Involvement of CCR5 on interstitial macrophages in the development of lung fibrosis in severe asthma.

CCR5 may be involved in the pathogenesis of asthma; however, the underlying mechanisms remain unclear. In comparison with a mild asthma model, subepithelial fibrosis was more severe and CCR5 gene expression in the lungs was significantly higher in our recently developed murine model of steroid-resistant severe asthma. Treatment with the CCR5 antagonist, maraviroc, significantly suppressed the development of subepithelial fibrosis in bronchi, whereas dexamethasone did not. On the other hand, increases in leukocytes related to type 2 inflammation, eosinophils, Th2 cells, and group 2 innate lymphoid cells in the lungs were not affected by the treatment with maraviroc. Increases in neutrophils and total macrophages were also not affected by the CCR5 antagonist. However, increases in transforming growth factor (TGF)-ß-producing interstitial macrophages (IMs) were significantly reduced by maraviroc. The present results confirmed increases in CCR5-expressing IMs in the lungs of the severe asthma model. In conclusion, CCR5 on IMs plays significant roles in the development of subepithelial fibrosis in severe asthma through TGF-ß production in the lungs.

Pronecroptotic Therapy Using Ceramide Nanoliposomes Is Effective for Triple-Negative Breast Cancer Cells.

Regulated necrosis, termed necroptosis, represents a potential therapeutic target for refractory cancer. Ceramide nanoliposomes (CNLs), considered potential chemotherapeutic agents, induce necroptosis by targeting the activating protein mixed lineage kinase domain-like protein (MLKL). In the present study, we examined the potential of pronecroptotic therapy using CNLs for refractory triple-negative breast cancer (TNBC), for which there is a lack of definite and effective therapeutic targets among the various immunohistological subtypes of breast cancer. MLKL mRNA expression in tumor tissues was significantly higher in TNBC patients than in those with non-TNBC subtypes. Similarly, among the 50 breast cancer cell lines examined, MLKL expression was higher in TNBC-classified cell lines. TNBC cell lines were more susceptible to the therapeutic effects of CNLs than the non-TNBC subtypes of breast cancer cell lines. In TNBC-classified MDA-MB-231 cells, the knockdown of MLKL suppressed cell death induced by CNLs or the active substance short-chain C6-ceramide. Accordingly, TNBC cells were prone to CNL-evoked necroptotic cell death. These results will contribute to the development of CNL-based pronecroptotic therapy for TNBC.

Ceramide Nanoliposomes as Potential Therapeutic Reagents for Asthma.

Ceramides are an emerging class of anti-inflammatory lipids, and nanoscale ceramide-delivery systems are potential therapeutic strategies for inflammatory diseases. This study investigated the therapeutic effects of ceramide nanoliposomes (CNL) on type 2 inflammation-based asthma, induced by repeated ovalbumin (OVA) challenges. Asthmatic mice intratracheally treated with ceramide-free liposomes (Ghost) displayed typical airway remodeling including mucosal accumulation and subepithelial fibrosis, whereas, in CNL-treated mice, the degree of airway remodeling was significantly decreased. Compared to the Ghost group, CNL treatment unexpectedly failed to significantly influence formation of type 2 cytokines, including IL-5 and IL-13, known to facilitate pathogenic production of airway mucus predominantly comprising MUC5AC mucin. Interestingly, CNL treatment suppressed OVA-evoked hyperplasia of MUC5AC-generating goblet cells in the airways. This suggests that CNL suppressed goblet cell hyperplasia and airway mucosal accumulation independently of type 2 cytokine formation. Mechanistically, CNL treatment suppressed cell growth and EGF-induced activation of Akt, but not ERK1/2, in a human lung epithelial cell culture system recapitulating airway goblet cell hyperplasia. Taken together, CNL is suggested to have therapeutic effects on airway remodeling in allergic asthma by targeting goblet cell hyperplasia. These findings raise the potential of ceramide-based therapies for airway diseases, such as asthma.

Cellular concentrations of plasmalogen species containing a polyunsaturated fatty acid significantly increase under hypoxia in human colorectal cancer, Caco2 cells.

