A novel sphingosine kinase inhibitor induces autophagy in tumor cells

Abstract

The sphingolipids ceramide, sphingosine, and sphingosine 1-phosphate (S1P) regulate cell signaling, proliferation, apoptosis, and autophagy. Sphingosine kinase-1 and -2 (SK1 and SK2) phosphorylate sphingosine to form S1P, shifting the balanced activity of these lipids toward cell proliferation. We have previously reported that pharmacological inhibition of SK activity delays tumor growth in vivo. The present studies demonstrate that the SK2-selective inhibitor 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide (ABC294640) induces nonapoptotic cell death that is preceded by microtubule-associated protein light chain 3 cleavage, morphological changes in lysosomes, formation of autophagosomes, and increases in acidic vesicles in A-498 kidney carcinoma cells. ABC294640 caused similar autophagic responses in PC-3 prostate and MDA-MB-231 breast adenocarcinoma cells. Simultaneous exposure of A-498 cells to ABC294640 and 3-methyladenine, an inhibitor of autophagy, switched the mechanism of toxicity to apoptosis, but decreased the potency of the SK2 inhibitor, indicating that autophagy is a major mechanism for tumor cell killing by this compound. Induction of the unfolded protein response by the proteasome inhibitor N-(benzyloxycarbonyl)leucinylleucinylleucinal Z-Leu-Leu-Leu-al (MG-132) or the heat shock protein 90 inhibitor geldanamycin synergistically increased the cytotoxicity of ABC294640 in vitro. In severe combined immunodeficient mice bearing A-498 xenografts, daily administration of ABC294640 delayed tumor growth and elevated autophagy markers, but did not increase terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in the tumors. These data suggest that ABC294640 promotes tumor cell autophagy, which ultimately results in nonapoptotic cell death and a delay of tumor growth in vivo. Consequently, ABC294640 may effectively complement anticancer drugs that induce tumor cell apoptosis.

Fig. 1.

ABC294640 induces nonapoptotic cell death in A-498 cells. A, chemical formula of ABC294640. B, cells were exposed to the indicated concentrations of ABC294640 and fixed at 24, 48 or 72 h. Nuclei were harvested and stained with PI, and the DNA content was analyzed by flow cytometry as described under Materials and Methods. C, cells were exposed to the indicated concentrations of ABC294640 for 72 h, and caspase 3/7 activity was measured by luminescence as described under Materials and Methods. Cisplatin (cis-DDP) was used as a control (black bar). Data represent mean ± standard error for three experiments. D, cells were exposed to the indicated concentrations of ABC294640 for 72 h and stained with PI and Annexin-V-FITC before analysis by flow cytometry as described under Materials and Methods. For each panel, the bottom left corner indicates Annexin V- and PI-negative cells; the bottom right corner indicates Annexin V-positive cells (apoptotic cells); the top-right corner indicates dead cells with membranes permeable to PI (PI-positive cells) stained with Annexin V; and the top left corner indicates dissociated nuclei. The percentage of total cells is indicated in each quadrant.

Fig. 2.

Effects of SK inhibitors on LC3 cleavage and formation of autophagosomes in A-498 cells. A, cells were treated with the indicated concentrations of ABC294640 or 5 μM ceramide for the indicated times. The generation of LC3-II was then examined by Western blotting as described under Materials and Methods. Equal loading was confirmed by stripping and reprobing with anti-actin antibody. B, cells were exposed to the indicated concentrations of SKI-2 or 5 μM DMS, and the LC3 cleavage was assessed by Western blotting of cell lysates at 24, 48 or 72 h as described under Materials and Methods. C, A-498 cells were exposed to the vehicle, ABC294640, SKI-2, or DMS for 48 h, and then fixed and immunostained for LC3 as described under Materials and Methods. D, quantification of LC3-positive cells. Cells were exposed to 50 μM ABC294640, 30 μM SKI-2, or 5 μM DMS for 48 h. Data represent mean ± S.E. for three experiments. The numbers below the Western blots indicate the relative LC3-II/actin ratios compared with the vehicle-treated cells ± S.E.

Fig. 3.

