Aminomethylphosphonic Acid and Methoxyacetic Acid Induce Apoptosis in Prostate Cancer Cells

Aminomethylphosphonic acid (AMPA) and its parent compound herbicide glyphosate are analogs to glycine, which have been reported to inhibit proliferation and promote apoptosis of cancer cells, but not normal cells. Methoxyacetic acid (MAA) is the active metabolite of ester phthalates widely used in industry as gelling, viscosity and stabilizer; its exposure is associated with developmental and reproductive toxicities in both rodents and humans. MAA has been reported to suppress prostate cancer cell growth by inducing growth arrest and apoptosis. However, it is unknown whether AMPA and MAA can inhibit cancer cell growth. In this study, we found that AMPA and MAA inhibited cell growth in prostate cancer cell lines (LNCaP, C4-2B, PC-3 and DU-145) through induction of apoptosis and cell cycle arrest at the G1 phase. Importantly, the AMPA-induced apoptosis was potentiated with the addition of MAA, which was due to downregulation of the anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2), leading to activation of caspases 7 and 3. These results demonstrate that the combination of AMPA and MAA can promote the apoptosis of prostate cancer cells, suggesting that they can be used as potential therapeutic drugs in the treatment of prostate cancer.     

Plasma sphingolipids in patients with sickle cell disease: Multiple-site vaso-occlusive crises could be associated with lower sphingolipid levels

Although sickle cell disease (SCD) and its manifestations have been associated with various lipid alterations, there are a few studies exploring the impact of sphingolipids in SCD. In this study, we determined plasma ceramide (Cer) and sphingomyelin (CerPCho) species and investigated their association with the crisis in SCD. SCD patients (N = 27) suffering from vaso-occlusive crisis (VOC) or acute chest syndrome (ACS) were involved in this study. Blood samples were drawn at crisis and later at steady state periods. Clinical history, white blood cell count (WBC), C-reactive protein and lactate dehydrogenase (LDH) levels were recorded. 16:0, 18:0, 20:0, 22:0 Cer and 16:0, 18:0, 24:0 CerPCho were measured via LC–MS/MS. All measured Cer and CerPCho levels of SCD patients at crisis and steady-state were found to be similar. Inflammation-related parameters were significantly higher in patients with ACS compared to single-site VOC. Patients with multiple-site VOC were found to have significantly lower sphingolipid levels compared with those with single-site VOC, at crisis (16, 18, 24 CerPCho and 18, 22 Cer) and at steady-state (24:0 CerPCho and 18 Cer). Our results show that sphingolipid levels in SCD patients are similar during crisis and at steady state. However, lower sphingolipid levels appear to be associated with the development of multiple-site VOC. Since the differences were observed at both crisis and steady-state, sphingolipid level could be an underlying factor associated with crisis characteristics in patients with SCD.     

Decreased Serum Levels of Sphingomyelins and Ceramides in Sickle Cell Disease Patients

Limited data are available on the serum levels of different sphingomyelin (CerPCho) and ceramide (CER) species in sickle‐cell disease (SCD). This study was aimed at identifying the levels of C16–C24 CerPCho and C16–C24 CER in serum obtained from SCD patients and controls. Circulating levels of neutral sphingomyelinase (N‐SMase) activity, ceramide‐1‐phosphate (C1P), and sphingosine‐1‐phosphate (S1P) were also determined. Blood was collected from 35 hemoglobin (Hb)A volunteers and 45 homozygous HbSS patients. Serum levels of C16–C24 CerPCho and C16–C24 CER were determined by an optimized multiple reaction monitoring (MRM) method using ultrafast liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Serum activity of N‐SMase was assayed by standard kit methods, and C1P and S1P levels were determined by enzyme‐linked immunosorbent assay. A significant decrease was observed in the serum levels of C18–C24 CerPCho in patients with SCD compared to controls. No significant difference was found in C16 CerPCho levels between the two groups. Very‐long‐chain C22–C24 CER were significantly decreased in SCD, while long‐chain C16–C20 CER levels showed no significant difference between SCD patients and controls. Significant positive correlation was found between the serum total cholesterol levels and C18–C24 CerPCho and C22–C24 CER in SCD patients. Patients with SCD had significantly elevated serum activity of N‐SMase as well as increased circulating levels of C1P and S1P compared to controls. The decrease in serum levels of C18–C24 CerPCho in patients with SCD was accompanied by decreased levels of C22–C24 CER. Future studies are needed to understand the role of decreased CerPCho and CER in the pathophysiology of SCD.     

