To determine the effect of retinoic acid (RA) in neuroblastoma we treated RA sensitive neuroblastoma cell lines with 9-cis RA or ATRA for 9 days, or for 5 days followed by absence of RA for another 4 days. Both isomers induced apoptosis and reduced cell density as a result of cell differentiation and/or apoptosis. Flow cytometry revealed that 9-cis RA induced apoptosis more effectively than ATRA. The expression profile of apoptosis and survival pathways was cell line specific and depended on the isomer used.

Cancer cells acquire resistance to cytotoxic therapies targeting major survival pathways by adapting their metabolism. The AKT pathway is a major regulator of human pancreatic adenocarcinoma progression. The mechanisms of adaptation to long-term silencing of AKT isoforms of pancreatic cancer cells were studied. Following silencing, cancer cells remained quiescent for long periods of time, after which they recovered proliferative capacities. Adaptation caused profound proteomic changes largely affecting mitochondrial biogenesis, energy metabolism, and acquisition of a number of distinct cancer stem cell (CSC) characteristics depending on the AKT isoform that was silenced.The adaptation to AKT1 silencing drove most de-differentiation and acquisition of stemness through C-MYC down-modulation and NANOG up-regulation, which were required for survival of adapted CSCs. The changes associated to adaptation sensitized cancer cells to inhibitors targeting regulators of oxidative respiration and mitochondrial biogenesis. In vivo pharmacological coinhibition of AKT and mitochondrial metabolism effectively controlled pancreatic adenocarcinoma growth in pre-clinical models.

Neuroblastoma is a malignant embryonal childhood tumor of the peripheral nervous system, which often shows poor clinical prognosis. p53 mutations are rare at diagnosis of neuroblastoma but may be acquired at relapse, as well as chemoresistance. We have previously detected a mutation in exon 5 of the p53 gene (codon 135; Cys to Phe) in the SK-N-FI neuroblastoma cell line. To test whether this mutation affects p53 function driving the cells to a higher chemoresistant phenotype, we analyzed the action of doxorubicin, etoposide, cisplatin, melphalan and retinoic acid on cell survival, apoptosis induction and cell cycle distribution in these cells. SK-N-FI cells were incubated in the presence of doxorubicin, etoposide, cisplatin, melphalan and 9-cis or all-trans retinoic acid at five different concentrations ranging from 10-4 to 10-8 M. After 72 h of treatment, cell survival was estimated by the MTT method. Quantification of apoptosis and cell cycle analysis were carried out by flow cytometry. With this purpose, fixation of SK-N-FI cells was conducted after 12, 24, 72 h and 5 days of treatment with the previously mentioned drugs. All tested compounds were toxic at the micromolar range except for the retinoic acid isomers which were active at higher doses. The smaller GI50 values were found with doxorubicin, followed by melphalan, etoposide and cisplatin. A statistically significant rise in apoptosis was observed in all cases at day 5. This increase was even noticeable at 72 h for doxorubicin, 9-cis retinoic acid and cisplatin. With regard to the cell cycle distribution, a decrease of the G0/G1 cell population at day 5, accompanied by a time course increase in the Sub-G1 phase, was detected for every tested drug but the retinoic acid isomers. Melphalan also decreased S phase cells. Moreover, while for doxorubicin treatment reduction of the G0/G1 cell population was preceded by an increase in S and G2/M cells at shorter periods, for etoposide and cisplatin only the reduction in the G0/G1 cells in the long term was observed. Finally, 9-cis retinoic acid but not all-trans retinoic acid treatment provoked an initial accumulation of G0/G1 cells that was followed by an increment in the Sub-G1 phase. Despite bearing a mutation of the p53 gene, SK-N-FI cells respond to doxorubicin, cisplatin, etoposide, melphalan, 9-cis and all-trans retinoic acid. All mentioned drugs were able to induce apoptosis, affected to the cell cycle distribution of the cells in a drug specific manner and, except for the retinoic acid isomers, displayed cytotoxic activity at the micromolar range. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3663.