Antineoplastic Properties by Proapoptotic Mechanisms Induction of Inula viscosa and Its Sesquiterpene Lactones Tomentosin and Inuviscolide.

Cancer is a complex disease including approximately 200 different entities that can potentially affect all body tissues. Among the conventional treatments, radiotherapy and chemotherapy are most often applied to different types of cancers. Despite substantial advances in the development of innovative antineoplastic drugs, cancer remains one of the most significant causes of death, worldwide. The principal pitfall of successful cancer treatment is the intrinsic or acquired resistance to therapeutic agents. The development of more effective or synergistic therapeutic approaches to improve patient outcomes and minimize toxicity has become an urgent issue. Inula viscosa is widely distributed throughout Europe, Africa, and Asia. Used as a medicinal plant in different countries, I. viscosa has been characterized for its complex chemical composition in order to identify the bioactive compounds responsible for its biological activities, including anticancer effects. Sesquiterpene lactones (SLs) are natural, biologically active products that have attracted considerable attention due to their biological activities. SLs are alkylating agents that form covalent adducts with free cysteine residues within enzymes and key proteins favoring cancer cell cytotoxicity. They are effective inducers of apoptosis in several cancer cell types through different molecular mechanisms. This review focuses on recent advances in the cytotoxic effects of I. viscosa and SLs in the treatment of neoplastic diseases, with a special emphasis on their proapoptotic molecular mechanisms.

Tomentosin a Sesquiterpene Lactone Induces Antiproliferative and Proapoptotic Effects in Human Burkitt Lymphoma by Deregulation of Anti- and Pro-Apoptotic Genes.

(1) Tomentosin is the most representative sesquiterpene lactone extracted by I. viscosa. Recently, it has gained particular attention in therapeutic oncologic fields due to its anti-tumor properties. (2) In this study, the potential anticancer features of tomentosin were evaluated on human Burkitt's lymphoma (BL) cell line, treated with increasing tomentosin concentration for cytotoxicity screening. (3) Our data showed that both cell cycle arrest and cell apoptosis induction are responsible of the antiproliferative effects of tomentosin and may end in the inhibition of BL cell viability. Moreover, a microarray gene expression profile was performed to assess differentially expressed genes contributing to tomentosin activity. Seventy-five genes deregulated by tomentosin have been identified. Downregulated genes are enriched in immune-system pathways, and PI3K/AKT and JAK/STAT pathways which favor proliferation and growth processes. Importantly, different deregulated genes identified in tomentosin-treated BL cells are prevalent in molecular pathways known to lead to cellular death, specifically by apoptosis. Tomentosin-treatment in BL cells induces the downregulation of antiapoptotic genes such as BCL2A1 and CDKN1A and upregulation of the proapoptotic PMAIP1 gene. (4) Overall, our results suggest that tomentosin could be taken into consideration as a potential natural product with limited toxicity and relevant anti-tumoral activity in the therapeutic options available to BL patients.

Clarifying the molecular mechanism of tomentosin­induced antiproliferative and proapoptotic effects in human multiple myeloma via gene expression profile and genetic interaction network analysis.

