Mehrdad Rahmaniyan

Identification of Dihydroceramide Desaturase as a Direct in Vitro Target for Fenretinide

The dihydroceramide desaturase (DES) enzyme is responsible for inserting the 4,5-trans-double bond to the sphingolipid backbone of dihydroceramide. We previously demonstrated that fenretinide (4-HPR) inhibited DES activity in SMS-KCNR neuroblastoma cells. In this study, we investigated whether 4-HPR acted directly on the enzyme in vitro. N-C8:0-d-erythro-dihydroceramide (C8-dhCer) was used as a substrate to study the conversion of dihydroceramide into ceramide in vitro using rat liver microsomes, and the formation of tritiated water after the addition of the tritiated substrate was detected and used to measure DES activity. NADH served as a cofactor. The apparent Km for C8-dhCer and NADH were 1.92 ± 0.36 μm and 43.4 ± 6.47 μm, respectively; and the Vmax was 3.16 ± 0.24 and 4.11 ± 0.18 nmol/min/g protein. Next, the effects of 4-HPR and its metabolites on DES activity were investigated. 4-HPR was found to inhibit DES in a dose-dependent manner. At 20 min, the inhibition was competitive; however, longer incubation times demonstrated the inhibition to be irreversible. Among the major metabolites of 4-HPR, 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) showed the highest inhibitory effect with substrate concentration of 0.5 μm, with an IC50 of 1.68 μm as compared with an IC50 of 2.32 μm for 4-HPR. N-(4-Methoxyphenyl)retinamide (4-MPR) and 4-Oxo-N-(4-methoxyphenyl)retinamide (4-oxo-4-MPR) had minimal effects on DES activity. A known competitive inhibitor of DES, C8-cyclopropenylceramide was used as a positive control. These studies define for the first time a direct in vitro target for 4-HPR and suggest that inhibitors of DES may be used as therapeutic interventions to regulate ceramide desaturation and consequent function.

The sphingosine kinase 2 inhibitor ABC294640 reduces the growth of prostate cancer cells and results in accumulation of dihydroceramides in vitro and in vivo

Despite recent advances in the development of novel therapies against castration-resistant prostate cancer, the advanced form of the disease remains a major treatment challenge. Aberrant sphingolipid signaling through sphingosine kinases and their product, sphingosine-1-phosphate, can promote proliferation, drug resistance, angiogenesis, and inflammation. The sphingosine kinase 2 inhibitor ABC294640 is undergoing clinical testing in cancer patients, and in this study we investigated the effects this first-in-class inhibitor in castration-resistant prostate cancer. In vitro, ABC294640 decreased prostate cancer cell viability as well as the expression of c-Myc and the androgen receptor, while lysosomal acidification increased. ABC294640 also induced a greater than 3-fold increase in dihydroceramides that inversely correlated with inhibition of dihydroceramide desaturase (DEGS) activity. Expression of sphingosine kinase 2 was dispensable for the ABC294640-mediated increase in dihydroceramides. In vivo, ABC294640 diminished the growth rate of TRAMP-C2 xenografts in syngeneic hosts and elevated dihydroceramides within tumors as visualized by MALDI imaging mass spectroscopy. The plasma of ABC294640-treated mice contained significantly higher levels of C16- and C24:1-ceramides (but not dihydro-C16-ceramide) compared with vehicle-treated mice. In summary, our results suggest that ABC294640 may reduce the proliferative capacity of castration-resistant prostate cancer cells through inhibition of both sphingosine kinase 2 and dihydroceramide desaturase, thereby providing a foundation for future exploration of this small-molecule inhibitor for the treatment of advanced disease. Mol Cancer Ther; 14(12); 2744–52. ©2015 AACR.

Dihydroceramide desaturase activity is modulated by oxidative stress

Oxidative stress has been implicated previously in the regulation of ceramide metabolism. In the present study, its effects on dihydroceramide desaturase were investigated. To stimulate oxidative stress, HEK (human embyronic kidney)-293, MCF7, A549 and SMS-KCNR cells were treated with H2O2, menadione or tert-butylhydroperoxide. In all cell lines, an increase in dihydroceramide was observed upon oxidative stress as measured by LC (liquid chromatography)/MS. In contrast, total ceramide levels were relatively unchanged. Mechanistically, dihydroceramide desaturase activity was measured by an in situ assay and decreased in a time- and dose-dependent fashion. Interestingly, no detectable changes in the protein levels were observed, suggesting that oxidative stress does not induce degradation of dihydroceramide desaturase. In summary, oxidative stress leads to potent inhibition of dihydroceramide desaturase resulting in significant elevation in dihydroceramide levels in vivo.

Cell density-dependent reduction of dihydroceramide desaturase activity in neuroblastoma cells.