Plasmalogen localized in the raft of mammalian cell membranes plays a role in the storage of polyunsaturated fatty acid (PUFA), and exists to a higher extent in malignant cells that survive, and even grow in hypoxic conditions. The biosynthesis of plasmalogen in mammalian cells has been reported to depend on aerobic conditions. Using liquid chromatography-tandem mass spectrometry, we found that the intracellular concentration of plasmalogen species containing a PUFA at the sn-2-position did not change for two days from the start of hypoxic culture in human colorectal cancer-derived Caco2 cells. At the third day of hypoxia, Caco2 cells showed the average increase rate of 2.6 times in ethanolamine plasmalogen and 2.9 times in choline plasmalogen depending on the molecular species compared with those in the second day of hypoxia. In normoxic culture, there was little quantitative change in any species of both ethanolamine and choline plasmalogens for three days. The up-regulations of mRNA of Ca2+-independent phospholipase A2ß and cytoplasmic phospholipase A2γ as well as the down-regulation of lysoplasmalogenase observed in hypoxia were suggested to be responsible for the increase of plasmalogen in Caco2 cells under hypoxia.

Involvement of small extracellular vesicle-derived TIE-1 in the chemoresistance of ovarian cancer cells.

BACKGROUND: Ovarian cancer is the most lethal gynecologic malignancy due to the tumor's acquisition of chemoresistance to platinum-based chemotherapy. To solve this problem, we conducted RNAi-based large-scale screening and determined that tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE-1) is a key molecule involved in the platinum resistance of ovarian cancer cells. Recently, a variety of studies have investigated that small extracellular vesicles (sEVs) contribute to the communication between cancer cells, including the development of chemoresistance in ovarian cancer. The purpose of our study is to determine if sEVs-derived TIE-1 is involved in the chemoresistance of ovarian cancer cells. MATERIALS AND METHODS: TIE-1-overexpressed TOV112D cells, termed TOV112DTIE-1 cells, were established, and sEVs were isolated from TOV112DTIE-1 cells supernatants by ultracentrifugation. We assessed cisplatin sensitivity in recipient cells with TOV112DTIE-1-derived sEVs by cell-Titer Glo kit. We also asked whether sEV-derived TIE-1 suppressed the DNA damage response in recipient cells and evaluated the DNA damage response by counting cells positive for DNA damage foci. RESULTS: TIE-1 was contained within sEVTIE-1 derived from the cellular supernatant of TOV112DTIE-1. We showed that sEV-derived TIE-1 decreased chemosensitivity to cisplatin by suppressing the DNA damage response in recipient cells. CONCLUSION: Our findings suggest that sEV-derived TIE-1 could be a new therapeutic target for refractory ovarian cancer.

Ceramide synthase 2-C24:1 -ceramide axis limits the metastatic potential of ovarian cancer cells.

Regulation of sphingolipid metabolism plays a role in cellular homeostasis, and dysregulation of these pathways is involved in cancer progression. Previously, our reports identified ceramide as an anti-metastatic lipid. In the present study, we investigated the biochemical alterations in ceramide-centered metabolism of sphingolipids that were associated with metastatic potential. We established metastasis-prone sublines of SKOV3 ovarian cancer cells using an in vivo selection method. These cells showed decreases in ceramide levels and ceramide synthase (CerS) 2 expression. Moreover, CerS2 downregulation in ovarian cancer cells promoted metastasis in vivo and potentiated cell motility and invasiveness. Moreover, CerS2 knock-in suppressed the formation of lamellipodia required for cell motility in this cell line. In order to define specific roles of ceramide species in cell motility controlled by CerS2, the effect of exogenous long- and very long-chain ceramide species on the formation of lamellipodia was evaluated. Treatment with distinct ceramides increased cellular ceramides and had inhibitory effects on the formation of lamellipodia. Interestingly, blocking the recycling pathway of ceramides by a CerS inhibitor was ineffective in the suppression of exogenous C24:1 -ceramide for the formation of lamellipodia. These results suggested that C24:1 -ceramide, a CerS2 metabolite, predominantly suppresses the formation of lamellipodia without the requirement for deacylation/reacylation. Moreover, knockdown of neutral ceramidase suppressed the formation of lamellipodia concomitant with upregulation of C24:1 -ceramide. Collectively, the CerS2-C24:1 -ceramide axis, which may be countered by neutral ceramidase, is suggested to limit cell motility and metastatic potential. These findings may provide insights that lead to further development of ceramide-based therapy and biomarkers for metastatic ovarian cancer.

Study Profile of the Tohoku Medical Megabank Community-Based Cohort Study.

