Synergistic Cytotoxicity of Arsenic Trioxide and Sulindac Against Neuroblastoma

Choi, Du Young

Abstract

PURPOSE
Recent clinical studies have shown that arsenic trioxide(As2O3) at low concentrations induces complete remission without significant toxicity in patients with refractory acute promyelocytic leukemia(APL). Like APL cells, neuroblastoma(NB) cells are thought to be arrested at an early stage of differentiation, and can respond with retinoic acid treatment. Sulindac, a nonsteroidal antiinflammatory drug, was reported to induce antitumor activity against various tumors through induction of apoptosis and inhibition of angiogenesis. There was no data with the effect of sulindac on NB, and the concentration of sulindac for antitumor effect could not be safely used in the clinical field. Therefore, we at first focused the synergistic cytotoxicity of two drugs using CHLA-15, CHLA-20 NB cells with different resistance to anticancer drugs, and then studied the mechanism of synergistic cytotoxicity at a clinically safe concentration of two drugs. METHODS: CHLA-15 and CHLA-20 cells were cultured in IMDM and treated with As2O3 and/or sulindac. Cell viability was measured by methilthiazol-2-yl-2, 5-diphenyl, tetrazolium bromide(MTT) assay. Apoptosis was assessed by DNA fragmentation assay. Apoptotic machinaries including FAS, caspase-3, PARP[poly(ADP-ribose) polymerase] were determined by Western blot analysis. RESULTS: As2O3 caused significant cytotoxicity on both CHLA-15 and CHLA-20 cell lines in a dose dependent manner, whereas sulindac had no demonstrable cytotoxic effects. Both drugs in combination induced synergistic cytotoxicity on two cell lines. Especially on a clinically therapeutic level with 2micrometer of As2O3 and 10micrometer of sulindac in combination, the viability of CHLA-15 and CHLA-20 cells was decreased to 15%, 60% more than arsenic alone, respectively. The synergistic cytotoxicity occurred by apoptosis through up-regulation of the Fas receptor, activation of caspase-3 and cleavage of PARP, which was a central pathway of induction of apoptosis. CONCLUSION: As2O3 and sulindac induced synergistic cytotoxicity and apoptosis on NB through up- regulation of the FAS receptor, activation of caspase-3 and cleavage of PARP. Taken together, these results indicate that As2O3 and sulindac are therapeutic agent candidates for treatment of NB.