Multiple myeloma (MM) is an aggressive B cell malignancy. Substantial progress has been made in the therapeutic context for patients with MM, however it still represents an incurable disease due to drug resistance and recurrence. Development of more effective or synergistic therapeutic approaches undoubtedly represents an unmet clinical need. Tomentosin is a bioactive natural sesquiterpene lactone extracted by various plants with therapeutic properties, including anti­neoplastic effects. In the present study, the potential antitumor activity of tomentosin was evaluated on the human RPMI­8226 cell line, treated with increasing tomentosin concentration for cytotoxicity screening. The data suggested that both cell cycle arrest and cell apoptosis could explain the antiproliferative effects of tomentosin and may result in the inhibition of RPMI­8226 cell viability. To assess differentially expressed genes contributing to tomentosin activity and identify its mechanism of action, a microarray gene expression profile was performed, identifying 126 genes deregulated by tomentosin. To address the systems biology and identify how tomentosin deregulates gene expression in MM from a systems perspective, all deregulated genes were submitted to enrichment and molecular network analysis. The Protein­Protein Interaction (PPI) network analysis showed that tomentosin in human MM induced the downregulation of genes involved in several pathways known to lead immune­system processes, such as cytokine­cytokine receptor interaction, chemokine or NF­κB signaling pathway, as well as genes involved in pathways playing a central role in cellular neoplastic processes, such as growth, proliferation, migration, invasion and apoptosis. Tomentosin also induced endoplasmic reticulum stress via upregulation of cyclic AMP­dependent transcription factor ATF­4 and DNA damage­inducible transcript 3 protein genes, suggesting that in the presence of tomentosin the protective unfolded protein response signaling may induce cell apoptosis. The functional connections analysis executed using the Connectivity Map tool, suggested that the effects of tomentosin on RPMI­8226 cells might be similar to those exerted by heat shock proteins inhibitors. Taken together, these data suggested that tomentosin may be a potential drug candidate for the treatment of MM.

Transmucosal Solid Lipid Nanoparticles to Improve Genistein Absorption via Intestinal Lymphatic Transport.

Genistein (GEN) is a soy-derived isoflavone that exhibits several biological effects, such as neuroprotective activity and the prevention of several types of cancer and cardiovascular disease. However, due to its poor water solubility and the extensive first-pass metabolism, the oral bioavailability of GEN is limited. In this work, solid lipid nanoparticles (SLN) were developed to preferentially reach the intestinal lymphatic vessels, avoiding the first-pass metabolism of GEN. GEN-loaded SLN were obtained by a hot homogenization process, and the formulation parameters were chosen based on already formulated studies. The nanoparticles were characterized, and the preliminary in vitro chylomicron formation was evaluated. The cell uptake of selected nanocarriers was studied on the Caco-2 cell line and intestinal mucosa. The SLN, characterized by a spherical shape, showed an average diameter (about 280 nm) suitable for an intestinal lymphatic uptake, good stability during the testing time, and high drug loading capacity. Furthermore, the intestinal mucosa and Caco-2 cells were found to uptake SLN. The approximately two-fold increase in particle size suggested a possible interaction between SLN and the lipid components of chylomicrons like phospholipid; therefore, the results may support the potential for these SLN to improve oral GEN bioavailability via intestinal lymphatic absorption.

Antiproliferative and proapoptotic effects of Inula viscosa extract on Burkitt lymphoma cell line.

Burkitt lymphoma is a very aggressive B-cell non-Hodgkin lymphoma. Although remarkable progress has been made in the therapeutic scenario for patients with Burkitt lymphoma, search and development of new effective anticancer agents to improve patient outcome and minimize toxicity has become an urgent issue. In this study, the antitumoral activity of Inula viscosa, a traditional herb obtained from plants collected on the Asinara Island, Italy, was evaluated in order to explore potential antineoplastic effects of its metabolites on Burkitt lymphoma. Raji human cell line was treated with increasing Inula viscosa extract concentration for cytotoxicity screening and subsequent establishment of cell cycle arrest and apoptosis. Moreover, gene expression profiles were performed to identify molecular mechanisms involved in the anticancer activities of this medical plant. The Inula viscosa extract exhibited powerful antiproliferative and cytotoxic activities on Raji cell line, showing a dose- and time-dependent decrease in cell viability, obtained by cell cycle arrest in the G2/M phase and an increase in cell apoptosis. The treatment with Inula viscosa caused downregulation of genes involved in cell cycle and proliferation (c-MYC, CCND1) and inhibition of cell apoptosis (BCL2, BCL2L1, BCL11A). The Inula viscosa extract causes strong anticancer effects on Burkitt lymphoma cell line. The molecular mechanisms underlying such antineoplastic activity are based on targeting and downregulation of genes involved in cell cycle and apoptosis. Our data suggest that Inula viscosa natural metabolites should be further exploited as potential antineoplastic agents against Burkitt lymphoma.

