Su-Fern Tan

Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia

The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C₆-ceramide (C₆) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C₆-ceramide may be a promising therapeutic approach for a fatal leukemia.

Therapeutic efficacy of FTY720 in a rat model of NK-cell leukemia

NK-cell leukemia is a clonal expansion of NK cells. The illness can occur in an aggressive or chronic form. We studied cell lines from human and rat NK-cell leukemias (aggressive NK-cell leukemia) as well as samples from patients with chronic NK-cell leukemia to investigate pathogenic mechanisms. Here we report that Mcl-1 was overexpressed in leukemic NK cells and that knockdown of Mcl-1 induced apoptosis in these leukemic cells. In vitro treatment of human and rat NK leukemia cells with FTY720 led to caspase-dependent apoptosis and decreased Mcl-1 expression in a time- and-dose-dependent manner. These biologic effects could be inhibited by blockade of reactive oxygen species generation and the lysosomal degradation pathway. Lipidomic analyses after FTY720 treatment demonstrated elevated levels of sphingosine, which mediated apoptosis of leukemic NK cells in vitro. Importantly, systemic administration of FTY720 induced complete remission in the syngeneic Fischer rat model of NK-cell leukemia. Therapeutic efficacy was associated with decreased expression of Mcl-1 in vivo. These data demonstrate that therapeutic benefit of FTY720 may result from both altered sphingolipid metabolism as well as enhanced degradation of a key component of survival signaling.

Novel off-target effect of tamoxifen — Inhibition of acid ceramidase activity in cancer cells

Acid ceramidase (AC), EC 3.5.1.23, a lysosomal enzyme, catalyzes the hydrolysis of ceramide to constituent sphingoid base, sphingosine, and fatty acid. Because AC regulates the levels of pro-apoptotic ceramide and mitogenic sphingosine-1-phosphate, it is considered an apt target in cancer therapy. The present study reveals, for the first time, that the prominent antiestrogen, tamoxifen, is a pan-effective AC inhibitor in the low, single digit micromolar range, as demonstrated in a wide spectrum of cancer cell types, prostate, pancreatic, colorectal, and breast. Prostate cancer cells were chosen for the detailed investigations. Treatment of intact PC-3 cells with tamoxifen produced time- and dose-dependent inhibition of AC activity. Tamoxifen did not impact cell viability nor did it inhibit AC activity in cell-free assays. In pursuit of mechanism of action, we demonstrate that tamoxifen induced time-, as early as 5min, and dose-dependent, as low as 5μM, increases in lysosomal membrane permeability (LMP), and time- and dose-dependent downregulation of AC protein expression. Assessing various protease inhibitors revealed that a cathepsin B inhibitor blocked tamoxifen-elicited downregulation of AC protein; however, this action failed to restore AC activity unless assayed in a cell-free system at pH4.5. In addition, pretreatment with tamoxifen inhibited PC-3 cell migration. Toremifene, an antiestrogen structurally similar to tamoxifen, was also a potent inhibitor of AC activity. This study reveals a new, off-target action of tamoxifen that may be of benefit to enhance anticancer therapies that either incorporate ceramide or target ceramide metabolism.

Targeting glucosylceramide synthase synergizes with C6-ceramide nanoliposomes to induce apoptosis in natural killer cell leukemia

Abstract Natural killer (NK) cell leukemia is characterized by clonal expansion of CD3 − NK cells and comprises both chronic and aggressive forms. Currently no effective treatment exists, thus providing a need for identification of novel therapeutics. Lipidomic studies revealed a dysregulated sphingolipid metabolism as evidenced by decreased levels of overall ceramide species and increased levels of cerebrosides in leukemic NK cells, concomitant with increased glucosylceramide synthase (GCS) expression. GCS, a key enzyme of this pathway, neutralizes pro-apoptotic ceramide by transfer of a uridine diphosphate (UDP)-glucose. Thus, we treated both rat and human leukemic NK cells in combination with: (1) exogenous C6-ceramide nanoliposomes in order to target mitochondria and increase physiological pro-apoptotic levels of long chain ceramide, and (2) 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of GCS. Co-administration of C6-ceramide nanoliposomes and PPMP elicited an increase in endogenous long-chain ceramide species, which led to cellular apoptosis in a synergistic manner via the mitochondrial intrinsic cell death pathway in leukemic NK cells.

