High-throughput discovery of novel small-molecule inhibitors of acid Ceramidase.

Ceramide has a key role in the regulation of cellular senescence and apoptosis. As Ceramide levels are lowered by the action of acid ceramidase (AC), abnormally expressed in various cancers, the identification of AC inhibitors has attracted increasing interest. However, this finding has been mainly hampered by the lack of formats suitable for the screening of large libraries. We have overcome this drawback by adapting a fluorogenic assay to a 384-well plate format. The performance of this optimised platform has been proven by the screening a library of 4100 compounds. Our results show that the miniaturised platform is well suited for screening purposes and it led to the identification of several hits, that belong to different chemical classes and display potency ranges of 2-25 µM. The inhibitors also show selectivity over neutral ceramidase and retain activity in cells and can therefore serve as a basis for further chemical optimisation.

Inhibition of acid ceramidase elicits mitochondrial dysfunction and oxidative stress in pancreatic cancer cells.

Although the inhibition of acid ceramidase (AC) is known to induce antitumor effects in various cancers, there are few reports in pancreatic cancer, and the underlying mechanisms remain unclear. Moreover, there is currently no safe administration method of AC inhibitor. Here the effects of gene therapy using siRNA and shRNA for AC inhibition with its mechanisms for pancreatic cancer were investigated. The inhibition of AC by siRNA and shRNA using an adeno-associated virus 8 (AAV8) vector had antiproliferative effects by inducing apoptosis in pancreatic cancer cells and xenograft mouse model. Acid ceramidase inhibition elicits mitochondrial dysfunction, reactive oxygen species accumulation, and manganese superoxide dismutase suppression, resulting in apoptosis of pancreatic cancer cells accompanied by ceramide accumulation. These results elucidated the mechanisms underlying the antitumor effect of AC inhibition in pancreatic cancer cells and suggest the potential of the AAV8 vector to inhibit AC as a therapeutic strategy.

N-acylsphingosine amidohydrolase 1 promotes melanoma growth and metastasis by suppressing peroxisome biogenesis-induced ROS production.

OBJECTIVE: Metabolic deregulation is a key hallmark of cancer cells and has been shown to drive cancer growth and metastasis. However, not all metabolic drivers of melanoma are known. Based on our finding that N-acylsphingosine amidohydrolase 1 (ASAH1) is overexpressed in melanoma, the objective of these studies was to establish its role in melanoma tumor growth and metastasis, understand its mechanism of action, and evaluate ASAH1 targeting for melanoma therapy. METHODS: We used publicly available melanoma datasets and patient-derived samples of melanoma and normal skin tissue and analyzed them for ASAH1 mRNA expression and ASAH1 protein expression using immunohistochemistry. ASAH1 was knocked down using short-hairpin RNAs in multiple melanoma cell lines that were tested in a series of cell culture-based assays and mouse-based melanoma xenograft assays to monitor the effect of ASAH1 knockdown on melanoma tumor growth and metastasis. An unbiased metabolomics analysis was performed to identify the mechanism of ASAH1 action. Based on the metabolomics findings, the role of peroxisome-mediated reactive oxygen species (ROS) production was explored in regard to mediating the effect of ASAH1. The ASAH1 inhibitor was used alone or in combination with a BRAFV600E inhibitor to evaluate the therapeutic value of ASAH1 targeting for melanoma therapy. RESULTS: We determined that ASAH1 was overexpressed in a large percentage of melanoma cells and regulated by transcription factor E2F1 in a mitogen-activated protein (MAP) kinase pathway-dependent manner. ASAH1 expression was necessary to maintain melanoma tumor growth and metastatic attributes in cell cultures and mouse models of melanoma tumor growth and metastasis. To identify the mechanism by which ASAH1 facilitates melanoma tumor growth and metastasis, we performed a large-scale and unbiased metabolomics analysis of melanoma cells expressing ASAH1 short-hairpin RNAs (shRNAs). We found that ASAH1 inhibition increased peroxisome biogenesis through ceramide-mediated PPARγ activation. ASAH1 loss increased ceramide and peroxisome-derived ROS, which in turn inhibited melanoma growth. Pharmacological inhibition of ASAH1 also attenuated melanoma growth and enhanced the effectiveness of BRAF kinase inhibitor in the cell cultures and mice. CONCLUSIONS: Collectively, these results demonstrate that ASAH1 is a druggable driver of melanoma tumor growth and metastasis that functions by suppressing peroxisome biogenesis, thereby inhibiting peroxisome-derived ROS production. These studies also highlight the therapeutic utility of ASAH1 inhibitors for melanoma therapy.