Antiproliferative activity of vanadium compounds: effects on the major malignant melanoma molecular pathways.

Malignant melanoma (MM) is the most fatal skin cancer, whose incidence has critically increased in the last decades. Recent molecular therapies are giving excellent results in the remission of melanoma but often they induce drug resistance in patients limiting their therapeutic efficacy. The search for new compounds able to overcome drug resistance is therefore essential. Vanadium has recently been cited for its anticancer properties against several tumors, but only a few data regard its effect against MM. In a previous work we demonstrated the anticancer activity of four different vanadium species towards MM cell lines. The inorganic anion vanadate(v) (VN) and the oxidovanadium(iv) complex [VO(dhp)2] (VS2), where dhp is 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate, showed IC50 values of 4.7 and 2.6 µM, respectively, against the A375 MM cell line, causing apoptosis and cell cycle arrest. Here we demonstrate the involvement of Reactive Oxygen Species (ROS) production in the pro-apoptotic effect of these two V species and evaluate the activation of different cell cycle regulators, to investigate the molecular mechanisms involved in their antitumor activity. We establish that VN and VS2 treatments reduce the phosphorylation of extracellular-signal regulated kinase (ERK) by about 80%, causing the deactivation of the mitogen activated protein kinase (MAPK) pathway in A375 cells. VN and VS2 also induce dephosphorylation of the retinoblastoma protein (Rb) (VN 100% and VS2 90%), together with a pronounced increase of cyclin-dependent kinase inhibitor 1 p21 (p21Cip1) protein expression up to 1800%. Taken together, our results confirm the antitumor properties of vanadium against melanoma cells, highlighting its ability to induce apoptosis through generation of ROS and cell cycle arrest by counteracting MAPK pathway activation and strongly inducing p21Cip1 expression and Rb hypo-phosphorylation.

NF90 regulation of immune factor expression in response to malaria antigens.

Nuclear factor 90 (NF90) is a dual DNA- and RNA-binding protein expressed ubiquitously in mammalian cells, including monocytes. Here, to elucidate the function of NF90 in the immune response, we analyzed systematically its influence on gene expression programs in the human monocytic cell line THP-1 expressing normal or reduced NF90 levels. RNA sequencing analysis revealed many mRNAs showing differential abundance in NF90-silenced cells, many of them encoding proteins implicated in the response to immune stimuli and malaria infection. The transcription of some of them (e.g. TNF, LILRB1, and CCL2 mRNAs) was modulated by silencing NF90. Ribonucleoprotein immunoprecipitation (RIP) analysis further revealed that a subset of these mRNAs associated directly with NF90. To understand how NF90 influenced globally the immune response to malaria infection, lysates of red blood cells infected with Plasmodium falciparum (iRBC lysates) or uninfected/mock-infected (uRBC lysates) were used to treat THP-1 cells as a surrogate of malaria infection. NF90 affected the stability of a few target mRNAs, but influenced more generally the translation and secretion of the encoded cytokines after treatment with either uRBC or iRBC lysates. Taken together, these results indicate that NF90 contributes to repressing the immune response in cells responding to P. falciparum infection and suggest that NF90 can be a therapeutic target in malaria.

Increasing protective activity of genistein by loading into transfersomes: A new potential adjuvant in the oxidative stress-related neurodegenerative diseases?

