Changes In Ceramide Levels Research Articles

The role of ceranib-2 and its nanoform on the decrease of telomerase levels in human non-small cell cancer.

Ceranib-2, an acid ceramidase (AC) inhibitor, can inhibit cancer cell proliferation and tumor development. However, poor water solubility and low cellular bioavailability limit its efficacy in cancer treatment. This study aimed to investigate the cell death induced by ceranib-2 and its solid lipid nanoformulation (ceranib-2-SLN) produced by the hot homogenization technique and the synergistic relationship between ceramide and telomerase in vitro and in silico. Furthermore, this study proved the possible mechanism of ceranib-2-induced AC inhibition by in silico studies. The effective cytotoxic concentrations of ceranib-2, telomerase level, and changes in ceramide levels were measured by MTT colorimetric cytotoxicity assay, ELISA, and LC/MS/MS methods, respectively. TEM results showed that ceranib-2-SLN was 13-fold smaller than the size of ceranib-2. Ceranib-2 and ceranib-2-SLN had IC50 concentrations of 31.62 (± 2.1) and 27.69 (± 1.75) µM in A549, and 48.79 (± 1.56) and 67.98 (± 2.33) in Beas-2B cells. These compounds simultaneously increased ceramide levels and decreased telomerase levels in A549 cells. Ceranib-2 increased telomerase levels while decreasing ceramide levels in Beas-2B cells. It was shown how the synergistic impact of ceranib-2-induced ceramide production and ceramide-induced telomerase level reduction on cytotoxicity in A549 cells. Ceranib-2-SLN was discovered to be more cytotoxic on cancer cells than ceranib-2, suggesting that it could be a promising option for the development of a new anti-cancer agent.

A review of the mechanisms of abnormal ceramide metabolism in type 2 diabetes mellitus, Alzheimer's disease, and their co-morbidities.

The global prevalence of type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD) is rapidly increasing, revealing a strong association between these two diseases. Currently, there are no curative medication available for the comorbidity of T2DM and AD. Ceramides are structural components of cell membrane lipids and act as signal molecules regulating cell homeostasis. Their synthesis and degradation play crucial roles in maintaining metabolic balance in vivo, serving as important mediators in the development of neurodegenerative and metabolic disorders. Abnormal ceramide metabolism disrupts intracellular signaling, induces oxidative stress, activates inflammatory factors, and impacts glucose and lipid homeostasis in metabolism-related tissues like the liver, skeletal muscle, and adipose tissue, driving the occurrence and progression of T2DM. The connection between changes in ceramide levels in the brain, amyloid β accumulation, and tau hyper-phosphorylation is evident. Additionally, ceramide regulates cell survival and apoptosis through related signaling pathways, actively participating in the occurrence and progression of AD. Regulatory enzymes, their metabolites, and signaling pathways impact core pathological molecular mechanisms shared by T2DM and AD, such as insulin resistance and inflammatory response. Consequently, regulating ceramide metabolism may become a potential therapeutic target and intervention for the comorbidity of T2DM and AD. The paper comprehensively summarizes and discusses the role of ceramide and its metabolites in the pathogenesis of T2DM and AD, as well as the latest progress in the treatment of T2DM with AD.

A simple, highly sensitive, and facile method to quantify ceramide at the plasma membrane

The role of ceramide in biological functions is typically based on the elevation of cellular ceramide, measured by LC-MS in the total cell lysate. However, it has become increasingly appreciated that ceramide in different subcellular organelles regulates specific functions. In the plasma membrane, changes in ceramide levels might represent a small percentage of the total cellular ceramide, evading MS detection but playing a critical role in cell signaling. Importantly, there are currently no efficient techniques to quantify ceramide in the plasma membrane. Here, we developed a method to measure the mass of ceramide in the plasma membrane using a short protocol that is based on the hydrolysis of plasma membrane ceramide into sphingosine by the action of exogenously applied bacterial recombinant neutral ceramidase. Plasma membrane ceramide content can then be determined by measuring the newly generated sphingosine at a stoichiometry of 1:1. A key step of this protocol is the chemical fixation of cells to block cellular sphingolipid metabolism, especially of sphingosine to sphingosine 1-phosphate. We confirmed that chemical fixation does not disrupt the lipid composition at the plasma membrane, which remains intact during the time of the assay. We illustrate the power of the approach by applying this protocol to interrogate the effects of the chemotherapeutic compound doxorubicin. Here we distinguished two pools of ceramide, depending on the doxorubicin concentration, consolidating different reports. In summary, we have developed the first approach to quantify ceramide in the plasma membrane, allowing the study of new avenues in sphingolipid compartmentalization and function.

