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.

A New Glucocerebrosidase Chaperone Reduces α-Synuclein and Glycolipid Levels in iPSC-Derived Dopaminergic Neurons from Patients with Gaucher Disease and Parkinsonism.

UNLABELLED: Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT: Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.

Glucosylceramide biosynthesis is involved in Golgi morphology and protein secretion in plant cells.

Lipids have an established role as structural components of membranes or as signalling molecules, but their role as molecular actors in protein secretion is less clear. The complex sphingolipid glucosylceramide (GlcCer) is enriched in the plasma membrane and lipid microdomains of plant cells, but compared to animal and yeast cells, little is known about the role of GlcCer in plant physiology. We have investigated the influence of GlcCer biosynthesis by glucosylceramide synthase (GCS) on the efficiency of protein transport through the plant secretory pathway and on the maintenance of normal Golgi structure. We determined that GlcCer is synthesized at the beginning of the plant secretory pathway [mainly endoplasmic reticulum (ER)] and that D,L-threo-1-phenyl-2-decanoyl amino-3-morpholino-propanol (PDMP) is a potent inhibitor of plant GCS activity in vitro and in vivo. By an in vivo confocal microscopy approach in tobacco leaves infiltrated with PDMP, we showed that the decrease in GlcCer biosynthesis disturbed the transport of soluble and membrane secretory proteins to the cell surface, as these proteins were partly retained intracellularly in the ER and/or Golgi. Electron microscopic observations of Arabidopsis thaliana root cells after high-pressure freezing and freeze substitution evidenced strong morphological changes in the Golgi bodies, pointing to a link between decreased protein secretion and perturbations of Golgi structure following inhibition of GlcCer biosynthesis in plant cells.

Ceramide metabolism determines glioma cell resistance to chemotherapy.

Tumor cell resistance to chemotherapy constitutes a major problem in the treatment of malignant tumors. We here investigated the role of ceramide metabolism for the resistance of glioma cells to treatment with the chemotherapeutic drug, gemcitabine. Gemcitabine triggers a marked release of ceramide in drug-sensitive cells, while glioma cells that are resistant to gemcitabine, fail to accumulate ceramide. While the release of ceramide is very similar in gemcitabine-sensitive and resistant glioma cells upon stimulation, resistant glioma cells rapidly consume ceramide upon gemcitabine treatment or exogenous sphingomyelinase stimulation. Pharmacologic or genetic inhibition of glucosyltransferases prevents ceramide consumption in resistant cells and restores sensitivity of resistant glioma cells to gemcitabine. These data suggest that glioma cell resistance to at least some chemotherapeutic drugs is mediated by rapid consumption of ceramide to prevent cell death.

New synthetic seven-membered 1-azasugars displaying potent inhibition towards glycosidases and glucosylceramide transferase.

Several members of a new family of seven-membered azasugars, which can be seen as 1-azasugar ring homologues, have been obtained by simple chemical transformations starting from a sugar-derived azidolactol. Unlike their piperidine counterparts, these molecules are chemically stable when they possess a hydroxy group at the pseudo-C-2 position. Biological assays with a range of carbohydrate-processing enzymes have revealed interesting potential for these compounds. A trihydroxymethyl-substituted azepane displayed strong competitive inhibition on almond beta-glucosidase (K(i)=2.5 microM) while a trihydroxylated carboxylic acid derivative proved to be a potent and selective L-fucosidase inhibitor (K(i)=41 nM). N-Butylation of these seven-membered 1-azasugars generated derivatives with some activity towards the Gaucher's disease-related glucosylceramide transferase (IC(50) 75 microM) that did not interact significantly with digestive glucosidases.

Membrane disruption and cytotoxicity of hydrophobic N-alkylated imino sugars is independent of the inhibition of protein and lipid glycosylation.

