a long N-terminal prodomain that, after activation by alternative stimuli, facilitates recruitment and interaction with other caspase proteins6,7. The effector caspases could be activated by initiator caspases. The activated effector caspases, in turn, cleave a number of cytoplasmic and nuclear substrates such as poly ADP-ribose polymerase (PARP), lamin B, protein kinase C, and inhibitor of caspase activated DNAse (ICAD), which will result in the execution of cell death5. Study regarding the mechanism of paclitaxel-induced head and neck cancer cell death is rare. Thus paclitaxel acting on different cell mechanism it is of interest to follow the kinetic of paclitaxel penetration in cancerous cell in order to establish the drug activation cascade. The ability of confocal Raman microscope to follow
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