Therapeutic Potential of Ceramide in Cancer Treatment

Abstract

Ceramides are a family of wax-like lipids that fall under the broader category of sphingolipids. A ceramide is composed of a sphingosine side chain coupled to a fatty acid via an amide linkage. Distinct from complex sphingolipids, the head of ceramide is a simple alcohol rather than a phosphate, phosphocholine, sugar, or more. The fatty acid chains of ceramide can also vary in chain length and degree of saturation. The degree of saturation may determine the biological activity of the ceramide species. Ceramides are highly abundant within the cell membrane of eukaryotic cells and are appreciated for their structural roles in these cells. Moreover, ceramides are well-known for their biological activity including as regulators of apoptosis, senescence, the cell cycle, and differentiation. This review discusses pathways of ceramide, roles of ceramide in various diseases, targeting ceramide metabolism in the treatment of cancer, as well as ceramide-delivering nanotechnologies.

Ceramide Metabolism

Ceramide metabolism consists of a complex network of interconnected pathways with many origins and many downstream biological implications (Figure 1) [1]. Broadly, ceramide metabolism can be classified into de novo synthesis and catabolism, including recycling, salvage, and degradation.

Figure 1. Basic pathway of ceramide metabolism.

De novo ceramide synthesis initially generates dihydrosphingolipid species prior to desaturation. Salvage ceramide synthesis can include turnover by the activities of acidic/lysosomal-localized catabolic enzymes, which generates sphingosine that leaves the lysosome and is re-acylated to ceramide. Glucosylceramide synthase (GCS); cerebrosidase (GBA); sphingomyelin synthase (SGMS); ceramide kinase (CERK), ceramide-1-phosphate phosphatase (C1PP); ceramidases (CDase); ceramide synthase (CERS); sphingosine kinase (SPHK); S1P phosphatase (S1PP).

De novo synthesis

De novo synthesis of ceramide occurs in the endoplasmic reticulum [1]. It starts with the condensation of the amino acid serine and palmitoyl-CoA by serine palmitoyltransferase, which produces 3-keto-dihydrosphingosine. This intermediate is reduced to dihydrosphingosine (also known as sphinganine) by 3-ketosphinganine reductase. The ceramide synthase (CERS) family catalyzes the N-acylation of dihydrosphingosine to dihydroceramide. Dihydroceramide desaturase next introduces a 4,5-trans-double bond into the backbone of dihydroceramide, producing ceramide.

Six ceramide synthases (CERS1–6) have been identified in humans (Table 1) [1]. Each CERS enzyme prefers different chain lengths of the fatty acyl-CoA substrate involved in the N-acylation of dihydrosphingosine. The resulting dihydroceramide may contain a fatty acid of varying lengths depending on the CERS enzyme utilized. The chain lengths of the fatty acyl-CoA used tend to vary from 14 to 36 carbons. Ceramides with different chain compositions impact cell physiology differently [2], as has been observed with contrast death/survival roles reported for C16 and C18 ceramides [3,4]. In addition, the various CERS enzymes can have different tissue distribution patterns. For example, CERS1 is highly expressed in the brain and skeletal muscle, which correlates with an abundance of C18 ceramide in those tissues. CERS2 is highly expressed in the liver and kidneys and favors very-long chain fatty acids (C22-C24) [5]. CERS3 is highly expressed in the skin and testis and is selective toward very-long (C22-C24) and ultra-long chains (≥ C26). CERS4 is highly expressed in the skin and leukocytes and catalyzes formation of ceramide with a selectivity towards long and very-long chains (C18-C22) [6]. CERS5 is more commonly found in the muscle and brain. It mainly produces ceramides incorporating C16 fatty acyl-CoA [5]. CERS6 also uses C16 fatty acyl-CoA as a substrate and is commonly found in the brain [7].

Table 1.

Classification and distribution of ceramide synthases

Ceramide synthase	Chain length of the fatty acyl-CoA substrate	Tissue distribution
CERS1	C18	Brain and skeletal muscle
CERS2	C22-C24	Liver and kidneys
CERS3	C22-C24, ≥ C26	Skin and testis
CERS4	C18-22	Skin, leukocytes
CERS5	C16	Muscle and brain
CERS6	C16	Brain