We applied a metabolic approach to investigate the role of sphingolipids in cell density-induced growth arrest in neuroblastoma cells. Our data revealed that sphingolipid metabolism in neuroblastoma cells significantly differs depending on the cells’ population context. At high cell density, cells exhibited G0/G1 cell-cycle arrest and reduced ceramide, monohexosylceramide, and sphingomyelin, whereas dihydroceramide was significantly increased. In addition, our metabolic-labeling experiments showed that neuroblastoma cells at high cell density preferentially synthesized very long chain (VLC) sphingolipids and dramatically decreased synthesis of sphingosine-1-phosphate (S1P). Moreover, densely populated neuroblastoma cells showed increased message levels of both anabolic and catabolic enzymes of the sphingolipid pathway. Notably, our metabolic-labeling experiments indicated reduced dihydroceramide desaturase activity at confluence, which was confirmed by direct measurement of dihydroceramide desaturase activity in situ and in vitro. Importantly, we could reduce dihydroceramide desaturase activity in low-density cells by applying conditional media from high-density cells, as well as by adding reducing agents, such as DTT and l-cysteine to the media. In conclusion, our data suggest a role of the sphingolipid pathway, dihydroceramides desaturase in particular, in confluence-induced growth arrest in neuroblastoma cells.

Increased killing of SCCVII squamous cell carcinoma cells after the combination of Pc 4 photodynamic therapy and dasatinib is associated with enhanced caspase-3 activity and ceramide synthase 1 upregulation

Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1-and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of ceramide synthase 1 mRNA but not the production of ceramide. The data imply potential significance of the combination for cancer treatment.

Bioactive Sphingolipids in Neuroblastoma

Neuroblastoma is a solid tumor cancer that originates in the nerve tissue of the neck, chest, abdomen, or pelvis, but most commonly in the adrenal gland. It is the third most common pediatric cancer and accounts for ~15% of all childhood cancer deaths. Neuroblastoma has one of the lowest survival rates of all pediatric cancers. It is the most common solid tumor diagnosed during infancy. This tumor may involute spontaneously in infants, or it may sometimes mature to a benign ganglioneuroma. On the other hand, older children often present with advanced stage disease at the time of diagnosis. While survival in patients with favorable biological features may exceed 90%; the survival rates for children with high risk neuroblastoma have historically been under 40%. However, advances made in recent years in understanding the biology of neuroblastoma have led to the identification of patient groups with higher risk disease and to the rise of new modalities for combating this disease. Patients with high-risk neuroblastoma undergo aggressive multi-modal therapies including, chemotherapy, immunotherapy, surgery, stem cell transplantation, and radiation. Survivors also experience many treatment related toxicities and long term side effects. Therefore, new approaches are needed for these patients (Maris, 2010) One of the molecules that can stimulate differentiation of neuroblastoma cells is Ceramide (Fig. 1), a biologically active effector molecule composed of a fatty acyl chain bound to a sphingoid backbone via an amide linkage (Kraveka & Hannun, 2009). The study and modulation of sphingolipids and their effects has emerged as an attractive therapeutic target due to their potent anti-proliferative effects (Hannun & Obeid, 2008). Ceramide is the central building block for sphingolipids. It serves as a precursor for the synthesis of more complex sphingolipids, and is generated by multiple pathways (Fig. 2). Sphingolipids comprise a class of lipids that share the presence of a sphingosine (or related sphingoid) base in the backbone of their structures. Research in the past two decades has shown that sphingolipids, in addition to their roles as structural components of cell membranes, play important roles as regulators of signal transduction in cell differentiation, cell proliferation, inflammation and apoptosis. Ceramide mediates important cellular activities such as induction of cell differentiation, growth arrest, senescence, and apoptosis. Sphingosine-1phosphate has the opposite effect of ceramide stimulating cell proliferation and is involved in angiogenesis and inflammation (Kolesnick, 2002). In this chapter we will review the roles

Abstract 2479: Curcumin mediated apoptosis in human neuroblastoma cells via ROS and sphingolipid generation

