Home practice and quality of life among patients with neurofibromatosis randomized to a mind-body intervention.

OBJECTIVES: The purpose of this study was to summarize home practice in patients with neurofibromatosis (NF) randomized to an 8-week group mind-body intervention, the Relaxation Response Resiliency Program for NF (3RP-NF). We further examined the association between home practice and changes in four domains of quality of life (QoL). METHODS: Data are derived from a single-blind RCT of the 3RP-NF (N = 31) delivered via videoconferencing versus an attention placebo control. 3RP-NF participants submitted weekly home practice logs to the group leader prior to each weekly session, which included information regarding their engagement of relaxation response (RR)-eliciting skills and appreciation skills. Physical, psychological, social and environmental QoL were measured at baseline, post-intervention and at a 6-months follow up. RESULTS: Participants reported engaging in home practice of RR-eliciting skills on average 28.55 days (SD = 10.79) and appreciation skills on average 24.39 days (SD = 13.48) over the 49-day treatment period. Participants reported an average of 383.42 (SD = 274.38) minutes of RR-eliciting skills home practice and an average of 49.13 (SD = 41.90) appreciations skills home practice. A significant association was observed between frequency of RR-eliciting skills home practice and physical QoL at the 6-month follow-up (r = .383, p = .034). CONCLUSIONS: Participants with NF are able and willing to practice RR-eliciting skills and appreciation skills outside of treatment sessions. Frequency of RR-eliciting skills home practice may be associated with improvement in physical QoL. Future research should replicate these efforts with larger samples, and attempt to identify additional factors that predict optimal response to mind-body interventions other than home practice.

Ceramide inhibition of NF-kappaB activation involves reverse translocation of classical protein kinase C (PKC) isoenzymes: requirement for kinase activity and carboxyl-terminal phosphorylation of PKC for the ceramide response.

Protein kinase C (PKC) is known to activate NF-kappaB whereas the lipid mediator ceramide was recently shown to inhibit activation of this transcription factor (1, 2). In this study, the mechanisms by which ceramide interferes with this pathway were examined in Jurkat leukemia and MCF-7 breast cancer cells. Both exogenous and endogenous ceramide inhibited selectively PKC-mediated activation of NF-kappaB by reverting PKC translocation to the membrane. Next, confocal and immunofluorescence studies were performed to evaluate the direct effects of ceramide on PKC. These studies showed that ceramide inhibited translocation of a green fluorescent protein (GFP)-PKCbeta2 fusion protein in response to PMA. A mutant PKC in which autophosphorylation sites were mutated to alanine (PKC-DA) was resistant to ceramide. A kinase-inactive mutant (PKC-KR) was also resistant to ceramide action, and the results were supported using kinase inhibitors of the enzyme. Finally, overexpression of PKC-DA prevented, at least partly, the ability of ceramide to inhibit activation of NF-kappaB. Taken together, these studies show that ceramide has acute effects on translocation of PKC by inducing reverse translocation, and this reversal requires both the kinase activity of PKC and phosphorylation of the autophosphorylation sites.

Role of ceramide in mediating the inhibition of telomerase activity in A549 human lung adenocarcinoma cells.

This study was designed to analyze whether ceramide, a bioeffector of growth suppression, plays a role in the regulation of telomerase activity in A549 cells. Telomerase activity was inhibited significantly by exogenous C(6)-ceramide, but not by the biologically inactive analog dihydro-C(6)-ceramide, in a time- and dose-dependent manner, with 85% inhibition produced by 20 microm C(6)-ceramide at 24 h. Moreover, analysis of phosphatidylserine translocation from the inner to the outer plasma membrane by flow cytometry and of poly(ADP-ribose) polymerase degradation by Western blotting showed that ceramide treatment (20 microm for 24 h) had no apoptotic effects. Trypan blue exclusion, [(3)H]thymidine incorporation, and cell cycle analyses, coupled with clonogenic cell survival assay on soft agar, showed that ceramide treatment with a 20 microm concentration at 24 h resulted in the cell cycle arrest of the majority of the cell population at G(0)/G(1) with no detectable cell death. These results suggest that the inhibition of telomerase by ceramide is not a consequence of cell death but is correlated with growth arrest. Next, to determine the role of endogenous ceramide in telomerase modulation, A549 cells were transiently transfected with an expression vector containing the full-length bacterial sphingomyelinase cDNA (b-SMase). The overexpression of b-SMase, but not exogenously applied purified b-SMase enzyme, resulted in significantly decreased telomerase activity compared with controls, showing that the increased endogenous ceramide is sufficient for telomerase inhibition. Moreover, treatment of A549 cells with daunorubicin at 1 microm for 6 h resulted in the inhibition of telomerase, which correlated with the elevation of endogenous ceramide levels and growth arrest. Finally, stable overexpression of human glucosylceramide synthase, which attenuates ceramide levels by converting ceramide to glucosylceramide, prevented the inhibitory effects of C(6)-ceramide and daunorubicin on telomerase. Therefore, these results provide novel data showing for the first time that ceramide is a candidate upstream regulator of telomerase.