Salvage, recycling, and degradation

In addition to de novo synthesis, ceramide can be “salvaged” from complex sphingolipid reservoirs, recycled to/from other sphingolipids, or degraded into simple molecules [1]. These pathways, alongside de novo synthesis, regulate the cellular levels of ceramide. The salvage of ceramide from complex sphingolipids provides for more than half of ceramide biosynthesis. One such example is the catabolism of glycosphingolipids [1]. During the breakdown of large glycosphingolipids, sugar groups are cleaved off which leads to the formation of either glucosylceramide or galactosylceramide. In turn, specific β-glucosidases and galactosidases hydrolyze these lipids to form ceramide [8]. In addition to the formation of ceramide from larger sphingolipids such as glycosphingolipids, ceramide can be formed via the hydrolysis of sphingomyelin by various sphingomyelinases (SMase) [5], which are defined by their pH optima. Ceramide can also act as a substrate to build more complex sphingolipids. For instance, ceramide can be converted to sphingomyelin, a large component of the fatty myelin sheath, via the action of sphingomyelin synthase (SGMS). Similarly, ceramide can be utilized by glucosylceramide synthase (GCS) to generate glucosylceramide. Glucosylceramide, as well as galactosylceramide, can go on to form more complex sphingolipids in the Golgi apparatus, which include gangliosides, globosides, and sulfatides. These can then be moved to the plasma membrane, the major reservoir of these lipids [9,10]. The degradation of ceramide is also a necessary part of maintaining lipid homeostasis. Ceramides present in endothelial cells can be de-acylated to yield sphingosine by ceramidases (CDase), which are also defined by their pH optima. Sphingosine can then be used to generate sphingosine-1-phosphate (S1P) or be re-acylated to again form ceramide [11]. This re-acylation process is further important to complex sphingolipid catabolism in the lysosome as the ceramide that is initially generated is catabolized by acid CDase to sphingosine, which can subsequently be re-acylated by CERS to re-form ceramide. In a similar mechanism, short-chain ceramide analogs can be catabolized by acid CDase to sphingosine and then re-acylated to yield ceramide with physiological chain lengths [12].

Ceramide and S1P

In addition to ceramide, S1P is an important sphingolipid to highlight. Ceramide can be converted into S1P via two enzymatic reactions [1]. Typically, acid CDase (or another CDase) hydrolyzes ceramide to sphingosine and then a sphingosine kinase (SPHK), typically SPHK1, phosphorylates sphingosine to generate S1P. Although S1P and ceramide share structural similarities and are easily interconverted, the two exhibit opposite effects. Whereas ceramide is well appreciated for its role in the stimulation of apoptosis, S1P is appreciated as a pro-survival signal, leading to increased cell growth and viability [13]. S1P’s pro-survival effects are beneficial to tumors and thus an excess of S1P in tissues is associated with tumor neogenesis. S1P predominantly signals through G protein-coupled receptors (GPCRs) known as S1P receptors (S1PRs). Activation of S1PRs often leads to subsequent activation of the Raf-Mek-Erk pathway, a classic mitogenic pathway. Thus, activation of these GPCRs leads to increased tumor cell viability. The control of the titer of ceramide and S1P in tumors is thought to be a potential therapeutic target [14].

Basic role of ceramide in skin disease, metabolic syndrome, infection, and cancer

Structurally diverse ceramides can provide platforms for signaling events and protein/lipid trafficking along cellular membranes [15]. Thus, changes in ceramide levels can alter the biophysical properties of the membrane. In this way, ceramide and its metabolism can function as a sensor, affect the distribution of transmembrane proteins, and impact a diversity of other cellular processes [16]. Consequently, abnormalities in ceramide homeostasis can contribute to various pathologies. This is important when considering inflammatory links between conditions including metabolic disease or infection and cancer, or even the persistence of cancer within the skin.

Skin diseases

Skin ceramide composition, which plays crucial roles in maintaining the barrier function of the stratum corneum, is altered in patients with atopic dermatitis [17]. It is accompanied by a change of ceramide synthase isoform expression from CERS3 to CERS4, leading to a barrier repair response [18]. Atopic dermatitis, sometimes referred to as eczema, is an inflammatory condition linked with a compromised epidermal barrier [19,20]. Topical treatment of atopic dermatitis with pseudo-ceramide lotion, which contains 3% Cetyl-PG hydroxyethyl palmitamide, has been found to reduce symptoms such as pruritis. Additionally, a decrease in trans-epidermal water loss and increased water content has been shown with pseudo-ceramide treatment. Those changes are associated with a distinct switch of the ceramide profile from an atopic dermatitis to a healthy skin phenotype without any increased level of endogenous ceramides [21]. Furthermore, increased ceramide content has been shown to result in acceleration of epidermal barrier restoration after acute damage to the skin [22]. Collectively, this is noteworthy as pseudo-ceramide has become a major and well-advertised component of various skin creams.