The N-alkyl moiety of N-alkylated imino sugars is crucial for therapeutic activities of these compounds as inhibitors of glycosphingolipid (GSL) biosynthesis and as antivirals. The improved potency afforded by a long N-alkyl moiety is coincident with increased compound-induced cytotoxicity. Therefore, in the present study, we examined the mechanism of this cytotoxicity in detail. Despite N-butyl-deoxynojirimycin and N-butyl-deoxygalactonojirimycin inhibiting the glycosylation of ceramide to glucosylceramide, ceramide levels did not increase in HL60 cells treated with these compounds. Long-chain N-alkylated imino sugars were toxic to cells at concentrations considerably lower than the critical micellar concentrations for these compounds and consequently did not solubilize radioactively labelled cellular proteins and lipids. However, membrane disruption and cell fragmentation did increase in a concentration- and chain-length-dependent manner. These results are consistent with previously proposed interactions between surface-active amphiphiles and protein-containing lipid membranes when drug concentrations are below the critical micellar concentration. Taken together, these results demonstrate that the cellular toxicity of hydrophobic N-alkylated imino sugars is due to cell lysis and cell fragmentation and, most importantly, is not related to the beneficial therapeutic effects of these compounds on protein and in lipid glycosylation. This information will aid in the future development of more selective imino sugar therapeutics for the treatment of human disease.

Modulators of ceramide metabolism sensitize colorectal cancer cells to chemotherapy: a novel treatment strategy.

Irinotecan is a first-line chemotherapeutic agent for patients with metastatic colorectal cancer (CRC). Response rates of less than 40% underscore the problem of treating CRC with irinotecan. Our studies have shown that chemosensitization correlates with high levels of ceramide, whereas resistance correlates with high levels of glucosylceramide (GlcCer). The purpose of this study was to characterize the role of ceramide in irinotecan-mediated CRC cell death. We used four human CRC cell lines to assess ceramide metabolism, cell viability, and apoptosis after treatment with irinotecan. Fumonisin B(1) (FB(1)) and 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) were used to inhibit de novo ceramide synthesis and GlcCer production, respectively. L-threo-dihydrosphingosine (safingol) was used to inhibit secondary proliferative pathways mediated by an atypical protein kinase C that is activated by ceramide. Irinotecan elicited dose- and time-dependent increases in ceramide, which preceded apoptosis. When FB(1) was added to irinotecan, CRC cell death was significantly decreased. A significant increase in intracellular levels of GlcCer also was noted after treatment with irinotecan. When GlcCer production was blocked by treating cells with PPMP in addition to irinotecan, ceramide levels increased to 228% of control values and cell death increased by 88%, compared to irinotecan alone. When irinotecan was combined with both PPMP and safingol, cell death was increased by 225% to 325%, compared to irinotecan lone. CRC cell death due to irinotecan is mediated, at least in part, by the de novo synthesis of ceramide. Blocking further metabolism of ceramide can enhance this cytotoxicity. Targeting ceramide pathways is a novel strategy for the treatment of patients with CRC.

The T lymphocyte structure CD60 contains a sialylated carbohydrate epitope that is expressed on both gangliosides and glycoproteins.

The CD60 antigen is expressed on a majority of T cells in autoimmune lesions, and anti-CD60 can activate T lymphocytes. CD60 has been defined as the GD3 ganglioside, and subsequently as the 9-O-acetylated form of GD3. However, other evidence suggests that anti-CD60 recognizes a glycoprotein or family of glycoproteins expressed by T lymphocytes. The current studies were undertaken to better define the identity of the CD60 antigen on both T cells and non-T cells. Treatment of intact cells with neuraminidases of various specificities confirmed that detection of the CD60 epitope depends on expression of an alpha2, 8-disialic acid carbohydrate linkage, as is found in GD3 and related gangliosides. However, the sialicacid polymer colominic acid inhibited anti-GD2 and anti-GD3, but not anti-CD60 from binding to cell surfaces. Expression of CD60 did not correlate with expression of GD3 on a variety of cell lines and T cell populations. Expression of CD60 and 9-O-acetyl-GD3 was roughly parallel on some non-T cell lines such as melanoma cells, but on T cells expression of CD60 was consistently greater. Antibodies to GD2, GD3 and 9-O-acetyl-GD3 were ineffective at inhibiting binding of anti-CD60 to CD60+ cells. Activation responses of T cells to anti-CD60 were inducible in either the presence or absence of a response to anti-GD3. A novel inhibitor of glucosyl ceramide synthesis, D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (D-t-P4) reduced expression of GD3 much more than CD60 on activated T lymphocytes. Following biotinylation of HUT78 T cells, anti-CD60 immunoprecipitated a 70 kDa antigen. Taken together, the present data and previous findings suggest that anti-CD60 can recognize both a modified form of the GD3 ganglioside and a carbohydrate-dependent complex epitope present on one or more glycoproteins. This glycoprotein epitope may be the more abundant and functionally significant CD60 antigen on T lymphocytes, while 9-O-acetyl-GD3 is likely to be the principal structure recognized by anti-CD60 on melanoma cells. These findings emphasize the complexity of understanding the functional roles of carbohydrate epitopes in cell activation.