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

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. The 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

ABSTRACT Introduction: Acute myeloid leukemia (AML) is the most common adult leukemia. Only a fraction of AML patients will survive with existing chemotherapy regimens. Hence, there is an urgent and unmet need to identify novel targets and develop better therapeutics in AML. In the past decade, the field of sphingolipid metabolism has emerged into the forefront of cancer biology due to its importance in cancer cell proliferation and survival. In particular, acid ceramidase (AC) has emerged as a promising therapeutic target due to its role in neutralizing the pro-death effects of ceramide. Areas covered: This review highlights key information about AML biology as well as current knowledge on dysregulated sphingolipid metabolism in cancer and AML. We describe AC function and dysregulation in cancer, followed by a review of studies that report elevated AC in AML and compounds known to inhibit the enzyme. Expert opinion: AML has a great need for new drug targets and better therapeutic agents. The finding of elevated AC in AML supports the concept that this enzyme represents a novel and realistic therapeutic target for this common leukemia. More effort is needed towards developing better AC inhibitors for clinical use and combination treatment with existing AML therapies.

Ceramide-tamoxifen regimen targets bioenergetic elements in acute myelogenous leukemia

The objective of our study was to determine the mechanism of action of the short-chain ceramide analog, C6-ceramide, and the breast cancer drug, tamoxifen, which we show coactively depress viability and induce apoptosis in human acute myelogenous leukemia cells. Exposure to the C6-ceramide-tamoxifen combination elicited decreases in mitochondrial membrane potential and complex I respiration, increases in reactive oxygen species (ROS), and release of mitochondrial proapoptotic proteins. Decreases in ATP levels, reduced glycolytic capacity, and reduced expression of inhibitors of apoptosis proteins also resulted. Cytotoxicity of the drug combination was mitigated by exposure to antioxidant. Cells metabolized C6-ceramide by glycosylation and hydrolysis, the latter leading to increases in long-chain ceramides. Tamoxifen potently blocked glycosylation of C6-ceramide and long-chain ceramides. N-desmethyltamoxifen, a poor antiestrogen and the major tamoxifen metabolite in humans, was also effective with C6-ceramide, indicating that traditional antiestrogen pathways are not involved in cellular responses. We conclude that cell death is driven by mitochondrial targeting and ROS generation and that tamoxifen enhances the ceramide effect by blocking its metabolism. As depletion of ATP and targeting the “Warburg effect” represent dynamic metabolic insult, this ceramide-containing combination may be of utility in the treatment of leukemia and other cancers.

Vitamin D decreases STAT phosphorylation and inflammatory cytokine output in T-LGL Leukemia

ABSTRACT Large granular lymphocyte leukemia (LGLL) is a rare incurable chronic disease typically characterized by clonal expansion of CD3+ cytotoxic T-cells. Two signal transducer and activator of transcription factors, STAT1 and STAT3, are constitutively active in T-LGLL. Disruption of this activation induces apoptosis in T-LGLL cells. Therefore, considerable efforts are focused on developing treatments that inhibit STAT activation. Calcitriol, the active form of vitamin D, has been shown to decrease STAT1 and STAT3 phosphorylation in cancer cell lines and autoimmune disease mouse models. Thus, we investigated whether calcitriol could be a valid therapeutic for T-LGLL. Calcitriol treatment of the TL-1 cell line (model of T-LGLL) led to decreased phospho-Y701 STAT1 and phospho-Y705 STAT3 and increased vitamin D receptor (VDR) levels. Doses of 10 and 100 nM calcitriol also significantly decreased the inflammatory cytokine IFN-γ in the TL-1 cell line. The overall cell viability did not change when the TL-1 cell line was treated with 0.1 to 1000 nM calcitriol. Studies with primary T-LGLL patient peripheral blood mononuclear cells showed that the majority of T-LGLL patients have detectable VDR and activated STATs in contrast to normal donor controls. Treatment of primary T-LGLL patient cells with calcitriol recapitulated findings from the TL-1 cell line. Overall, our results suggest that calcitriol may reprogram T-cells to decrease essential STAT activation and pro-inflammatory cytokine output. These data support further investigation into calcitriol as an experimental therapeutic for T-LGLL.

