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A. MTS assay showing percent metabolic viability after treating Des1+/+ and Des1−/− cells with 20 µM etoposide at 24 hours and 48 hours of post-treatment. MTS assay measures metabolic viability that also indicates cell viability. After 48 hours of etoposide treatment, significant increase in metabolic viability is observed in Des1−/− cells compared to Des1+/+ cells (*P<0.05, n = 3). B. Percentage of dead cells (PI positive cells) measured using PI (propidium iodide) staining after treating MEFs with etoposide. At 24 hours of post-etoposide treatment, percentage of PI positive cells is higher in Des1 +/+ cells compared to Des1−/− cells (# P<0.05, n = 3), However, after 48 hours significantly high cell death is observed in Des1 +/+cells compared to Des1−/− cells (P<0.005, n = 3). C. Activation of caspase 3 & 7 as measured by luminescence produced from luminogenic substrate of caspase 3&7. At both 24 hours and 48 hours, caspase 3/7 activation is significantly higher in Des1 +/+ cells (*P<0.05, n = 3). D. PI staining of MEFs after treatment with 20 µM etoposide, 20 µM etoposide + 100 µM C2 ceramide, and 100 µM C2 ceramide (as a control to confirm ceramide's toxicity) for 24 hours. Addition of exogenous ceramide demise the pro-survival property of the Des1−/− cells in presence of etoposide (#P = 0.87, n = 3, difference is not significant) when comparing between etoposide treated Des1+/+ cells (that harbor endogenous ceramide) and Des1−/− cells treated with etoposide+exogenous ceramide (both panels are marked with #). Additional experiments with etoposide and ceramide show the efficacy of these two compounds to induce cell death (control +/+ vs. etoposide-treated +/+ or ceramide-treated +/+ and control−/− vs. etoposide-treated −/− or ceramide-treated −/−).
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Sphingolipid biosynthesis is potently upregulated by factors associated with cellular stress, including numerous chemotherapeutics, inflammatory cytokines, and glucocorticoids. Dihydroceramide desaturase 1 (Des1), the third enzyme in the highly conserved pathway driving sphingolipid biosynthesis, introduces the 4,5-trans-double bond that typifies m...
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Citations
... DEGS1 shows mainly Δ4-desaturase activity, whereas DEGS2 demonstrates both Δ4-desaturase and C4 monooxygenase activities [60,87]. DEGS1 is ubiquitously expressed whereas DEGS2 is expressed only in certain tissues such as the skin, liver, and intestines [87][88][89][90]. DEGS1 was identified as a direct in vitro target for fenretinide, a synthetic retinoid that is used in cancer treatment. ...
... DES2 is highly expressed in skin, intestine and kidney [97]. The deletion of DES1 and DES2 shifts sphingolipid synthesis pathway toward the sphingolipid lacking the double bond introduced by DES1 and DES2, such as dhS1P, dhSph, dhsphingomyelin (dhSM) and especially dhCer [99]. In Des1 −/− mice, the inability to form ceramide leads to highly elevated dihydroceramide, low levels of ceramide, multi-organ dysfunction, and failure to thrive. ...
: Molecular targeting strategies have been used for years to control cancer progression and are often based on targeting various enzymes involved in metabolic pathways. Keeping that in mind, it is essential to know the role of each enzyme in a particular metabolic pathway. In this review, we are providing in-depth knowledge of various enzymes such as ceramidase, sphingosine kinase, sphingomyelin synthase, dihydroceramide desaturase, and ceramide synthase, which are associated with multiple types of cancer that depend on ceramide metabolism. Focus has also been given to discussing physicochemical properties of well-studied inhibitors of natural products origin and their related structures for these enzymes. Targeting ceramide metabolism exhibited promise in mono and combination therapies at preclinical stages to prevent cancer progression and paved the way for the significance of sphingolipid metabolism in cancer treatments. Targeting ceramide metabolizing enzymes will help the medicinal chemist to design potent and selective small molecules for treating cancer progression at various levels.
... For example, saturated C18:0 and C16:0 ceramides were more toxic to HeLa cells compared to C18:1 and C24:1 ceramides [13]. Of note, the sphingoid base of the sphingolipids is also a crucial determinant of its toxicity, as dihydroceramide, which lacks a double bond in its sphingoid base in comparison to ceramides, does not share similar cytotoxicity as observed in ceramides [14,15]. ...
