Sphingolipid Metabolism

Sphingolipids are the major structural components of the eukaryotic cell membranes with members including ceramide (Cer), sphingosine (Sph), sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P) and glucosylceramide (GC). In addition to their roles in the membranes, they have been found to regulate various important cellular functions such as cell growth, differentiation, senescence, apoptosis and inflammation by regulating intracellular signaling pathways.

Sphingolipid metabolism (anabolism/catabolism) is a complex pathway in which different enzymes subjected to strict regulation are involved. All pathways involved are connected to each other and Cer is the central molecule in both anabolic and catabolic reactions. Ceramide is synthesized by two main pathways which are de novo pathway and hydrolysis of complex lipids such as sphingomyelin (SM).

The de novo pathway begins with the condensation of serine and palmitoyl-CoA to produce 3-keto-dihydrosphingosine, which is catalyzed by serine palmitoyl transferase (SPT). The production of sphingolipids starts at endoplasmic reticulum (ER) where heterodimeric phosphate-bound SPT localized. In mammals, SPT consists of two large subunits (SPTLC1 and SPTLC2).

3-keto-dihydrosphingosine is reduced to form dihydrosphingosine (sphinganine) which is converted to dihodroceramide (dhCer) and Cer by Cer synthases (CerS) via N-acetylation reaction. Six Cer synthase genes (CerS1-6) have been identified to synthesize Cers with different chain lengths. The resulting Cer is primarily used for the synthesis of SM by adding phosphocholine headgroup from phosphatidylcholine by SM synthases with the generation of diacylglycerol (DAG).

Ceramide can be also phosphorylated to C1P by ceramide kinase (CK) and converted to glucosyl or galactosylceramide. In the hydrolytic pathway of Cer synthesis, SM is cleaved by sphingomyelinases (SMases) to produce phosphocholine and Cer. Ceramide can be also released through the hydrolysis of glycosphingolipids, glucosylceramide or galactosylceramide by the function specific beta-glucosidases and galactosidases such as glucosyl ceramide synthase (GCS). GCS transfers glucose molecules to ceramide to produce glucosylceramide (GC). This enzyme is found in golgi apparatus in which ceramide is glycosylated and transported by ceramide transport proteins.

Cer is metabolized to Sph by ceramidases (CDases) and Sph can be recycled to synthesize Cer by CerS or S1P by sphingosine kinases (SK). SKs have two isoforms called SK-1 and SK-2 which are encoded by SPHKL1 and SPHKL2, respectively. SK-1 and SK-2 have similarity in their protein sequences except for Ser225 phosphorylation site which is conserved and required for SK-1 activation [79]. SK-1 is located in cytoplasm under normal conditions.

However, the presence of growth factors and cytokines might alter its localization from cytoplasm to plasma membrane. SK-2 is normally located in the nucleus and cytoplasm. However, its location can be changed to ER during cellular responses where S1P phosphatases is located. S1P is available to regenerated Sph by S1P phospahateses.


Sphingolipid Metabolism in Cancer

Sphingolipid metabolism and the roles of sphingolipids have been extensively investigated in cancer. In particular, Cer, Sph and their phosphorylated forms affect many physiological and pathological conditions such as regulation of fever and sugar metabolism and cancer in the cell and they act as a secondary messenger to determine the cell fate.

The intracellular balance between sphingosine (or S1P) and ceramide is crucial for the cells to determine either they survive or die, which is called ‘’sphingolipid rheostat’’ [85]. If this balance is disrupted due to external factors towards ceramide, intrinsic or extrinsic apoptosis is activated. On the other hand, the conversion of Cer by CDases to Sph is associated with cell proliferation and division. Morever, S1P directly or indirectly by binding to G-protein coupled receptor (GPCRs) induces PI3K and PLC (Phospholipase C) pathways to induce cell proliferation and division. Therefore, Cer is considered as an apoptotic lipid while Sph and S1P act as antiapoptotic molecules.

