Nocodazole is an anticancer agent, well-known for its antimetastatic activity that acts on microtubules, microfilaments and extracellular matrix proteins. Hela, Hep G2, A549, L929, and NIH/3T3 cell lines were cultivated in alpha-MEM with 3microM or 30microM nocodazole. To investigate the mechanism of nocodazole preventing tumor cell metastasis, the influences of nocodazole on the amounts of glycoprotein, fibronectin, laminin and actin were investigated using PAS staining and PAP technique at light microscopic level. Two designed models ; coverglass and 3-day-old rat heart fragments models, were used in observing the invasiveness of cancer cells. Partitularly the three-dimensional model coculturing cell lines and heart fragment was used in evaluating the migration and/or proliferation or the invasiveness of cell around the fragment, and observed under inverted or bright field light microscope. The amount of glycoprotein of all cell-lines increased in cells of groups treated with nocedazole for 1, 2 and 3 day. The amounts of fibronectin usually increased in cells of groups treated with nocodazole for 1, 2 and 3 day. The amounts of laminin increased in cells of groups treated with nocodazole. The amounts of actin usually increased in cells of groups treated with nocodazole for 1, 2 and 3 days. With the prolonging of nocodazole-treatment time in two dimensional model using coverglass, the cells of control group except Hep G2 cells formed monolayer in cell-free zone according to migration or proliferation of many cells. But only a few cells of experimental groups migrated or proliferated into cell-free zone. In rat heart fragment model the cells of control group showed the invasiveness into the fragment but few or none of the cells from experimental groups attached around the fragment. Taken together, nocodazole increased the synthesis of fibronectin and laminin in cells in place of depolymerizing microtubules. Therefore, the amounts of extracellular matrix proteins in the extracellular space increased. And the increase amounts of actin connected to the extracellular matrix proteins through the integrin of plasma membrane seemed to strengthen cell attachment because of accordance between the orientation of actin and extracellular matrix proteins. Since it is important for cancer cells` metastasis to secrete various enzymes to pass through extracellular matrix proteins, it is expected more difficult for the cells to metastasize into other regions due to the increase of extracellular matrix proteins. As a result of confirmation of antiinvasive actions using two kinds of model, nocodazole seems to be a valuable anti-metastatic agent by supressing the cell motility and consequently, the invasiveness into the fragment. Nocodazole at concentration of 3microM will be probably anticipated antimetastatic activity reflecting that the effects of nocodazole between 3microM and 30microM groups had no differences.
Actin Cytoskeleton ; Actins ; Animals ; Cell Line* ; Cell Membrane ; Cell Movement ; Extracellular Matrix Proteins* ; Extracellular Matrix* ; Extracellular Space ; Fibronectins ; Glycoproteins ; Heart ; Hep G2 Cells ; Laminin ; Microtubules ; Neoplasm Metastasis ; Nocodazole* ; Rats

Nocodazole is an anticancer agent that acts on microtubules or filaments. HeLa, Hep G2, A549, L929 and NIH/3T3 cell lines were cultivated in alpha-MEM with 3micrometer or 30micrometer nocodazole. To elucidate the associations between nocodazole`s antitumor actions and these effects, the influences of nocodazole on the cellular morphology and the organelles involving synthesis, secretion and destruction of proteins were investigated under light and electron microscopes. The changes of intermediate filaments such as pancytokeratins and vimentins that maybe suggest antimetastatic action of nocodazole were observed using immunocytochemical technique, PAP at light microscopic level. Rounded or micronucleate cells were induced by treatment with 3micrometer and 30micrometer nocodazole for 2 hours to 4 days. Multimicronucleate cells appeared in experimental groups of all cell lines. Nuclear foldings occurred in cells of experimental groups treated with nocodazole for 2-3 days. The numerical increases of rough endoplasmic reticulum were observed in HeLa cells treated with nocodazole for 3 days and the dilatation or numerical increases in L929 cells treated with nofodazole for 1-3 days. The fragmentations or dispersion of Golgi complex were observed in cells treated with nocodazole for 1-3 days. The amount of filaments increased in cells treated with nocodazole for 1-3 days. The number of lysosomes increased in cells treated with nocodazole for 1-3 days. The number of liposomes also increased in Hep G2 cells treated with 30micrometer nocodazole for 3 days and in 3micrometer & 30micrometer, 3 days group of 3T3 cells. The amount of pancytokeratins and vimentins increased in cells treated with nocodazole for 1-3 days. Taken together, depolymerization of microlubules was induced by nocodazole, and then the organization of cells was disintegrated. As a result, the rounded cells, the cells having multimicronuclei, and the changes of golgi complexes occurred. But there were relatively no great changes of rough endoplasmic reticulum. The amount of intermediate filaments that maintain the differentiated states of cells increased by nocodazole treatment. It was suggested that morphological changes of cells could be used in evaluation of actions of nocodazole. Especially, the increase of amount of intermediate filaments by nocodazole changed cells of each cell line from undifferentiated state to differentiated, and therefore the author hope that the changes in amount of intermediate filaments provide an important clue in anticancer and antimetastatic actions of nocodazole.
3T3 Cells ; Animals ; Cell Line ; Dilatation ; Endoplasmic Reticulum, Rough ; Golgi Apparatus ; HeLa Cells ; Hep G2 Cells ; Hope ; Humans ; Intermediate Filaments ; Liposomes ; Lysosomes ; Mice ; Microtubules ; Nocodazole* ; Organelles ; Vimentin