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. 2006 Apr 15;395(2):311-8.
doi: 10.1042/BJ20051184.

Lack of Ceramide Generation and Altered Sphingolipid Composition Are Associated With Drug Resistance in Human Ovarian Carcinoma Cells

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Free PMC article

Lack of Ceramide Generation and Altered Sphingolipid Composition Are Associated With Drug Resistance in Human Ovarian Carcinoma Cells

Alessandro Prinetti et al. Biochem J. .
Free PMC article

Abstract

PTX (Paclitaxel) is an antimitotic agent used in the treatment of a number of major solid tumours, particularly in breast and ovarian cancer. This study was undertaken to gain insight into the molecular alterations producing PTX resistance in ovarian cancer. PTX treatment is able to induce apoptosis in the human ovarian carcinoma cell line, CABA I. PTX-induced apoptosis in CABA I cells was accompanied by an increase in the cellular Cer (ceramide) levels and a decrease in the sphingomyelin levels, due to the activation of sphingomyelinases. The inhibition of acid sphingomyelinase decreased PTX-induced apoptosis. Under the same experimental conditions, PTX had no effect on Cer and sphingomyelin levels in the stable PTX-resistant ovarian carcinoma cell line, CABA-PTX.The acquisition of the PTX-resistant phenotype is accompanied by unique alterations in the complex sphingolipid pattern found on lipid extraction. In the drug-resistant cell line, the levels of sphingomyelin and neutral glycosphingolipids were unchanged compared with the drug-sensitive cell line. The ganglioside pattern in CABA I cells is more complex compared with that of CABA-PTX cells. Specifically, we found that the total ganglioside content in CABA-PTX cells was approximately half of that in CABA I cells, and GM3 ganglioside content was remarkably higher in the drug-resistant cell line. Taken together our findings indicate that: i) Cer generated by acid sphingomyelinase is involved in PTX-induced apoptosis in ovarian carcinoma cells, and PTX-resistant cells are characterized by their lack of increased Cer upon drug treatment, ii) PTX resistance might be correlated with an alteration in metabolic Cer patterns specifically affecting cellular ganglioside composition.

Figures

Figure 1
Figure 1. PTX-induced apoptosis in detached CABA I cells and CABA-PTX cells
(A) PTX-sensitive CABA I cells (black bars) and resistant CABA-PTX (white bars) cells were treated with PTX 0.3, 0.8 and 2 μg/ml, or vehicle control (Ve), for 24, 48 and 72 h (106 cells/10 cm dish). Apoptotic nuclear fragmentation was evaluated by propidium iodide staining and FACScan analysis. Data are means±S.D. for 3 independent experiments. (B) Agarose gel analysis of DNA fragmentantion induced by PTX. After 72 h of treatment with 2 μg/ml PTX only floating cells (F) show DNA fragmentation compared with adherent cells (A) under the same conditions, or cells treated with vehicle alone (Ve).
Figure 2
Figure 2. Cer detection: CABA I (lanes 1–3) and CABA-PTX (lanes 4–5) cells were treated with vehicle (lanes 1 and 4) or 2 μg/ml PTX for 72 h (lanes 2, 3 and 5)
In the case of PTX-treated CABA I cells, both adherent (lane 2) and floating (lane 3) cells were collected separately and analysed for their lipid content. Radioactive lipids were detected by digital autoradiography (250 d.p.m. applied on a 3 mm line; acquisition time, 70 h). (A) Solvent system, chloroform/methanol/water (55:20:3, by vol.); (B) solvent system, hexane/chloroform/acetone/acetic acid (10:35:10:1, by vol.). The position of pure standard lipids is indicated on the left of each panel. Experiments were performed in triplicate.
Figure 3
Figure 3. SMase activities in CABA I and CABA-PTX cells. CABA I (lanes 1–3) and CABA-PTX (lanes 4–5) cells were treated with vehicle (lanes 1 and 4) or 2 μg/ml PTX for 24 h (lanes 2, 3 and 5)
In the case of PTX-treated CABA I cells, both adherent (lane 2) and floating (lane 3) cells were collected separately and assayed. Acid (A) and neutral (B) SMase activities were assayed in cell homogenates as described in the Materials and methods section. Negative controls were heat-inactivated cell homogenates. The reaction mixture was analysed by HPTLC. Data are expressed as Cer pmoles formed/min per mg of cell protein and are the means±S.D. for 3 independent experiments.
Figure 4
Figure 4. Effect of inhibitors in PTX-induced apoptosis in CABA I cells
Parental cells were treated with PTX (2 μg/ml) for 72 h in the presence or absence of several inhibitors. Data obtained for FB1 (50 μM), GW4869 (20 μM) and IMP (50 μM) are shown as flow cytometric plots, and data from GSH (10 mM), ManA (1 μM), monensin (20 μM) and D609 (50 μg/ml) are shown as histograms. The percentage of hypodiploid nuclei are reported for each condition. The results are the means±S.D. for 3 independent experiments. Mon, monensin.
Figure 5
Figure 5. Ganglioside patterns of CABA I and CABA-PTX cells
(A) CABA I (lanes 1–4) and CABA-PTX (lanes 5–7) cells were treated with vehicle (lanes 1, 2, 5 and 6) or 2 μg/ml PTX for 72 h (lanes 3, 4 and 7). In the case of PTX-treated CABA I cells, both adherent (lane 3) and floating (lane 4) cells were separately collected and analysed for their lipid content. For ganglioside characterization, aliquots of the aqueous phase from vehicle-treated CABA I and CABA-PTX cells were treated with Vibrio cholerae sialidase (lanes 2 and 6). The position of pure standard lipids is indicated on the left of the panel. (B) Two-dimensional ganglioside patterns in CABA I and CABA-PTX cells. The position of GM3, the main ganglioside in the mixtures, is indicated by arrows. Radioactive lipids were detected by digital autoradiography (250 d.p.m. applied on a 3 mm line; acquisition time 70 h). Patterns were representative of those obtained from 3 independent experiments.

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