Exercise Alters Ceramide Metabolism in Melanoma

IntroductionWe previously demonstrated that exercise enhanced the efficacy of chemotherapy against B16F10 melanoma in mice. Exercise likely impacts both the tumor microenvironment and the tumor cells. Exercise has been demonstrated to modulate ceramide metabolism in healthy tissue, but the relationship between exercise and ceramide in tumors has not been evaluated. Ceramides are essential sphingolipid metabolites involved in cell death and survival pathways. Low content of ceramides in melanoma is a potential mechanism for drug resistance. Thus, increasing ceramides content in tumor cells is likely to increase sensitivity to chemotherapy. We hypothesized that exercise would increase accumulation of ceramide in tumor by regulating ceramide metabolism.MethodsC57BL/6 male mice were subcutaneously injected with B16F10 or BP melanoma cells and randomly assigned into sedentary control (No Ex) and exercise (Ex) groups. After tumor establishment, Ex mice performed treadmill running at 12 meter/min for 45 min for 5 consecutive exercise days with two days off then 5 additional exercise days. Ceramides were measured in tumor homogenates by liquid chromatography‐tandem mass spectrometry (LC/MS). Ceramide metabolizing enzymes were analyzed in tumors by immunofluorescence staining and western blotting. Cultured B16F10 or BP cells were treated with serum collected from corresponding melanoma tumor bearing mouse to examine effect of circulating factors on ceramide regulating enzymes.ResultsSeveral ceramide species were increased in Ex compared to No Ex B16F10 tumors, with the largest exercise‐induced increases in C16‐, dihydro‐C16‐, and C20‐ ceramide. Significantly increased expression of ceramide synthase 6 (CerS6), which generates C16‐ceramide using sphinganine or sphingosine, was observed in Ex B16F10 tumors by immunofluorescence staining and western blotting. Surprisingly, there was a trend toward an increase in the ceramide degrading enzyme acid ceramidase (aCDase) in Ex tumors. This may suggest a feedback loop. In contrast, Ex significantly suppressed expression of sphingosine kinase 1 (Sphk1), which converts sphingosine to the key survival sphingolipid S1P, in B16F10 tumors. Likewise, Sphk1 protein expression decreased in B16F10 cells treated in vitro with serum from Ex. Unlike the findings in B16F10 tumors and cultured B16F10 cells, no change in C16‐Ceramide, CerS6, or Sphk1 was observed in Ex BP tumors and in in vitro treatment of BP cells with Ex mice serum.DiscussionOur data suggests that exercise may contribute to B16F10 tumor cell death in response to chemotherapy by modulating ceramide metabolism toward a pro‐apoptotic ceramide/sphingosine‐1‐phosphate balance. However, these results were not consistent in BP tumors. This raises the important finding that exercise can have different effects between tumors of different patient or mouse origin, even with the same diagnosis (i.e. B16F10 vs BP melanoma models). Future work to determine the extent to which exercise regulates ceramide‐sphingolipid signaling in other melanoma tumor models, and in patients, is necessary. Future work will also determine whether exercise‐induced changes in ceramide levels are responsible for the previously reported exercise‐induced increased chemotherapy efficacy against melanoma tumors.

Endogenous levels of 1-O-acylceramides increase upon acidic ceramidase deficiency and decrease due to loss of Dgat1 in a tissue-dependent manner

Except for epidermis and liver, little is known about endogenous expression of 1-O-acylceramides (1-OACs) in mammalian tissue. Therefore, we screened several organs (brain, lung, liver, spleen, lymph nodes, heart, kidney, thymus, small intestine, and colon) from mice for the presence of 1-OACs by LC-MS2. In most organs, low levels of about 0.25–1.3 pmol 1-OACs/mg wet weight were recorded. Higher levels were detected in liver, small and large intestines, with about 4–13 pmol 1-OACs/mg wet weight. 1-OACs were esterified mainly with palmitic, stearic, or oleic acids. Esterification with saturated very long-chain fatty acids, as in epidermis, was not observed. Western-type diet induced 3-fold increased 1-OAC levels in mice livers while ceramides were unaltered. In a mouse model of Farber disease with a decrease of acid ceramidase activity, we observed a strong, up to 50-fold increase of 1-OACs in lung, thymus, and spleen. In contrast, 1-OAC levels were reduced 0.54-fold in liver. Only in lung 1-OAC levels correlated to changes in ceramide levels - indicating tissue-specific mechanisms of regulation. Glucosylceramide synthase deficiency in liver did not cause changes in 1-OAC or ceramide levels, whereas increased ceramide levels in glucosylceramide synthase-deficient small intestine caused an increase in 1-OAC levels. Deficiency of Dgat1 in mice resulted in a reduction of 1-OACs to 30% in colon, but not in small intestine and liver, going along with constant free ceramides levels. From these data, we conclude that Dgat1 as well as lysosomal lipid metabolism contribute in vivo to homeostatic 1-OAC levels in an organ-specific manner.