Enzymes of sphingolipid metabolism: from modular to integrative signaling.

Many enzymes of sphingolipid metabolism are regulated in response to extra- and intracellular stimuli and in turn serve as regulators of levels of bioactive lipids (such as sphingosine, ceramide, sphingosine 1-phosphate, and diacylglycerol), and as such, they serve a prototypical modular function in cell regulation. However, lipid metabolism is also closely interconnected in that a product of one enzyme serves as a substrate for another. Moreover, many cell stimuli regulate more than one of these enzymes, thus adding to the complexity of regulation of lipid metabolism. In this paper, we review the status of enzymes of sphingolipid metabolism in cell regulation and propose a role for these enzymes in integration of cell responses, a role that builds on the modular organization while also taking advantage of the complexity and interconnectedness of lipid metabolism, thus providing for a combinatorial mechanism of generating diversity in cell responses. This may be a general prototype for the involvement of metabolic pathways in cell regulation.

Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria.

B-cells, triggered via their surface B-cell receptor (BcR), start an apoptotic program known as activation-induced cell death (AICD), and it is widely believed that this phenomenon plays a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been proposed to be involved in AICD, the contribution of either and the exact molecular events through which AICD commences are still unknown. Here we show that in Ramos B-cells, BcR-triggered cell death is associated with an early rise of C16 ceramide that derives from activation of the de novo pathway, as demonstrated using a specific inhibitor of ceramide synthase, fumonisin B1 (FB1), and using pulse labeling with the metabolic sphingolipid precursor, palmitate. There was no evidence for activation of sphingomyelinases or hydrolysis of sphingomyelin. Importantly, FB1 inhibited several specific apoptotic hallmarks such as poly(A)DP-ribose polymerase cleavage and DNA fragmentation. Electron microscopy revealed morphological evidence of mitochondrial damage, suggesting the involvement of mitochondria in BcR-triggered apoptosis, and this was inhibited by FB1. Moreover, a loss of mitochondrial membrane potential was observed in Ramos cells after BcR cross-linking, which was inhibited by the addition of FB1. Interestingly, benzyloxycarbonyl-Val-Ala-dl-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentation. These results suggest a crucial role for de novo generated C16 ceramide in the execution of AICD, and they further suggest an ordered and more specific sequence of biochemical events in which de novo generated C16 ceramide is involved in mitochondrial damage resulting in a downstream activation of caspases and apoptosis.

Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of C. neoformans.

Cryptococcus neoformans is a leading cause of life-threatening fungal infection in immunocompromised patients. Inositol-phosphoryl ceramide synthase 1 (Ipc1) is a fungus-specific enzyme, encoded by the essential IPC1 gene, that catalyzes the formation of complex sphingolipids and may also regulate the levels of phytoceramide and diacylglycerol. Here, we investigated the functions of this essential gene by modulating its expression in C. neoformans using a galactose-inducible promoter. Down-regulation of IPC1 significantly lowers the expression of certain virulence traits such as melanin pigmentation and, remarkably, impairs pathogenicity of C. neoformans in an established rabbit model. Interestingly, we found that IPC1 down-regulation significantly decreases the intracellular growth of C. neoformans in the J774.16 murine macrophage-like cells. Finally, we studied the effect of IPC1 expression under different stress conditions and found that down-regulation of IPC1 confers a defect on in vitro growth at low pH. Because this environment is similar to that in the phagolysosome of J774.16 macrophage-like cells, our findings indicate that down-regulation of IPC1 confers a growth defect in vivo through a pH-dependent mechanism. In conclusion, our study is the first to define a novel and crucial function of Ipc1 in fungal pathogenesis.

