Modification of sphingolipid metabolism by tamoxifen and N-desmethyltamoxifen in acute myelogenous leukemia—Impact on enzyme activity and response to cytotoxics

Abstract

The triphenylethylene antiestrogen, tamoxifen, can be an effective inhibitor of sphingolipid metabolism. This off-target activity makes tamoxifen an interesting ancillary for boosting the apoptosis-inducing properties of ceramide, a sphingolipid with valuable tumor censoring activity. Here we show for the first time that tamoxifen and metabolite, N –desmethyltamoxifen (DMT) block ceramide glycosylation and inhibit ceramide hydrolysis (by acid ceramidase, AC) in human acute myelogenous leukemia (AML) cell lines and in AML cells derived from patients. Tamoxifen (1-10 μM) inhibition of AC in AML cells was accompanied by decreases in AC protein expression. Tamoxifen also depressed expression and activity of sphingosine kinase 1 (SphK1), the enzyme catalyzing production of mitogenic sphingosine 1-phosphate (S1-P). Results from mass spectroscopy showed that tamoxifen and DMT, i ) increased the levels of endogenous C16:0- and C24:1 ceramide molecular species, ii) nearly totally halted production of respective glucosylceramide (GC) molecular species, iii ) drastically reduced levels of sphingosine ( to 9% of control), and iv ) reduced levels of S1-P by 85%, in vincristine-resistant HL-60/VCR cells. Co-administration of tamoxifen with either N-(4-hydroxyphenyl)retinamide (4-HPR), a ceramide-generating retinoid, or a cell-deliverable form of ceramide, C6-ceramide, resulted in marked decreases in HL-60/VCR cell viability that far exceeded single agent potency. Combination treatments resulted in synergistic apoptotic cell death as gauged by increased Annexin V binding and DNA fragmentation and activation of caspase-3. These results show the versatility of adjuvant triphenylethylene with ceramide-centric therapies for magnifying therapeutic potential in AML. Such drug regimens could serve as effective strategies, even in the multidrug resistant setting.

The emergence of acid ceramidase as a therapeutic target for acute myeloid leukemia

“…Both tamoxifen and its metabolite N- desmethyltamoxifen specifically reduce ceramide glycosylation and inhibit acid ceramidase independent of known anti-estrogen mechanisms. Co-treatment with tamoxifen and exogenous ceramide or a ceramide-generating drug induced synergistic apoptosis in AML cells 77 .…”

Ceramide-tamoxifen regimen targets bioenergetic elements in acute myelogenous leukemia

“…Apropos in this context, tamoxifen can suppress ceramide metabolism via glycosylation (13) and inhibit hydrolysis (14), thus reducing S1-P formation (15). Therefore, when used in combination with a cell-deliverable ceramide, such as C6-ceramide, or ceramide-generating drugs, beneficial actions from a therapeutic standpoint can be realized.…”

“…In earlier work in HL-60/VCR cells, we demonstrated that tamoxifen enhanced the cytotoxicity of fenretinide, a ceramide-generating retinoid (16), and C6-ceramide; however, mechanisms supporting this enhancement had not been elucidated (15). Herein, we demonstrate that the mechanisms promoting C6-ceramide-tamoxifen cytotoxicity in AML cells encompass a reduction in mitochondrial membrane potential, inhibition of complex I respiration, generation of reactive oxygen species (ROS), reduced glycolytic capacity, and decreases in ATP.…”

“…Apoptosis was assessed using the ApoDETECT Annexin V-FITC Kit (Life Technologies) as previously described, to detect annexin V binding by flow cytometry (15).…”

Role of P-glycoprotein inhibitors in ceramide-based therapeutics for treatment of cancer

“…In addition to inhibition of ceramide glycosylation [21], tamoxifen also exhibits a number of estrogen receptor-independent actions, including circumvention of multidrug resistance, downregulation of survivin, inhibition of Acyl-CoA: cholesterol acyl transferase (ACAT) [22], and downregulation of acid ceramidase [15]. The capacity to block ceramide glycosylation has made tamoxifen an object of myriad investigations into its use as an adjuvant with ceramide-centric therapies, including 4-HPR [23], short-chain ceramides [24], and short-chain ceramides in combination with paclitaxel [25]. Although tamoxifen is not a direct inhibitor of GCS, it limits intracellular production of GC by blocking GC transport into the Golgi, a process that requires Golgi-resident P-gp [22].…”