Sphingosine kinase mediates resistance to the synthetic retinoid N-(4-hydroxyphenyl)retinamide in human ovarian cancer cells

Abstract

A2780 human ovarian carcinoma cells respond to treatment with the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) with the production of dihydroceramide and with a concomitant reduction of cell proliferation and induction of apoptosis. The derived HPR-resistant clonal cell line, A2780/HPR, is less responsive to HPR in terms of dihydroceramide generation. In this report, we show that the production of sphingosine 1-phosphate (S1P) is significantly higher in A2780/HPR versus A2780 cells due to an increased sphingosine kinase (SK) activity and SK-1 mRNA and protein levels. Treatment of A2780 and A2780/HPR cells with a potent and highly selective pharmacological SK inhibitor effectively reduced S1P production and resulted in a marked reduction of cell proliferation. Moreover, A2780/HPR cells treated with a SK inhibitor were sensitized to the cytotoxic effect of HPR, due to an increased dihydroceramide production. On the other hand, the ectopic expression of SK-1 in A2780 cells was sufficient to induce HPR resistance in these cells. Challenge of A2780 and A2780/HPR cells with agonists and antagonists of S1P receptors had no effects on their sensitivity to the drug, suggesting that the role of SK in HPR resistance in these cells is not mediated by the S1P receptors. These data clearly demonstrate a role for SK in determining resistance to HPR in ovarian carcinoma cells, due to its effect in the regulation of intracellular ceramide/S1P ratio, which is critical in the control of cell death and proliferation.

FIGURE 1.

Growth of A2780 and A2780/HPR cells. At different times after seeding, the mitochondrial metabolic activity of A2780 (triangle) and A2780/HPR (square) cells was measured by the MTT reduction assay (A), and the cell number was determined by counting trypsinized cells using a Bürker counting chamber (B). Data are the means ± S.D. of three different experiments. *, p < 0.001 versus A2780 cells.

FIGURE 2.

SK activity and SK isoform expression in A2780 and A2780/HPR cells. A, A2780 and A2780/HPR cells were lysed, and cell extracts were employed for SK activity determination as described under “Experimental Procedures.” Data represent the mean ± S.D. of three independent experiments performed in duplicate. The difference between A2780 and A2780/HPR cells was statistically different by the Student's t test (*, p < 0.01). B, semi-quantitative PCR analysis of SK1 and SK2 mRNA expression levels was performed in A2780 and A2780/HPR cells by simultaneous amplification of the housekeeping gene β-actin. Data are normalized versus β-actin expression and utilizing individual SK isoforms of the A2780 specimen set as 1. Data are means ± S.D. of three independent experiments performed in triplicate. C, cell extracts from A2780 and A2780/HPR cells were employed for Western analysis using anti-SK1 and anti-SK2 antibodies. Left panel: a blot representative of three independent experiments is shown. Right panel: the histogram represents mean densitometric quantification (n = 3) of SK1 and SK2 versus β-actin, reported as a percentage relative to the intensity of the band corresponding to A2780 specimen set as 100. Statistical significance was determined by the Student's t test (*, p < 0.01).

FIGURE 3.

Effect of SK inhibitor on the proliferation of A2780 and A2780/HPR cells and on HPR sensitivity in A2780/HPR cells. A, 12 h after seeding, A2780 and A2780/HPR cells were treated with SK inhibitor (gray), and cell viability was evaluated after 96 h as mitochondrial metabolic activity measured by the MTT reduction assay. Data are expressed as percentage of the control treated with vehicle (white). B, 12 h after seeding, A2780/HPR cells were treated with SK inhibitor in the presence of different HPR concentrations and, after 96 h, the mitochondrial metabolic activity was measured by the MTT reduction assay (B, left), and cell number was determined by counting trypsinized cells using a Bürker counting chamber (B, right). Data are the means ± S.D. of three different experiments. *, p < 0.001 versus controls, cells treated with vehicle only.

FIGURE 4.

Effect of SK inhibition on HPR sensitivity and on ceramide and dihydroceramide production in A2780/HPR cells. 12 h after seeding, A2780/HPR cells were treated with SK inhibitor in the presence of different HPR concentrations and, after 96 h, the number of dead cells was evaluated by the Trypan blue exclusion assay (A, left). Data are the means ± S.D. of three different experiments. *, p < 0.01 versus controls. Non-fragmented (B, left) and fragmented DNA (B, right) was extracted as described under “Experimental Procedures.” The samples were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining (in B, right, genomic DNA was loaded on the same gel as control). C, MS analysis of ceramide and dihydroceramide content in A2780 cells resistant to HPR. Ceramide (light gray) and dihydroceramide (dark gray) content expressed as nanomoles/10⁶ cells in 1, A2780/HPR control; 2, A2780/HPR plus HPR; 3, A2780/HPR plus SK inhibitor; 4, A2780/HPR plus HPR plus SK inhibitor; 5, SK1-overexpressing A2780; and 6, SK1-overexpressing A2780 plus HPR. Data are the means ± S.D. of three different experiments. *, p < 0.01 versus control cells. #, p < 0.025 versus SK1-overexpressing A2780.

FIGURE 5.

Expression and effect of agonists or antagonists of S1PRs on proliferation and HPR resistance in A2780 and A2780/HPR cells. A, quantitative real-time PCR analysis was performed in A2780 and A2780/HPR by simultaneous amplification of the target S1P₁, S1P₂, S1P₃, S1P₄, and S1P₅ genes together with the housekeeping gene 18 S rRNA. The mRNA quantization was based on the comparative 2^−ΔΔC_t method, utilizing for each receptor its expression in A2780 cells as calibrator. Data are means ± S.D. of three independent experiments performed in triplicate. Inset: cell extracts from A2780 and A2780/HPR were employed for Western analysis using anti-S1P₁ or anti-S1P₃ antibodies. Equally loaded protein was checked by expression of β-actin. B, 12 h after seeding, A2780 and A2780/HPR cells were treated with different antagonists of S1PRs, and cell proliferation was evaluated after 96 h as mitochondrial metabolic activity measured by the MTT reduction assay. Data are the means ± S.D. of three different experiments. C, 12 h after seeding, A2780 cells were treated with different agonists of S1PRs alone or in the presence of 1 μm HPR (left panel), and A2780/HPR cells were treated with different antagonists of S1PRs alone or in the presence of 10 μm HPR (right panel); after 96 h, the mitochondrial metabolic activity was measured by the MTT reduction assay. Data are the means ± S.D. of three different experiments.

FIGURE 6.

Effect of SK-1 overexpression on HPR sensitivity in A2780 cells. A2780 cells were transfected with the empty expression vector (mock transfected) or with pcDNA3-hSK1^WT plasmid (SK1-overexpressing cells). A, Western blot analysis of FLAG expression in A2780 transfectants. Equal amount of proteins of A2780, mock, and SK1-overexpressing cells were loaded onto SDS-PAGE and then blotted onto a polyvinylidene difluoride membrane, and the overexpressed SK1 tagged with FLAG M2 epitope was detected by anti-FLAG M2 antibody. B, 12 h after seeding, A2780 and A2780 transfectants (one mock and 3 SK1-overexpressing clones, 1, 2, and 3) were treated with 1 μm HPR. After 48 h, the total cell number was determined by counting trypsinized cells using a Bürker counting chamber, and the number of dead cells was evaluated by the Trypan blue exclusion test. Data are presented as the percentage of the total counted cells and are the means ± S.D. of three different experiments. *, p < 0.01 versus A2780 cells.