Identification of ISC1 (YER019w) as inositol phosphosphingolipid phospholipase C in Saccharomyces cerevisiae.

Sphingolipids have emerged as novel bioactive mediators in eukaryotic cells including yeast. It has been proposed that sphingomyelin (SM) hydrolysis and the concomitant generation of ceramide are involved in various stress responses in mammalian cells. The yeast Saccharomyces cerevisiae has inositol phosphosphingolipids (IPS) instead of SM and glycolipids, and synthesis of IPS is indispensable to its growth. Although the genes responsible for the synthesis of IPS have been identified, the gene(s) for the degradation of IPS has not been reported. Here we show that ISC1 (YER019w), which has homology to bacterial neutral sphingomyelinase (SMase), encodes IPS phospholipase C (IPS-PLC). First, we observed that overexpression of ISC1 greatly increased neutral SMase activity, and this activity was dependent on the presence of phosphatidylserine. Cells deleted in ISC1 demonstrated negligible neutral SMase activity. Because yeast cells have IPS instead of SM, we investigated whether IPS are the physiologic substrates of this enzyme. Lysates of ISC1-overexpressing cells demonstrated very high PLC activities on IPS. Deletion of ISC1 eliminated endogenous IPS-PLC activities. Labeling yeast cells with [(3)H]dihydrosphingosine showed that IPS were increased in the deletion mutant cells. This study identifies the first enzyme involved in catabolism of complex sphingolipids in S. cerevisiae.

Differential effects of sphingomyelin hydrolysis and resynthesis on the activation of NF-kappa B in normal and SV40-transformed human fibroblasts.

The precise role of ceramide in NF-kappaB signaling remains unclear. The recent observation of differential sphingomyelin synthase (SMS) activity in normal (low SMS) versus SV40-transformed (high SMS) WI38 human lung fibroblasts provides an opportunity to assess the involvement of ceramide and SMS in NF-kappaB activation. Treatment of normal WI38 fibroblasts with bacterial sphingomyelinase resulted in a 4-fold elevation of ceramide and blocked NF-kappaB activation by serum stimulation. Such inhibition was not observed in SV40-transformed fibroblasts. Under regular growth conditions, after sphingomyelinase was washed out, normal WI38 did not show SM re-synthesis nor NF-kappaB activation. In SV40-WI38, on the other hand, sphingomyelinase washout induced resynthesis of SM due to the action of SMS on ceramide generated at the plasma membrane. NF-kappaB activation correlated with SM resynthesis. This activation was abrogated by D609, which inhibited SM resynthesis but not the initial formation of ceramide. The differential activity of SMS may explain the effects of ceramide in NF-kappaB signaling: in the absence of significant SMS activity, ceramide inhibits NF-kappaB, whereas with high SMS, the conversion of the ceramide signal to a diacylglycerol signal by the action of SMS stimulates NF-kappaB. These results also suggest a role for SMS in regulating NF-kappaB.

Rapid replenishment of sphingomyelin in the plasma membrane upon degradation by sphingomyelinase in NIH3T3 cells overexpressing the phosphatidylinositol transfer protein beta.