<Emphasis Type="Italic">N</Emphasis>-Acyl Taurines are Anti-Proliferative in Prostate Cancer Cells

Endocannabinoids have been implicated in cancer development and cause heterogenous effects in tumor cells, by inducing apoptosis, reducing migration, causing anti-angiogenic activity and alterations in the cell cycle resulting in growth arrest. Recently, several novel amides of fatty acids that are structurally related to endocannabinoids have been isolated from mammalian sources, although the functions of these fatty amides are not well studied. One group of these novel fatty acid amides are the N-acyl taurines (fatty acids conjugated to the amino acid taurine). This study examined if N-acyl taurines, specifically N-arachidonoyl taurine and N-oleoyl taurine could function in a similar way to endocannabinoids and result in cell cycle alterations or growth arrest in the human prostate adenocarcinoma cell line PC-3. PC-3 cells were treated with various concentrations of N-arachidonoyl taurine and N-oleoyl taurine and cell proliferation and viability was measured using resazurin and colony formation assays. Effects of N-acyl taurines on the cell cycle was measured using FACS analysis. Treatment with N-arachidonoyl taurine and N-oleoyl taurine resulted in a significant reduction in proliferation of PC-3 cells, even at concentrations as low as 1 μM. Treatment with N-oleoyl taurine resulted in an increased number of cells in the subG1 population, suggesting apoptosis, and a lower number of cells in S-phase of the cell cycle. In summary, our results show that novel biologically active lipids, the N-acyl taurines, result in reduced proliferation in PC-3 cells.     

Krill Oil Perturbs Proliferation and Migration of Mouse Colon Cancer Cells in vitro by Impeding Extracellular Signal-Regulated Protein Kinase Signaling Pathway

The prevalence of colorectal cancer (CRC) continues to increase. Treatment of CRC remains a significant clinical challenge, and effective therapies for advanced CRC are desperately needed. Increasing attention and ongoing research efforts have focused on krill oil that may provide health benefits to the human body. Here we report that krill oil exerts in vitro anticancer activity through a direct inhibition on proliferation, colony formation, migration, and invasion of mouse colon cancer cells. Krill oil inhibited the proliferation and colony formation of CT-26 colon cancer cells by causing G0/G1 cell cycle arrest and apoptosis. Cell cycle arrest was attributable to reduction of cyclin D1 levels in krill oil-treated cells. Further studies revealed that krill oil induced mitochondrial-dependent apoptosis of CT-26 cells, including loss of mitochondrial membrane potential, increased cytosolic calcium levels, activation of caspase-3, and downregulation of anti-apoptotic proteins MCL-1 and BCL-XL. Krill oil suppressed migration of CT-26 cells by disrupting the microfilaments and microtubules. Extracellular signal-regulated protein kinase (ERK) plays crucial roles in regulating proliferation and migration of cancer cells. We found that krill oil attenuated the activation of ERK signaling pathway to exert the effects on cell cycle, apoptosis, and migration of colon cancer cells. We speculate that polyunsaturated fatty acids of krill oil may dampen ERK activation by decreasing the phospholipid saturation of cell membrane. Although findings from in vitro studies may not necessarily translate in vivo, our study provides insights into the possibility that krill oil or its components could have therapeutic potential in colon cancer.     

