1,4-Benzoquinone (PBQ) induced toxicity in lung epithelial cells is mediated by the disruption of the microtubule network and activation of caspase-3.
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.
Das
Amlan
A
Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB, India 700019.
Chakrabarty
Subhendu
S
Choudhury
Diptiman
D
Chakrabarti
Gopal
G
D017209:Apoptosis / Q000187:drug effects; D016227:Benzoquinones / Q000009:adverse effects*; D053148:Caspase 3 / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000166:cytology*; D008870:Microtubules / Q000378:metabolism*
D016227:Benzoquinones; C004532:quinone; D053148:Caspase 3