In order to study the in vivo function of the phosphatidylinositol transfer protein beta (PI-TPbeta), mouse NIH3T3 fibroblasts were transfected with cDNA encoding mouse PI-TPbeta. Two stable cell lines were isolated (SPIbeta2 and SPIbeta8) in which the levels of PI-TPbeta were increased 16- and 11-fold respectively. The doubling time of the SPIbeta cells was about 1.7 times that of the wild-type (wt) cells. Because PI-TPbeta expresses transfer activity towards sphingomyelin (SM) in vitro, the SM metabolism of the overexpressors was investigated. By measuring the incorporation of [methyl-(3)H]choline chloride in SM and phosphatidylcholine (PtdCho), it was shown that the rate of de novo SM and PtdCho synthesis was similar in transfected and wt cells. We also determined the ability of the cells to resynthesize SM from ceramide produced in the plasma membrane by the action of bacterial sphingomyelinase (bSMase). In these experiments the cells were labelled to equilibrium (60 h) with [(3)H]choline. At relatively low bSMase concentrations (50 munits/ml), 50% of [(3)H]SM in wt NIH3T3 cells was degraded, whereas the levels of [(3)H]SM in SPIbeta cells appeared to be unaffected. Since the release of [(3)H]choline phosphate into the medium was comparable for both wt NIH3T3 and SPIbeta cells, these results strongly suggest that breakdown of SM in SPIbeta cells was masked by rapid resynthesis of SM from the ceramide formed. By increasing the bSMase concentrations to 200 munits/ml, a 50% decrease in the level of [(3)H]SM in SPIbeta cells was attained. During a recovery period of 6 h (in the absence of bSMase) the resynthesis of SM was found to be much more pronounced in these SPIbeta cells than in 50% [(3)H]SM-depleted wt NIH3T3 cells. After 6 h of recovery about 50% of the resynthesized SM in the SPIbeta cells was available for a second hydrolysis by bSMase. When monensin was present during the recovery period, the resynthesis of SM in bSMase-treated SPIbeta cells was not affected. However, under these conditions 100% of the resynthesized SM was available for hydrolysis. On the basis of these results we propose that, under conditions where ceramide is formed in the plasma membrane, PI-TPbeta plays an important role in restoring the steady-state levels of SM.

Ceramide in the eukaryotic stress response.

Several extracellular agents and stress stimuli, such as tumour necrosis factor alpha, chemotherapeutic agents and heat, cause ceramide accumulation. They do this by regulating enzymes involved in its metabolism. Ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, cell-cycle arrest and cell senescence.

Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?

Sphingomyelin synthase (SMS), an enzyme involved in sphingomyelin (SM) and ceramide metabolism, can potentially regulate, in opposite directions, the levels of ceramide and diacylglycerol. In this study SMS activity was investigated in normal and SV40-transformed human lung fibroblasts (WI38). The addition of [3H]C2-ceramide to cells resulted in a time-dependent formation of [3H]C2-SM. At 24 h after treatment, normal WI38 cells cleared 17% of [3H]C2-ceramide producing [3H]C2-SM, which accounted for 13% of total radioactivity. On the other hand, SV40-transformed cells cleared 45% of [3H]C2-ceramide and produced C2-SM, which accounted for 24% of total radioactivity. This enhanced production of C2-SM was also supported by an increase in the total SMS activity of cells (measured in vitro), such that SV40-transformed cells had SMS activity of 222 pmol/mg of protein/h, whereas wild type cells had 78 pmol/mg of protein/h of activity. Additional studies aimed at examining the SMS activity directed at ceramide produced in the plasma membrane. Treatment of cells with exogenous bacterial sphingomyelinase (SMase) for 25 min resulted in cleavage of 90-95% of total SM and the concomitant generation of ceramide. After bacterial SMase treatment, wild type WI38 cells cleared ceramide very slowly (19.2 pmol of ceramide/nmol of phosholipid Pi after 6 h of incubation) and hardly regenerated any SM. On the other hand, SV40-transformed cells cleared ceramide much faster (41.1 pmol/nmol of Pi after 6 h of incubation) and regenerated approximately 80% of the original SM. These results show that the enhanced SMS activity of transformed cells is particularly pronounced when ceramide is produced in the plasma membrane. Finally, several observations led us to consider the relationship of SMS to the "putative" phosphatidylcholine-specific phospholipase C (PC-PLC). We, therefore, tested the effects of D609, a purported PC-PLC-specific inhibitor on the activity of SMS. D609 inhibited SMS activity in vitro. In addition, cellular studies showed that SMS activity was dramatically inhibited by concentrations of D609 used previously to study PC-PLC (10-50 microg/ml). These results suggest SMS as an important biochemical target for D609, and they raise the distinct possibility that many of the roles of PC-PLC, especially in cell transformation, may be attributable to SMS.

