Effects of Nocodazole on Fine Structures in Co-cultured Vascular Endothelial Cells and Cancer Cell Lines

Kim, Joo Young; Song, In Hwan; Lee, Jong Yong; Sung, Eon Ki; Lee, Yungchang

Abstract

Endothelial cells were isolated from the aortic intima of Sprague-Dawley species, rat. These cells and each cancer cell line (HeLa, Hep G2, A549, L929 and NIH/3T3 cells) were co-cultivated in alpha-MEM with 3 micrometer or 30 micrometer nocodazole. To investigate the influences induced by nocodazole, the morphological changes were observed under inverted microscope and transmission electron microscope, the amounts of fibronectin produced by vascular endothelial cells and cancer cell lines and the activities of nitric oxide synthetase synthesized mainly by endothelial cells were analyzed in the aspects associated with fine structural changes. The vascular endothelial cells of control group at the 1st, 2nd and 3rd days extended the cell processes, which contacted with cells from all cell lines investigated, but the endothelial cells of nocodazole-treated groups didn't possess the processes. All cell lines in nocodazole-treated groups had a large number of micronucleated cells, but endothelial cells didn't show micronuclei. Compared with control group, the endothelial cells of nocodazole-treated groups at the 1st, 2nd and 3rd days showed the decrease of amounts of fibronectin because of the increase of heterochromatin area. The amounts of fibronectin increased in all cell lines of nocodazole-treated groups at the 2nd and 3rd days whereas the nuclear folding or the dilatation/numerical increase of rough endoplasmic reticulum didn't appear. The activities of nitric oxide synthetase heightened in endothelial cells of nocodazole-treated groups, and therefore the considerable changes in fine structures such as vesicles, lysosomes, liposomes, pyknosis and cell lysis occurred even though the extent of changes differed among the cell lines. Taken together, the materials such as fibronectin or nitric oxide synthetase produced by endothelial cells directly or indirectly acted on cancer cells, and the amounts of fibronectin and the vesicles, lysosomes, liposomes and cell lysis seemed to be much more increased or enforced. Therefore, co-culture system seemed to work better for the investigation of actions of nocodazole and the role of endothelial cells in cancer cells research. Also, the co-culture system was closer to the in vivo state and more favorable in studies for proliferation or metastasis of cancer cells.