Elevated glucosylsphingosine in Gaucher disease induced pluripotent stem cell neurons deregulates lysosomal compartment through mammalian target of rapamycin complex 1.

Gaucher disease (GD) is a lysosomal storage disorder caused by mutations in GBA1, the gene that encodes lysosomal ß-glucocerebrosidase (GCase). Mild mutations in GBA1 cause type 1 non-neuronopathic GD, whereas severe mutations cause types 2 and 3 neuronopathic GD (nGD). GCase deficiency results in the accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). GlcSph is formed by deacylation of GlcCer by the lysosomal enzyme acid ceramidase. Brains from patients with nGD have high levels of GlcSph, a lipid believed to play an important role in nGD, but the mechanisms involved remain unclear. To identify these mechanisms, we used human induced pluripotent stem cell-derived neurons from nGD patients. We found that elevated levels of GlcSph activate mammalian target of rapamycin (mTOR) complex 1 (mTORC1), interfering with lysosomal biogenesis and autophagy, which were restored by incubation of nGD neurons with mTOR inhibitors. We also found that inhibition of acid ceramidase prevented both, mTOR hyperactivity and lysosomal dysfunction, suggesting that these alterations were caused by GlcSph accumulation in the mutant neurons. To directly determine whether GlcSph can cause mTOR hyperactivation, we incubated wild-type neurons with exogenous GlcSph. Remarkably, GlcSph treatment recapitulated the mTOR hyperactivation and lysosomal abnormalities in mutant neurons, which were prevented by coincubation of GlcSph with mTOR inhibitors. We conclude that elevated GlcSph activates an mTORC1-dependent pathogenic mechanism that is responsible for the lysosomal abnormalities of nGD neurons. We also identify acid ceramidase as essential to the pathogenesis of nGD, providing a new therapeutic target for treating GBA1-associated neurodegeneration.

Design, Synthesis, and Biological Evaluation of a Series of Oxazolone Carboxamides as a Novel Class of Acid Ceramidase Inhibitors.

Acid ceramidase (AC) is a cysteine hydrolase that plays a crucial role in the metabolism of lysosomal ceramides, important members of the sphingolipid family, a diversified class of bioactive molecules that mediate many biological processes ranging from cell structural integrity, signaling, and cell proliferation to cell death. In the effort to expand the structural diversity of the existing collection of AC inhibitors, a novel class of substituted oxazol-2-one-3-carboxamides were designed and synthesized. Herein, we present the chemical optimization of our initial hits, 2-oxo-4-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a, which resulted in the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent AC inhibitor with optimal physicochemical and metabolic properties, showing target engagement in human neuroblastoma SH-SY5Y cells and a desirable pharmacokinetic profile in mice, following intravenous and oral administration. 32b enriches the arsenal of promising lead compounds that may therefore act as useful pharmacological tools for investigating the potential therapeutic effects of AC inhibition in relevant sphingolipid-mediated disorders.

Does tamoxifen have a therapeutic role outside of breast cancer? A systematic review of the evidence.

