Effects of Nocodazole on Extracellular Matrix Proteins of Cancer Cell Lines

Jung, Byung Wook; Kim, Joo Young; Lee, Yun Chanl

Abstract

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.