Antagonism of etoposide cytotoxicity for neuroblastoma by retinoic acid is mediated by the anti-apoptotic bcl-2 family proteins.

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Retinoic acid (as 13-cis-retinoic acid = 13-cis-RA) is given at completion of cytotoxic therapy to control minimal residual disease in neuroblastoma. In acute promyelocytic leukemia, retinoic acid (as all-trans-retinoic acid = ATRA) was most effective when combined directly with cytotoxic chemotherapy. We evaluated the interaction between retinoic acid and cytotoxic chemotherapy in neuroblastoma cell lines and its relation to the bcl-2 family of proteins. Neuroblastoma cell lines (SMS-KCNR, SMS-LHN, SMS-SAN, CHLA-90, CHLA-79) were tested for cytotoxic response to drugs + ATRA or 13-cis-RA with a fluorescence-based digital imaging microscopy cytotoxicity assay (DIMSCAN). Apoptosis was assessed as DNA fragmentation and sub-genomic DNA content by flow cytometry; loss of mitochondrial membrane potential employed flow cytometryand the fluorochrome JC-1. Real-time quantitative RT-PCR (TAQMAN) was used to measure mRNA expression; Western blot was used to follow the intrinsic apoptotic pathway. Direct combination of 13-cis-RA or ATRA with etoposide, topotecan, carboplatin, cisplatin, melphalan and doxorubicin markedly antagonized the cytotoxicity of those agents for all tested neuroblastoma cell lines, increasing the fraction of cell survival by 1 to 3 logs. The concentration of drugs lethal for 99% of cells (LC99) was increased by 13-cis-RA from clinically achievable levels to non-achievable levels: 100 to 300-fold (doxorubicin) to > 1000-fold (cisplatin and etoposide). 13-cis-RA protected cells from late apoptosis, defined as DNA fragmentation and loss of membrane integrity, in response to etoposide. By quantitative RT-PCR, 13-cis-RA increased the expression of bcl-2 and bcl-xL, but decreased the expression of MYCN (p>0.03). Western blot data showed that 13-cis-RA pre-treated cells activated p53, but did not release Cytochrome c in response to etoposide. Pre-treatment of neuroblastoma cell lines with 13-cis-RA reduced the fraction of cells with loss of mitochondrial membrane potential in response to etoposide from 38% to 8% (basal level = 12%), but this was restored to 52% by a small molecule inhibitor of the bcl-2 family of proteins (ABT-737). Similar data were obtained for early apoptosis (at 16 hrs). 13-cis-RA pre-treatment decreased apoptotic (sub-G1) cells in response to etoposide from 58% to 18% (basal 17%), treating with ABT-737 reversed the antagonistic effect resulting in 74% apoptosis. Directly combining retinoic acid with cytotoxic chemotherapy in neuroblastoma markedly reduced the cytotoxic effect of the chemotherapy, mediated at least in part via the anti-apoptotic bcl-2 family of proteins.