INTRODUCTION: Tamoxifen is a widely used hormonal based therapy for breast cancer in the adjuvant and metastatic setting, prolonging overall and recurrence-free survival. There has been increasing interest in the potential for novel "off-target" effects of tamoxifen and its metabolite N-desmethyltamoxifen across a number of cancer types. We aim to review the current literature regarding the potential use of tamoxifen in other primary malignancies. METHOD: A qualitative systematic review was performed according to the PRISMA guidelines using pre-set search criteria across the PubMed, Cochrane and Scopus databases from 1985 to 2019. Additional results were generated from included papers references. RESULTS: A total of 324 papers were identified, of which 47 were included; a further 29 articles were obtained from additional referencing to give a total of 76 articles. Clinical trials have demonstrated benefits with the use of tamoxifen in isolation and combination, specifically in patients with advanced non-resectable malignancy, however results are not consistent across the literature. In vivo data consistently suggests that off target effects of tamoxifen are mediated through the ceramide pathway or through inhibition of protein kinase C (PKC). CONCLUSIONS: With increased focus upon the potential of repurposing drugs, tamoxifen may be a candidate for repurposing in the wider cancer setting. There is evidence to suggest that the ceramide or PKC pathway could act as a therapeutic target for tamoxifen or alternative chemotherapeutics and merits further investigation.

N-Metallocenoylsphingosines as targeted ceramidase inhibitors: Syntheses and antitumoral effects.

Three N-metallocenoylsphingosines with variance in the central metal (Fe, Co, Ru), the charge (neutral or cationic), and the arene ligands (Cp2, Cp*Ph) were synthesized from serine and metallocene carboxylic acids as substrate-analogous inhibitors of human acid ceramidase (AC). Their inhibitory potential was examined using the recombinant full length ASAH1 enzyme, expressed and secreted from High Five insect cells, and the fluorescent substrate Rbm14-12. All complexes inhibited AC, most strongly so ruthenium(II) complex 13a. Some antitumoral effects of the complexes, such as the interference with the microtubular and F-actin cytoskeleton of cancer cells, were correlated to their AC-inhibition, whereas others, e.g. their cytotoxicity and their induction of caspase-3/-7 activity in cancer cells, were not. All complexes accumulated preferentially in the lysosomes of cancer cells like their target AC, arrested the cells in G1 phase of the cell cycle, and displayed cytotoxicity with mostly single-digit micromolar IC50 values while inducing cancer cell apoptosis.

Tamoxifen is a candidate first-in-class inhibitor of acid ceramidase that reduces amitotic division in polyploid giant cancer cells-Unrecognized players in tumorigenesis.

Polyploid giant cancer cells (PGCC) represent a poorly understood, small subpopulation of tumor cells that are increasingly being recognized for their critical role in therapy resistance, metastasis, and cancer recurrence. PGCC have the potential to generate progeny through primitive or cleavage-like division, which allows them to evade antimitotic insults. We recently demonstrated that the sphingolipid enzyme acid ceramidase (ASAH1) is required for this process. Since specific ASAH1 inhibitors are not clinically available, we investigated whether tamoxifen, which interferes with ASAH1 function via off-target effects, has a potential clinical benefit independent of estrogen signaling. Our results show that tamoxifen inhibits generation of PGCC offspring in prostate cancer, glioblastoma, and melanoma cells. Analysis of two state-level cancer registries revealed that tamoxifen improves survival outcomes for second, nonbreast cancers that develop in women with early stage breast cancer. Our results suggest that tamoxifen may have a clinical benefit in a variety of cancers that is independent of estrogen signaling and could be due to its inhibition of acid ceramidase. Thus the distinct application of tamoxifen as potentially a first-in-class therapeutic that inhibits the generation of PGCC offspring should be considered in future clinical trials.

Lead Optimization of Benzoxazolone Carboxamides as Orally Bioavailable and CNS Penetrant Acid Ceramidase Inhibitors.