421 - Changes in Ceramide Levels in Various Diseases Are Associated with Oxidative Stress

Ceramide is a bioactive lipid that induces cellular apoptosis, proliferation, and differentiation. Deficiency of acid sphingomyelinase, which synthesizes ceramide, has been reported to result in smaller aortic root lesions in Western diet-fed Ldl -/- mice. Thus, ceramide is predicted to play a role in atherosclerosis pathogenesis. Therefore, using animal models, we investigated whether ceramide levels were affected by oxidative stress in diseases such as atherosclerosis, diabetes, and fulminant hepatitis. The experiments were performed under acute oxidative stress caused by carbon tetrachloride (CCl 4 ) intoxication and chronic oxidative stress in diabetic rats and in aged apolipoprotein E knock out (apoE -/- ) mice. Total ceramide concentrations significantly increased in the liver, brain, kidney, and plasma of CCl 4 -intoxicated rats. This increase was primarily derived from an increase in neutral sphingomyelinase activity in the liver 2 hours after intoxication with CCl 4 and was consistent with the decrease in the GSH/GSSG ratio in the liver. In contrast, secretory sphingomyelinase was stimulated during prolonged oxidative stress associated with diabetes and atherosclerosis. After 2 weeks of streptozotocin administration, plasma secretory sphingomyelinase activity was significantly increased, resulting in elevated ceramide concentrations, whereas neutral sphingomyelinase activity was unchanged in the kidney and liver. Plasma ceramide concentrations and secretory sphingomyelinase activity also increased in aged apoE -/- mice. In conclusion, ceramide levels increased in both acute and chronic oxidative stress-related diseases; however, the enzymes responsible for these changes differed. Based on these results, we propose that antioxidants can prevent ceramide production and ameliorate the exacerbation of oxidative stress-related diseases.

Quantitative analysis of ceramides using a novel lipidomics approach with three dimensional response modelling

In the outermost layer of the skin, the stratum corneum (SC), ceramides form a diverse and essential pool of lipids. Due to their diversity and the limited availability of synthetic standards it is challenging to quantitatively analyse all SC ceramides independently. We aim to perform a detailed analysis of ceramides on SC harvested from in vivo and ex vivo skin, therefore, a LC/MS method was developed in which all steps from sample acquisition until data analysis were examined and optimized. Improving extraction efficiency of ceramides resulted in an increase in efficiency from 71.5% to 99.3%. It was shown that sample harvesting by tape-stripping in vivo was accurate and precise. A full scan MS method was developed, compatible with all sample types, enabling simultaneously qualitative and quantitative data analysis. A novel three dimensional response model was constructed to quantify all detected ceramides from full scan data using a limited amount of synthetic ceramides. The application is demonstrated on various SC sample types. When ex vivo SC was regenerated during human skin culture, increases are observed in the amount of the ceramide sphingosine subclasses, in mono unsaturated ceramides (which have an cis-double bond in the acyl chain), and ceramides with a short C34 carbon chain (ceramides with a total carbon chain of 34 carbon atoms), compared with native human skin. These changes in ceramide levels are also often encountered in diseased skin.

Disruption of sphingolipid metabolism augments ceramide-induced autophagy in preeclampsia