BACKGROUND: We established a community-based cohort study to assess the long-term impact of the Great East Japan Earthquake on disaster victims and gene-environment interactions on the incidence of major diseases, such as cancer and cardiovascular diseases. METHODS: We asked participants to join our cohort in the health check-up settings and assessment center based settings. Inclusion criteria were aged 20 years or over and living in Miyagi or Iwate Prefecture. We obtained information on lifestyle, effect of disaster, blood, and urine information (Type 1 survey), and some detailed measurements (Type 2 survey), such as carotid echography and calcaneal ultrasound bone mineral density. All participants agreed to measure genome information and to distribute their information widely. RESULTS: As a result, 87,865 gave their informed consent to join our study. Participation rate at health check-up site was about 70%. The participants in the Type 1 survey were more likely to have psychological distress than those in the Type 2 survey, and women were more likely to have psychological distress than men. Additionally, coastal residents were more likely to have higher degrees of psychological distress than inland residents, regardless of sex. CONCLUSION: This cohort comprised a large sample size and it contains information on the natural disaster, genome information, and metabolome information. This cohort also had several detailed measurements. Using this cohort enabled us to clarify the long-term effect of the disaster and also to establish personalized prevention based on genome, metabolome, and other omics information.

Potential of Tyrosine Kinase Receptor TIE-1 as Novel Therapeutic Target in High-PI3K-Expressing Ovarian Cancer.

Tyrosine kinase receptor TIE-1 plays a critical role in angiogenesis and blood-vessel stability. In recent years, increased TIE-1 expression has been observed in many types of cancers; however, the biological significance and underlying mechanisms remain unknown. Thus, in the present study, we investigated the tumor biological functions of TIE-1 in ovarian cancer. The treatment of SKOV3 ovarian-cancer cells with siRNA against TIE-1 decreased the expression of key molecules in the PI3K/Akt signaling pathway, such as p110α and phospho-Akt, suggesting that TIE-1 is related to the PI3K/Akt pathway. Furthermore, the knockdown of TIE-1 significantly decreased cell proliferation in high-PI3K-expressing cell lines (SKOV3, CAOV3) but not low-PI3K-expressing cell lines (TOV112D, A2780). These results suggested that inhibition of TIE-1 decreases cell growth in high-PI3K-expressing cells. Moreover, in low-PI3K-expressing TOV112D ovarian-cancer cells, TIE-1 overexpression induced PI3K upregulation and promoted a PI3K-mediated cell proliferative phenotype. Mechanistically, TIE-1 participates in cell growth and proliferation by regulating the PI3K/Akt signaling pathway. Taken together, our findings strongly implicate TIE-1 as a novel therapeutic target in high-PI3K-expressing ovarian-cancer cells.

C6-ceramide Inhibits the Motility of Anaplastic Thyroid Carcinoma Cells.

BACKGROUND: Anaplastic thyroid carcinoma (ATC) is an aggressive type of thyroid cancer, and its metastasis requires cell motility. Ceramide is involved in a variety of biological processes, including inflammation, cell signaling, cell motility, and induction of apoptosis, however has not previously been reported to inhibit the motility of ATC cells. We evaluated the effect of short chain C6-ceramide on motility of ATC cells. METHODS: Cell motility of 8305C thyroid carcinoma cell line treated with C6-ceramide was assessed using a transwell migration assay and a pseudopodia formation assay. RESULTS: Treatment with 10 µM C6-ceramide resulted in significantly fewer migratory cells than control treatment in a transwell migration assay (P < 0.002). In condition medium, 82.6% of C6-ceramide-treated cells formed lamellipodia. Importantly, treatment with 10 µM C6-ceramide drastically decreased the number of cells forming lamellipodia by 17.6% (P < 0.01). CONCLUSION: Our results suggest that treatment with a low concentration of ceramide may prevent metastasis and recurrence of ATC by inhibiting cell motility. Further studies are necessary to investigate the mechanism of inhibition of cell motility by ceramide. Ceramide shows promise as a therapeutic treatment for ATC.

Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice.

Type 1 regulatory T (Tr1) cells are CD4+ T cells that produce a large amount of IL-10, an anti-inflammatory cytokine. However, it has not been fully elucidated whether Tr1 cells suppress allergic asthma. In this study, the effects of adoptive transfer of in vitro-induced Tr1 cells on allergic asthma were evaluated. Splenocytes from ovalbumin (OVA)-sensitized BALB/c mice were cultured with OVA, IL-21, IL-27, and TGF-ß. After culture, IL-10-producing CD4+ T cells were isolated by Dynabeads mouse CD4 and IL-10 secretion assay, and analyzed by flow cytometry. Purified Tr1 cells (IL-10+ CD4+ T cells) were intravenously injected into OVA-sensitized BALB/c mice. The recipient mice were intratracheally challenged with OVA. Airway hyperresponsiveness to methacholine was assessed by the forced oscillation technique, followed by bronchoalveolar lavage (BAL). Almost all of the induced IL-10-producing CD4+ T cells were negative for interferon-γ, IL-4, IL-17A, and forkhead box P3, suggesting that the cells were Tr1 cells. The adoptive transfer of Tr1 cells significantly suppressed the development of airway hyperresponsiveness, and increases in IL-5, eosinophils, and neutrophils in BAL fluid. In conclusion, we demonstrated that Tr1 cells suppressed allergic asthma in mice.

