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 function in processes relevant to carcinogenesis, tumor cell metastasis, and apoptosis. Safingol, an optical isomer (the L-threo enantiomer) of dihydrosphingosine, is a specific inhibitor of PKC and might represent a novel agent for anticancer therapy. Preclinical animal studies show that safingol alone has a minimal effect on tumor cell growth, but combining this compound with conventional chemotherapy agents dramatically potentiates their antitumor effects. It has been suggested that many chemotherapeutic agents exert their antitumor effects by inducing apoptosis.


OBJECTIVE
We wanted to determine the extent to which safingol, alone or in combination with a standard chemotherapeutic agent (mitomycin 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 their drug-resistance status.


METHODS
SK-GT-5 (p53-deficient and MMC-resistant) and MKN-74 (p53 wild-type and MMC-sensitive) gastric cancer cells were exposed to either no drug, safingol (50 microM) alone, MMC (5 micrograms/mL) alone, or a combination of safingol (50 microM) and MMC (5 micrograms/mL). In some experiments, cells were exposed simultaneously to safingol and the PKC activator, 3-phorbol 12-myristate 13-acetate (PMA), prior to treatment with MMC. Apoptosis was measured by two methods: 1) quantitative fluorescence microscopy of nuclear chromatin condensation in cells stained with the dye, bisbenzamide trihydrochloride (Hoechst-33258), and 2) terminal deoxynucleotidyl transferase (TdT) labeling of the 3'-OH ends of DNA fragments produced in apoptotic cells.


RESULTS
As determined by quantitative fluorescence microscopy, exposure of SK-GT-5 cells to safingol alone induced apoptosis in 2% +/- 1% (mean +/- SD) of the cells, and MMC alone increased that level to 18% +/- 1%. However, 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 the 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 safingol-mediated enhancement of MMC-induced apoptosis.


CONCLUSIONS
The PKC inhibitor safingol enhances the cytotoxic effect of the chemotherapeutic agent MMC in gastric cancer cells by promoting drug-induced apoptosis. The induction of apoptosis occurs regardless of the p53 status or the drug-resistance status of the cells.
Schwartz
G K
GK
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Haimovitz-Friedman
A
A
Dhupar
S K
SK
Ehleiter
D
D
Maslak
P
P
Lai
L
L
Loganzo
F
F
Kelsen
D P
DP
Fuks
Z
Z
Albino
A P
AP
D017209:Apoptosis / Q000187:drug effects; D004351:Drug Resistance; D004357:Drug Synergism; D006801:Humans; D016685:Mitomycin / Q000627:therapeutic use*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013237:Stereoisomerism; D013274:Stomach Neoplasms / Q000737:chemistry / Q000188:drug therapy* / Q000503:physiopathology*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis
D016159:Tumor Suppressor Protein p53; D016685:Mitomycin; D011493:Protein Kinase C; D013110:Sphingosine; C005682:safingol