Bioactive sphingolipids including ceramides are involved in a variety of pathophysiological processes by regulating cell death and survival. The objective of the current study was to examine ceramide metabolism in preeclampsia, a serious disorder of pregnancy characterized by oxidative stress, and increased trophoblast cell death and autophagy. Maternal circulating and placental ceramide levels quantified by tandem mass spectrometry were elevated in pregnancies complicated by preeclampsia. Placental ceramides were elevated due to greater de novo synthesis via high serine palmitoyltransferase activity and reduced lysosomal breakdown via diminished ASAH1 expression caused by TGFB3-induced E2F4 transcriptional repression. SMPD1 activity was reduced; hence, sphingomyelin degradation by SMPD1 did not contribute to elevated ceramide levels in preeclampsia. Oxidative stress triggered similar changes in ceramide levels and acid hydrolase expression in villous explants and trophoblast cells. MALDI-imaging mass spectrometry localized the ceramide increases to the trophophoblast layers and syncytial knots of placentae from pregnancies complicated by preeclampsia. ASAH1 inhibition or ceramide treatment induced autophagy in human trophoblast cells via a shift of the BOK-MCL1 rheostat toward prodeath BOK. Pharmacological inhibition of ASAH1 activity in pregnant mice resulted in increased placental ceramide content, abnormal placentation, reduced fetal growth, and increased autophagy via a similar shift in the BOK-MCL1 system. Our results reveal that oxidative stress-induced reduction of lysosomal hydrolase activities in combination with elevated de novo synthesis leads to ceramide overload, resulting in increased trophoblast cell autophagy, and typifies preeclampsia as a sphingolipid storage disorder.

Induction of a Hardening Phenomenon and Quantitative Changes of Ceramides in Stratum Corneum

BackgroundHardening phenomenon of human skin after repeated exposure to the irritants is well-known, but the precise mechanism remains elusive.ObjectiveTo modify the previous experimental model of hardening phenomenon by repeated applications of two different concentrations of sodium lauryl sulfate (SLS) solutions to Korean healthy volunteers and to investigate the quantitative changes of ceramides in stratum corneum before and after chronic repeated irritation.MethodsEight hundred microliters of distilled water containing 0.1% and 2% SLS was applied for 10 minutes on the forearm of 41 healthy volunteers for 3 weeks. After an intervening 3-week rest, 24-hour patch tests with 1% SLS were conducted on previously irritated sites. Transepidermal water loss (TEWL), erythema index and quantity of ceramide were measured in the stratum corneum before and after irritation.ResultsTEWL values on the sites preirritated with 2% SLS were lower than those with 0.1% SLS. Hardening phenomenon occurred in 24 volunteers at day 44. The changes in ceramide levels were not significantly higher in the hardened skin than in the non-hardened skin.ConclusionRepetitive stimulation with a higher concentration of SLS can more easily trigger skin hardening.

TGF-β Induces Microvascular Endothelial Cell Migration and PP-2A Inactivation (B65)

Abstract An increasing number of anti-tumor therapies have focused on angiogenesis pathways. Targeting various aspects of angiogenesis has been shown to disrupt tumor growth and reduce tumor progression and recurrence. Our studies show that treatment of microvascular endothelial cells with TGF-β leads to the reduction in activity of the protein phosphatase PP-2A, which results in increased motility. The mechanism by which TGF-β signaling results in PP-2A inactivation remains unknown. Our studies will show that neither change in mRNA nor protein levels of PP-2A subunits is associated with this decreased activity. However, TGF-β induced changes in ceramide levels may play a role in reduced activity. Furthermore, the role of TGF-β signaling on the protein inhibitors of PP-2A, I1PP2A and I2PP2A, will be demonstrated. The goal of these studies is to identify mechanisms by which TGF-β signaling induces PP-2A inactivation and, in turn, increased cellular motility in the tumor microenvironment. Elucidation of these pathways will provide novel cellular targets to interfere with tumor angiogenesis and metastasis.

Cyclooxygenase inhibitors block cell growth, increase ceramide and inhibit cell cycle.

We have shown previously in a model of metastatic breast cancer that murine mammary tumor cells express both cyclooxygenase-1 (Cox-1) and Cox-2 isoforms. Growth and metastasis of these tumors in syngeneic hosts are inhibited by either selective Cox-1 (SC560) or selective Cox-2 (celecoxib) inhibitors. To gain insight into the relevant mechanisms involved in the therapeutic response, we determined the effect of Cox inhibitors on tumor cell behavior in vitro. We now report that either selective Cox-1 or Cox-2 drugs inhibited cell replication, but only at concentrations that are no longer selective for either isoform. Growth delay by either nonselective or selective inhibitors was associated with changes in cell morphology including cell rounding; these changes were reversed upon removal of drug. Unlike many other cell types examined, treatment of these mammary tumor cells with Cox inhibitors was not associated with detectable apoptosis. Growth inhibition, induced by either selective or nonselective Cox inhibitors, was accompanied by increased intracellular levels of the sphingolipid ceramide by 1.7-2.6-fold in comparison to vehicle-treated cells. Ceramide changes are associated with cell cycle arrest and we observed that all the Cox inhibitors examined increased significantly the number of cells in G0/G1 and reduced the S phase fraction. Likewise, addition of a cell-permeable form of ceramide (C6-ceramide) could mimic the effect of Cox inhibitors on both cell cycle and cell growth inhibition. Thus, mammary tumor cells are growth restricted by Cox inhibitors. These effects are associated with changes in ceramide levels and a block in cell cycle progression.