Sphingolipids (SphLs) are a diverse class of molecules that are regulated by a complex network of enzymatic pathways. A disturbance in these pathways leads to lipid accumulation and initiation of several SphL-related disorders. Acid ceramidase is one of the key enzymes that regulate the metabolism of ceramides and glycosphingolipids, which are important members of the SphL family. Herein, we describe the lead optimization studies of benzoxazolone carboxamides resulting in piperidine 22m, where we demonstrated target engagement in two animal models of neuropathic lysosomal storage diseases (LSDs), Gaucher's and Krabbe's diseases. After daily intraperitoneal administration at 90 mg kg-1, 22m significantly reduced the brain levels of the toxic lipids glucosylsphingosine (GluSph) in 4L;C* mice and galactosylsphingosine (GalSph) in Twitcher mice. We believe that 22m is a lead molecule that can be further developed for the correction of severe neurological LSDs where GluSph or GalSph play a significant role in disease pathogenesis.

Altering Sphingolipid Metabolism Attenuates Cell Death and Inflammatory Response After Myocardial Infarction.

BACKGROUND: Sphingolipids have recently emerged as a biomarker of recurrence and mortality after myocardial infarction (MI). The increased ceramide levels in mammalian heart tissues during acute MI, as demonstrated by several groups, is associated with higher cell death rates in the left ventricle and deteriorated cardiac function. Ceramidase, the only enzyme known to hydrolyze proapoptotic ceramide, generates sphingosine, which is then phosphorylated by sphingosine kinase to produce the prosurvival molecule sphingosine-1-phosphate. We hypothesized that Acid Ceramidase (AC) overexpression would counteract the negative effects of elevated ceramide and promote cell survival, thereby providing cardioprotection after MI. METHODS: We performed transcriptomic, sphingolipid, and protein analyses to evaluate sphingolipid metabolism and signaling post-MI. We investigated the effect of altering ceramide metabolism through a loss (chemical inhibitors) or gain (modified mRNA [modRNA]) of AC function post hypoxia or MI. RESULTS: We found that several genes involved in de novo ceramide synthesis were upregulated and that ceramide (C16, C20, C20:1, and C24) levels had significantly increased 24 hours after MI. AC inhibition after hypoxia or MI resulted in reduced AC activity and increased cell death. By contrast, enhancing AC activity via AC modRNA treatment increased cell survival after hypoxia or MI. AC modRNA-treated mice had significantly better heart function, longer survival, and smaller scar size than control mice 28 days post-MI. We attributed the improvement in heart function post-MI after AC modRNA delivery to decreased ceramide levels, lower cell death rates, and changes in the composition of the immune cell population in the left ventricle manifested by lowered abundance of proinflammatory detrimental neutrophils. CONCLUSIONS: Our findings suggest that transiently altering sphingolipid metabolism through AC overexpression is sufficient and necessary to induce cardioprotection post-MI, thereby highlighting the therapeutic potential of AC modRNA in ischemic heart disease.

Control of lysosomal TRPML1 channel activity and exosome release by acid ceramidase in mouse podocytes.

The transient receptor potential mucolipin 1 (TRPML1) channel has been reported to mediate lysosomal Ca2+ release that is involved in Ca2+-dependent lysosome trafficking and autophagic flux. However, this regulatory mechanism of lysosomal TRPML1 channel activity in podocytes remains poorly understood. In the present study, we tested whether the TRPML1 channel in podocytes mediates lysosome trafficking, which is essential for multivesicular body (MVB) degradation by lysosomes. We first demonstrated the abundant expression of TRPML1 channel in podocytes. By GCaMP3 Ca2+ imaging, we characterized the lysosomal specificity of TRPML1 channel-mediated Ca2+ release in podocytes. Given the important role of acid ceramidase (AC) in lysosome function and podocyte injury, we tested whether AC regulates this TRPML1 channel-mediated Ca2+ release and consequent lysosome-dependent MVB degradation in podocytes. Pharmacologically, it was found that TRPML1 channel activity was remarkably attenuated by the AC inhibitor carmofur. Sphingosine, as an AC product, was demonstrated to induce TRPML1-mediated Ca2+ release, which was inhibited by a TRPML1 blocker, verapamil. Using a Port-a-Patch planar patch-clamp system, we found that AC-associated sphingolipids, sphingomyelin, ceramide, and sphingosine had different effects on TRPML1 channel activity in podocytes. Functionally, the inhibition of AC or blockade of TRPML1 channels was found to suppress the interaction of lysosomes and MVBs, leading to increased exosome release from podocytes. These results suggest that AC is critical for TRPML1 channel-mediated Ca2+ release, which controls lysosome-MVB interaction and exosome release in podocytes.