Metabolic syndrome

Ceramide contributes to lipotoxicity that underlies metabolic syndrome and related diseases such as type 2 diabetes, as well as various types of cardiovascular disease including coronary artery disease, hypertension, and stroke [23]. The species of ceramide typically associated with cardiovascular disease progression are C16 ceramide (d18:1/16:0), C18 ceramide (d18:1/18:0), and C24:1 ceramide (d18:1/24:1). These ceramides have been shown to lead to abnormalities in glucose metabolism by blocking insulin signaling. Additionally, ceramides dysregulate lipid metabolism by promoting adipose inflammation. In contrast, C24 ceramide (d18:1/24:0) has been associated with good cardiovascular health [24]. Interestingly, skeletal muscle fatty acid oxidation was promoted alongside a decrease in whole-body fat accumulation following oral treatment with P053, which targets CERS1 [25].

Infection

The structural domains formed by different ceramides can cause changes in membrane curvature. These changes in membrane morphology may be exploited by pathogens, leading to host cell infection [26]. Bile acids and ceramides can assist the entry of viruses and to facilitate their subsequent replication in jejunal enteroids. Some viral pathogens can invade the epithelial barrier and replicate in the human small intestine. For instance, in the case of norovirus strains, ceramide can assist their release into the cytoplasm by acting as a binding factor [27]. Conversely, it has been shown that ceramide can mediate neutrophil extracellular trap formation to boost their bactericidal capacity [28]. Interestingly, studies have shown that the glucosylceramide-ceramide cycle can regulate cellular entry of certain viruses including the influenza virus [29,30]. Glucosylceramide can be generated from ceramide as well as broken down to ceramide. In one study, manipulation of glucosylceramide synthesis revealed that glucosylceramide can facilitate influenza viral binding and entry into host cells [29]. A follow-up study instead manipulated the breakdown of glucosylceramide to ceramide to demonstrated that this process was key to mediating endocytosis of influenza and other enveloped viruses [30].

Cancer

Ceramides of various chain lengths have been found to be dysregulated in different cancer types [13]. Ceramides have been shown to either mediate apoptosis via mitochondrial membrane perturbation or to exert their effects by influencing cell death signaling. Conversely, some reports have suggested that certain ceramide species may exert anti-apoptotic, protective roles [13]. The specific effect of a given species of ceramide towards cancer may depend on its molecular structure or the cellular context [31]. For example, C16 ceramide generation has been shown to be decreased in human ovarian carcinoma cells [32]. Whereas the overexpression of CERS2, which increases the generation of C24 ceramide, was shown to protect Hela cells from ionizing radiation (IR)-induced apoptosis [33]. Therefore, these reports may suggest that cancer cell survival may be due to a lack of C16 ceramide or a prevalence of C24 ceramide, or alternatively due entirely to the cancer cell type independent of the ceramide species. This later perspective may be further supported by a study that showed elevations of C14 ceramide, C16 ceramide, and C18 ceramide in breast cancer tissue as compared to normal tissue [34]. Suffice to say, while most roles attributed to ceramides are pro-stress or pro-death, there are instances where this traditional dogma does not apply for reasons that deserve further investigation. Notably though, various anticancer therapeutics have been shown to increase cellular levels of ceramides or alter the expression or activity of different CERS, potentially contributing to their anti-neoplastic activity (Table 2). Importantly, an increase or decrease in the expression or activity of a given CERS will not always manifest in an increase or decrease in the associated ceramide species alone. This is because those ceramide species may be converted to any number of ceramide metabolites, such as sphingomyelins or glycosphingolipids. Therefore, changes in CERS can potentially alter ceramide species or any of their metabolic derivatives.

Table 2.