Cell Adhesion Regulates Expression of the Androgen Receptor and Coregulators in Different Prostate Cancer Cells

Prostate cancer cells adhere to a tumor basement membrane, while secretory epithelial cells reside in a suprabasal cell compartment. Since tumor cells are derived from suprabasal epithelial cells, they experience de-novo substratum adhesion in the context of oncogenesis. We therefore analyzed whether cell-matrix adhesion could affect the protein expression and activity of the AR. In this study, AR protein expression declined upon suspension of BPH-1-AR cells, but not in PC-3-AR cells shown by Western blot. In a time course study, BPH-1 cell lost AR expression within 6 hours, and the synthetic androgen, R1881 reduced the loss of AR expression. We further explored the mechanism of AR loss in suspended BPH-1 cells. BPH-1-AR cells underwent apoptosis (anoikis) when suspended for 2 – 5 hours. Suspension did not induce significant apoptosis or decreasing of AR expression in PC-3 cells. Inhibition of apoptosis in suspended BPH-1-AR cells, either by expression of Bcl-2 or Bcl-xl or by treatment with Z-VAD, a caspase inhibitor, prevented loss of AR protein. In contrast, the calpain protease inhibitor, ALLN, accelerated the loss of AR protein expression. Additionally, cell-matrix adhesion changed the expression of coregulators of AR in the mRNA level of prostate cancer cells. Our results demonstrate that AR protein expression was reduced through activation of cell death pathways, and thus indirectly through cell suspension in BPH-AR cells. The activity of AR can also be regulated by adhesion in PC-3-AR and LNCaP cells through affecting the coregulators level.     

Myocardial Infarction Changes Sphingolipid Metabolism in the Uninfarcted Ventricular Wall of the Rat

It is known that the ratio, the level of sphingosine-1-phosphate (S1P)/the level of ceramide (CER) determines survival of the cells. The aim of the present study was to examine the effect of myocardial infarction on the level of different sphingolipids in the uninfarcted area. The experiments were carried out on male Wistar rats: 1, control; 2, after ligation of the left coronary artery (infarct) and 3, sham operated. Samples of the uninfarcted area of the left ventricle were taken in 1, 6 and 24 h after the surgery. The level of sphingolipids, S1P, CER, sphingosine (SPH), sphinganine-1-phosphate (SPA1P) and sphinganine (SPA) was determined. The control values were (ng/mg), S1P-0.33 ± 0.03, SPH-1.02 ± 0.13, SPA1P-0.11 ± 0.01, SPA-0.28 ± 0.04, total CER-20.3 ± 1.8. In infarct, the level of S1P in the uninfarcted area was reduced by ~3 times in 1 and 6 h and decreased further in 24 h. The level of SPH decreased in 1 h and returned to the control thereafter. The total level of CER decreased in 6 h after the infarction. Sham surgery also produced changes in the level of certain sphingolipids. The ratio, the level of S1P/the level of CER was markedly reduced at each time point after the infarction. It is concluded that the reduction in the S1P/CER ratio could be responsible for increased apoptosis in the uninfarcted area after the myocardial infarction in the rat.     

Sphingolipid Content in the Human Uterus and Pair-Matched Uterine Leiomyomas Remains Constant

In the present work we sought to investigate the content of sphingolipids (sphingosine, sphinganine, sphingosine-1-phosphate and ceramide) in human fibroids and pair-matched healthy uterus tissue. We demonstrated that, in uterine leiomyomas, the contents of sphingosine, sphinganine, sphingosine-1-phosphate and ceramide remains quite constant. However, a trend towards elevation of ceramide and simultaneous reduction of sphingosine-1 phosphate levels was also noticed. Additionally, in uterine leiomyomas we found relevant activation of both PTEN and MAPK(ERK1/2) signaling pathways with only a minor change in AKT activity and relatively absent HIF-1α/AMPK activation. In conclusion, rather modest changes in sphingolipids are correlated with the activation of PTEN and MAPK(ERK1/2) signaling proteins in human uterine leiomyomas.     