Genetic and pharmacological inhibition of acid ceramidase prevents asymmetric cell division by neosis.

Radiation treatment failure or relapse after initial response to chemotherapy presents significant clinical challenges in cancer patients. Escape from initial courses of treatment can involve reactivation of embryonic developmental stages, with the formation of polynuclear giant cancer cells (PGCCs). This strategy of dedifferentiation can insulate cancer cells from a variety of treatments and allows a residual subpopulation to reestablish tumors after treatment. Using radiation or docetaxel chemotherapy, we generated PGCCs from prostate cancer cells. Here, we show that expression of acid ceramidase (ASAH1), an enzyme in the sphingolipid pathway linked to therapy resistance and poor outcomes, is elevated in PGCCs. Targeting ASAH1 with shRNA or treatment with the ASAH1 inhibitor, LCL-521, did not impair the formation of PGCCs, but prevented the formation of PGCC progeny that arise through an asymmetric cell division called neosis. Similar results were obtained in lung cancer cells that had been exposed to radiation or cisplatin chemotherapy as stressors. In summary, our data suggest that endoreplication occurs independent of ASAH1 while neosis is ASAH1-dependent in both prostate and lung cancer cells. Because ASAH1 knockout is embryonic lethal but not deleterious to adult animals, targeting this enzyme has the potential to be highly specific to cells undergoing the dedifferentiation process to escape cancer treatments. Pharmacological inhibition of ASAH1 is a potentially powerful strategy to eliminate cells that could otherwise serve as seed populations for recurrence.

Activity-Based Imaging of Acid Ceramidase in Living Cells.

Acid ceramidase (AC) hydrolyzes ceramides into sphingoid bases and fatty acids. The enzyme is overexpressed in several types of cancer and Alzheimer's disease, and its genetic defect causes different incurable disorders. The availability of a method for the specific visualization of catalytically active AC in intracellular compartments is crucial for diagnosis and follow-up of therapeutic strategies in diseases linked to altered AC activity. This work was undertaken to develop activity-based probes for the detection of AC. Several analogues of the AC inhibitor SABRAC were synthesized and found to act as very potent (two-digit nM range) irreversible AC inhibitors by reaction with the active site Cys143. Detection of active AC in cell-free systems was achieved either by using fluorescent SABRAC analogues or by click chemistry with an azide-substituted analogue. The compound affording the best features allowed the unprecedented labeling of active AC in living cells.

Acid Ceramidase: A Novel Therapeutic Target in Cancer.

Sphingolipids are important constituents of the eukaryotic cell membrane which govern various signaling pathways related to different aspects of cell survival. Ceramide and Sphingosine are interconvertible sphingolipid metabolites, out of which Ceramide is pro-apoptotic and sphingosine is anti-apoptotic in nature. The conversion of ceramide to sphingosine is mediated by Acid Ceramidase (ASAH1) thus maintaining a rheostat between a tumor suppressor and a tumor promoter. This rheostat is completely altered in many tumors leading to uncontrolled proliferation. This intriguing property of ASAH1 can be used by cancer cells to their advantage, by increasing the expression of the tumor promoter, sphingosine inside cells, thus creating a favorable environment for cancer growth. The different possibilities through which this enzyme serves its role in formation, progression and resistance of different types of cancers will lead to the possibility of making Acid Ceramidase a promising drug target. This review discusses the current understanding of the role of acid ceramidase in cancer progression, metastasis and resistance, strategies to develop novel natural and synthetic inhibitors of ASAH1 and their usefulness in cancer therapy.