Induction of autophagic vacuolization or apoptosis by ABC294640 in A-498 cells. A and B, cells were treated with 50 μM ABC294640 (A) or vehicle (B) for 24 h, and electron micrographs were generated as described under Materials and Methods. Representative micrographs at low magnification (left, bar = 10 μm) or high magnification (right, bar = 0.5 μm) depicting the ultrastructure of the cells are shown. Arrows indicate autophagic vacuoles. C and D, A-498 cells were treated with ABC294640 or 3-MeA alone or in combination for 72 h, and then processed for analyses of their DNA content (C) or caspase 3/7 activity (D) as described under Materials and Methods. The black bar represents caspase 3/7 activity in cells treated with the vehicle, the dark gray bar represents caspase 3/7 activity in cells treated with 90 μM ABC294640, the light gray bar represents caspase 3/7 activity in cells treated with 5 mM 3-MeA, and the white bar represents caspase 3/7 activity in cells treated with both after 72 h. E, A-498 cells were treated with either ABC294640 (squares) or DMS (triangles) alone (filled symbols) or in combination with 5 mM 3-MeA (open symbols) for 48 h, and cell survival was assessed by SRB staining. F, A-498 wild-type cells (filled symbols) or SK2 shRNA-transfected A-498 cells (open symbols) were treated with cisplatin (circles) or ceramide (squares) for 48 h, and their survival was assessed by the standard SRB assay.

Fig. 4.

Activation of autophagy in PC-3 and MDA-MB-231 cells. A, PC3 cells (left) or MDA-MB-231 cells (right) were treated with vehicle or 90 μM ABC294640 for 72 h. The cells were then fixed in ethanol and stained with PI and analyzed by flow cytometry as described under Materials and Methods. B, PC3 (left) or MDA-MB-231 (right) cells were exposed to the indicated concentrations of ABC294640 (μM) for 24 h before lysis and immunoblotting. The arrow indicates LC3-II. C, PC3 (left) or MDA-MB-231 (right) cells were exposed to 50 μM ABC294640 for 24 h and stained with MitoTracker green and LysoTracker red dyes, and confocal imaging was performed as described under Materials and Methods.

Fig. 5.

Effects of ABC294640 on signaling pathways and combination effects of ABC294640 with geldanamycin or MG-132. A, cells were exposed to the indicated concentrations of ABC294640 for 48 h, fractionated by SDS-PAGE, and probed for the indicated proteins by Western blotting as described under Materials and Methods. Beclin 1 expression at 24 and 72 h is also shown. The numbers below the Western blots indicate either the relative phosphorylated protein/total protein ratios compared with the vehicle-treated cells ± standard error or the relative beclin-1/actin ratios compared with the vehicle-treated cells ± standard error. B and C, A-498 cells were exposed to the indicated concentrations of ABC294640 alone (gray bars), geldanamycin (B, white bars), MG-132 (C, white bars), or combinations of ABC294640 and geldanamycin or MG-132 (black bars), and survival was assessed by the SRB assay at 72 h. Combination indexes (CI) calculated by CalcuSyn for the respective combinations are shown on the right. CI values between 0 and 1 indicate synergism, and CI values above 1 indicate antagonism. Strong synergism is indicated by values close to 0. *, p < 0.05.

Fig. 6.

Effects of ABC294640 on tumor growth in mice and histological characterization of ABC294640-treated tumors. A, female SCID mice (n = 8 per group) were injected subcutaneously with A-498 cells suspended in PBS. After palpable tumors were formed, the animals were treated every day by oral gavage with 0 (♦) or 50 mg/kg ABC294640 (○). Values represent the mean ± S.E. tumor volume normalized to treatment day 1 for each mouse. *, p < 0.05; **, p < 0.01. Mice were sacrificed on day 27 after the beginning of treatment, and tumors were fixed in formalin and sections from tumors were stained for TUNEL and immunostained for LC3 and beclin 1 as described under Materials and Methods. B, quantification of TUNEL-positive cells. The black bar represents vehicle-treated tumors, and the white bar represents ABC294640-treated tumors. Data represent mean ± S.E. for three experiments. C, histologic characterization of ABC294640-treated tumors.

Fig. 7.

A model for tumor cell death in response to ABC294640. A, under normal growth conditions, the MAPK and Akt pathways are active, resulting in high cell proliferation and low apoptosis. There is a basal level of autophagy to provide metabolites to support the rapid proliferation and energy demand of the cells. B, upon exposure to ABC294640, MAPK and Akt pathways are down-regulated, thereby inhibiting proliferation and attenuating the restriction on apoptosis. Concurrently, ABC294640-induced production of ceramide (via inhibition of SK) accelerates autophagy (manifested as increased production of autophagosomes) to an unsustainable point, resulting in autophagic death. C, in the presence of 3-MeA, autophagy is blocked, which drives the tumor cells into apoptosis, indicated by DNA fragmentation and caspase activation.