BACKGROUND: Genistein is a soy-derived isoflavone and phytoestrogen with antioxidant and neuroprotective activity. Genistein has intrinsically low oral bioavailability that affects its dose-response activities. PURPOSE: Nanotechnologies were used to obtain the delivery of genistein to the brain: lipid-based nanovesicles, transfersomes, loaded with the phytoestrogen were developed as potential therapeutic or preventive strategy against neurodegenerative diseases by intranasal administration. METHODS: Phosphatidylcholine from soybean and different edge activators were used to prepare transfersomes. The effect of selected nanovesicles on the oxidative damage was studied in PC12 cell line. RESULTS: Suitable nanovesicles as carrier of genistein were obtained; their composition affects deformability, drug permeation behavior and cytotoxicity. In particular, the formulation containing Span 80, GEN-TF2, showed efficiency of internalization into the cell and it was able to attenuate ROS formation and to reduce the amount of apoptotic cells generated by H2O2 treatment compared to genistein. CONCLUSION: GEN-TF2 was able to reduce the oxidative damage suggesting a possible antioxidant role of this drug delivery system. These obtained data confer to GEN-TF2 a potential antioxidant activity and then it could be used as adjuvant therapy in oxidative stress-related neurodegenerative diseases.

Radio Electric Asymmetric Conveyer (REAC) technology to obviate loss of T cell responsiveness under simulated microgravity.

Alterations of the gravitational environment are likely to modify cell behavior. Several studies have proven that T cells are sensitive to gravity alterations and that microgravity conditions may induce immunosuppression and weakened T cell immune response in humans during spaceflights. The aim of this work was to elucidate if a specific treatment of Radio Electric Asymmetric Conveyer (REAC) technology could restore, after mitogenic activation (Con A), a correct expression of cytokine IL2 gene and its receptor IL2R alpha, which are inhibited in T cells under microgravity conditions, as demonstrated in several studies. The results of this study, conducted in microgravity simulated with Random Positioning Machine (RPM), confirm the T cell activation recovery and offer the evidence that REAC technology could contribute to the understanding of T cell growth responsiveness in space, reducing the impact of weightlessness on the immune system experienced by humans in long duration space missions.

Effect of exposure to CeO2 nanoparticles on ram spermatozoa during storage at 4 °C for 96 hours.

BACKGROUND: Cerium oxide nanoparticles (CeO2 NPs) are able to store and release oxygen, conferring them scavenger activity against oxidative stress. However, their effects in reproductive systems are not yet well understood. The aim of the study was to investigate the effects of exposure of refrigerated ram semen to CeO2 NPs for 96 h on the main structural and kinematic parameters of spermatozoa. METHODS: The ejaculates of 5 Sarda rams were collected, pooled and diluted in a soybean lecithin extender. Samples were exposed to increasing doses of CeO2 NPs (0, 44 and 220 µg/mL) and stored at 4 °C for 96 h. Analyses of kinematic parameters (computer assisted sperm analysis, CASA), integrity of membranes (PI/PSA staining), ROS production (H2DCFDA staining) and DNA damage (sperm chromatin structure assay with acridine orange, SCSA) were performed every 24 h (0, 24, 48, 72 and 96 h of incubation). The experiment was carried out in 6 replicates. Data were analysed by repeated measures ANOVA with Bonferroni's as post hoc test. When the assumption of normality was not met (ROS), non-parametric Kruskal-Wallis rank test was carried out. RESULTS: Exposure of ram spermatozoa to increasing doses of CeO2 NPs had a beneficial effect on the main motility parameters from 48 h of incubation onward. Velocity of sperm cells was enhanced in the groups exposed to CeO2 NPs compared to the control. Incubation with NPs had beneficial effects on the integrity of plasma membranes of spermatozoa, with higher percentage of damaged cells in the control group compared to the exposed ones. Production of ROS was not affected by exposure to NPs and its levels rose at 96 h of incubation. The integrity of DNA remained stable throughout the 96 h of storage regardless of co-incubation with NPs. CONCLUSIONS: We reported beneficial effects of CeO2 NPs on kinematic and morphologic parameters of ram semen, such as motility and membrane integrity following 96 h of exposure. Furthermore, we also proved no genotoxic effects of CeO2 NPs. These effects could not be related to an antioxidant activity of CeO2 NPs, since ROS levels in exposed cells were similar to those of unexposed ones.