Acid ceramidase, an emerging target for anti-cancer and anti-angiogenesis.

Sphingolipid metabolism plays an important role in determining the fate of a cell. Among several sphingolipid metabolites, ceramide is a key player in intracellular signal transduction. Ceramide is usually converted to various metabolites such as sphingomyelin, sphingosine, ceramide-1-phosphate, and glucosylceramide. If ceramide is accumulated in the cell, it induces apoptosis. On the other hand, its metabolite sphingosine is converted to sphingosine-1-phosphate (S1P), which promotes angiogenesis via G protein coupled receptor signaling. Therefore, the equilibrium in ceramide and S1P levels in cells plays an important role in angiogenesis as well as cell death. Acid ceramidase (AC) is a promising target protein in the development of multi-targeted anticancer drugs as its inhibition can simultaneously inhibit angiogenesis via the Akt and ERK 1/2 pathway and limit cancer growth through ceramide-induced apoptosis. Although some inhibitors of AC have been reported, they have not been proven effective for human therapy. Recent advancement in the elucidation of AC structure will facilitate the development of better inhibitors for treating human diseases.

A Guanidine-Based Synthetic Compound Suppresses Angiogenesis via Inhibition of Acid Ceramidase.

Angiogenesis generates new blood vessels from pre-existing vessels. Tumors induce the formation of new blood vessels to ensure sufficient oxygen and nutrients for their growth. Normally, angiogenesis is induced by various pro-angiogenesis factors, including vascular endothelial growth factor (VEGF). Inhibition of VEGF is a promising approach to cancer treatment. A guanidine-based synthetic compound, E2, was identified as a potent hit from 68 guanidine-based derivatives by screening for angiogenesis inhibitors showing antiproliferative activity in human umbilical vein endothelial cells (HUVECs). To explore the mode of action of E2, target proteins were investigated using phage display biopanning, and acid ceramidase 1 (ASAH1) was identified as an E2-binding protein. Drug affinity responsive target stability (DARTS) and ASAH1 activity assays revealed the direct binding of E2 to ASAH1. Moreover, siRNA knockdown of ASAH1 demonstrated its role as an angiogenesis factor. Consequently, E2 inhibited chemoinvasion and tube formation of HUVECs in a dose-dependent manner. E2 also potently suppressed neo-vascularization of chorioallantoic membranes in vivo. Collectively, these data suggest that E2 is a novel angiogenesis inhibitor and ASAH1 is proposed to be a new antiangiogenesis target.

Molecular Mechanism of Inhibition of Acid Ceramidase by Carmofur.

Human acid ceramidase (AC) is a lysosomal cysteine amidase, which has received a great deal of interest in recent years as a potential target for the development of new therapeutics against melanoma and glioblastoma tumors. Despite the strong interest in obtaining structural information, only the structures of the apo-AC enzyme in its zymogen and activated conformations are available. In this work, the crystal structure of AC in complex with the covalent carmofur inhibitor is presented. Carmofur is an antineoplastic drug containing an electrophilic carbonyl reactive group that targets the catalytic cysteine. This novel structural data explains the basis of the AC inhibition, provides insights into the enzymatic properties of the protein, and is a great aid toward the structure-based drug design of potent inhibitors for AC, providing the detailed mechanism, which has eluded the scientific community for more than 30 years, of carmofur's mysterious 5-fluorouracil-independent antitumor activity.

Dose dependent actions of LCL521 on acid ceramidase and key sphingolipid metabolites.