Anticancer therapeutics that affect ceramide generation

Name of anticancer therapeutic	Mechanism of action	Effect on ceramide metabolism	Cancer type	Citation
Chemotherapy agents
Camptothecin	Inhibiting DNA synthesis	Increase de novo ceramide synthesis	Follicular thyroid carcinoma	Rath et al., 2009 [35]
Carboplatin	Interfering with DNA replication	Increase CERS1 expression	Embryonic kidney cells	Min et al., 2007 [36]
Cisplatin	Interfering with DNA replication	Increase CERS1, CERS6 expression	Embryonic kidney cells, Oral squamous carcinoma	Li et al., 2018 [37]; Min et al. 2007 [36]
Crenolanib	Tyrosine kinase inhibitor against FLT3, PDGFRα and PDGFRβ	Increase CERS1 expression	Acute myeloid leukemia	Dany et al. 2016 [38]
Dasatinib	Tyrosine kinase inhibitor against Bcr-Abl	Increase CERS1, CERS5, CERS6 expression	Chronic myeloid leukemia	Gencer et al., 2011 [39]
Daunorubicin	DNA intercalation	Increase CERS expression	Leukemia	Bose et al., 1995 [40]
Doxorubicin	DNA intercalation	Increase CERS1, CERS2, CERS5 expression	Embryonic kidney cells, Bladder cancer, Breast cancer	Fan et al., 2013 [41]; Huang et al., 2018 [42]; Min et al., 2007 [36]
Etoposide	Topoisomerase II inhibitor	Increase CERS2, CERS3, CERS4 expression	Embryonic fibroblasts	Siddique et al., 2012 [43]
Fludarabine	Purine analog; interferes with DNA synthesis	Increase CERS2 expression	Chronic lymphocytic leukemia	Biswal et al., 2000 [44]
Fluorouracil (5-FU)	Thymidylate synthase inhibitor, inhibiting DNA replication	Decrease CERS5 expression	Colon cancer	Brachtendorf et al., 2018 [45]
Gemcitabine	Inhibiting DNA synthesis	Increase CERS2 expression	Pancreatic cancer	Modrak et al., 2009 [46]
Imatinib	Tyrosine kinase inhibitor against Bcr-Abl	Increase CERS1 expression	Chronic myeloid leukemia	Baran et al., 2007 [47]
Irinotecan	Inhibiting DNA synthesis	Block formation of glucosyl-ceramide	Colon cancer	Litvak et al. 2003 [48]
Methotrexate	Inhibiting the synthesis of DNA, RNA, via inhibition of thymidylate	Increase CERS6 expression	Liver cancer, Lung cancer	Fekry et al., 2016 [49]
Oxaliplatin	Inhibiting DNA synthesis	Increase CERS5 expression	Colon cancer	Brachtendorf et al., 2018 [45]
Quizartinib	Tyrosine kinase inhibitor against FLT3/STK1, CSF1R/FMS, SCFR/KIT, and PDGFRs	Increase CERS1 expression	Acute myeloid leukemia	Dany et al. 2016 [38]
Sorafenib	Kinase inhibitor against VEGFR, PDGFR and Raf kinases.	Increase CERS1 expression	Acute myeloid leukemia	Dany et al. 2016 [38]
Vincristine	Inhibitor of mitosis at metaphase	Increase CERS1, CERS5 expression	Embryonic kidney cells	Min et al., 2007 [36]
Radiation
Ionizing radiation	Damaging DNA	Increase CERS2, CERS5, CERS6 expression	Cervical cancer, multiple cancer types	Mesicek et al., 2010 [33]
UV-B radiation	Damaging DNA	Increase CERS1 expression	Lung cancer	Sridevi et al., 2009 [50]
Alternative medicine
Staurosporine	Inhibitor of protein kinases against PKC	Increase CERS2, CERS6 expression	Glioma	Jensen et al., 2014 [51]
Hormone inhibitors
Anastrozole	Aromatase inhibitor, inhibits the production of estrogens	Decrease CERS4, CERS5 expression	Endometrial cancer	Mojakgomo et al., 2015 [52]
Tamoxifen	Selective estrogen receptor modulator	Inhibit ceramide glycosylation	Breast cancer	Wang et al., 2003 [53]
Adjuvant drugs
R(+)-meth anandamide	Synthetic long-lasting anandamide analog	Increase CERS6 expression	Mantle cell lymphoma	Gustafsson et al., 2009 [54]
Tetrahydro cannabinol (THC)	Partial agonist activity at cannabinoid receptors	Increase CERS5, CERS6 expression	Glioma	Hernández-Tiedra et al., 2016 [55]
Anti-inflammatory drugs
Celecoxib	COX-2 inhibitor	Increase CERS6 expression	Colon cancer	Schiffmann et al., 2010 [56]
Clinical trial drugs
Cladososide C2	Inducing apoptosis	Increase CERS6 expression	Leukemia	Yun et al., 2015 [57]
Fenretinide	Inducing apoptosis	Activate serine palmitoyl transferase	Prostate cancer, relapsed and refractory T-cell lymphoma, ovarian cancer, neuroblastoma, head and neck squamous cell carcinoma, recurrent small cell lung cancer, kidney cancer, breast cancer	Wang et al., 2003 [53]; Clinicaltrials.gov identifiers: NCT04234048, NCT00026091, NCT00646230, NCT00006471, NCT00009971, NCT00011973, NCT00003099
IL-24	Inducing apoptosis and angiogenesis	Increase CERS6 expression	Glioblastoma	Yacoub et al., 2010 [58]
Resveratrol	Not yet clear	Inhibition of dihydroceramide desaturase	Gastric cancer	Signorelli et al., 2009 [59]
Stichoposide D	Inducing apoptosis	Increase CERS6 expression	Leukemia	Yun et al., 2015 [57]
TRAIL(Apo-2L)	Inducing TNF-related apoptosis	Increase CERS6 expression	Colon cancer	White-Gilbertson et al., 2009 [60]