Intrahepatic bile duct primary cilia in biliary atresia.

AIM: The etiopathogenesis of non-syndromic biliary atresia (BA) is obscure. The primary aim was to investigate intrahepatic bile duct cilia (IHBC) in BA at diagnosis and its correlation with clinical outcome. The secondary aim was to analyze IHBC in routine paraffin-embedded liver biopsies using conventional scanning electron microscopy (SEM). METHODS: Surgical liver biopsies taken at diagnosis from 22 BA infants (age range, 39-116 days) and from eight children with non-BA chronic cholestasis (age range, 162 days -16.8 years) were evaluated for IHBC by immunofluorescence (IF) and SEM. A minimum 18-month follow-up after surgery was available for all patients. RESULTS: By IF, cilia were present in 6/8 (75%) non-BA but only in 3/22 (14%) BA cases, and cilia were reduced or absent in 19/22 (86%) BA and 2/8 (25%) non-BA livers (P < 0.01). In BA, cilia presence was found to be associated with clearance of jaundice at 6-month follow-up (P < 0.05). However, high overall survival rates with native liver, >90% at 12 months, and >70% at 24 months post-surgery, were recorded regardless of cilia presence/absence at diagnosis. Electron microscopy was able to detect bile ducts and cilia in routine liver biopsies, revealing significant abnormalities in 100% BA livers. CONCLUSIONS: The presence of IHBC in BA livers at the diagnosis was associated with resolution of cholestasis, although was not predictive of short-term survival with native liver. Scanning electron microscopy represents a powerful new tool to study routine liver biopsies in biliary disorders. Cilia dysfunction in BA pathogenesis and/or disease progression warrants further investigation.

Human interferon regulatory factor 5 homologous epitopes of Epstein-Barr virus and Mycobacterium avium subsp. paratuberculosis induce a specific humoral and cellular immune response in multiple sclerosis patients.

BACKGROUND: A large number of reports indicate the association of Epstein-Barr virus (EBV), and Mycobacterium avium subsp. paratuberculosis (MAP) with multiple sclerosis (MS). OBJECTIVE: To gain a better understanding of the role of these two pathogens, we investigated the host response induced by selected antigenic peptides. METHODS: We examined both humoral and cell-mediated responses against peptides deriving from EBV tegument protein BOLF1, the MAP_4027 and the human interferon regulatory factor 5 (IRF5424-434) homolog in several MS patients and healthy controls (HCs). RESULTS: Antibodies against these peptides were highly prevalent in MS patients compared to HCs. Concerning MS patients, BOLF1305-320, MAP_402718-32 and IRF5424-434 peptides were able to induce mainly Th1-related cytokines secretion, whereas Th2-related cytokines were down-regulated. Flow cytometry analyses performed on a subset of MS patients highlighted that these peptides were capable of inducing the release of pro-inflammatory cytokines: IFN-γ and TNF-α by CD4(+) and CD8(+) T lymphocytes, and IL-6 and TNF-α by CD14(+) monocyte cells. CONCLUSION: Our data demonstrated that both EBV and MAP epitopes elicit a consistent humoral response in MS patients compared to HCs, and that the aforementioned peptides are able to induce a T-cell-mediated response that is MS correlated.

Molecular changes induced by the curcumin analogue D6 in human melanoma cells.

