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Acute myelogenous leukemia (AML) is a hematological malignancy marked by the accumulation of large numbers of immature myeloblasts in bone marrow. The overall prognosis in AML is poor; hence, there is a pressing need to improve treatment. Although the sphingolipid (SL) ceramide demonstrates known cancer suppressor properties, it’s mechanism of action is multifaceted. Our studies in leukemia and other cancers have demonstrated that when combined with the antiestrogen, tamoxifen, the apoptosis-inducting effect of ceramide is greatly enhanced. The goal of the present study was to establish whether a ceramide-tamoxifen regimen also affects autophagic-driven cellular responses in leukemia. Using the human AML cell line KG-1, we demonstrate that, unlike exposure to the single agents, combination C6-ceramide-tamoxifen upregulated LC3-II expression, inhibited the mTOR signaling pathway, and synergistically induced KG-1 cell death in an Atg5-dependent manner. In addition, colocalization of autophagosome and mitochondria, indicative of mitophagosome formation and mitophagy, was observed. Versatility of the drug regimen was confirmed by experiments in MV4-11 cells, a FLT3-ITD AML mutant. These results indicate that the C6-ceramide-tamoxifen regimen plays a pivotal role inducing autophagy in AML, and thus constitutes a novel therapeutic design.

The combination of daunorubicin (dnr) and cytarabine (Ara-C) is a cornerstone of treatment for acute myelogenous leukemia (AML); resistance to these drugs is a major cause of treatment failure. Ceramide, a sphingolipid (SL), plays a critical role in cancer cell apoptosis in response to chemotherapy. Here, we investigated the effects of chemotherapy selection pressure with Ara-C and dnr on SL composition and enzyme activity in the AML cell line HL-60. Resistant cells, those selected for growth in Ara-C- and dnr-containing medium (HL-60/Ara-C and HL-60/dnr, respectively), demonstrated upregulated expression and activity of glucosylceramide synthase, acid ceramidase (AC), and sphingosine kinase 1 (SPHK1); were more resistant to ceramide than parental cells; and displayed sensitivity to inhibitors of SL metabolism. Lipidomic analysis revealed a general ceramide deficit and a profound upswing in levels of sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) in HL-60/dnr cells versus parental and HL-60/Ara-C cells. Both chemotherapyselected cells also exhibited comprehensive upregulations in mitochondrial biogenesis consistent with heightened reliance on oxidative phosphorylation, a property that was partially reversed by exposure to AC and SPHK1 inhibitors and that supports a role for the phosphorylation system in resistance. In summary, dnr and Ara-C selection pressure induces acute reductions in ceramide levels and large increases in S1P and C1P, concomitant with cell resilience bolstered by enhanced mitochondrial remodeling. Thus, strategic control of ceramide metabolism and further research to define mitochondrial perturbations that accompany the drug-resistant phenotype offer new opportunities for developing therapies that regulate cancer growth., This work was supported by National Institutes of Health Grant P01 CA171983 and by a grant from the Brody Brothers Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors declare that they have no conflicts of interest with the contents of this article.

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.

Acid ceramidase (AC) occupies an important place in the control of cancer cell proliferation. We tested the influence of AC inhibition on the effects of PSC 833, a P-glycoprotein antagonist with potent ceramide-generating capacity, to determine whether AC could be a therapeutic target in pancreatic cancer.Ceramide metabolism was followed using (3)H-palmitate, and molecular species were determined by mass spectroscopy. Apoptosis was measured by DNA fragmentation, autophagy by acridine orange staining, and cell cycle was assessed by flow cytometry and RB phosphorylation. AC was measured in intact cells using fluorescent substrate.Exposure of human PANC-1 or MIA-PaCa-2 cells to PSC 833 promoted increases in de novo (dihydro)ceramides, (dihydro)glucosylceramides, and (dihydro)sphingomyelins, demarking ceramide generation and robust metabolism. Despite the multifold increases in (dihydro)ceramide levels, cells were refractory to PSC 833. However, PSC 833 produced a dose-dependent decrease in DNA synthesis and dose- and time-dependent decreases in RB phosphorylation, consistent with cell cycle arrest as demonstrated at G1. Cytostatic effects of PSC 833 were converted to cytotoxic end-point by acid ceramidase inhibition. Cytotoxicity was accompanied by formation of acridine orange-stained acidic vesicles and an increase in LC3 expression, indicative of autophagic response. Cell death was not reversed by preexposure to myriocin, which blocks PSC 833-induced ceramide generation.Although the role of ceramide in end-point cytotoxicity is unclear, our results suggest that acid ceramidase is a viable target in pancreatic cancer. We propose that AC inhibition will be effective in concert with other anticancer therapies.

Although ceramide has potent tumor suppressor properties, its use in cancer therapeutics has been limited; this is mainly due to rapid metabolism of ceramide by cancer cells, the inability to attain therapeutic levels when ceramide-generating agents are employed, and the inherent insolubility of ceramide as a therapeutic. Our approach circumvents these disadvantages and provides advantages over existing methodologies. For example, rather than promoting intracellular ceramide generation by application of exogenous drugs that activate de novo and sphingomyelinase pathways, we have chosen to administer ceramide exogenously in the form of short-chain C6-ceramide nanoliposomes; this overcomes solubility issues and improves uptake. In addition, nanoliposomes are efficient platforms for delivering other hydrophobic chemotherapeutics. The antiestrogen tamoxifen was selected for our studies because of its unusual “off-target” actions, which include inhibition of ceramide metabolism at glycosylation and hydrolysis. Blocking these junctures increases C6-ceramide intracellular residence time and diminishes production of sphingosine 1-phosphate, a mitogenic lipid derived via the action of ceramidase and sphingosine kinase. Hence, C6-ceramide and tamoxifen make up a versatile drug duo. Using in vitro models of human colon and breast cancer, acute mylogenous leukemia (AML), and melanoma, we show that simultaneous administration of nanoliposomal C6-ceramide and tamoxifen potently and synergistically decreased cell viability, compared to the single agents. The mix induced caspase-dependent apoptosis, cell cycle arrest at G1 and G2, upregulation of JNK, p38, and p53, and downregulation of Akt and survivin. In triple negative, hormone-insensitive breast cancer cells, C6-ceramide-tamoxifen induced cell cycle arrest, caspase-dependent apoptosis, and lysosomal and mitochondrial membrane permeability. Evaluation of single agents showed that tamoxifen elicited lysosomal membrane permeability and inhibited acid ceramidase. We have obtained similar results in in vitro models of human melanoma and AML. In an in vivo retroviral transduced murine AML model, the combination lowered leukemia burden in spleen and marrow. The combination also perpetuated synergistic increases in long-chain ceramides, likely compounding the overall ceramide effect. Of interest regarding in vivo utility, the major metabolites of tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen, were effective in combination with C6-ceramide. Our findings show that tamoxifen magnifies the antiproliferative effects of C6-ceramide via aggregate mechanisms. This proficient, ceramide-based nanoliposomal drug regimen may have potential as an effective anticancer therapeutic. Supported by NIGMS 77391; ABC's, Los Angeles; Fashion Footwear Association of New York Charitable Foundation. Citation Format: Samy A.F. Morad, Jonathan C. Levin, Noha Abdelmageed, Mark Kester, Sriram S. Shanmugavdlandy, Robert F. Paulson, Daniel W. Rosenberg, James P. Madigan, Myles C. Cabot. Tamoxifen amplifies antitumor impact of nanoformulated ceramide- pan efficacy in a myriad of tumor cell types. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2027. doi:10.1158/1538-7445.AM2013-2027