Ceramides are a class of sphingolipids which are implicated in skin disorders, obesity, and other metabolic diseases. As a class with pleiotropic effects, recent efforts have centred on discerning specific ceramide species and their effects on atopic dermatitis, obesity, type 2 diabetes, and cardiovascular diseases. This delineation has allowed the identification of disease biomarkers, with long acyl chain ceramides such as C16- and C18-ceramides linked to metabolic dysfunction and cardiac function decline, while ultra-long acyl chain ceramides (>25 carbon acyl chain) were reported to be essential for maintaining a functional skin barrier. Given the intricate link between free fatty acids with ceramides, especially the de novo synthetic pathway, intracellular lipid droplet formation is increasingly viewed as an important mechanism for preventing accumulation of toxic ceramide species. Here, we review recent reports of various ceramide species involved in skin abnormalities and metabolic diseases, and we propose that promotion of lipid droplet biogenesis can be seen as a potential protective mechanism against deleterious ceramides.
... Consequently, the significant decrease of CERS in dox-resistant cells supports the dominant depletion of CERS. Gene expression of both isoforms of dihydroceramide desaturase (DeS) transcripts was analyzed because of their crucial role in controlling the balance between SPLs and dihydrosphingolipids [53]. Interestingly, our qRT-PCR results demonstrated that DeS1 was remarkably expressed in dox-resistant cells, while DeS2 was completely absent in this cell line. ...
Drug resistance is responsible for the failure of many available anticancer drugs. Several studies have demonstrated the association between the alteration in sphingolipids (SPLs) and the development of drug resistance. To investigate the association between SPLs metabolism and doxorubicin (dox)-resistance in MCF-7 cells, a comparative sphingolipidomics analysis between dox-sensitive (parental) and-resistant MCF-7 cell lines along with validation by gene expression analysis were conducted. A total of 31 SPLs representing 5 subcategories were identified. The data obtained revealed that SPLs were clustered into two groups differentiating parental from dox-resistant cells. Eight SPLs were significantly altered in response to dox-resistance including SM (d18:1/16), SM (d18:1/24:2), SM (d18:1/24:0), SM (d18:1/20:0), SM (d18:1/23:1), Hex-Cer (d18:1/24:0), SM (d18:1/15:0), DHSM (d18:0/20:0). The current study is the first to conclusively ascertain the potential involvement of dysregulated SPLs in dox-resistance in MCF-7 cells. SPLs metabolism in dox-resistant MCF-7 cells is oriented toward the downregulation of ceramides (Cer) and the concomitant increase in sphingomyelin (SM). Gene expression analysis has revealed that dox-resistant cells tend to escape from the Cer-related apoptosis by the activation of SM-Cer and GluCer-LacCer-ganglio-side pathways. The enzymes that were correlated to the alteration in SPLs metabolism of dox-resistant MCF-7 cells and significantly altered in gene expression can represent potential targets that can represent a winning strategy for the future development of promising anticancer drugs.
... According to the recent data, dihydroceramide is unable to decrease the mitochondrial function, as opposed to bioactive ceramide (119)(120)(121). Ceramide desaturase 1 (Des1) ablation in ob/ob mice leads to the accumulation of sphinganine-based sphingolipid species (dihydrosphingolipids, missing double bond at C4-C5 position of sphingoid base) in both the liver and adipose tissue at the expense of sphingosine-based ceramide species. ...
