POTENTIATION OF APOPTOSIS BY TREATMENT WITH THE PROTEIN-KINASE C-SPECIFIC INHIBITOR SAFINGOL IN MITOMYCIN C-TREATED GASTRIC-CANCER CELLS

Background: Protein kinase C (PKC) is a family of enzymes that functio n in processes relevant to carcinogenesis, tumor cell metastasis, and apoptosis. Safingol, an optical isomer (the L-threo enantiomer) of dih ydrosphingosine, is a specific inhibitor of PKC and might represent a novel agent for anticancer therapy. Preclinical animal studies show th at safingol alone has a minimal effect on tumor cell growth, but combi ning this compound with conventional chemotherapy agents dramatically potentiates their antitumor effects. It has been suggested that many c hemotherapeutic agents exert their antitumor effects by inducing apopt osis. Purpose: We wanted to determine the extent to which safingol, al one or in combination with a standard chemotherapeutic agent (mitomyci n C [MMC]), would promote apoptosis in gastric cancer cells in vitro. Furthermore, we investigated whether the induction of apoptosis in the treated cells was affected by their p53 tumor suppressor status or th eir drug-resistance status. Methods: SK-GT-5 (p53-deficient and MMC-re sistant) and MKN-74 (p53 wild-type and MMC-sensitive) gastric cancer c ells were exposed to either no drug, safingol (50 mu M) alone, MMC (5 mu g/mL) alone, or a combination of safingol (50 mu M and MMC (5 mu g/ mL). In some experiments, cells were exposed simultaneously to safingo l and the PKC activator, 3-phorbol 12-myristate 13-acetate (PMA), prio r to treatment with MMC. Apoptosis was measured by two methods: 1) qua ntitative fluorescence microscopy of nuclear chromatin condensation in cells stained with the dye, bisbenzamide trihydrochloride (Hoechst-33 258), and 2) terminal deoxynucleotidyl transferase (TdT) labeling of t he 3'-OH ends of DNA fragments produced in apoptotic cells. Results: A s determined by quantitative fluorescence microscopy, exposure of SK-G T-5 cells to safingol alone induced apoptosis in 2% +/- 1% (mean +/- S D) of the cells, and MMC alone increased that level to 18% +/- 1%. How ever, the combination of safingol and MMC induced apoptosis in 39% +/- 1% of the cells (P<.001, for the drug combination versus MMC alone). With MKN-74 cells, safingol alone induced apoptosis in 8% +/- 3% of th e cells, whereas MMC alone induced apoptosis in 40% +/- 4% of treated cells and the combination of safingol and MMC induced apoptosis in 83% +/- 4% of the cells. Similar results were obtained with the TdT assay . Simultaneous exposure of cells to safingol and PMA abrogated the saf ingol-mediated enhancement of MMC-induced apoptosis. Conclusions: The PKC inhibitor safingol enhances the cytotoxic effect of the chemothera peutic agent MMC in gastric cancer cells by promoting drug-induced apo ptosis. The induction of apoptosis occurs regardless of the p53 status or the drug-resistance status of the cells.