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 glucosylcer-amide 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.

Endocrine- and chemo-resistance are major causes for failure of treatment in recurrent breast cancer. Normal apoptotic pathways targeted by chemotherapeutic agents become altered, diminishing their efficacy and increasing resistance mechanisms. The proapoptotic sphingolipid compound N-Octanoyl-D-erythro-sphingosine (C8-ceramide) mimics endogenous long chain ceramide and is a potent drug in endocrine-/chemo-resistant breast cancer cells. In this study, we examined basal expression levels of modifier enzymes for endogenous ceramide regulation across drug sensitive, chemoresistant and hormone therapy- resistant breast cancer cells lines. Differences were found among UDP-galactose ceramide galactosyltransferase, sphingosine kinase-1, and acid sphingomyelinase gene expression in: MCF-10A, MCF-7, MDA-MB-231 and MCF-7TN-R cell lines. Using C8-ceramide as the lead compound, 8 novel ceramide analogs were synthesized with either enol or amide backbone substitutions and tested for biological activity. The enol substitution was found to be the most potent, with (2S,3R,E)-2-((E)benzylideneamino)octadec-4-ene1,3-diol (Analog C) displaying the greatest reduction in proliferation. Analog B appeared to be the most potent cytotoxic drug in MCF-7 and MDA-MB-231 cell lines with IC50 values of 6.2uM and 3.0 uM respectively. In the cytotoxicity assay MCF-7TN-R cell line, Analog A appeared to be the most potent with an IC50 of 2.4uM. The clonogenic proliferation assay provided very different, yet promising results. Analog D had the lowest IC50 of 460nM in MDA-MB-231, Analog C had an IC50 of 100nM in MCF-7TN-R, and Analog A had an IC50 of 1.48uM in the MCF-7 breast cancer cell line. All of the IC50s for proliferation assays were at least an order of magnitude more potent than the control drug C8 (IC50 of 5uM). Due to the potency of these drugs, these results exemplify the potential of novel ceramide-targeted therapies in chemo-resistant and endocrine-resistant breast cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1031.