Background: Neuroblastoma is the most common solid tumor of infancy and third leading cause of cancer death in children. It has one of the lowest survival rates of all pediatric cancers with less than 50% survival in “high risk” disease. Curcumin is the active compound of the yellow spice turmeric. Curcumin induces apoptosis and inhibits proliferation, angiogenesis, invasion and metastasis in many human cancer cells. The mechanism of cytotoxicity in neuroblastoma is unclear. Objectives: In this study we investigated the effects of curcumin on SMS-KCNR and CHLA-20 human neuroblastoma cells. Methods: SMS-KCNR and CHLA-20 neuroblastoma, cells were treated with increasing concentrations of curcumin. Cell viability was determined by Alamar Blue assays. Real-time PCR analysis of ER stress markers and apoptotic pathway genes was performed. Western Blotting was also performed to examine the downstream signaling pathways. Measurement of endogenous sphingolipids was performed by LC/MS. Sphingolipid pathway enzyme activities were also determined. Results: Curcumin was cytotoxic to both cell lines at 10 and 20 uM concentrations. PARP cleavage was noted at 24 hours, but cleavage of caspases 3, 8 and 9 was not observed. Treatment with the pancaspase inhibitor z-VAD did not reverse the cytotoxicity in curcumin treated cells, confirming that curcumin induced cell death was caspase-independent. Since perturbation in complex sphingolipid levels is associated with apoptosis, LC/MS measurement of endogenous sphingolipids was performed and showed increases in both dihydroceramides and ceramides. The increases in endogenous dihydroceramides, indicated that the dihydroceramide desaturase (DEGS-1) enzyme was inhibited. DEGS-1 activity was inhibited in-situ in a dose dependent manner in SMS-KCNR cells. There was no change in the mRNA or protein levels of DEGS-1, supporting that curcumin inhibited this enzyme indirectly. Next, the mechanism of ceramide generation was investigated by measuring the activity of sphingomyelin synthase (SMS) glycosylceramide synthase (GCS), acid ceramidase, neutral ceramidase, acid sphingomyelinase and neutral sphingomyelinase (SMase). At 6hrs, curcumin downregulated SMS activity by 30% and 54% GCS activity by 40% and 42% at concentrations of 10 and 20uM respectively. Curcumin has been demonstrated to induce ROS generation. Pre-treatment with the antioxidants N-acetylcysteine or glutathione abrogated curcumin mediated apoptosis and sphingolipid generation in SMS-KNCR cells. Furthermore, curcumin mediated SMS and GCS inhibition was blocked by these antioxidants. Conclusions: ROS plays a key role in sphingolopid and curcumin induced-cytotoxicity in neuroblastoma cells. Modulation of sphingolipid signaling pathways may provide a more effective and novel approach for the treatment of pediatric solid tumors. Curcumin is a potential novel therapy for neuroblastoma. Citation Format: Mehrdad Rahmaniyan, Li Li, Julio Garcia, A A. Qudeimat, Jacqueline M. Kraveka. Curcumin mediated apoptosis in human neuroblastoma cells via ROS and sphingolipid generation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2479.

Abstract B18: Role of sphingolipids and ER stress in curcumin mediated cytotoxicity In human neuroblastoma cells

Background: Neuroblastoma is the most common solid tumor of infancy and third leading cause of cancer death in children. It has one of the lowest survival rates of all pediatric cancers with less than 50% survival in “high risk” disease. Curcumin is the active compound of the yellow spice turmeric. Curcumin induces apoptosis and inhibits proliferation, angiogenesis, invasion and metastasis in many human cancer cells. The mechanism of cytotoxicity in neuroblastoma is unclear. Objectives: In this study we investigated the effects of curcumin on SMS-KCNR and CHLA-20 human neuroblastoma cells. Methods: SMS-KCNR and CHLA-20 neuroblastoma, cells were treated with increasing concentrations of curcumin. Cell viability was determined by Alamar Blue assays. Real-time PCR analysis of ER stress markers and apoptotic pathway genes was performed. Western Blotting was also performed to examine the downstream signaling pathways. Measurement of endogenous sphingolipids was performed by LC/MS. Sphingolipid pathway enzyme activities were also determined. Results: Curcumin was cytotoxic to both cell lines at 10 and 20 uM concentrations. PARP cleavage was noted at 24 hours, but cleavage of caspases 3, 8 and 9 was not observed. Treatment with the pancaspase inhibitor z-VAD did not reverse the cytotoxicity in curcumin treated cells, confirming that curcumin induced cell death was caspase-independent. ER stress markers of activation of the unfolded protein response were then examined and increase in the expression of CHOP (9 fold and 16 fold) and GRP78 (3 and 2 fold) mRNA levels was noted at 6 and 24 hours respectively. Increases in protein levels of CHOP and GRP-78 were also observed. ER stress may play the main role in curcumin induced-cytotoxicity in neuroblastoma. Since perturbation in complex sphingolipid levels is associated with ER stress and apoptosis, LC/MS measurement of endogenous sphingolipids was performed and showed increases in both dihydroceramides and ceramides. The increases in endogenous dihydroceramides, indicated that the dihydroceramide desaturase (DEGS-1) enzyme was inhibited. DEGS-1 activity was inhibited in-situ in a dose dependent manner in SMS-KCNR cells. There was no change in the mRNA or protein levels of DEGS-1, supporting that curcumin inhibited this enzyme indirectly. Next, the mechanism of ceramide generation was investigated by measuring the activity of sphingomyelin synthase (SMS) glycosylceramide synthase (GCS), acid ceramidase, neutral ceramidase, acid sphingomyelinase and neutral sphingomyelinase (SMase). At 6hrs, curcumin downregulated SMS activity by 30% and 54% GCS activity by 40% and 42% at concentrations of 10 and 20uM respectively. Conclusions: ER stress plays a key role in curcumin induced-cytotoxicity in neuroblastoma cells. Modulation of sphingolipid signaling pathways may provide a more effective and novel approach for the treatment of pediatric solid tumors. Curcumin is a potential novel therapy for neuroblastoma. Citation Format: Mehrdad Rahmaniyan, Li Li, Amr Qudeimat, Julio Garcia, Jacqueline M. Kraveka. Role of sphingolipids and ER stress in curcumin mediated cytotoxicity In human neuroblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B18.