Safingol is a sphingolipid with promising anticancer potential, which is currently in phase I clinical trial. Yet, the underlying mechanisms of its action remain largely unknown. We reported here that safingol-induced primarily accidental necrotic cell death in MDA-MB-231 and HT-29 cells, as shown by the increase in the percentage of cells stained positive for 7-aminoactinomycin , collapse of mitochondria membrane potential and depletion of intracellular ATP. Importantly, safingol treatment produced time- and concentration-dependent reactive oxygen species (ROS) generation. Autophagy was triggered following safingol treatment, as reflected by the formation of autophagosomes, acidic vacuoles, increased light chain 3-II and Atg biomarkers expression. Interestingly, scavenging ROS with N-acetyl--cysteine could prevent the autophagic features and reverse safingol-induced necrosis. Our data also suggested that autophagy was a cell repair mechanism, as suppression of autophagy by 3-methyladenine or bafilomycin A1 significantly augmented cell death on 2-5 μ safingol treatment. In addition, Bcl-xL and Bax might be involved in the regulation of safingol-induced autophagy. Finally, glucose uptake was shown to be inhibited by safingol treatment, which was associated with an increase in p-AMPK expression. Taken together, our data suggested that ROS was the mediator of safingol-induced cancer cell death, and autophagy is likely to be a mechanism triggered to repair damages from ROS generation on safingol treatment.

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Our study provided proof-of-concept evidence to deliver synergistic combination of bioactive lipid to achieve complete remission in AML.

Abstract. Colon cancer represents one of the most common solid tumors in adults. Although 5-fluorouracil (5-FU) and irinotecan have been frequently administered in colon cancer patients, low response rates to these single drug therapies were reported. It is therefore imperative to search for new targeted combination therapies that are effective. In this study, we investigated the anti-cancer effect of safingol as a single agent or in combination with irinotecan using HT-29 and LS-174T colon cancer cells as our in vitro models. As a single agent, safingol was more potent than irinotecan and 5-FU, with IC 50 values of 2.5±1.1 μM and 3.4±1.0 μM achieved in HT-29 and LS-174T cells, respectively. However, protein kinase C (PKC) was not inhibited with concentrations of safingol which could induce substantial cell kill. The combination of safingol/irinotecan at 1:1 molar ratio was found to be additive in HT-29 cells (CI=0.94) and synergistic in LS-174T cells (CI=0.68), and resulted in concentration-and time-dependent down-regulation of p-PKC and p-MARCKS. The drug effect of the safingol/irinotecan combination was further modulated in the presence of a PKC stimulator (phorbol 12-myristate 13-acetate) or a PKC inhibitor (staurosporine). Furthermore, the 1:1 safingol/irinotecan combination inhibited the adhesion of colon cancer cells to the extracellular matrix 4-h post-treatment. Taken together, modulation of the PKC pathway could be a possible molecular basis for the observed synergism of the safingol/irinotecan combination, and these results demonstrate the therapeutic potential of this drug combination in colon cancer treatment.