Targeting ceramide metabolism for cancer therapy

Considering the known effects of ceramide and its dysregulation in many cancers, developing ceramide-based therapies for cancer is very attractive. Various therapies have aimed to target ceramide metabolism as a method to promote apoptosis in cancer cells (Table 3). These therapies upregulate endogenous production of ceramide or downregulate ceramide neutralization pathways. Enzymes that neutralize ceramide convert ceramide to non-ceramide sphingolipids. Examples include GCS, SGMS, CDases, and SPHKs (Figure 1). The goal of these therapies is to raise the intracellular concentration of ceramide to direct cancer cells towards apoptosis. In addition to augmenting endogenous ceramide synthesis via pharmacological interventions, increasing ceramide levels through delivery of exogenous analogs has been explored in various cancer models. Various analogs or inhibitors of ceramide signaling have also shown efficacy in clinical trials. Targeting ceramide is also not the only sphingolipid-based strategy that is being explored for their potential to treat cancer. For example, fingolimod (FTY720 or Gilyena^™), a structural analog of sphingosine (or S1P once phosphorylated) and regulator of S1PR signaling, has been approved for clinical use for the treatment of multiple sclerosis and has also shown potential anticancer efficacy in various preclinical models [61]. Mechanistically, fingolimod suppresses tumor growth via S1PR-dependent or receptor-independent mechanisms in colon and lung cancer, as well as acute myeloid leukemia, chronic lymphocytic and large granular lymphocytic leukemia [62–67]. Most of these effects are thought to be dependent on its activity towards S1PR1, but antagonistic effects towards other S1PRs such as S1PR5 have been reported in preclinical studies of its use to treat large granular lymphocytic leukemia [64]. Fingolimod has also shown potential to treat imatinib-refractory chronic myeloid leukemia by inhibiting stem cell proliferation and expansion in vitro [68].

Table 3.