Insulin resistance is defined as a complex pathological condition of abnormal cellular and metabolic response to insulin. Obesity and consumption of high-fat diet lead to ectopic accumulation of bioactive lipids in insulin-sensitive tissues. Intracellular lipid accumulation is regarded as one of the major factors in the induction of insulin resistance and type 2 diabetes (T2D). A significant number of studies have described the involvement of ceramides and other sphingolipids in the inhibition of insulin-signaling pathway in both skeletal muscles and the liver. Adverse effects of sphingolipid accumulation have recently been linked to the activation of protein kinase Cζ (PKCζ) and protein phosphatase 2A (PP2A), which, in turn, negatively affect phosphorylation of serine/threonine kinase Akt [also known as protein kinase B (PKB)], leading to decreased glucose uptake in skeletal muscles as well as increased gluconeogenesis and glycogenolysis in the liver. Sphingolipids, in addition to their direct impact on the insulin signaling pathway, may be responsible for other negative aspects of diabetes, namely mitochondrial dysfunction and deficiency. Mitochondrial health, which is characterized by appropriate mitochondrial quantity, oxidative capacity, controlled oxidative stress, undisturbed respiratory chain function, adenosine triphosphate (ATP) production and mitochondrial proliferation through fission and fusion, is impaired in the skeletal muscles and liver of T2D subjects. Recent findings suggest that impaired mitochondrial function may play a key role in the development of insulin resistance. Mitochondria stay in contact with the endoplasmic reticulum (ER), Golgi membranes and mitochondria-associated membranes (MAM) that are the main places of sphingolipid synthesis. Moreover, mitochondria are capable of synthesizing ceramide though ceramide synthase (CerS) activity. Recently, ceramides have been demonstrated to negatively affect mitochondrial respiratory chain function and fission/fusion activity, which is also a hallmark of T2D. Despite a significant correlation between sphingolipids, mitochondrial dysfunction, insulin resistance and T2D, this subject has not received much attention compared to the direct effect of sphingolipids on the insulin signaling pathway. In this review, we focus on the current state of scientific knowledge regarding the involvement of sphingolipids in the induction of insulin resistance by inhibiting mitochondrial function.
... In fact, reports demonstrate opposing roles of DEGS1 in apoptosis [56], whereby its polyubiquitination results in 'gain of function' with pro-survival effects [57]. Indeed, inhibiting DEGS1 activity with drugs other than HPR, siRNA, or gene depletion causes resistance to apoptosis by various stimuli [58][59][60][61]. Meanwhile, depending on cell types, dhCer might not have a role in HPR-triggered apoptosis [62], suggesting that the apoptogenic potential depends on the ratio of dhCer to Cer, rather than dhCer alone [55,62]. ...
Ceramide (Cer) is a bioactive cellular lipid with compartmentalized and tightly regulated levels. Distinct metabolic pathways lead to the generation of Cer species with distinguishable roles in oncogenesis. Deregulation of Cer pathways has emerged as an important mechanism for acquired chemotherapeutic resistance. Adult T-cell leukemia (ATL) cells are defective in Cer synthesis. ATL is an aggressive neoplasm that develops following infection with human T-cell lymphotropic virus-1 (HTLV-1) where the viral oncogene Tax contributes to the pathogenesis of the disease. ATL cells, resistant to all-trans-retinoic acid, are sensitive to pharmacologically achievable concentrations of the synthetic retinoid ST1926. We studied the effects of ST1926 on Cer pathways in ATL cells. ST1926 treatment resulted in early Tax oncoprotein degradation in HTLV-1-treated cells. ST1926 induced cell death and a dose- and time-dependent accumulation of Cer in malignant T cells. The kinetics and degree of Cer production showed an early response upon ST1926 treatment. ST1926 enhanced de novo Cer synthesis via activation of ceramide synthase CerS(s) without inhibiting dihydroceramide desaturase, thereby accumulating Cer rather than the less bioactive dihydroceramide. Using labeling experiments with the unnatural 17-carbon sphinganine and measuring the generated Cer species, we showed that ST1926 preferentially induces the activities of a distinct set of CerS(s). We detected a delay in cell death response and interruption of Cer generation in response to ST1926 in Molt-4 cells overexpressing Bcl-2. These results highlight the potential role of ST1926 in inducing Cer levels, thus lowering the threshold for cell death in ATL cells.
... In this work, we show for the first time that ABTL0812 treatment of human pancreatic, endometrial, adenocarcinoma and squamous NSCLC cancer cells resulted in impaired DEGS1 activity (Figure 6(c,d)). Consequently, ABTL0812 induced an increase on the levels of cellular long-chain dihydroceramides in those cells (Figures 5(b-d) and 6(g,h)) without affecting ceramide levels at short treatment times, similar to that observed for short-time treatment with the DEGS1 specific inhibitor GT11 (Figure 7(a)) or for the cancer cells where DEGS1 was silenced [37,38]. Importantly, DEGS1 inhibitor GT11 recapitulated ABTL0812 mechanism of action, since it induced ER stress-dependent autophagy and selective cytotoxicity (non-tumor cells are less sensitive than tumor cells) (Figure 7). ...