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

Graphical abstract Figure. No Caption available. ABSTRACT The anticancer properties of ceramide, a sphingolipid with potent tumor‐suppressor properties, can be dampened via glycosylation, notably in multidrug resistance wherein ceramide glycosylation is characteristically elevated. Earlier works using the ceramide analog, C6‐ceramide, demonstrated that the antiestrogen tamoxifen, a first generation P‐glycoprotein (P‐gp) inhibitor, blocked C6‐ceramide glycosylation and magnified apoptotic responses. The present investigation was undertaken with the goal of discovering non‐anti‐estrogenic alternatives to tamoxifen that could be employed as adjuvants for improving the efficacy of ceramide‐centric therapeutics in treatment of cancer. Herein we demonstrate that the tamoxifen metabolites, desmethyltamoxifen and didesmethyltamoxifen, and specific, high‐affinity P‐gp inhibitors, tariquidar and zosuquidar, synergistically enhanced C6‐ceramide cytotoxicity in multidrug resistant HL‐60/VCR acute myelogenous leukemia (AML) cells, whereas the selective estrogen receptor antagonist, fulvestrant, was ineffective. Active C6‐ceramide‐adjuvant combinations elicited mitochondrial ROS production and cytochrome c release, and induced apoptosis. Cytotoxicity was mitigated by introduction of antioxidant. Effective adjuvants markedly inhibited C6‐ceramide glycosylation as well as conversion to sphingomyelin. Active regimens were also effective in KG‐1a cells, a leukemia stem cell‐like line, and in LoVo human colorectal cancer cells, a solid tumor model. In summary, our work details discovery of the link between P‐gp inhibitors and the regulation and potentiation of ceramide metabolism in a pro‐apoptotic direction in cancer cells. Given the active properties of these adjuvants in synergizing with C6‐ceramide, independent of drug resistance status, stemness, or cancer type, our results suggest that the C6‐ceramide‐containing regimens could provide alternative, promising therapeutic direction, in addition to finding novel, off‐label applications for P‐gp inhibitors.

Abstract 761: Acid ceramidase promotes drug resistance in acute myeloid leukemia through P-gp upregulation mediated by NF- kB activation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background. Acute myeloid leukemia (AML) is the most common form of adult leukemia that affects the differentiation and survival of myeloid precursors. AML patients have elevated immature leukemic cells known as blasts. Failure to achieve remission commonly revolves around the drug resistance advantage of AML blasts. P-glycoprotein (P-gp), an ATP-binding cassette transporter (MDR1/ABCB1), is highly expressed in AML blasts. However, P-gp inhibitors have failed to increase overall survival in AML patients. Sphingolipids are a class of lipids that have a wide variety of functions in cellular processes. Acid ceramidase (AC), an enzyme in the sphingolipid network, catalyzes ceramide breakdown to generate sphingosine. Sphingosine can then undergo phosphorylation by sphingosine kinases to form sphingosine -1-phosphate (S1P). Aims. Our aims were to determine whether AC is elevated in AML, is essential for AML drug resistance and to identify the mechanism through which AC maintains drug resistance in AML. Methods. We used microarray and fluorogenic AC substrate to quantify AC expression and activity levels. Quantitative real-time PCR, Western blotting and flow cytometry were used to determine P-gp expression and activity. AC inhibitor LCL204 and AC shRNA were used in human AML cell lines HL-60, HL-60/VCR and HL-60/ABT to uncover the role of AC in drug resistance. We created an AC-overexpressing HL-60 cell line to study AC-mediated drug resistance. NF-kB inhibitor JSH-23 was used to test the pathways involvement in AC-mediated drug resistance in AML. Results. We demonstrate that AC expression and activity are elevated in AML patient samples compared to normal donors. P-gp expression is also elevated in AML patient samples compared to normal PBMC and cord blood. Using human HL-60 cells and drug-resistant derivatives HL-60/VCR and HL-60/ABT as our model, we found that P-gp expression is high in both HL-60/VCR and HL-60/ABT. AC activity in both drug resistant cell lines is also higher than the parental HL-60. Targeting AC with pharmacological inhibitor LCL204 in HL-60/VCR cells decreases P-gp levels and increases sensitivity to both chemotherapeutic drugs mitoxantrone and daunorubicin. Lentiviral AC overexpression in HL-60 upregulates P-gp expression and confers resistance to mitoxantrone and daunorubicin. Conversely, AC knockdown in HL-60/VCR and HL-60/ABT cells decreased P-gp expression. AC overexpression in HL-60 increases P-gp mRNA levels, which suggests involvement of a transcriptional regulatory mechanism. NF-κB inhibition by JSH-23 decreases p65, P-gp and AC expression and warrants further investigation into the mechanism for AC-mediated regulation of P-gp expression. Summary. Our data supports an important role for AC in regulating P-gp expression in AML and presents a novel target to overcome drug resistance in AML. Citation Format: Su-Fern Tan, Xin Liu, Kenichiro Doi, Hong-Gang Wang, Myles Cabot, David Feith, Thomas P. Loughran. Acid ceramidase promotes drug resistance in acute myeloid leukemia through P-gp upregulation mediated by NF- kB activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 761. doi:10.1158/1538-7445.AM2014-761