Overexpression of sphingosine-1-phosphate lyase or inhibition of sphingosine kinase in Dictyostelium discoideum results in a selective increase in sensitivity to platinum-based chemotherapy drugs

Abstract

The efficacy of the chemotherapy drug cisplatin is often limited due to resistance of the tumors to the drug, and increasing the potency of cisplatin without increasing its concentration could prove beneficial. A previously characterized Dictyostelium discoideum mutant with increased resistance to cisplatin was defective in the gene encoding sphingosine-1-phosphate (S-1-P) lyase, which catalyzes the breakdown of S-1-P, an important regulatory molecule in cell function and development and in the regulation of cell fate. We hypothesized that the increased resistance to cisplatin was due to an elevation of S-1-P and predicted that lowering levels of S-1-P should increase sensitivity to the drug. We generated three strains that stably overexpress different levels of the S-1-P lyase. The overexpressor strains have reduced growth rate and, confirming the hypothesis, showed an expression-dependent increase in sensitivity to cisplatin. Consistently, treating the cells with D-erythro-N,N,-dimethylsphingosine, a known inhibitor of sphingosine kinase, increased the sensitivity of mutant and parent cells to cisplatin, while addition of exogenous S-1-P or 8-Br-cyclic AMP made the cells more resistant to cisplatin. The increased sensitivity of the overexpressors to cisplatin was also observed with the cisplatin analog carboplatin. In contrast, the response to doxorubicin, 5-flurouracil, or etoposide was unaffected, indicating that the involvement of the sphingolipid metabolic pathway in modulating the response to cisplatin is not part of a global genotoxic stress response. The augmented sensitivity to cisplatin appears to be the result of an intracellular signaling function of S-1-P, because D. discoideum does not appear to have endothelial differentiation growth (EDG/S1P) receptors. Overall, the results show that modulation of the sphingolipid pathway at multiple points can result in increased sensitivity to cisplatin and has the potential for increasing the clinical usefulness of this important drug.

FIG. 1.

Schematic presentation of the last steps in the sphingomyelin degradation pathway. The S-1-P lyase and sphingosine kinase enzymes shown in shaded boxes are the primary focus of this study. The compounds that were used in this study (DMS and cAMP) and their effects on sphingosine kinase are shown. sglAOE is the S-1-P lyase overexpressor strain, and sglAΔ is the S-1-P lyase null strain.

FIG. 2.

Expression of the SglA protein in D. discoideum. Cells of each of the sglA-myc transformants were harvested, washed, and lysed in denaturing buffer, and the protein concentration was determined. Fifty micrograms of total protein per lane was separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis. (A) Total protein, stained with colloidal Coomassie blue. Note that the samples have equal amounts of protein. Vector1 and Vector2 are vector control transformants. Ax3-ORF⁺ is the untransformed parental cells. MW, molecular size standards. (B) Proteins were transferred to nitrocellulose membranes and were probed with anti-c-myc antibodies. (C) The exposed film shown in panel B was scanned and quantitated. The image shows relative units of expression, using sglAOE-3 as a reference (1).

FIG. 3.

Growth rate of the sglAOE strains. The three sglAOE strains, the vector control transformation strains, and the parent strain were plated on SM agar plates in association with K. aerogenes. Plates were scanned daily, and the diameter of the plaques was measured. (A) Photographs of the plates at day 4. (B) Plaque size. The results are presented as the area of the plaque over 3 days, and each point is the average of 10 randomly selected plaques. Inset is a visual representation of the results at day 3, when the plaques were still very small. (C) The three sglAOE strains, the vector control transformation strain, and the parent strain were inoculated at a density of 10⁵ cells/ml in 20 ml of HL5 medium and were grown with shaking at 200 rpm at 22°C. Cultures were counted daily. When the cultures reached approximately 5 × 10⁶ cells/ml they were diluted down to 5 × 10⁴ cells/ml so that the culture could be followed through three consecutive passages. The last passage was allowed to go through stationary phase. (D) Cells of the three sglAOE strains, the vector control transformation strain, and the parent strain were harvested from HL5 medium at a density of 3 × 10⁵ (mid-log phase) and washed, and 100 μl was allowed to settle on coverslips, where they were fixed with 3.7% formaldehyde. The cells were stained with 20 μg of DAPI/ml. The number of nuclei per cell was counted in 300 cells from each strain on a Zeiss IM microscope with a Chroma Technology Corp. lucifer yellow filter. Results are presented as the percentages of the total number of cells.