ABTL0812 is a first-in-class small molecule with anticancer activity currently in Phase 2a clinical evaluation in patients with advanced endometrial and squamous NSCLC. We have previously described that ABTL0812 induces TRIB3 pseudokinase expression, resulting in inhibition of the Akt-mTORC1 axis and autophagy-mediated cancer cell death. However, classical PI3K/Akt/mTOR inhibitors do not induce an autophagy as strong as ABTL0812 does, therefore we aimed to further elucidate the molecular mechanism responsible for the cytotoxic autophagy which causes ABTL0812 anticancer activity. ABTL0812 induced UPR hallmarks ATF4, CHOP and TRIB3 in vitro in lung, endometrial and pancreatic cancer cell lines, as well as in non-tumor cells. Nevertheless, therapeutic concentrations of ABTL0812 did not induce cytotoxic autophagy in non-tumor cells. Furthermore, genetic or pharmacological inhibition of the UPR resulted in impaired ABTL0812 cytotoxicity in cancer cells. Expression of UPR markers (ATF4, CHOP and TRIB3) in response to ABTL0812 treatment were also validated in xenograft models. In order to uncover the precise molecular mechanism involved in ABTL0812-induced UPR and cytotoxic autophagy we undertook a comprehensive sphingolipidomic analysis, since changes in sphingolipids have been reported to contribute to activation of both UPR and autophagy. ABTL0812 treatment resulted in increased levels of long chain dihydroceramides in cultured cancer cells as well as in vivo. Mechanistically, ABTL0812 impaired desaturase-1 activity (Des-1), the enzyme that introduces a 4,5-trans-double bond in the sphingolipid backbone of dihydroceramides to generate ceramides. Accordingly, in vitro incubation of cancer cells with dihydroceramides resulted in activation of UPR, autophagy and cytotoxicity. Of interest, we observed that tumor cells showed higher Des-1 expression levels than non-tumor cells. Finally, we showed that Des-1 inhibition (GT11) and mTORC1 inhibition (everolimus) collaborate to promote autophagy and cancer cell death, simulating ABTL0812 activity. Furthermore, we have validated the increased expression of CHOP and TRIB3 mRNA levels in blood samples from ABTL0812 treated patients. These biomarkers are currently used as pharmacodynamic biomarkers in the ongoing phase 2 clinical trial in patients with squamous NSCLC and endometrial cancer. To our knowledge, this is the first time that UPR markers are reported to change in human blood in response to any drug treatment. In conclusion, we have shown that ABTL0812 triggers a sustained ER stress and UPR activation mediated by the impairment of Des-1 activity which collaborates with mTORC1 inhibition to induce cytotoxic autophagy in cancer cells, which offers improved anticancer activity over just inhibiting mTORC1.
Citation Format: Pau Muñoz-Guardiola, Josefina Casas, Elisabet Megías-Roda, Hector Perez-Montoyo, Sonia Solé-Sánchez, Marc Yeste-Velasco, Tatiana Erazo, Nora Diéguez-Martínez, Sergio Espinosa-Gil, Guillermo Yoldi, Cristina Muñoz-Pinedo, Miguel F. Segura, Jose Alfon, Gemma Fabriàs, Guillermo Velasco, Carles Domenech, Jose M. Lizcano. The anticancer drug ABTL0812 induces cancer cell death by impairing Akt/mTORC1 axis and inducing ER stress-mediated cytotoxic autophagy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1234.
... In this work, we show for the first time that ABTL0812 treatment of human pancreatic, endometrial, adenocarcinoma and squamous NSCLC cancer cells resulted in impaired DEGS1 activity (Figure 6(c,d)). Consequently, ABTL0812 induced an increase on the levels of cellular long-chain dihydroceramides in those cells (Figures 5(b-d) and 6(g,h)) without affecting ceramide levels at short treatment times, similar to that observed for short-time treatment with the DEGS1 specific inhibitor GT11 (Figure 7(a)) or for the cancer cells where DEGS1 was silenced [37,38]. Importantly, DEGS1 inhibitor GT11 recapitulated ABTL0812 mechanism of action, since it induced ER stress-dependent autophagy and selective cytotoxicity (non-tumor cells are less sensitive than tumor cells) (Figure 7). ...
ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased DDIT3 and TRIB3 mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer.
Abbreviations:
4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ9-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.
... However, DES1 activation by palmitic acid activated DES1 leading to cell death, 31 and DES1 ablation conferred resistance to etoposide-induced apoptosis. 32 Thus, the effects of DES1 inhibition are variable depending on the cellular context and degree of inhibition. 33 The role of DES1 in VTC treatment remains to be determined and further analysis is needed. ...
Resveratrol (RSV) has recently attracted keen interest because of its pleiotropic effects. It exerts a wide range of health-promoting effects. In addition to health-promoting effects, RSV possesses anti-carcinogenic activity. However, a non-physiological concentration is needed to achieve an anti-cancer effect, and its in vivo bioavailability is low. Therefore, the clinical application of phytochemicals requires alternative candidates that induce the desired effects at a lower concentration and with increased bioavailability. We previously reported a low IC50 of vaticanol C (VTC), an RSV tetramer, among 12 RSV derivatives (Ito T. et al, 2003). However, the precise mechanism involved remains to be determined. Here, we screened an in-house chemical library bearing RSV building blocks ranging from dimers to octamers for cytotoxic effects in several leukemia and cancer cell lines and their anti-cancer drug-resistant sublines. Among the compounds, VTC exhibited the highest cytotoxicity, which was partially inhibited by a caspase 3 inhibitor, Z-VAD-FMK. VTC decreased the expression of sphingosine kinase 1, sphingosine kinase 2 and glucosylceramide synthase by transcriptional or post-transcriptional mechanisms, and increased cellular ceramides/dihydroceramides and decreased sphingosine 1-phosphate (S1P). VTC-induced sphingolipid rheostat modulation (the ratio of ceramide/S1P) is thought to be involved in cellular apoptosis. Indeed, exogenous S1P addition modulated VTC cytotoxicity significantly. A combination of SPHK1, SPHK2, and GCS chemical inhibitors induced sphingolipid rheostat modulation, cell growth suppression, and cytotoxicity similar to that of VTC. These results suggest the involvement of sphingolipid metabolism in VTC-induced cytotoxicity, and indicate VTC is a promising prototype for translational research.
... In this work, we show for the first time that ABTL0812 treatment of human pancreatic, endometrial, adenocarcinoma and squamous NSCLC cancer cells resulted in impaired DEGS1 activity (Figure 6(c,d)). Consequently, ABTL0812 induced an increase on the levels of cellular long-chain dihydroceramides in those cells (Figures 5(b-d) and 6(g,h)) without affecting ceramide levels at short treatment times, similar to that observed for short-time treatment with the DEGS1 specific inhibitor GT11 (Figure 7(a)) or for the cancer cells where DEGS1 was silenced [37,38]. Importantly, DEGS1 inhibitor GT11 recapitulated ABTL0812 mechanism of action, since it induced ER stress-dependent autophagy and selective cytotoxicity (non-tumor cells are less sensitive than tumor cells) (Figure 7). ...
Gene dosage is a key defining factor to understand cancer pathogenesis and progression, which requires the development of experimental models that aid better deconstruction of the disease. Here, we model an aggressive form of prostate cancer and show the unconventional association of LKB1 dosage to prostate tumorigenesis. Whereas loss of Lkb1 alone in the murine prostate epithelium was inconsequential for tumorigenesis, its combination with an oncogenic insult, illustrated by Pten heterozygosity, elicited lethal metastatic prostate cancer. Despite the low frequency of LKB1 deletion in patients, this event was significantly enriched in lung metastasis. Modeling the role of LKB1 in cellular systems revealed that the residual activity retained in a reported kinase-dead form, LKB1K78I, was sufficient to hamper tumor aggressiveness and metastatic dissemination. Our data suggest that prostate cells can function normally with low activity of LKB1, whereas its complete absence influences prostate cancer pathogenesis and dissemination.