De novo-synthesized ceramide is involved in cannabinoid-induced apoptosis

Delta(9)-Tetrahydrocannabinol (THC) and other cannabinoids have been shown to induce apoptosis of glioma cells via ceramide generation. In the present study, we investigated the metabolic origin of the ceramide responsible for this cannabinoid-induced apoptosis by using two subclones of C6 glioma cells: C6.9, which is sensitive to THC-induced apoptosis; and C6.4, which is resistant to THC-induced apoptosis. Pharmacological inhibition of ceramide synthesis de novo, but not of neutral and acid sphingomyelinases, prevented THC-induced apoptosis in C6.9 cells. The activity of serine palmitoyltransferase (SPT), which catalyses the rate-limiting step of ceramide synthesis de novo, was remarkably enhanced by THC in C6.9 cells, but not in C6.4 cells. However, no major changes in SPT mRNA and protein levels were evident. Changes in SPT activity paralleled changes in ceramide levels. Pharmacological inhibition of ceramide synthesis de novo also prevented the stimulation of extracellular-signal-regulated kinase and the inhibition of protein kinase B triggered by cannabinoids. These findings show that de novo-synthesized ceramide is involved in cannabinoid-induced apoptosis of glioma cells.

De novo-synthesized ceramide is involved in cannabinoid-induced apoptosis

Δ9-Tetrahydrocannabinol (THC) and other cannabinoids have been shown to induce apoptosis of glioma cells via ceramide generation. In the present study, we investigated the metabolic origin of the ceramide responsible for this cannabinoid-induced apoptosis by using two subclones of C6 glioma cells: C6.9, which is sensitive to THC-induced apoptosis; and C6.4, which is resistant to THC-induced apoptosis. Pharmacological inhibition of ceramide synthesis de novo, but not of neutral and acid sphingomyelinases, prevented THC-induced apoptosis in C6.9 cells. The activity of serine palmitoyltransferase (SPT), which catalyses the rate-limiting step of ceramide synthesis de novo, was remarkably enhanced by THC in C6.9 cells, but not in C6.4 cells. However, no major changes in SPT mRNA and protein levels were evident. Changes in SPT activity paralleled changes in ceramide levels. Pharmacological inhibition of ceramide synthesis de novo also prevented the stimulation of extracellular-signal-regulated kinase and the inhibition of protein kinase B triggered by cannabinoids. These findings show that de novo-synthesized ceramide is involved in cannabinoid-induced apoptosis of glioma cells.

Changes in ceramide levels upon catechins-induced apoptosis in LoVo cells

It has been demonstrated that there is difference in the induction of apoptosis in LoVo cells by (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), and (−)-epicatechin (EC). In this study, we explored changes in ceramide levels upon the three catechins-induced apoptosis in LoVo cells. Addition of C 2- and C 6-ceramide to LoVo cells mimicked EGCG or EGC in leading to apoptotic death. Further measurement of intracellular ceramide content showed that the treatment of LoVo cells with EGCG or EGC resulted in a rapidly transient increase in ceramide content, and then back gradually to base line level, whereas the action of EC was just opposite to that of EGCG or EGC. These results suggest there is difference in the generation of intracellular ceramide by the three catechins and ceramide may take part in the regulation of EGCG- or EGC-induced apoptosis in LoVo cells.

Increases in ceramide levels in normal human mesangial cells subjected to different cellular stresses result from changes in distinct enzyme activities and can influence cellular responses to other stimuli.

Sphingolipids, ceramide in particular, have come to be regarded as having roles in cellular signaling, most recently being associated with stress and the cellular responses to stress. In the present study we first examined the mechanisms involved in the changes in cellular ceramide levels in normal human mesangial cells (NHMC) in the growth, quiescent, and senescent phases as well as those resulting from environmental stimuli. We found that in NHMC total ceramide levels increase in response to cellular stresses as a result of a combination of enzyme activities. Furthermore, different stresses cause different alterations in various enzyme activities, with age and growth influencing acidic enzymes, but cell density affecting neutral, resulting in final ceramide level increases which most likely are associated with distinct pools of ceramide. Secondly, we examined the influence of changes in ceramide levels on apoptosis induced by sphingosine and its methylated derivative N, N-dimethylsphingosine. We found that increases in cellular ceramide levels prohibited the apoptosis and caused a quiescent state in the cells. The data presented here provide additional insight into the roles of ceramide and related enzymes in cellular responses to stress and suggest a possible relevance to in vivo disease states.

