3-Ketone-4,6-diene ceramide analogs exclusively induce apoptosis in chemo-resistant cancer cells

Abstract

Multidrug-resistance is a major cause of cancer chemotherapy failure in clinical treatment. Evidence shows that multidrug-resistant cancer cells are as sensitive as corresponding regular cancer cells under the exposure to anticancer ceramide analogs. In this work we designed five new ceramide analogs with different backbones, in order to test the hypothesis that extending the conjugated system in ceramide analogs would lead to an increase of their anticancer activity and selectivity towards resistant cancer cells. The analogs with the 3-ketone-4,6-diene backbone show the highest apoptosis-inducing efficacy. The most potent compound, analog 406, possesses higher pro-apoptotic activity in chemo-resistant cell lines MCF-7TN-R and NCI/ADR-RES than the corresponding chemo-sensitive cell lines MCF-7 and OVCAR-8, respectively. However, this compound shows the same potency in inhibiting the growth of another pair of chemo-sensitive and chemo-resistant cancer cells, MCF-7 and MCF-7/Dox. Mechanism investigations indicate that analog 406 can induce apoptosis in chemo-resistant cancer cells through the mitochondrial pathway. Cellular glucosylceramide synthase assay shows that analog 406 does not interrupt glucosylceramide synthase in chemo-resistant cancer cell NCI/ADR-RES. These findings suggest that due to certain intrinsic properties, ceramide analogs' pro-apoptotic activity is not disrupted by the normal drug-resistance mechanisms, leading to their potential use for overcoming cancer multidrug-resistance.

Keywords: Anti-cancer drugs; Ceramide; Glucosylceramide synthase (GCS); Multidrug resistance; P-glycoprotein.

Figure 1

The structure of C₈-ceramide, 4,6-diene-ceramide, analogs 3 and 406. C₈-ceramide is a short-chain ceramide widely used as a positive control in the studies of ceramide analogs.

Figure 2

¹³C NMR spectrum sections for the two diastereomers3-up and 3-down. In each spectrum, two sets of carbon peaks are observed, suggesting the presence of two conformational isomers for each diastereomer. The complete spectral data are provided in the Supplementary Materials.

Figure 3

Hypothesized conformations for the conformational isomers of compound 3. For easy observation two methyl groups in 2-position of the oxazolidineare not shown up in the projections. The bulky group –COCH₂SOPh is located above the carbamate group plane in the half-chair form, while below the amide plane in the envelope form. In both conformations α-H is in an axial position.

Figure 4

Effects of ceramide analogs on breast cancer intrinsic cell death. MCF-7TN-R cells were treated with double IC₅₀ concentrations (the IC₅₀ values determined from MTT viability assay) for 24 h. (A) Treatment with analog 406 induced a 4.30 ± 1.10 fold (*p <0.05) increase in apoptosis compared to vehicle control. (B) Treatment with analog 406 induced a 3.59 ± 0.45 fold (*p <0.05) increase in caspase-9 activity compared to vehicle control. DMSO, vehicle control; Taxol and C₈-Cer, positive control. The values are the mean ± SE of three independent experiments.

Figure 5

Ceramide analog 406 effectively eliminates drug-resistant cancer cells in ovarian and breast cancers. Error bars represent the standard errors of three independent experiments. Cells were treated with ceramide analogs for 72 h. *p <0.01 compared with in cells treated with analog 3. The IC₅₀ values of analogs in each cell line are indicated. (A) Drug-sensitive OVCAR-8 human ovarian cancer cells. (B) Drug-resistant NCI/ADR-RES human ovarian cancer cells. (C) Drug-sensitive MCF-7 human breast cancer cells. (D) Drug-resistant MCF-7/Dox human breast cancer cells.

Figure 6

Fluorescent chromatograms of lipid components in OVCAR-8 (A) and NCI/ADR-RES (B) cells. Cells were grown for 24 h in 6-well plates with a 10% FBS RPMI-1640 medium, and then exposed to blank control (DMSO), analog 3, or analog 406 in 5% FBS RPMI-1640 medium for 4 h at 37 °C. Cells were switched to 1% BSA RPMI-1640 medium containing NBD C₆-Cer complexed to BSA. After 2 h of incubation at 37 °C, lipids were extracted using chloroform. Fluorescent chromatograms were captured by the AlphaImager, and were inverted using the Adobe Photoshop CS2 Program. Cer, ceramide; GluCer, glucosylceramide.

Scheme 1

Synthetic routes for analogs 401, 402, 403, 404, and 406. Reagents and conditions: (a) methyl phenyl sulfoxide, LDA/THF; (b) triphenylphosphine, NBS/CH₂Cl₂; (c) K₂CO₃/DMF; (d) DIBAL-H/THF; (e) trifluoroacetic acid/CH₂Cl₂; (f) phenylacetyl chloride, NaHCO₃/CH₂Cl₂.