Induction of Endoplasmic Reticulum Stress-Mediated Apoptosis by Aminosteroid RM-581 Efficiently Blocks the Growth of PC-3 Cancer Cells and Tumors Resistant or Not to Docetaxel

Aminosteroid derivative RM-581 was previously identified as an endoplasmic-reticulum (ER) stress inducer with potent in vitro and in vivo anticancer activities. We report its evaluation in androgen-independent prostate cancer (PC-3) cells. RM-581 efficiently blocks PC-3 cell proliferation with stronger activity than that of a selection of known antineoplastic agents. This later also showed a synergistic effect with docetaxel, able to block the proliferation of docetaxel-resistant PC-3 cells and, contrary to docetaxel, did not induce cell resistance. RM-581 induced an increase in the expression level of ER stress-related markers of apoptosis, potentially triggered by the presence of RM-581 in the ER of PC-3 cells. These in vitro results were then successfully translated in vivo in a PC-3 xenograft tumor model in nude mice, showing superior blockade than that of docetaxel. RM-581 was also able to stop the progression of PC-3 cells when they had become resistant to docetaxel treatment. Concomitantly, we observed a decrease in gene markers of mevalonate and fatty acid pathways, and intratumoral levels of cholesterol by 19% and fatty acids by 22%. Overall, this work demonstrates the potential of an ER stress inducer as an anticancer agent for the treatment of prostate cancers that are refractory to commonly used chemotherapy treatments.     

Comprehensive Quantitation Using Two Stable Isotopically Labeled Species and Direct Detection of <Emphasis Type="Italic">N</Emphasis>-Acyl Moiety of Sphingomyelin

Sphingomyelin (ceramide‐phosphocholine, CerPCho) is a common sphingolipid in mammalian cells and is composed of phosphorylcholine and ceramide as polar and hydrophobic components, respectively. In this study, a qualitative liquid chromatography‐electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS/MS) analysis is proposed in which CerPCho structures were assigned based on product ion spectra corresponding to sphingosylphosphorylcholine and N‐acyl moieties. From MS/MS/MS analysis of CerPCho, we observed product ion spectra of the N‐acyl fatty acids as [RCO₂]^? ions as well as sphingosylphosphorylcholine. A calibration curve for CerPCho was constructed using two stable isotopically labeled CerPCho species and then used to quantify the CerPCho species in HeLa cells as a proof‐of‐principle study. The present study proposes an accurate method for quantifying and assigning structures to each CerPCho species in crude biologic samples by LC–ESI–MS/MS/MS analysis.     

三七总皂甙对大鼠肺动脉血管平滑肌细胞增殖和凋亡的影响及其机制

目的探讨三七总皂甙(Panax notoginseng saponins,PNS)对大鼠肺动脉血管平滑肌细胞(Pulmonary artery smoothmuscle cell,PASMC)增殖和凋亡的影响及其机制。方法将PASMC分为5组:对照组(不给药)、bFGF组(8 ku/L)、bFGF+低、中、高剂量PNS组(bFGF 8 ku/L+300、450、600μg/L PNS),给药24 h后,采用MTT法检测PASMC的增殖活力;流式细胞仪检测细胞周期分布和细胞凋亡率;Westernblot检测细胞中胱天蛋白酶-3(caspase-3)蛋白的表达。结果 PNS干预可抑制PASMC增殖,阻遏bFGF诱导的PASMC进入S期,提高滞留在G0/G1期细胞的比例,并使PASMC凋亡率和caspase-3蛋白表达上调,PASMC凋亡率达(18.70%±0.33%)～(28.40%±0.79%),是bFGF诱导组的10～14倍。结论 PNS能显著抑制bFGF诱导的PASMC增殖,促进其凋亡,其机制可能与其调控细胞周期转换、激活caspase-3蛋白的表达有关。     

A Dual Anti-Inflammatory and Anti-Proliferative 3-Styrylchromone Derivative Synergistically Enhances the Anti-Cancer Effects of DNA-Damaging Agents on Colon Cancer Cells by Targeting HMGB1-RAGE-ERK1/2 Signaling

