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.
Parabenzoquinone (1,4-benzoquinone) (PBQ) is a bioactve quinone present in cigarette smoke and diesel smoke, which causes severe genotoxic effects both in vitro and in vivo. In the previous study, we showed that the microtubules are one of the major targets of cigarette smoke-induced damage of lung epithelium cells. In the present study, we have investigated the effect of PBQ on cellular microtubules using human type II lung epithelial cells (A549) and also on purified tubulin. Cell viability experiments using A549 cells indicated a very low IC(50) value (approximately 7.5 microM) for PBQ. PBQ inhibited cell cycle progression and induced apoptosis of A549 cells. PBQ also induced the contraction and shrinkage of the A549 cells in a time- and concentration-dependent manner, which is proved to be a direct effect of the damage of the microtubule cytoskeleton network, and that was demonstrated by a immunofluorescence study. PBQ also inhibited the assembly of tubulin in lung cells and a in cell free system (IC(50) approximately 5 microM). Treatment with PBQ resulted in the degradation of tubulin in lung cells without affecting the actin network, and this was confirmed by a Western blot experiment. Upregulation of pro-apoptotic proteins such as p53 and Bax and downregulation of antiapoptotic protein Bcl-2 were observed in PBQ-treated A549 cells. Simultaneously, loss of mitochondrial membrane potential and activation of caspase-3 were also observed in the PBQ treated lung epithelium cells. Fluorescence and circular dichroism studies demonstrated that the denaturation of tubulin in a cell free system was caused by PBQ. However, in the presence of N-acetyl cysteine (NAC), damage of the microtubule network in A549 cells by PBQ was prevented, which led to a significant increase in the viability of A549 cells. These results suggest that microtubule damage is one of the key mechanisms of PBQ induced cytotoxity in lung cells.
Naphthazarin (DHNQ, 5,8-dihydroxy-l,4-naphthoquinone) is a naturally available 1,4-naphthoquinone derivatives. In this study, we focused on elucidating the cytotoxic mechanism of naphthazarin in A549 non-small cell lung carcinoma cells. Naphthazarin reduced the A549 cell viability considerably with an IC(50) of 16.4 ± 1.6 μM. Naphthazarin induced cell death in a dose- and time-dependent manner by activating apoptosis and autophagy pathways. Specifically, we found naphthazarin inhibited the PI3K/Akt cell survival signalling pathway, measured by p53 and caspase-3 activation, and PARP cleavage. It also resulted in an increase in the ratio of Bax/Bcl2 protein levels, indicating activation of the mitochondrial apoptotic pathway. Similarly naphthazarin triggered LC3II expression and induced autophagic flux in A549 cells. We demonstrated further that naphthazarin is a microtubule inhibitor in cell-free system and in A549 cells. Naphthazarin treatment depolymerized interphase microtubules and disorganised spindle microtubules and the majority of cells arrested at the G(2)/M transition. Together, these data suggest that naphthazarin, a microtubule depolymerizer which activates dual cell death machineries, could be a potential novel chemotherapeutic agent.
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