Abstract
Purpose: :
To establish the mechanism by which combretastatin A4 phosphate (CA4P) inhibits wound healing and cell proliferation of primary and immortalised lens epithelial cells.
Methods: :
Primary bovine lens epithelial cells (BLEC) and the immortalised human lens epithelial cell line FHL124 were grown in serum-containing media on plastic and exposed to 1-10µM of CA4P. Colchicine, vinblastine and taxol were included given that CA4P has been shown to affect microtubule function in other cell types. Wound healing was measured using a scratch assay and live cell imaging to quantify the rate of wound closure in the presence or absence of the drugs. Cell viability was quantified using the Cell Titer 96R AQueous Assay 3 days after treatment. Treated and untreated cells were also processed for immunofluorescence microscopy and the effects of the various drugs upon the distribution of the actin, vimentin and tubulin networks determined by confocal immunofluorescence microscopy. Untreated cells served as controls and data from three independent experiments were analysed using the independent two-tailed t-test, with any P value below 0.05 being considered to indicate significance.
Results: :
The viability of both primary BLEC and human FHL124 was efficiently prevented by 1µM CA4P. 10µM CA4P significantly decreased cell number. Although both vinblastine and taxol significantly decreased cell number when compared to controls, BLECs were more sensitive to vinblastine and FHL124 more sensitive to taxol. For both cell lines, colchicine was the least effective. In scratch wound assays, both 1µM CA4P and vinblastine were equally effective at delaying wound closure. Confocal immunofluorescence microscopy confirmed extensive depolymerisation of the microtubule network. An increased pool of soluble tubulin was detected in the CA4P treated cells. In contrast, the microtubule network was mostly unchanged in colchicine and taxol treated cells. Both CA4P and vinblastine also appeared to cause condensation of the vimentin network, whereas the actin network was mostly unaffected.
Conclusions: :
The potential PCO-preventing drug CA4P is an effective inhibitor of cell viability of lens epithelial cells, comparable in efficacy to taxol and vinblastine. CA4P was also effective at preventing wound closure. CA4P binds to and competes for the colchicine binding site on tubulin. These data suggest that CA4P is a more effective inhibitor of microtubule function in lens epithelial cells.
Keywords: cytoskeleton • microscopy: light/fluorescence/immunohistochemistry • aging