In glioblastoma cell lines, the association between Cer and Fas-mediated extrinsic apoptosis was investigated and Cer was responsible for the downregulation of FLICE inhibitory protein (FLIP), negative regulator of Fas-FasL signaling. In a study, serum-levels of C16 ceramide and S1P have become a diagnostic marker for hepatocellular carcinoma. In the study performed in glioblastomas, S1P was observed 9-fold higher and Cer was observed 5-fold lower compared to normal gray matter.

SK-1 has been upregulated in many cancers and SK-1 inhibition has reduced proliferation, angiogenesis and metastasis and increased apoptosis by using pharmacological inhibitors or genetic silencing. S1P and sphingolipid pathway played an important role in the pathogenesis and resistance of ovarian cancer. In addition, the conversion of ceramide to S1P, GC and SM in ovarian cancer has a mitogenic effect and inhibits apoptotic pathway.

In a study conducted in hepatocellular carcinoma, melatonin increased the amount of ceramide by regulating ceramide synthesis pathways and inhibition of SPT with myriocin inhibited melanin-related autophagy.

SK-1 overexpression resulting in increased S1P levels inhibited apoptosis in NIH3T3 fibroblasts and HEK293 kidney cells. Similarly, overexpression of SK-1 and S1P production has been proven to cause cell proliferation in many cancer types. SK/S1P/S1PR pathway modulates pro-survival cellular responses via autocrine and paracrine manner by activating GPCR family S1P receptor 1-5 (S1PR1-5). S1P inhibited intrinsic apoptotic pathway activation by inhibiting cytochrome c and Smac /DIABLO release from mitochondria in AML cells. In non-small cell lung cancer, S1P was found to activate the oncogenic signal by activating PI3K.

It was determined that MOLT-4 T-ALL cells were arrested at the G0 /G1 phase due to the accumulation of ceramide produced by SM hydrolysis after exposure to serum starvation. In neuroblastoma cells, dihydroceramide arrested the cell cycle progression at G0/G1. In a study, ceramide arrested G1/S transition by dephosphorylating p21 and Rb through p53 dependent and independent manner. In addition, several studies have shown that ceramide affects autophagy by regulating autophagy related players. For instance, melatonin increased ceramide levels via de novo and salvage patyway which led to autophagy related cell death in hepatocarcinoma cells. In this study, SPT inhibition prevented autophagy while SPT inhibition induced cell death.

Ceramide caused cell cycle arrest by dephosphorylating Rb gene, activating p21 inhibitor, and inhibiting cyclin dependent kinase 2 (CDK2) in breast cancer. S1P has been found to have an important role in cell migration and matrix metalloproteinase-9 expression, also induce Epithelial-Mesenchymal Transition (EMT) in breast cancer. In another study, S1P and S1P receptors were found to be positive regulators of angiogenesis and metastasis in breast cancer cells. In human glioblastoma cells, S1P initiated metastasis by secreting matrix metalloproteinase to degrade extracellular matrix. Ceramide increased sensitivity of chemoresistant breast cancer cells to chemotherapy.

Abnormal GCS expression in cancer is associated with prognosis. Inhibition of GCS, either molecularly or pharmacologically, eliminated resistance to chemotherapy. For instance, upregulated MDR1 expression is associated with overexpressed GCS in breast, ovary, cervical and colon cancer cells. Targeting GCS by genetically reversed drug resistant these cancer cells to doxorubicin.

Effect of Sphingolipid Metabolism in Leukemia

The effect of sphingolipid metabolism in leukemia has been investigated intensively as compared to solid tumors. In T-ALL cells, dihydroceramides increased retinoid-induced cytotoxicity [111] and inhibition of sphingomyelin synthase (SMS) increased the amount of Fas-associated ceramide and triggered caspase-9 activation in human Jurkat leukemia cells. SMS and glycosyl ceramide synthase (GCS) activities have made AML and CML patients resistant to chemotherapy by decreasing ceramide levels and increasing leukemic blasts.