[Analyses of Foxp3+ Treg cells and Tr1 cells in subcutaneous immunotherapy-treated allergic individuals in humans and mice].

Subcutaneous immunotherapy (SCIT) is a causative treatment for allergic diseases. More recently, it has become clear that regulatory T (Treg) cells are increased by SCIT. Treg cells are generally divided into two main groups: 1) CD25+ Foxp3+ CD4+ T cells (Foxp3+ Treg cells) and 2) IL-10-producing Foxp3- CD4+ T cells (Tr1 cells). We demonstrated that the number of Tr1 cells in peripheral blood mononuclear cells in SCIT-treated pollinosis patients were significantly higher than that in non-SCIT-treated patients, but Foxp3+ Treg cells were not. Consistent with the results of human peripheral blood, Tr1 cells were increased in the lungs of asthmatic mice by SCIT, but Foxp3+ Treg cells were not. Moreover, in vitro-induced Tr1 cells were responded to the antigen to produce a large amount of IL-10 in in vitro and in vivo. Adoptive transfer of the induced Tr1 cells significantly suppressed the development of asthma. In any species of human and mouse, the increase in Tr1 cells rather than Foxp3+ Treg cells could be important for the effects of SCIT. The increased Tr1 cells by SCIT functionally suppressed allergic asthma probably via production of IL-10 in response to the specific antigen. Therefore, analyses of the induction mechanisms of Tr1 cells and search for compounds which induce Tr1 cells are thought to lead to development of more efficient SCIT.

La-Related Protein 4 as a Suppressor for Motility of Ovarian Cancer Cells.

The La-related proteins (LARPs) are a family of RNA binding proteins that control the degradation and stabilization of RNAs. As emerging research reveals the biology of each LARP, it is evident that LARPs are dysregulated in some types of cancer. Upregulation of cell motility potentiates the metastatic potential of ovarian cancer cells; however, the roles of LARPs in cell motility remain unknown. In the present study, we investigated the roles of LARPs in the progression of ovarian cancer using SKOV3 human ovarian cancer cells and a public database that integrates microarray-based gene expression data and clinical data. To explore the involvement of LARPs in the cell motility, we performed RNA interference screening for LARP mRNAs in SKOV3 cells. The screening identified LARP4 as a potential suppressor of the formation of lamellipodia. Conversely, enforced expression of LARP4 suppressed the formation of lamellipodia. Moreover, cell migration was significantly increased in LARP4-depleted SKOV3 cells. Mechanistically, LARP4 depletion was associated with the decrease in RhoA protein expression. These results suggest that LARP4 may limit RhoA-dependent cell motility. In a mouse xenograft model with SKOV3 cells, LARP4 depletion potentiated peritoneal metastasis. Upon analysis of a public database of patients with ovarian cancer, the LARP4 mRNA-high expression group (n = 166) showed longer overall survival compared with the LARP4 mRNA-low expression group (n = 489), implying a positive correlation of LARP4 mRNA levels in ovarian cancer tissues with patient prognosis. Taken together, we propose that LARP4 could suppress motility and metastatic potential of ovarian cancer cells.

Tyrosine kinase receptor TIE-1 mediates platinum resistance by promoting nucleotide excision repair in ovarian cancer.

Platinum resistance is one of the most challenging problems in ovarian cancer treatment. High-throughput functional siRNA screening identified tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE-1) as a gene that confers cells resistant to cisplatin. Conversely enforced over-expression of TIE-1 was validated to decrease cisplatin sensitivity in multiple ovarian cancer cell lines and up-regulation of TIE-1 was correlated with poor prognosis and cisplatin resistance in patients with ovarian cancer. Mechanistically, TIE-1 up-regulates the nucleotide excision repair (NER) system mediated by xeroderma pigmentosum complementation group C (XPC), thereby leading to decreased susceptibility to cisplatin-induced cell death without affecting cisplatin uptake and excretion. Importantly potentiation of therapeutic efficacy by TIE-1 inhibition was selective to DNA-adduct-type chemotherapeutic platinum reagents. Therefore, TIE-1 is suggested to promote XPC-dependent NER, rendering ovarian cancer cells resistant to platinum. Accompanied with novel findings, TIE-1 could represent as a novel therapeutic target for platinum-resistant ovarian cancer.

