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A subset of four synthetic sphingoid marine compound analogs was chosen from a preliminary in vitro cytotoxicity study for further analysis. The selected analogs were initially screened in monolayer cultures for their anticancer potential against a panel of eight human tumor cell lines, ovarian, colon and lung cancer, squamous cell carcinoma and leukemia producing IC50 values ranging from 1.5 to 6.9 μM. In a secondary screening, the sphingoid analogs were evaluated against multilayered postconfluent cultures of A2780 ovarian cancer and WiDr colon cancer cells. In this model, compounds 5 and 8 were the most active derivatives showing EC50 values in the range 25-32 μM. The performance of 5 and 8 against both cell lines was not dependent on the cell culture model as shown with resistance factor values in the range 8-12. Cell cycle studies in HL60 leukemia cells showed an arrest in G0/G1 at a low drug concentration (3 μM) but accumulation in S phase at a high drug concentration (9 μM). It can be concluded that the analogs showed a cell line independent activity, with an apparent selectivity against cells grown in more physiological three-dimensional condition compared to standard anticancer drugs.