Sphingosine kinase catalyzes synthesis of sphingosine -1-phosphate (S-1-P), which promotes cell survival. We previously reported that some multi-drug resistant (MDR) neuroblastoma cell lines showed high basal expression of sphingosine kinase I mRNA (SPHK1) levels relative to drug-sensitive neuroblastoma lines, and that SPHK1 mRNA expression correlated significantly (P= 0.005) with the LC90 (concentration lethal for 90% of cells) for etoposide. We studied 3 MDR neuroblastoma cell lines with high basal sphingosine kinase mRNA levels (CHLA-90, LA-N-6, and SK-N-RA). For all three cell lines, SPHK-1 mRNA expression was significantly increased (P < 0.05) after vincristine (3.5-10 fold), doxorubicin (1.8- 4.1 fold), and etoposide (1.8-3.5 fold) treatment, while SPHK-2 mRNA expression was not significantly altered. Topotecan (1.3-2.1 fold), paclitaxel (1.2 -2.5 fold), and mephalan (1.3-1.7 fold) also increased SPHK-1 mRNA after drug treatment but carboplatin did not increase SPHK-1 mRNA. Vincristine or doxorubicin treatment also increased sphingosine kinase enzyme activity, and pretreatment with sphingosine-1-phosphate (product of SPHK), antagonized etoposide cytotoxicity in the drug-sensitive SMS-KCNR neuroblastoma cell line. L-threo-dihydrosphingosine (safingol) (0 - 3 μM) and N-N-dimethylsphingosine (DMS) (0 - 5 μM) were employed as sphingosne kinase inhibitors in fixed-ratio combinations with etoposide (0 to 20 μM), melphalan (0 - 34 μM), cisplatin (0 - 34 μM), carboplatin (0 - 34 μM), topotecan (0 - 34 μM), paclitaxel (0 - 500 nM), 5-fluouracil (0-180 μM), vincristine (0 - 1 μM), or doxorubicin (0-500 nM). Cytotoxicity was measured by a fluorescence digital imaging microscopy (DIMSCAN) assay. Synergy was determined by combination index (CI), where CI < 1 indicates synergy and >1 indicates antagonism. Vincristine, doxorubicin, or etoposide in combination with DMS or safingol showed significant synergistic cytotoxicity (CI < 0.5) in all 3 cell lines, while mephalan, topotecan, or paxlitaxel exhibited moderate synergy in CHLA-90 and slight synergy or additive effect in other cell lines. Moderate synergy was observed with high-dose 5-fluouracil and cisplatin in SK-N-RA and LA-N-6 cell lines. SPHK inhibitors did not enhance carboplatin cytotoxicity in any tested cell lines. Thus, sphingosine kinase inhibitors can enhance the response of MDR neuroblastoma cell lines to etoposide, doxorubicin, and vincristine by 1-2 logs of cell kill. The striking induction of SPHK-1 mRNA by vincristine and the syngergistic interaction with SPHK inhibitors suggest that SPHK-1 induction by vincristine treatment might play a role in neuroblastoma drug resistance and that SPHK is a therapeutic target for modulating drug resistance in neuroblastoma.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]