The function of acid ceramidase (ACDase), whose congenital deficiency leads to Farber disease, has been recognized to be vital to tumor cell biology, and inhibition of its activity may be beneficial in cancer therapy. Therefore, manipulation of the activity of this enzyme may have significant effect, especially on cancer cells. LCL521, Di-DMG-B13, is a lysosomotropic inhibitor of ACDase. Here we define complexities in the actions of LCL521 on ACDase. Systematic studies in MCF7 cells showed dose and time divergent action of LCL521 on ACDase protein expression and sphingolipid levels. Low dose of LCL521 (1 µM) effectively inhibited ACDase in cells, but the effects were transient. A higher dose of LCL521 (10 µM) caused a profound decrease of sphingosine and increase of ceramide, but additionally affected the processing and regeneration of the ACDase protein, with biphasic and reversible effects on the expression of ACDase, which paralleled the long term changes of cellular sphingosine and ceramide. Finally, the higher concentrations of LCL521 also inhibited Dihydroceramide desaturase (DES-1). In summary, LCL521 exhibits significant effects on ACDase in a dose and time dependent manner, but dose range and treatment time need to be paid attention to specify its future exploration on ACDase targeted cancer treatment.

Acid ceramidase inhibition sensitizes human colon cancer cells to oxaliplatin through downregulation of transglutaminase 2 and ß1 integrin/FAK-mediated signalling.

Acid ceramidase (ASAH1) has been implicated in the progression and chemoresistance in different cancers. Its role in colon cancer biology and response to standard chemotherapy has been poorly addressed so far. Here, we have investigated ASAH1 expression at the protein level in human colon cancer cell lines and tissues from colon cancer patients, and have examined in vitro the possible link between ASAH1 expression and functional activity of p53 protein whose inactivation is associated with the progression from adenoma to malignant tumour in colon cancer. Finally, we have explored the role of ASAH1 in response and resistance mechanisms to oxaliplatin (OXA) in HCT 116 colon cancer cells. We have demonstrated that human colon cancer cells and colorectal adenocarcinoma tissues constitutively express ASAH1, and that its expression is higher in tumour tissues than in normal colonic mucosa. Furthermore, we found an inverse correlation between ASAH1 expression and p53 functional activity. Obtained data revealed that ASAH1 was involved in HCT 116 cell response to OXA and that anti-proliferative, pro-apoptotic, anti-migratory and anti-clonogenic effects of OXA could be significantly increased by combination treatment with ASAH1 inhibitor carmofur. Increased OXA sensitivity was associated with downregulation of signalling involved in acquired resistance to OXA in colon cancer, in particular transglutaminase 2 and ß1 integrin/FAK, which resulted in the suppression of NF-κB and Akt. Thus, combination of OXA with ASAH1 inhibitors could be a promising strategy to counter chemoresistance and improve treatment outcome in advanced colon cancer.

Anticancer actions of lysosomally targeted inhibitor, LCL521, of acid ceramidase.

Acid ceramidase, which catalyzes ceramide hydrolysis to sphingosine and free fatty acid mainly in the lysosome, is being recognized as a potential therapeutic target for cancer. B13 is an effective and selective acid ceramidase inhibitor in vitro, but not as effective in cells due to poor access to the lysosomal compartment. In order to achieve targeting of B13 to the lysosome, we designed lysosomotropic N, N-dimethyl glycine (DMG)-conjugated B13 prodrug LCL521 (1,3-di-DMG-B13). Our previous results indicated the efficient delivery of B13 to the lysosome resulted in augmented effects of LCL521 on cellular acid ceramidase as evaluated by effects on substrate/product levels. Our current studies indicate that functionally, this translated into enhanced inhibition of cell proliferation. Moreover, there were greater synergistic effects of LCL521 with either ionizing radiation or Tamoxifen. Taken together, these results clearly indicate that compartmental targeting for the inhibition of acid ceramidase is an efficient and valuable therapeutic strategy.