Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide metabolism in cancer cell lines and assessed metabolic junctures for enhancing efficacy. MDA-MB-231 breast cancer cells decreased the amount of C6-ceramide metabolized to C6-sphingomyelin (C6-SM) and increased the amount metabolized to C6-glucosylceramide (C6-GC) in response to increasing concentrations. A similar trend was seen in DU-145 (prostate cancer), PANC-1 (pancreatic cancer), and LoVo (colorectal cancer) cells. KG-1 leukemia cells favored C6-SM synthesis at low (0.6 μM) and high-dose (12 μM) C6-ceramide. Partnering C6-ceramide with tamoxifen, a P-glycoprotein antagonist that impedes ceramide glycosylation, was an effective regimen for enhancing cytotoxicity in cells. Experiments to assess the mechanism of cell death using KG-1 cells showed that tamoxifen inhibited synthesis of C6-GC and C6-SM from C6-ceramide by 80% and 50%, respectively, which was accompanied by enhanced apoptosis. Radiolabeling of KG-1 cells with [3H]palmitic acid produced a 2-fold increase in 3H-long-chain ceramides when unlabeled C6-ceramide was added and a 9-fold increase when C6-ceramide and tamoxifen were added. The increase in 3H-palmitate radiolabeling of long-chain ceramides was blocked by inclusion of a ceramide synthase inhibitor; however, inhibiting synthesis of long-chain ceramide did not rescue cells. These studies show that tamoxifen enhances the apoptotic effects of C6-ceramide. The proposed mechanism involves blocking short-chain ceramide anabolism to favor hydrolysis and generation of sphingosine. We propose that use of tamoxifen and other P-glycoprotein antagonists can be an effective means for enhancing cytotoxic potential of short-chain ceramides in the treatment of cancer.

This study shows that 4-HPR cytotoxicity is enhanced in a synergistic fashion by inclusion of the AC inhibitor DM102, by a mechanism that enlists generation of ROS, and thus provides a system to raise 4-HPR therapeutic potential. The role of ceramide however in the cytotoxic response is not clear, as blocking ceramide generation failed to rescue PC-3 cells from 4-HPR/DM102 cytotoxicity.

Poor prognosis in patients with later stage colorectal cancer (CRC) necessitates the search for new treatment strategies. Ceramide, because of its role in orchestrating death cascades in cancer cells, is a versatile alternative. Ceramide can be generated by exposure to chemotherapy or ionizing radiation, or it can be administered in the form of short-chain analogs (C6-ceramide). Because intracellular P-glycoprotein (P-gp) plays a role in catalyzing the conversion of ceramide to higher sphingolipids, we hypothesized that administration of P-gp antagonists with C6-ceramide would magnify cell death cascades. Human CRC cell lines were employed, HCT-15, HT-29, and LoVo. The addition of either tamoxifen, VX-710, verapamil, or cyclosporin A, antagonists of P-gp, enhanced C6-ceramide cytotoxicity in all cell lines. In depth studies with C6-ceramide and tamoxifen in LoVo cells showed the regimen induced PARP cleavage, caspase-dependent apoptosis, mitochondrial membrane permeabilization (MMP), and cell cycle arrest at G1 and G2. At the molecular level, the regimen, but not single agents, induced time-dependent upregulation of tumor suppressor protein p53; however, introduction of a p53 inhibitor staved neither MMP nor apoptosis. Nanoliposomal formulations of C6-ceramide and tamoxifen were also effective, yielding synergistic cell kill. We conclude that tamoxifen is a favorable adjuvant for enhancing C6-ceramide cytotoxicity in CRC, and demonstrates uniquely integrated effects. The high frequency of expression of P-gp in CRC presents an adventitious target for complementing ceramide-based therapies, a strategy that could hold promise for treatment of resistant disease.

P-glycoprotein (P-gp) antagonists inhibit ceramide metabolism at the juncture of glycosylation. The purpose of this study was to test whether targeting P-gp would be a viable alternative to targeting glucosylceramide synthase (GCS) for enhancing ceramide cytotoxicity. A2780 wild-type, and multidrug resistant 2780AD and NCI/ADR-RES human ovarian cancer cell lines and the cell-permeable ceramide analog, C6-ceramide (C6-cer), were employed. Compared to P-gp-poor A2780 cells, P-gp-rich 2780AD cells converted 3.7-fold more C6-cer to nontoxic C6-glucosylceramide (C6-GC), whereas cell-free GCS activities were equal. 2780AD cells displayed resistance to C6-cer (10 μM) that was reversed by inclusion of the P-gp antagonist tamoxifen (5 μM) but not by inclusion of a GCS inhibitor. Co-administration of C6-cer and P-gp antagonists was also effective in NCI/ADR-RES cells. For example, C6-cer, VX-710 (Biricodar), and cyclosporin A (cyc A) exposure resulted in viabilities of ~90% of control; however, C6-cer/VX-710 and C6-cer/cyc A additions were synergistic and resulted in viabilities of 22 and 17%, respectively. Further, whereas C6-ceramide and cyc A imparted 1.5- and zero-fold increases in caspase 3/7 activity, the combination produced a 3.5-fold increase. Although the upstream elements of cell death have not been elucidated, the novel C6-ceramide/P-gp antagonist combination merits further study and assessment of clinical translational potential.

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