Synthetic drugs that target sphingolipid metabolism or signaling for cancer

Synthetic drug	Mechanism of action	Cancer type effected	Citation
AB1	S1PR2 antagonist	Neuroblastoma	Li et al., 2015 [69]
ABC294640	Inhibitor of sphingosine kinase 2	Pancreatic cancer, prostate cancer, multiple myeloma, lymphoma	Britten et al., 2017 [70] ; Lewis et al., 2016 [71] ; Qin et al., 2014 [72] ; Venant et al., 2015 [73]; Venkata et al., 2014 [74]
Analog 315 and 403	Ceramide analog	Lymphoma, breast cancer	Chen et al., 2020 [75]
Analog 406	C8-Ceramide analog	Breast cancer, ovarian cancer	Ponnapakam et al., 2014 [76]
C16-serinol	C16-Ceramide analog	Neuroblastoma	Bieberich et al., 2000 [77]
Ceranib-2	Inhibitor of acid ceramidase	Breast cancer	Draper et al., 2011 [78]
D-MAPP, B13	Inhibitors of acid ceramidase	Colon cancer	Selzner et al., 2001 [79]
Fingolimod (FTY720 or Gilenya™)	S1PR antagonist (predominantly S1PR1)	Colon cancer, lung cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, large granular lymphocytic leukemia	Chen et al., 2014 [62]; Liang et al., 2013 [63]; Liao et al., 2011 [64]; Liu et al., 2008 [65]; Neviani et al., 2013 [68]; Saddoughi et al., 2013 [66]; Young et al., 2019 [67]
LCL29	C6-Ceramide analog	Head and neck squamous cell carcinoma	Separovic et al., 2011 [80]
LCL30	C16-Ceramide analog	Head and neck squamous cell carcinoma	Separovic et al., 2011 [80]
LCL85	C16-Ceramide analog	Colon cancer, breast cancer	Paschall et al., 2014 [81]
LCL124	C6-Ceramide analog	Pancreatic cancer	Beckham et al., 2013 [82]
LCL204	Inhibitor of acid ceramidase	Head and neck squamous cell carcinoma, acute myeloid leukemia	Elojeimy et al., 2007 [83] ; Tan et al., 2016 [84]
LCL385	Inhibitor of acid ceramidase	Prostate tumors	Mahdy et al., 2009 [85]
LCL521	Inhibitor of acid ceramidase	Head and neck squamous cell carcinoma	Korbelik et al., 2016 [86]
MP-A08	Inhibitor of sphingosine kinase 1	Acute myeloid leukemia	Powell et al., 2017 [87]
PF-543	Inhibitor of S1P	Head and neck squamous cell carcinoma, colon cancer	Ju et al., 2016 [88]; Schnute et al., 2016 [89]
SACLAC	Inhibitor of acid ceramidase	Acute myeloid leukemia	Pearson et al., 2020 [90]
SK1-I	Inhibitor of sphingosine kinase 1	Glioblastoma	Kapitonov et al., 2009 [91]
SKI-178	Inhibitor of sphingosine kinase 1	Acute myeloid leukemia	Hengst et al., 2017 [92]
Sonepcizumab	S1P monoclonal antibody	Renal cell carcinoma	Pal et al., 2017 [93]
VPC03090	S1PR1/3 antagonist	Breast cancer	Kennedy et al., 2011 [94]

Nanotechnology for ceramide delivery

One of the issues with ceramide-based therapies is the lipid’s delivery in vivo. Since ceramide is highly hydrophobic, a suitable carrier is needed for its successful delivery without restricting its pharmacological effects [95]. Recent advances in nanotechnology have helped to develop novel drug delivery platforms that overcome many typical pharmacological limitations. Nanoparticles, measuring up to 400 nm in size, have demonstrated efficacy for carrying and delivering therapeutic molecules with diverse physiological properties. Several delivery materials have been explored to increase ceramide-based drug performance (Table 4). Importantly, the use of nanoparticles may allow for selective targeting of tumors due to the enhanced permeation and retention (EPR) effect [95,96]. The EPR effect is due to the propensity of tumor vasculature to be leaky. This is largely due to the disorganized manner that tumor-mediated neovascularization occurs. Gaps between vascular endothelium provide potential for passive targeting of anticancer nanotherapies to tumors. Nanoparticles are small enough to leave the vasculature at these points of disorganization and leakiness. In theory, this facet of tumors allows for passive targeting of cancerous tissues by nanomedicines [95,96]. A particularly optimistic aspect for the use of nanotechnology in ceramide delivery is the potential for non-toxic therapy. One of the largest issues confronting cancer therapeutics is the toxic nature of many of these drugs. Unlike many standard of care therapeutics, the C6-ceramide nanoliposome has shown promising results in terms of toxicity. In dog and murine models, the C6-ceramide nanoliposome was minimally toxic [95]. More recently, the ceramide nanoliposome completed a phase I clinical trial with solid tumor patients [97]. Importantly, despite evaluating a dose range from 36 mg/m² to 323 mg/m², there were no dose-limiting toxicities observed and a maximum tolerated dose was not reached [97]. For this trial, the starting dose of 36 mg/m² was chosen based on prior canine studies yet the human trial rapidly increased beyond this dose when no dose-limiting toxicities were observed [97]. Beyond nanoliposomes, other nanotechnologies may likewise be able to effective deliver ceramide to cancer cells. Unfortunately, most nanoparticles are composed of highly toxic materials [92]. One exception, beyond nanoliposomes, is the calcium phosphosilicate nanoparticle. This platform offers many of the same advantages as nanoliposomes in that it can delivery both hydrophobic and lipophilic agents including ceramide [96, 98]. Intriguingly, calcium phosphosilicate nanoparticles can deliver ceramide of varying chain lengths, is composed of non-toxic materials, and offers controlled release inside target cells [96, 98]. Overall, the use of nanotechnology in drug delivery may lead to a new generation of cancer therapeutics, one in which toxicity is not the norm for standard of care therapies.

Table 4.

