Growth suppression of a tumorigenic rat liver cell line by the anticancer

Purpose: This study was undertaken to elucidate the potential mechanism of the antitumor activity of ET-18-O-CH3, a synthetic analogue of lysophosphatidyl choline, and a known antitumor agent and specific inhibitor of phosphoinositide phospholipase C (PI-PLC). Methods: A normal rat liver epithelial ''oval'' cell line (WB-F344) was neoplastically transformed by the H-ras oncogene (WB-ras2) and treated with a series of ET-18- O-CH3 concentrates for a number of days. Cell growth, morphological ''differentiation'', cell cycle regulation, karyotypic changes, growth in soft agar (anchorage-in- dependent growth) and the expression of cdk2, cdc2 and ERK genes were studied to determine the effect of ET-18- O-CH3 on these neoplastic cells. Results: ET-18-O-CH3 at 5 and 10 lg/ml was found to cause an increase in cell size, suppress cell growth, reduce the colony-forming efficiency and inhibit the anchorage-independent growth of the WB-ras2 cells. Significantly, flow-cytometric analysis revealed that while control cells and cells treated with concentrations of ET-18-O-CH3 below 5 lg/ml were diploid, cell populations treated with 5 and 10 lg/ml ET-18-O-CH3 comprised 33-37% diploid cells and over 60% tetraploid cells (4n-8n cycle cells). ET-18-O-CH3 was found to induce aberrant cytokinesis as evidenced by the presence of a high frequency of enlarged cells, which were binucleated or multinucleated and mitotic cells with 4n and 8n numbers of chromosomes. ET-18-O-CH3 was also capable of inhibiting both the expression of cdk2 and cdc2 and the activation of ERK1/2, while no effect was found on the expression of p21 ras. Conclusions: The effect of ET-18-O-CH3 on neoplastically transformed H-ras rat liver cells has been interpreted as the result of an altered phenotype characterized by an enlarged and flattened cell morphology with ploidy changes caused by inhibition of cytokinesis.