BACKGROUND: In a previous report, we described the in vitro and in vivo antiproliferative and proapoptotic activity of a hydroxylated biphenyl (D6), a structural analogue of curcumin, on malignant melanoma and neuroblastoma tumours. In this paper, we investigated the molecular changes induced by such a compound, underlying cell growth arrest and apoptosis in melanoma cells. RESULTS: To shed light on the mechanisms of action of D6, we firstly demonstrated its quick cellular uptake and subsequent block of cell cycle in G2/M phase transition. A gene expression profile analysis of D6-treated melanoma cells and fibroblasts was then carried out on high density microarrays, to assess gene expression changes induced by this compound. The expression profile study evidenced both an induction of stress response pathways and a modulation of cell growth regulation mechanisms. In particular, our data suggest that the antiproliferative and proapoptotic activities of D6 in melanoma could be partially driven by up-regulation of the p53 signalling pathways as well as by down-regulation of the PI3K/Akt and NF-kB pathways. Modulation of gene expression due to D6 treatment was verified by western blot analysis for single proteins of interest, confirming the results from the gene expression profile analysis. CONCLUSIONS: Our findings contribute to the understanding of the mechanisms of action of D6, through a comprehensive description of the molecular changes induced by this compound at the gene expression level, in agreement with the previously reported anti-tumour effects on melanoma cells.

In vitro activity of the αvß3 integrin antagonist RGDechi-hCit on malignant melanoma cells.

BACKGROUND: In malignant melanoma (MM), overexpression of αvß3 integrin is linked to a more metastatic phenotype. Development of anti-αvß3 agents able to counteract melanoma progression would be helpful for disease treatment. A new selective ligand of αvß3, RGDechi-hCit, has anti-angiogenic properties against endothelial cells in animal angiogenesis models. The aim of this study was to evaluate the in vitro effects of the RGDechi-hCit peptide on MM cell lines. MATERIALS AND METHODS: Cytofluorimetric analysis characterized the cell surface expression of αvß3 integrin on seven MM cell lines: A375, WM266-4, SK-Mel-28, Sbcl2, LB24Dagi, PR-Mel and PNP-Mel. Cell proliferation, adhesion, and migration assays were carried out using the αvß3-antagonist RGDechi-hCit. RESULTS: Proliferation was not significantly inhibited by RGDechi-hCit, although striking morphological changes were detected in MM cell lines highly expressing αvß3. Conversely, assays on fibronectin-coated plates showed a significant RGDechi-hCit dose-dependent inhibitory effect on both adhesion and migration. CONCLUSION: The data demonstrate anti-adhesion and anti-migration, but not antiproliferative, activities of RGDechi-hCit against MM cells.

The Rel/NF-κB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity.

This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in µg. Immunosuppression during spaceflight is a major barrier to safe, long-term human space habitation and travel. The goals of these experiments were to prove that µg was the cause of impaired T cell activation during spaceflight, as well as understand the mechanisms controlling early T cell activation. T cells from four human donors were stimulated with Con A and anti-CD28 on board the ISS. An on-board centrifuge was used to generate a 1g simultaneous control to isolate the effects of µg from other variables of spaceflight. Microarray expression analysis after 1.5 h of activation demonstrated that µg- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly, differentially down-regulated in µg. Importantly, several key immediate early genes were inhibited in µg. In particular, transactivation of Rel/NF-κB, CREB, and SRF gene targets were down-regulated. Expression of cREL gene targets were significantly inhibited, and transcription of cREL itself was reduced significantly in µg and upon anti-CD3/anti-CD28 stimulation in simulated µg. Analysis of gene connectivity indicated that the TNF pathway is a major early downstream effector pathway inhibited in µg and may lead to ineffective proinflammatory host defenses against infectious pathogens during spaceflight. Results from these experiments indicate that µg was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes.

Exposure to modeled microgravity induces metabolic idleness in malignant human MCF-7 and normal murine VSMC cells.

We investigated the effect of modeled microgravity (MMG) on normal vascular smooth muscle cells (VSMC) and neoplastic human breast cancer cells (MCF-7). In both cell types, MMG induced partial arrest in G2M and increased p14-3-3, HSP70, HSP60 and p21 expression. Cells synchronized by 24h starvation reentered the normal cycle within 24h if released in complete medium and exposed to normal gravity, but not if exposed to MMG. Similarly, MMG prevented VSMC and MCF-7 cells from overcoming growth arrest and re-synthesizing DNA. This study shows that cells adjust their metabolic rate in response to MMG.