The current anti-cancer treatments are not enough to eradicate tumors, and therefore, new modalities and strategies are still needed. Most tumors generate an inflammatory tumor microenvironment (TME) and maintain the niche for their development. Because of the critical role of inflammation via high-mobility group box 1 (HMGB1)–receptor for advanced glycation end-products (RAGE) signaling pathway in the TME, a novel compound possessing both anti-cancer and anti-inflammatory activities by suppressing the HMGB1-RAGE axis provides an effective strategy for cancer treatment. A recent work of our group found that some anti-cancer 3-styrylchromones have weak anti-inflammatory activities via the suppression of this axis. In this direction, we searched such anti-cancer molecules possessing potent anti-inflammatory activities and discovered 7-methoxy-3-hydroxy-styrylchromone (C6) having dual suppressive activities. Mechanism-of-action studies revealed that C6 inhibited the increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) under the stimulation of HMGB1-RAGE signaling and thereby suppressed cytokine production in macrophage-like RAW264.7 cells. On the other hand, in colorectal cancer HCT116 cells, C6 inhibited the activation of ERK1/2, cyclin-dependent kinase 1, and AKT, down-regulated the protein level of XIAP, and up-regulated pro-apoptotic Bax and caspase-3/7 expression. These alterations are suggested to be involved in the C6-induced suppression of cell cycle/proliferation and initiation of apoptosis in the cancer cells. More importantly, in cancer cells, the treatment of C6 potentiates the anti-cancer effects of DNA-damaging agents. Thus, C6 may be a promising lead for the generation of a novel class of cancer therapeutics.     

YKL-40转染对前列腺癌LNcap细胞增殖及侵袭活性的影响

目的探讨外源性YKL-40基因转染对前列腺癌LNcap细胞增殖、侵袭、迁移及黏附活性的影响。方法 RT-PCR法检测前列腺癌细胞LNcap、PC-3、DU-145 YKL-40基因内源性表达情况;用pcDNA3.1-YKL-40质粒转染LNcap细胞,分别经MTT法、Boyden小室法及黏附试验检测转染前后细胞增殖、侵袭、迁移及黏附活性,并进行体外药敏试验。结果仅DU-145细胞可内源性表达YKL-40基因;pcDNA3.1-YKL-40转染的LNcap细胞在第2～5天的A490值均显著高于对照组(P<0.05),其侵袭(75.11±4.40)和迁移穿膜细胞数(133.00±5.07)及黏附率(107.57%)均显著高于对照组(P<0.05),对5-FU、顺铂和依托泊苷的IC50值分别为(31.15±0.43)、(4.15±0.13)和(55.22±0.57)μmol/L,均显著高于对照组(P<0.05)。结论 YKL-40基因能促进LNcap细胞增殖,提高细胞侵袭、迁移及黏附活性,并使其对5-FU、顺铂和依托泊苷具有一定的耐药性。     

Pharmacological Effects of Guava (Psidium guajava L.) Seed Polysaccharides: GSF3 Inhibits PC-3 Prostate Cancer Cell Growth through Immunotherapy In Vitro

The inhibitory effects of purified fractions isolated from guava seed polysaccharides (GSPS) including guava seed polysaccharide fraction 1 (GSF1), GSF2, and GSF3 on prostate cancer cells remain unclear. To clarify the anti-prostate cancer potential, GSPS, GSF1, GSF2, and GSF3 were isolated using Sepharose 6B gel filtration chromatography to assay their inhibitory effects on prostate PC-3 cell growth with direct action or indirect immunotherapy using either splenocyte conditioned media (SCM) or macrophage conditioned media (MCM). Correlations between cytokine profiles in the conditioned media and pro-apoptotic gene expression levels in the corresponding treated PC-3 cells were analyzed. Results showed that GSPS, GSF1, GSF2, and GSF3, particularly GSF3, through either direct action or indirect treatments using SCM or MCM, significantly (p < 0.05) inhibited PC-3 cell growth. GSF3 direct treatments increased pro-apoptotic Bax/anti-apoptotic Bcl-2 mRNA expression ratios in corresponding treated PC-3 cells. Either SCM or MCM cultured with GSF3 increased Fas mRNA expression levels in corresponding treated PC-3 cells. Both Th2-polarized and anti-inflammatory cytokine IL-10 either secreted in SCM or MCM were positively correlated with Fas mRNA expression levels in corresponding treated PC-3 cells. Our results suggest that GSF3 is a potent biological response modifier to decrease PC-3 cell growth through inducing apoptosis.     