Glucosylceramide synthesis inhibitors block pharmacologically induced dispersal of the Golgi and anterograde membrane flow from the endoplasmic reticulum: implication of sphingolipid metabolism in maintenance of the Golgi architecture and anterograde membrane flow.

PDMP (D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) and PPMP (D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol), inhibitors of glucosylceramide synthesis, blocked brefeldin A (BFA)- and nordihydroguaiaretic acid-induced dispersal of the Golgi and trans Golgi network, and Golgi-derived vesicles were retained in the juxtanuclear region. PDMP and PPMP did not stabilize microtubules but blocked nocodazole-induced extensive fragmentation and dispersal of the Golgi, and large Golgi vesicles were retained in the juxtanuclear region. PPMP is a stronger inhibitor of glucosylceramide synthesis than PDMP, but PDMP showed a stronger activity against BFA-induced retrograde membrane flow. However, PPMP showed a stronger activity for Golgi disruption and inhibition of anterograde trafficking from the endoplasmic reticulum, and rebuilding of the Golgi architecture. Cumulatively, these results suggest that sphingolipid metabolism is implicated in maintenance of the Golgi architecture and anterograde membrane flow from the endoplasmic reticulum but not in Golgi dispersal induced by BFA.

Differential effects of glycolipid biosynthesis inhibitors on ceramide-induced cell death in neuroblastoma cells.

An in vitro model of Gaucher's disease in murine neuroblastoma x rat glioma NG108-15 cells was used to investigate the physiological effects of two specific inhibitors of glucosylceramide synthase, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d,l-PDMP) and N-butyldeoxynojirimycin (NB-DNJ), which have been suggested as agents for treatment of glycolipid storage disorders. Incubation of NG108-15 cells with conduritol-B-epoxide, a covalent inhibitor of glucosylceramidase, raised the intracellular concentration of glucosylceramide (GC) by more than fourfold, indicating a glycolipid composition equivalent to that of Gaucher's cells. The level of GC was decreased, and the cells were depleted of gangliosides by postincubation with d,l-PDMP or NB-DNJ. Treatment with d,l-PDMP, but not with NB-DNJ, resulted in a dose-dependent reduction of the growth rate and eventually caused cell death in NG108-15 cells on reaching confluency. An in situ detection assay using terminal nucleotidyltransferase indicated that cell degeneration was accompanied by apoptosis. Lipid analysis by high-performance TLC revealed that on incubation with d,l-PDMP, but not with NB-DNJ, the concentration of endogenous ceramide was elevated by threefold. Ceramide elevation and apoptosis were also observed when NG108-15 cells were incubated with daunorubicin, which was previously reported to induce programmed cell death by stimulation of ceramide synthesis. Structural characterization by HPLC and subsequent laser desorption mass spectrometry revealed that the endogenous ceramide contained fatty acids with chain lengths ranging from C14:0 to C24:0. The results indicate that elevation of levels of these ceramide species by incubation with d,l-PDMP or daunorubicin induces programmed cell death in NG108-15 cells. Because ceramide accumulation and cell death were not observed on incubation with NB-DNJ, its use is suggested to be less toxic than that of d,l-PDMP for treatment of Gaucher's disease and other sphingolipid storage disorders.

Modulation of carcinoembryonic antigen release by glucosylceramide--implications for HT29 cell differentiation.