Thus, inhibition of SMS or GCS may be a therapeutic approach in chemoresistant hematological malignancy. It was found that modulation of pro-apoptotic and pro-survival sphingolipids could contribute to overcome chemoresistance in HL-60 leukemia cells. Inhibiting GCS and SK-1 increased sensitivity resistant CML cells to nilotinib and resulted in cell death. The treatment of U937 leukemia cells with Bcl-2 family inhibitors and GCS inhibitor PDMP led to synergistic effect on cell death and PDMP treated imatinib resistant CML cells underwent cell death.

Disruption of sphingolipid rheostat toward S1P by SK-1 overexpression made K-562 cells imatinib resistant. However, suppression of SK-1 expression increased sensitivity to imatinib. In chemosensitive HL-60 cells, doxorubicin and etiposide treatment caused SK-1 inhibition and Cer accumulation. On the other hand, in doxorubin and etiposide resistant HL-60 cells, SK-1 activated and Cer levels decreased, which inhibited apoptosis through the prevention of cytochrome c release from mitochondria.

Interleukin-6 (IL-6) activated SK in human multiple myeloma cells resulted in upregulation of Mcl-1 which promotes cell proliferation and survival. SKI-II, SK-1 inhibitor, inhibited the cell growth and caused apoptosis in U937 and HL-60 AML cells by increasing intracellular ceramide level. The results of this study suggest that SKI-II may be a novel therapeutic agent in AML cells.

Tamoxifen and its metabolite caused cell death by blocking ceramide glycosylation, ceramide hydrolysis and SK1 activity in AML cell lines and AML patient samples. In Acute lymphoblastic leukemia, SK-2 has been shown to play an oncogenic role and modulates the regulation of the MYC oncogene. In the mouse model of ALL, SK-2 has caused the development of leukemia. However, the inhibition of SK-2 pharmacologically prolonged the survival of mouse.


Targeting Sphingolipid Metabolism

Sphingolipids have prominent roles for the determination of the cell fate and differences in expression levels of anti-apoptotic and pro-apoptotic lipids have been observed in many cancer cells. Dysregulations in sphingolipid metabolism might cause drug resistance. Thus, targeting sphingolipid metabolism has been paid attention in cancer therapy. Different approaches can be used to target sphingolipid metabolism including using synthetic ceramide analogs and small molecule inhibitors which increase ceramide anabolism and prevent its conversion into antiapoptotic sphingolipid specimens.

For instance, tumor promoting S1P effect can be eliminated by using SK inhibitors or by inactivating S1P receptor. Moreover, additional approaches might be used to reactivate some genes such as SMase and S1P phosphatase that are suppressed in cancer cells. The combination strategies including sphingolipid metabolism inhibitors and conventional cytotoxic chemotherapeutic agents to increase ceramide production have been studied in cancer.

Tamoxifen and sphingosine analog (FTY720) combination synergistically triggered apoptotic cell death as compared to each agent alone in drug-resistant ovarian cancer.Using SPT inhibitor (myriocin) and SK inhibitors reduced tumor volume in merkel cell carcinoma. Vincristine resistant HL-60 AML cells underwent apoptosis after treatment with P-glycoprotein inhibitors and C6-ceramide analog. Apoptotic event was associated with cytochrome c release and mitochondrial ROS production .

Acid ceramidases can be a target due to their contribution to metastasis and chemotherapy resistance. Therefore, targeting acid ceramidases by synthetic inhibitors may be a promising therapeutic strategy. For instance, inhibition of acid ceramidases increased ceramide levels and decreased S1P. Terefore, this strategy prevented cell proliferation in melanoma cells as compared to normal skin cells.

Targeting GCS as pharmacologically or genetically is another strategy to induce cell death or overcome drug resistance. In sorafenib resistant hepatoma cells, GSC inhibition increased the sensitivity to sorafenib. In cervical carcinoma cells using SK-2 inhibitor (ABC294640), apoptosis and cell cycle arrest in G1/S phase was induced. Moreover, SK-1 inhibition with the novel inibitors induced apoptosis in breast and prostate cancer cells.

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