Nanotechnology for ceramide delivery

Delivery material	Advantages	Citation
C6-ceramide nanoliposome	Overcomes the lack of solubility for lipophilic compounds (drugs) in physiological solutions, high ceramide encapsulation efficiency, stealth characteristics, can be actively targeted, ability to co-encapsulate/deliver hydrophobic and lipophilic drugs, increased systemic circulation, reduced toxicity Clinicaltrials.gov identifier: NCT02834611	Ciner et al., 2024 [97]; Kester et al., 2015 [95]; Stover and Kester, 2003 [99]
Ceramide-loaded calcium phosphosilicate nanoparticle	Encapsulates a wide range of compounds (hydrophobic and lipophilic), fully encapsulates active agents, can co-deliver multiple agents including imaging agents, stable at physiological pH for protected delivery, controlled release inside cells at acidic pH, stealth characteristics, can be actively targeted, increased systemic circulation, and reduced toxicity	Adair et al., 2010 [96]; Kester et al., 2008 [98]
Chitosan-ceramide graft copolymer	Sustained release, higher cellular uptake, improved stability and reduced toxicity under physiological conditions	Battogtokh and Ko, 2014a [100]; Battogtokh and Ko, 2014b [101]
Poly (D,L-lactide-co-glycolide) PLGA	Favorable anticancer drug delivery performance but insufficient tumor targeting	Graf et al., 2012 [102]
Solid lipid nanoparticles	Large surface area and high loading content for hydrophobic drugs, improved stability	Balakrishnan et al., 2016 [103]

Combined usage of ceramide and other drugs in the treatment of cancer

With the assistance of nanotechnology, exogenous ceramide delivery is being explored as a therapeutic strategy for various cancers. C6-ceramide has been found to prevent metastasis and recurrence of anaplastic thyroid carcinoma cell lines [104]. Moreover, the C6-ceramide nanoliposome slows the growth of liver tumors in mice by boosting T-cell activity [105]. In addition to being administered alone, exogenous ceramide can also be incorporated with other drugs as innovative cancer treatments [106]. The nanoliposome has potential as a delivery mechanism for combinatorial therapies because ceramide can be easily distributed into the hydrophobic membrane of nanoliposomes, alongside other lipophilic drugs, while hydrophilic therapies can be loaded into the nanoliposomal aqueous interior [95]. As studies demonstrate the ability of nano-formulated ceramide to improve the efficacy of other drugs, ceramide will likely gain more clinical interest as a combinatorial therapeutic than as a monotherapy (Table 5) [95].

Table 5.

Combination of ceramide and other drugs/agents in the treatment of cancer

Combination usage	Cancer type	Citation
7,8-Benzoflavone nanoliposome + C6-ceramide nanoliposome	Acute myeloid leukemia	Barth et al., 2014a [107]
Curcumin + C6-ceramide	Melanoma	Yu et al., 2010 [108]
Curcumin + C6-ceramide	Osteosarcoma	Dhule et al., 2014 [109]
Doxorubicin + C6-ceramide	Breast cancer, melanoma	Fonseca et al., 2014 [110]
Gemcitabine + C6-ceramide nanoliposome	Pancreatic cancer	Jiang et al., 2011 [111]
Ibrutinib + C6-ceramide nanoliposome	Chronic lymphocytic leukemia	Doshi et al., 2017 [112]
Myrisplatin + C6-ceramide	Ovarian cancer	Ganta et al., 2014 [113]
Paclitaxel + C6-ceramide	Melanoma	Carvalho et al., 2017 [114]
PDMP nanoliposome + C6-ceramide nanoliposome	Pancreatic cancer	Jiang et al., 2011 [111]
Safingol nanoliposome + C2-ceramide nanoliposome	Acute myeloid leukemia	Tan et al., 2014 [115]
Safingol nanoliposome + C6-ceramide nanoliposome	Acute myeloid leukemia	Brown et al., 2013 [116]
Sorafenib + C6-ceramide nanoliposome	Breast cancer, melanoma	Tran et al., 2008 [117]
Tamoxifen nanoliposome + C6-ceramide nanoliposome	Acute myeloid leukemia	Barth et al., 2014b [118]; Morad et al., 2016 [119]
Venetoclax + cytarabine + C6-ceramide nanoliposome	Acute myeloid leukemia	Khokhiatchev et al., 2022 [120]
Vinblastine nanoliposome + C6-ceramide nanoliposome	Hepatocellular carcinoma, colon cancer	Adiseshaiah et al., 2013 [121]
Vinblastine (free drug or nanoliposome) + C6-ceramide nanoliposome	Acute myeloid leukemia	Barth et al., 2019 [12]