Antioxidant and prooxidant role of beta-carotene in murine normal and tumor thymocytes: effects of oxygen partial pressure.

The effects of the partial pressure of oxygen (pO2) on antioxidant efficiency of beta-carotene in inhibiting radical-initiated lipid peroxidation were studied in murine normal and tumor thymocytes. At 150 mm Hg pO2 (the pressure of oxygen in normal air), beta-carotene acted as an antioxidant, inhibiting radical-induced lipid peroxidation in both normal and tumor thymocytes. At 760 mm Hg p02, beta-carotene lost its antioxidant activity in normal thymocytes and exhibited a dose-dependent prooxidant effect in tumor thymocytes. In these cells, the prooxidant effect of beta-carotene was also accompanied by an increase of endogenous alpha-tocopherol loss. beta-Carotene radical-trapping and autooxidation reactions were faster at 760 mm Hg pO2 than at 150 mm Hg pO2 in both normal and tumor thymocytes and the carotenoid was more rapidly consumed in tumor cells. These data point out a key role of the oxygen tension on the antioxidant effectiveness of beta-carotene. They also show a selective prooxidant effect of beta-carotene under 100% oxygen in tumor cells.

Antitumor effect of an oral administration of canthaxanthin on BALB/c mice bearing thymoma cells.

The antitumor effect of canthaxanthin in BALB/c mice bearing a transplantable thymoma was investigated. Male or female mice received two different doses of canthaxanthin (7 or 14 micrograms/g body wt/day) starting 15 days before tumor inoculation (7 x 10(7) cells i.p.). Canthaxanthin treatment delayed the appearance of macroscopic ascites and prolonged animal survival. This effect was dose dependent and more evident in females than in males. It appeared only when the carotenoid was administered before tumor transplantation. The antitumor efficacy of the carotenoid was related to its tissue incorporation. Canthaxanthin was incorporated in a dose-dependent manner in liver and thymoma cells and to a larger extent in females than in males. Our study shows the antitumor efficacy of canthaxanthin in vivo against a transplantable murine thymoma and points out the importance of dose, administration timing, and sex in the antitumor efficacy of this compound.

n-3 fatty acids induce oxidative modifications in human erythrocytes depending on dose and duration of dietary supplementation.

The present work was performed to study an optimal dose and duration of dietary n-3 polyunsaturated fatty acid (PUFA) supplementation that would not result in harmful modifications of oxidative cell metabolism. Forty healthy subjects were divided into four groups that received 2.5 g/d eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA), 5.1 g EPA + DHA/d, 7.7 g EPA + DHA/d, or placebo. Fatty acid composition, tocopherol status, and susceptibility to lipid peroxidation induced in vitro by 2,2'-azobis-(2-amidinopropane) (AAPH) were evaluated in human red blood cell (RBC) membranes on days 30 and 180. n-3 PUFA treatment increased EPA and DHA concentrations in RBC membranes in a time-dependent manner in all of the n-3 PUFA groups. These modifications occurred with concomitant dose- and time-dependent increases in the membrane unsaturation index. After 30 d of treatment with n-3 PUFAs, alpha-to-copherol significantly increased in RBC membranes of the intermediate- and high-dose groups. Because of the higher concentration of this antioxidant in these groups, the susceptibility of RBC membranes to peroxidation was decreased. However, after 180 d of treatment, alpha-tocopherol decreased to baseline values and AAPH-induced lipid peroxidation increased in a dose-dependent manner. These results show that high doses of dietary n-3 PUFAs, as well as long-time treatments, affect human RBC susceptibility to lipid peroxidation by changes in fatty acid composition and tocopherol content.

Effect of beta-carotene and canthaxanthin on tert-butyl hydroperoxide-induced lipid peroxidation in murine normal and tumor thymocytes.

