title: Unprecedented inhibition of tubulin polymerization directed by gold nanoparticles inducing cell cycle arrest and apoptosis.
abstract: The effect of gold nanoparticles (AuNPs) on the polymerization of tubulin has not been examined till now. We report that interaction of weakly protected AuNPs with microtubules (MTs) could cause inhibition of polymerization and aggregation in the cell free system. We estimate that single citrate capped AuNPs could cause aggregation of ∼10(5) tubulin heterodimers. Investigation of the nature of inhibition of polymerization and aggregation by Raman and Fourier transform-infrared (FTIR) spectroscopies indicated partial conformational changes of tubulin and microtubules, thus revealing that AuNP-induced conformational change is the driving force behind the observed phenomenon. Cell culture experiments were carried out to check whether this can happen inside a cell. Dark field microscopy (DFM) combined with hyperspectral imaging (HSI) along with flow cytometric (FC) and confocal laser scanning microscopic (CLSM) analyses suggested that AuNPs entered the cell, caused aggregation of the MTs of A549 cells, leading to cell cycle arrest at the G0/G1 phase and concomitant apoptosis. Further, Western blot analysis indicated the upregulation of mitochondrial apoptosis proteins such as Bax and p53, down regulation of Bcl-2 and cleavage of poly(ADP-ribose) polymerase (PARP) confirming mitochondrial apoptosis. Western blot run after cold-depolymerization revealed an increase in the aggregated insoluble intracellular tubulin while the control and actin did not aggregate, suggesting microtubule damage induced cell cycle arrest and apoptosis. The observed polymerization inhibition and cytotoxic effects were dependent on the size and concentration of the AuNPs used and also on the incubation time. As microtubules are important cellular structures and target for anti-cancer drugs, this first observation of nanoparticles-induced protein's conformational change-based aggregation of the tubulin-MT system is of high importance, and would be useful in the understanding of cancer therapeutics and safety of nanomaterials.
authors.affiliation: Department of Biotechnology and Dr B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, West Bengal, India 700019.
authors.forename: Diptiman
authors.initials: D
authors.lastname: Choudhury
authors.forename: Paulrajpillai Lourdu
authors.initials: PL
authors.lastname: Xavier
authors.forename: Kamalesh
authors.initials: K
authors.lastname: Chaudhari
authors.forename: Robin
authors.initials: R
authors.lastname: John
authors.forename: Anjan Kumar
authors.initials: AK
authors.lastname: Dasgupta
authors.forename: Thalappil
authors.initials: T
authors.lastname: Pradeep
authors.forename: Gopal
authors.initials: G
authors.lastname: Chakrabarti
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D006041:Goats; D006046:Gold / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D010316:Particle Size; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D016192:Resting Phase, Cell Cycle / Q000187:drug effects*; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: C494826:BAX protein, human; C495901:TP53 protein, human; D014404:Tubulin; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D006046:Gold; D011065:Poly(ADP-ribose) Polymerases