Exposure of human lymphocytes and lymphoblastoid cells to simulated microgravity strongly affects energy metabolism and DNA repair.

Exposure of freshly drawn lymphocytes and lymphoblastoid cells (LB and COR3) to simulated microgravity decreased the intracellular ATP concentration to 50%-40% of the value found in normal growth conditions. The decrease was reversible although recovery to normal values occurred only slowly both in lymphocytes and in lymphoblastoid cells. Poly(ADP-ribose) polymerase (PARP ) activity was increased indicating that cells exposed to conditions of reduced gravitation experience stress. Exposure to microgravity forces cells into a condition of metabolic quiescence in which they appear to be particularly sensitive to subsequent exposures to a genotoxic agent. Thus, treatment of cells with the strong redox agent potassium bromate under microgravity conditions, indicated an impairment in repair of DNA 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidized derivative of deoxyguanosine. We conclude that gravitational modulation of the kind routinely obtained under laboratory conditions and during spaceflights is a stressful process to which cells appear to be extremely sensitive. These effects may reflect the physiological alterations observed in astronauts and in animals following spaceflights or exposure to conditions of simulated microgravity.

Microgravity alters basal and insulin-mediated metabolic activity of normal and neoplastic cells.

In this paper we report the behaviour of normal vascular smooth muscle cells and transformed breast cancer cells under normal versus simulated microgravity conditions by comparing cell proliferation, Glucose transport, Methionine uptake and protein synthesis. Modeled microgravity profoundly affects cell growth (especially in normal cells) and Glucose or Methionine metabolism (although to different extent in the two cell lines). Since both cells own responsive insulin receptors, the comparison was extended to insulin-stimulated versus unstimulated conditions. We report that the detected metabolic changes were strongly enhanced when the cells were simultaneously stimulated with insulin and subjected to modeled microgravity stress. Such observations may have important returns for human health in space; they deserve further attention.

Creating conditions similar to those that occur during exposure of cells to microgravity induces apoptosis in human lymphocytes by 5-lipoxygenase-mediated mitochondrial uncoupling and cytochrome c release.

Creating conditions similar to those that occur during exposure of cells to microgravity induced a sixfold increase of apoptotic bodies and DNA fragments in human lymphocytes, paralleled by an early (within 2 h) fourfold increase in 5-lipoxygenase (5-LOX) activity and a fivefold decrease in mitochondrial membrane potential and increase in cytochrome c release (within 4 and 8 h, respectively). Similar membrane potential and cytochrome c release were observed in isolated mitochondria treated with physiological amounts of 5-LOX and were enhanced by creating conditions similar to those that occur during exposure of cells to microgravity. 5-LOX inhibitors, 5,8,11,14-eicosatetraynoic acid and caffeic acid, completely prevented apoptosis, whereas the phospholipase A(2) inhibitor methyl-arachidonoyl fluorophosphonate and the 5-LOX activating protein inhibitor MK886 reduced it to 65-70%. The intracellular calcium chelator EGTA-acetoxymethylester reduced 5-LOX activity and apoptosis to 30-40% of controls, whereas the p38 mitogen-activated protein kinase inhibitor SB203580 was ineffective. The caspase-3 and caspase-9 inhibitors Z-Asp(OCH(3))-Glu(OCH(3))-Val-Asp(OCH(3))-fluoromethylketone (FMK) and Z-Leu-Glu(OCH(3))-His-Asp(OCH(3))-FMK reduced apoptotic bodies to 25-30% of the control cells. Finally, creating conditions similar to those that occur during exposure of cells to microgravity did not induce apoptosis in human lymphoma U937 cells, which did not express an active 5-LOX.