Ceramide Profiling of Complex Lipid Mixtures by Electrospray Ionization Mass Spectrometry

Ceramides and sphingoid bases are important intracellular second messengers that play a role in the regulation of cell growth, differentiation, and programmed cell death. Until now, quantitative and qualitative analysis of ceramide second messengers has been limited by a lack of analytical methods capable of detecting endogenous levels of, and differentiating between, individual ceramide species. Here we report the use of electrospray ionization tandem mass spectrometry for the qualitative and quantitative analysis of ceramides. Collision-induced fragmentation resulted in characteristic product ions for the sphingosine and dihydrosphingosine (sphinganine) head groups atm/z264 and 282 andm/z266 and 284, respectively, regardless of the length of the fatty acyl chains, with spectra being reproducible at concentrations as low as 25 nm(25 fmol/μl). These reporter ions were used to detect both sphingosine- and sphinganine-based ceramides in complex mixtures using precursor ion scan analysis. We demonstrated the application of this method for profiling the composition of ceramides in a commercial preparation of bovine brain ceramides and, following minimal chromatographic separation, a lipid extract of cultured T cells. Furthermore, we easily detected relative differences in individual ceramide species levels by comparing the profiles of three related lymphocyte cell lines, Jurkat, U937, and WEHI 231. Finally, by the addition of a nonnaturally occurring internal standard, we show that the technique can be used to measure quantitative changes in ceramide levels in such biologically derived lipid preparations.

The effect of ionizing radiation on signal transduction: antibodies to EGF receptor sensitize A431 cells to radiation

What determines the degree of cell-resistance or sensitivity to ionizing radiation is not yet known. As a corollary to the ability of ceramide to induce apoptosis, some questions arise as to whether malignant cells escape apoptosis because of their inability to mount a ceramide response to inducers of apoptosis. To shed more light on the molecular mechanisms of tumor cell response to radiation, we tested whether exposure to ionizing radiation (of 200–1000 cGy) is associated with changes in ceramide levels in A431 tumor epithelial cells and whether the ability of ceramide to induce apoptosis is inhibited by protein kinase C (PKC) activation. Our studies demonstrate an immediate decrease in cellular levels of ceramide in response to radiation, while sphingosine levels increase. Under the same conditions the cellular 1,2-diacylglycerol (DAG) levels decrease as well, being accompanied by the translocation of PKC α from the membrane to the cytoplasm. Elevation of membrane PKC levels by 12- O-tetradecanoylphorbol 13-acetate (TPA) treatment had no effect on cell survival after irradiation, while treatment with EGF during and after irradiation augmented cell survival. Moreover, monoclonal antibodies to the EGF receptor (EGFR) sensitize cells to radiation by facilitating radiation-induced apoptosis. It is thus plausible that in human Squamous carcinoma cells, radiation activates predominantly the EGFR to induce resistance, while both sphingomyelin and PKC signal transduction pathways are deactivated and demonstrate no significant role in the modulation of the sensitivity or the resistance of A431 cells to ionizing radiation.

Role for ceramide as an endogenous mediator of Fas-induced cytotoxicity.

Triggering of the Fas/APO-1 cell-surface receptor induces apoptosis through an uncharacterized chain of events. Exposure of Fas-sensitive cells to an agonist monoclonal antibody induced cell death and a 200-300% elevation in endogenous levels of the sphingolipid ceramide, a proposed intracellular mediator of apoptosis. In contrast, similar treatment of Fas-resistant cells caused insignificant changes in ceramide levels. Because resistant cell lines expressed the Fas antigen, these results indicate that these cells have a defect in the proximal signaling events leading to ceramide generation. Exposure of the resistant cell lines to a synthetic analog of ceramide induced apoptosis, thus bypassing Fas resistance and indicating that the signaling pathways downstream of ceramide were intact. Furthermore, activation of protein kinase C with the diacylglycerol analog phorbol 12-myristate 13-acetate significantly reduced Fas-induced cytotoxicity, suggesting opposing roles for ceramide and protein kinase C in regulation of apoptosis. These results provide evidence for ceramide as a necessary and sufficient lipid mediator of Fas-mediated apoptosis and suggest this process may be modulated via activation of additional signal-transduction pathways.