Conclusions

Sphingolipids, as with other lipids, have been classically appreciated as being structural elements of cell membranes. While this is a truism, it is not the whole picture. Biologically active lipids play vital roles in the development and prevention of diseases. This is highlighted through an examination of ceramide, its metabolic pathways, as well as its demonstrative physiological effects. Ceramide plays large roles in many disease processes, including cancer. Many cancers experience a downregulation of ceramide and the enzymes responsible for its biosynthesis. This is because the presence of large quantities of endogenous ceramide is often associated with the induction of apoptosis. However, there have been contrasting findings that have challenged the dogma that ceramide exclusively promotes stress and apoptosis. For instance, by ascribing anti-apoptotic roles to specific ceramide species [4]. Moreover, the field’s perspective on the deleterious effects ascribed to certain ceramide species in cancer has evolved as elevations of various ceramide species have been observed in malignant cells and tissues [12,34]. Notably, in many lipidomic studies C16:0, C18:0, and C24:1 ceramide are the predominant ceramide species detected and so variations in these are frequently characterized. However, in many cases the change in these ceramide species can be identical and as such can sometimes be associated with similar biological effects. It is important though to recall that similar increases in multiple ceramide species may occur due to engagement of sphingomyelinases rather than an upregulation of de novo ceramide biosynthesis as the sphingomyelinases do not seem to be chain length specific. This highlights that the types of changes to endogenous ceramide levels may be driven by situationally unique molecular and cellular pathways. Furthermore, rather than changes to unique ceramide species there is emerging evidence that alterations in ratios of long-chain to very long-chain ceramide species may be more relevant to disease pathology [122]. This further underscores the cellular and molecular complexity associated with alterations to ceramide metabolism and its pathological or therapeutic relevance.

Interestingly a related sphingolipid, S1P, is highly bioactive but acts to achieve the opposite physiological effect of ceramide, promoting cell division and survival. More specifically, ceramide has antitumor therapeutic potential through its role in the stimulation of both mitochondrial mediated and death-receptor mediated apoptosis, as well as other mechanisms. The question of how to exploit the effect of endogenous ceramides is yet to be definitively answered. Some drugs, as mentioned, have aimed to control the metabolism of ceramide. The result of these therapies is an increased abundance of intracellular ceramide. They do this primarily by downregulating ceramide neutralization enzymes, which are responsible for the conversion of ceramide to non-ceramide sphingolipids. Within this category of therapeutics, therapies that aim to downregulate the activity of S1P, such as the numerous acid CDase and SPHK inhibitors or the anti-S1P monoclonal antibody Sonepcizumab, can be considered as they act to manipulate sphingolipid metabolism in an anticancer fashion. As S1P’s activity yields an opposite physiological effect of ceramide, the antagonism of S1PRs may also present therapeutic potential. However, the generation of S1P is not the sole metabolic outlet that malignant cells can use to neutralize ceramide. Moreover, the diversity of ceramide metabolic outlets may present a unique challenge to ceramide-based anticancer therapy due to a range of adaptive resistance that could develop [12,123].

Aside from pharmacological manipulations of sphingolipid metabolism, other approaches instead focus on introducing exogenous ceramide to cancer tissues. Although the goal of ceramide elevation is shared, these are distinct as delivery systems. In many ways, this is a more direct strategy to raise cellular levels of ceramide. However, there are issues that need to be solved before these ceramide-delivering therapies can be brought to the bedside. For instance, some therapies have had difficulty effectively targeting and delivering exogenous ceramide to cancer cells. The development of delivery systems such as the ceramide nanoliposome and the ceramide-loaded calcium phosphosilicate nanoparticle provide optimism for the next generation of cancer therapeutics as this platform is passively targeted by EPR, could be actively targeted, but importantly can encapsulate and protect ceramide during systemic delivery to exert limited off-target toxicity and maximal anticancer efficacy [95–98]. Most notably, the ceramide nanoliposome has completed a phase I clinical trial with solid tumor patients and no dose-limiting toxicities were observed despite evaluating an incredibly high dose of 323 mg/m² [97]. Although a promising field, ceramide-based therapies are continuing to improve and may eventually be best suited as combinatorial therapies alongside standard of care therapies. Improvements to these delivery systems will further reduce off-target toxicity, improve targeting, and co-load therapeutics, as to improve overall therapeutic efficacy. It is hoped that in time these novel ceramide-based therapies will efficiently treat a variety of malignancies.