Vitamin D3 Enriches Ceramide Content in Exosomes Released by Embryonic Hippocampal Cells

The release of exosomes can lead to cell–cell communication. Nutrients such as vitamin D3 and sphingolipids have important roles in many cellular functions, including proliferation, differentiation, senescence, and cancer. However, the specific composition of sphingolipids in exosomes and their changes induced by vitamin D3 treatment have not been elucidated. Here, we initially observed neutral sphingomyelinase and vitamin D receptors in exosomes released from HN9.10 embryonic hippocampal cells. Using ultrafast liquid chromatography tandem mass spectrometry, we showed that exosomes are rich in sphingomyelin species compared to whole cells. To interrogate the possible functions of vitamin D3, we established the optimal conditions of cell treatment and we analyzed exosome composition. Vitamin D3 was identified as responsible for the vitamin D receptor loss, for the increase in neutral sphingomyelinase content and sphingomyelin changes. As a consequence, the generation of ceramide upon vitamin D3 treatment was evident. Incubation of the cells with neutral sphingomyelinase, or the same concentration of ceramide produced in exosomes was necessary and sufficient to stimulate embryonic hippocampal cell differentiation, as vitamin D3. This is the first time that exosome ceramide is interrogated for mediate the effect of vitamin D3 in inducing cell differentiation.     

Subtype of Neuroblastoma Cells with High KIT Expression Are Dependent on KIT and Its Knockdown Induces Compensatory Activation of Pro-Survival Signaling

Neuroblastoma (NB) is a pediatric cancer with high clinical and molecular heterogeneity, and patients with high-risk tumors have limited treatment options. Receptor tyrosine kinase KIT has been identified as a potential marker of high-risk NB and a promising target for NB treatment. We investigated 19,145 tumor RNA expression and molecular pathway activation profiles for 20 cancer types and detected relatively high levels of KIT expression in NB. Increased KIT expression was associated with activation of cell survival pathways, downregulated apoptosis induction, and cell cycle checkpoint control pathways. KIT knockdown with shRNA encoded by lentiviral vectors in SH-SY5Y cells led to reduced cell proliferation and apoptosis induction up to 50%. Our data suggest that apoptosis induction was caused by mitotic catastrophe, and there was a 2-fold decrease in percentage of G2-M cell cycle phase after KIT knockdown. We found that KIT knockdown in NB cells leads to strong upregulation of other pro-survival growth factor signaling cascades such as EPO, NGF, IL-6, and IGF-1 pathways. NGF, IGF-1 and EPO were able to increase cell proliferation in KIT-depleted cells in an ERK1/2-dependent manner. Overall, we show that KIT is a promising therapeutic target in NB, although such therapy efficiency could be impeded by growth factor signaling activation.     

Plasma Ceramides and Sphingomyelins of Pediatric Patients Increase in Primary Ciliary Dyskinesia but Decrease in Cystic Fibrosis