FIG. 4.

sglAOE strains are sensitive to cisplatin. Cultures of 10⁶ cells/ml in HL5 medium were treated with increasing concentrations of cisplatin (0, 75, 150, and 300 μM) for 4 h, serially diluted, and plated for viability on SM agar in 24-well plates with K. aerogenes as the food source. Viability is expressed as the percentage of surviving cells relative to an untreated culture. sglAΔ is the SglA null strain. Because the sglAΔ and sglAOE strains had different parents, we established that the Ax3-ORF⁺ and Ax4 strains had identical sensitivities to cisplatin (data not shown).

FIG. 5.

Overexpression of sglA renders the cells more sensitive to cisplatin and carboplatin but not to other drugs. Ten milliliters of cultures of 10⁶ cells/ml in HL5 medium were treated with the indicated drugs for 5 h, serially diluted, and plated for viability measurements as described for Fig. 4. Survival was calculated as a percentage of the untreated culture. (A) 300 μM cisplatin; (B) 300 μM carboplatin; (C) 450 μM doxorubicin; (D) 400 μM 5-FU; (E) 300 μM etoposide. Strain names are the same as those shown in Fig. 4. P values (by Student's t test) for cisplatin and carboplatin were ≤0.05 and <0.001, respectively.

FIG. 6.

Pretreatment with DMS increased the cell response to cisplatin. Growing cultures of sglAOE-1, sglAΔ, and the parent strain at 10⁶ in HL5 medium were treated with 0, 2.5, 5, and 10 μM DMS for 1 h prior to adding 150 μM cisplatin. The cultures were assayed for viability at 5 and 24 h as described in the legend to Fig. 4. (A and B) parent strain; (C and D) sglAOE-1; (E and F) sglAΔ. Closed circles, 150 μM cisplatin with increasing concentrations of DMS. Open circles, increasing concentrations of DMS alone. (G) Photograph of the plaques of the viable cells at day 3 after plating. Note that increasing concentrations of DMS result in smaller plaques.

FIG. 7.

Pretreatment with S-1-P increases resistance of cells to cisplatin. Growing cultures at 10⁶ cells/ml in HL5 medium were treated with 0, 10, 20, and 50 μM S-1-P 1 h prior to the addition of 150 μM cisplatin. Cells were sampled at 5 and 24 h and were assayed for viability as described in the legend to Fig. 4. (A and B) Parental strain at 5 and 24 h, respectively. (C and D) sglAOE-1 at 5 and 24 h, respectively. Closed circles, 150 μM cisplatin with increasing concentrations of S-1-P. Open circles, increasing concentration of S-1-P alone.

FIG. 8.

Pretreatment with 8-Br-cAMP increases resistance of cell sensitivity to cisplatin. Growing cultures of 10⁶ cell/ml in HL5 medium were treated with 0, 2, and 5 mM 8-Br-cAMP for 1 h prior to the addition of 150 μM cisplatin. Viability and strain names are as described in the legend to Fig. 4. Closed circles, 150 μM cisplatin with increasing concentrations of 8-Br-cAMP. Open circles, increasing concentrations of 8-Br-cAMP alone.

FIG. 9.

Schematic model of the interactions that address the specificity of the resistance of cells to cisplatin with respect to sphingolipid signaling.