Protein phosphatase-2A restricts migration of Lewis lung carcinoma cells by modulating the phosphorylation of focal adhesion proteins.

Compared to metastatic Lewis lung carcinoma (LLC) cells, nonmetastatic LLC cells have increased levels of activity of the protein phosphatase PP-2A, which functions to limit their migration through transwell chambers. Inhibition of PP-2A in nonmetastatic LLC stimulates their transmigration to levels similar to those of metastatic LLC cells. Studies to define the signaling pathways intermediate between diminished PP-2A activity and stimulated migration showed that inhibiting PP-2A activity resulted in paxillin serine hyperphosphorylation and tyrosine dephosphorylation. Paxillin was important for the stimulated migration because the increased transmigration in response to PP-2A inhibition was dampened by expression of mutant paxillin at the LIM3 S457 and S481 residues. Inhibition of PP-2A also led to the dissolution of FAK/Src/paxillin focal adhesion complexes, which was also dependent on paxillin S457 and S481 residues. In addition, inhibition of PP-2A resulted in dephosphorylation of Src inhibitory Y527 residue, suggesting increased Src activity. The stimulated transmigration of cells with diminished PP-2A was in part dependent on this Src activity. These studies show the importance of PP-2A in limiting tumor cell migration through its modulation of proteins of the focal adhesions.

Effects of soybean extract on morphology and survival of Caco-2, SW620, and HT-29 cells.

Soybean consumption may be beneficial to prevention of certain human cancers. Low incidence of colon cancer in Asian countries is associated with consumption of soybean products. A limited number of human and animal studies suggested that soybean consumption might prevent colon cancer; other studies did not support this conclusion. Therefore, it is important to understand the biological effects of soybeans on colon cells. In the present study, cultures of Caco-2, SW620, and HT-29 cells were treated with soybean extract, the soluble fraction of a soybean product. The crude extract contains proteins and many soluble components of soybeans. After incubation with soybean extract (1-6%, vol/vol) for 24 h, most Caco-2 cells were found to contain numerous vacuoles within the cytoplasm and to become very flat. Exposure to > 6% soybean extract resulted in cell death and giant vacuoles. Soybean extract (0.25-2%) induced small vacuoles within the cytoplasm of SW620 cells. SW620 cells detached from culture dishes at > 2% soybean extract. Exposure to 0.5-2% soybean extract produced vacuoles within HT-29 cells similar to those observed in SW620 cells. Soybean extract significantly reduced density of Caco-2, SW620, and HT-29 cells. Reducing protein content of soybean extract reduced but did not abolish its effects on colon cells. Purified genistein (12.5 micrograms/ml) was capable of producing morphological changes similar to those observed after treatment of colon cells with soybean extract. Assays using annexin V-propidium iodide demonstrated that treatment of Caco-2 and SW620 cells with soybean extract increased cell death. Membranes of vacuoles in soybean-treated Caco-2 and SW620 cells were labeled with Texas red-conjugated wheat germ agglutinin, a cytological marker for the Golgi apparatus. Exposure to soybean extract enhanced protein levels of Rab6, a small GTP-binding protein that is involved in regulation of membrane traffic of the Golgi apparatus. Data from this study suggest that exposure to soybean extract or isoflavones affects morphology and survival of colon cancer cells and that the response to soybean extract varies depending on the cell lines examined.

Protein phosphatase-2A regulates endothelial cell motility and both the phosphorylation and the stability of focal adhesion complexes.

Solid cancers must stimulate expansion of the vascular network for continued growth. The process of angiogenesis involves endothelial cell migration so as to reorganize into vessel structures. The extent of cellular motility is regulated in part by the balance between serine/threonine kinases and protein phosphatases. In the present study, we show a decline in the activity of the serine/threonine phosphatase PP-2A in endothelial cells whose motility is stimulated by exposure to medium conditioned by either murine LLC cells or human HNSCC cells. Inhibition of endothelial cell PP-2A pharmacologically by treatment with okadaic acid also stimulated endothelial cell motility. Identification of mechanisms by which PP-2A inhibition might stimulate endothelial cell motility focused on proteins of the focal adhesions. Inhibition of PP-2A caused hyperphosphorylation of the paxillin serine residues and dephosphorylation of its tyrosine residues, dissolution of FAK/Src/paxillin complexes and decreased phosphorylation of the inhibitory Y529 residue of Src, suggesting increased Src activity. Inhibition of Src activity prevented the stimulation of PP-2A-inhibited cell motility. Our results suggest an interrelationship between tumor inhibition of PP-2A, dissolution of focal adhesion complexes and stimulated motility of endothelial cells.