Previous work suggested that glucosylceramide (GlcCer) plays a role in the regulation of cell differentiation of HT29 human colon tumor cells. In the present study, we investigated the role of GlcCer in the cellular release of carcinoembryonic antigen (CEA), a marker for cell differentiation. This was done by modulating the intracellular level of the glycolipid, according to two different approaches. The cells were treated with D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which resulted in a specific lowering of the cellular GlcCer pool. Alternatively, by exogenous addition of a short-chain analog of the lipid, hexanoyl(C6)-GlcCer, the cellular pool was enhanced. The results demonstrate that PDMP causes an increase in the release of CEA, while exogenous C6-GlcCer suppresses its release. Furthermore, the enhanced release of CEA in the presence of PDMP, could be completely reversed upon exogenous addition of C6-GlcCer. Control experiments reveal that a potential interference of the well-known modulator of cell physiology, ceramide (Cer), can be excluded. Long-term depletion of GlcCer resulted in a change in a morphological feature of differentiation of the cells, i.e. an increase in apical membrane surface with microvilli brush borders, accompanied by an enhanced expression of the cytoskeletal protein villin. These results, together with the observations on modulation of the differentiation marker CEA by GlcCer, provide support for the conclusion that GlcCer interferes with the differentiation of HT29 cells.

The synthetic pathway for glucosylsphingosine in cultured fibroblasts.

The synthesis of glucosylsphingosine (GlcSph), a glucosylceramide (GlcCer) analogue devoid of fatty acids, in cultured fibroblasts was studied by using conduritol beta epoxide (CBE), an inhibitor of beta-glucosidase, and 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glucosylceramide (GlcCer) synthase (glucosyltransferase). When CBE was added to the culture medium, the intracellular beta-glucosidase activity decreased, and both GlcCer and GlcSph accumulated in the cells. After the addition of PDMP, the concentration of GlcCer decreased, while the content of GlcSph increased. When CBE and PDMP were added together, the intracellular accumulation of GlcSph to decreased to less than when CBE alone was added. Based on these results, the synthetic pathway for GlcSph was thus considered to not only be through the glucosylation of sphingosine, but also through the deacylation of GlcCer. When GlcCer (d18:1, C12:0) was added to the culture medium, the intracellular accumulation of GlcSph (d18:1) was evident, and it was also more pronounced in the presence of CBE. In addition, when GlcCer (d18:0, C12:0) was used, apparent accumulation of GlcSph (d18:0) was also observed. In order to determine whether or not the deacylase of GlcCer is identical to acid ceramidase, a deacylase of ceramide, the same experiments were carried out using fibroblasts from two patients with Farber disease, in which acid ceramidase is genetically deficient. The accumulation of GlcSph in the Farber disease fibroblasts after the loading of GlcCer for 7 days was found to be one-fifth of the control level.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of D-[3H]threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthesis, and the synergistic action of an inhibitor of microsomal monooxygenase.

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) is an effective inhibitor of the glucosyltransferase that makes glucosylceramide. Virtually all of the hundreds of naturally occurring glycolipids are formed from this primary glycolipid, so the inhibitor acts to lower their concentrations by the process of attrition (hydrolytic catabolism). Trials with mice carrying ascites carcinoma cells showed that PDMP could produce a permanent cure in some of the animals and marked prolongation of life in the others (Inokuchi, J., I. Mason, and N.S. Radin. 1987. Cancer Lett. 38: 23-30). In order to maximize the effect, we studied the metabolism of PDMP by labeling it with [3H] on carbon one, using a labeling method that discriminated against the unwanted erythro-isomer. The active enantiomer of the inhibitor (D-) was isolated by chromatography of the camphanate esters, followed by methanolytic cleavage. Examination of the fate of the labeled drug after a single injection showed that it was very rapidly converted to several polar products that were rapidly excreted. The drug penetrated all of the organs readily and a small portion was oxidized at the C-1 position to yield 3H2O. From these findings it appeared likely that the amine is attacked by a mixed function oxidase based on cytochrome P450. This conclusion was confirmed by showing that the tissue levels of PDMP could be greatly elevated, for a much longer time, when the mice were pretreated with piperonyl butoxide or cimetidine. The amount of conversion to polar metabolites was substantially reduced and tissue levels of PDMP were maintained much longer. Analysis of mice injected with one or both drugs showed that piperonyl butoxide augmented the effects of PDMP on ceramide, glucosylceramide, and dihexosylceramide levels, as well as on the activity of glucosylceramide synthase. It is suggested that piperonyl butoxide be used as an adjuvant for the many useful drugs that are inactivated by the P450 system.