We investigated plasma sphingomyelin (CerPCho) and ceramide (Cer) levels in pediatric patients with cystic fibrosis (CF) and primary ciliary dyskinesia (PCD). Plasma samples were obtained from CF (n = 19) and PCD (n = 7) patients at exacerbation, discharge, and stable periods. Healthy children (n = 17) of similar age served as control. Levels of 16–24 CerPCho and 16–24 Cer were measured by LC–MS/MS. Concentrations of all CerPCho and Cer species measured at exacerbation were significantly lower in patients with CF than PCD. 16, 18, 24 CerPCho, and 22, 24 Cer in exacerbation; 18, 24 CerPCho, and 18, 20, 22, 24 Cer at discharge; 18, 24 CerPCho and 24 Cer at stable period were significantly lower in CF patients than healthy children (p < 0.001 and p < 0.05). All CerPCho and Cer levels of PCD patients were significantly higher except 24 CerPCho and 24 Cer during exacerbation, 24 CerPCho at discharge, and 18, 22 CerPCho levels at stable period (p < 0.001 and p < 0.05) compared with healthy children. There was no significant difference among exacerbation, discharge, and stable periods in each group for Cer and CerPCho levels. This is the first study measuring plasma Cer and CerPCho levels in PCD and third study in CF patients. The dramatic difference in plasma levels of most CerPCho and Cer species found between two diseases suggest that cilia pathology in PCD and CFTR mutation in CF seem to alter sphingolipid metabolism possibly in opposite directions.     

Ultraviolet (UV) and Hydrogen Peroxide Activate Ceramide-ER Stress-AMPK Signaling Axis to Promote Retinal Pigment Epithelium (RPE) Cell Apoptosis

Ultraviolet (UV) radiation and reactive oxygen species (ROS) impair the physiological functions of retinal pigment epithelium (RPE) cells by inducing cell apoptosis, which is the main cause of age-related macular degeneration (AMD). The mechanism by which UV/ROS induces RPE cell death is not fully addressed. Here, we observed the activation of a ceramide-endoplasmic reticulum (ER) stress-AMP activated protein kinase (AMPK) signaling axis in UV and hydrogen peroxide (H₂O₂)-treated RPE cells. UV and H₂O₂ induced an early ceramide production, profound ER stress and AMPK activation. Pharmacological inhibitors against ER stress (salubrinal), ceramide production (fumonisin B1) and AMPK activation (compound C) suppressed UV- and H₂O₂-induced RPE cell apoptosis. Conversely, cell permeable short-chain C6 ceramide and AMPK activator AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide) mimicked UV and H₂O₂’s effects and promoted RPE cell apoptosis. Together, these results suggest that UV/H₂O₂ activates the ceramide-ER stress-AMPK signaling axis to promote RPE cell apoptosis.     

The Tricyclodecan-9-yl-xanthogenate D609 Triggers Ceramide Increase and Enhances FasL-Induced Caspase-Dependent and -Independent Cell Death in T Lymphocytes

D609 is known to modulate death receptor-induced ceramide generation and cell death. We show that in Jurkat cells, non-toxic D609 concentrations inhibit sphingomyelin synthase and, to a lesser extent, glucosylceramide synthase, and transiently increase the intracellular ceramide level. D609 significantly enhanced FasL-induced caspase activation and apoptosis. D609 stimulated FasL-induced cell death in caspase-8-deficient Jurkat cells, indicating that D609 acts downstream of caspase-8. At high FasL concentration (500 ng/mL), cell death was significantly, but not completely, inhibited by zVAD-fmk, a broad-spectrum caspase inhibitor, indicating that FasL can activate both caspase-dependent and -independent cell death signaling pathways. FasL-induced caspase activation was abolished by zVAD-fmk, whereas ceramide production was only partially impaired. D609 enhanced caspase-independent ceramide increase and cell death in response to FasL. Also, D609 overcame zVAD-fmk-conferred resistance to a FasL concentration as low as 50 ng/mL and bypassed RIP deficiency. It is likely that mitochondrial events were involved, since Bcl-xL over-expression impaired D609 effects. In PHA-activated human T lymphocytes, D609 enhanced FasL-induced cell death in the presence or absence of zVAD-fmk. Altogether, our data strongly indicate that the inhibition of ceramide conversion to complex sphingolipids by D609 is accompanied by an enhancement of FasL-induced caspase-dependent and -independent cell death in T lymphocytes.