It has been reported that the anticarcinogenic effect of carotenoids could be related to an antioxidant mechanism. The antioxidant efficiency of beta-carotene and canthaxanthin was evaluated in murine normal and tumor thymocytes. Normal and tumor cells were exposed under air to tert-butyl hydroperoxide (t-BOOH) and lipid peroxidation was measured in the absence or in the presence of the two carotenoids. Our results show that: (a) Both carotenoids, added at effective and comparable concentrations (from 1 to 50 microM), were able to inhibit t-BOOH-induced malondialdehyde formation in a dose-dependent manner. (b) Canthaxanthin was a more potent antioxidant that beta-carotene. (c) The inhibition of lipid peroxidation was greater in tumor thymocytes. (d) Carotenoids were consumed differentially during the incubation with the prooxidant. beta-Carotene was consumed faster than canthaxanthin and in a larger amount in tumor than in normal thymocytes. The addition of the iron chelator deferoxamine or the SH group reducing agent dithiothreitol reduced t-BOOH-induced beta-carotene consumption in tumor cells but not in normal ones. (e) The loss of endogeneous alpha-tocopherol induced by t-BOOH was enhanced by the addition of beta-carotene, suggesting the possibility of oxidative interactions between the two antioxidants. These results confirmed the antioxidant effectiveness of carotenoids in normal and tumor cells, although differences depending on the kind of cells and carotenoids used were found.

Dietary fish oil inhibits human erythrocyte Mg,NaK-ATPase.

The effects of long-term treatment with a high dose (7.7 g/day) of n-3 polyunsaturated fatty acids (PUFA) were studied for human red blood cells (RBCs). RBCs isolated from healthy subjects treated for 30 and 180 days with n-3 PUFA showed the following modifications: (1) a time dependent modification of membrane fatty acid composition with a concomitant increase in membrane lipid unsaturation; (2) an increase in lipid peroxidation, expressed as malondialdehyde release, induced in vitro by t-butyl hydroperoxide (t-BOOH); (3) a time-dependent decrease in susceptibility to hemolysis, expressed as K+ leakage, induced in vitro by t-BOOH; (4) a time-dependent decrease in total and ouabain-insensitive Mg,NaK-ATPase activity. These results suggest that long term dietary supplementation with high doses of n-3 PUFA significantly modifies RBC structure and function that might lead to harmful side effects.

The effect of fatty acid unsaturation on the antioxidant activity of beta-carotene and alpha-tocopherol in hexane solutions.

The effect of fatty acid unsaturation on the antioxidant ability of beta-carotene and alpha-tocopherol to inhibit azobis-isobutyronitrile AIBN)-induced malondialdehyde (MDA) formation is investigated in a hexane solution. A positive correlation is shown between the fatty acid unsaturation and MDA production in homogeneous solutions. Both beta-carotene and alpha-tocopherol act as chain-breaking antioxidants in our model, effectively suppressing AIBN-induced MDA formation. When alpha-tocopherol is added to fatty acid solutions, a lag phase of about 30 min is observed and a propagation phase is produced at a rate dependent on the degree of unsaturation and similar to that observed in the absence of the antioxidant. A specific inhibition of initiation phase by alpha-tocopherol is confirmed by its total consumption after 30 min of incubation with AIBN. On the other hand, when beta-carotene is added, a lag period is not observed and the inhibition of propagation phase progressively increases in relation to the degree of fatty acid unsaturation. These data present different antioxidant roles for beta-carotene and alpha-tocopherol in AIBN-induced lipid peroxidation and suggest that beta-carotene can be a very effective antioxidant in highly unsaturated membranes, such as those enriched with n-3 polyunsaturated fatty acids (PUFA).

n-3 PUFA and alpha-tocopherol control of tumor cell proliferation.

Subjects at high risk for colon cancer received different doses of fish oil on a 30-day randomized double-blind trial to evaluate the chemopreventive effect of n-3 fatty acids against colorectal cancer. Using rectal mucosal proliferation, assessed with 3H-thymidine autoradiography, fish oil induced in the treated groups but not in the placebo group a change in the proliferative pattern, which resulted similar to that observed in low risk population; in the same groups rectal mucosal n-3 fatty acid content increased, where arachidonic acid level decreased. Moreover, n-3 PUFA treatment induced modifications of Vitamin E status. The results suggest that n-3 PUFA could protect high-risk subjects from colon cancer by a mechanism involving a modulation of Vitamin E.