Novel cooperative pathway of c-Myc and Furin, a pro-protein convertase, in cell proliferation as a therapeutic target in ovarian cancers.

c-Myc is a master regulator of various oncogenic functions in many types of human cancers. However, direct c-Myc-targeted therapy has not been successful in the clinic. Here, we explored a novel therapeutic target, which shows synthetic lethality in c-Myc-driven ovarian cancers, and examined the molecular mechanism of the synthetic lethal interaction. By high throughput siRNA screening with a library of 6,550 genes, Furin, a pro-protein convertase, was identified as the top hit gene. Furin inhibition by siRNA or a Furin inhibitor significantly suppressed cell proliferation in high c-Myc-expressing ovarian cancer cells compared with low c-Myc-expressing cells. Conversely, Furin overexpression in the presence of high c-Myc significantly promoted cell proliferation compared with only c-Myc or Furin overexpression. Notch1, one of the Furin substrates, was upregulated by c-Myc, and Notch1 cleaved by Furin increased cell proliferation of high c-Myc-expressing ovarian cancer cells. Notch1 was involved in the cooperative pathway of c-Myc and Furin in cell proliferation. In clinical ovarian cancer specimens, co-expression of c-Myc and Furin correlated with poor survival. In conclusion, we found that c-Myc cooperates with Furin to promote cell proliferation. Furin may be a promising therapeutic target in c-Myc-driven ovarian cancer.

Ceramide Nanoliposomes as a MLKL-Dependent, Necroptosis-Inducing, Chemotherapeutic Reagent in Ovarian Cancer.

Ceramides are bioactive lipids that mediate cell death in cancer cells, and ceramide-based therapy is now being tested in dose-escalating phase I clinical trials as a cancer treatment. Multiple nanoscale delivery systems for ceramide have been proposed to overcome the inherent toxicities, poor pharmacokinetics, and difficult biophysics associated with ceramide. Using the ceramide nanoliposomes (CNL), we now investigate the therapeutic efficacy and signaling mechanisms of this nanoscale delivery platform in refractory ovarian cancer. Treatment of ovarian cancer cells with CNL decreased the number of living cells through necroptosis but not apoptosis. Mechanistically, dying SKOV3 ovarian cancer cells exhibit activation of pseudokinase mixed lineage kinase domain-like (MLKL) as evidenced by oligomerization and relocalization to the blebbing membranes, showing necroptotic characteristics. Knockdown of MLKL, but not its upstream protein kinases such as receptor-interacting protein kinases, with siRNA significantly abolished CNL-induced cell death. Monomeric MLKL protein expression inversely correlated with the IC50 values of CNL in distinct ovarian cancer cell lines, suggesting MLKL as a possible determinant for CNL-induced cell death. Finally, systemic CNL administration suppressed metastatic growth in an ovarian cancer cell xenograft model. Taken together, these results suggest that MLKL is a novel pronecroptotic target for ceramide in ovarian cancer models. Mol Cancer Ther; 17(1); 50-59. ©2017 AACR.

Activation of p38 Mitogen-Activated Protein Kinase in Gaucher's Disease.

Gaucher's disease is caused by defects in acid ß-glucosidase 1 (GBA1) and has been also proposed as an inflammatory disease. GBA1 cleaves glucosylceramide to form ceramide, an established bioactive lipid, and defects in GBA1 lead to aberrant accumulation in glucosylceramide and insufficient formation of ceramide. We investigated if the pro-inflammatory kinase p38 is activated in Gaucher's disease, since ceramide has been proposed to suppress p38 activation. Three Gaucher's disease mouse models were employed, and p38 was found to be activated in lung and liver tissues of all Gaucher's disease mice. Most interestingly, neuronopathic Gaucher's disease type mice, but not non-neuronopathic ones, displayed significant activation of p38 and up-regulation of p38-inducible proinflammatory cytokines in brain tissues. In addition, all type of Gaucher's disease mice also showed increases in serum IL-6. As cellular signalling is believed to represent an in vivo inflammatory phenotype in Gaucher's disease, activation of p38 and possibly its-associated formation of proinflammatory cytokines were assessed in fibroblasts established from neuronopathic Gaucher's disease mice. In mouse Gaucher's disease cells, p38 activation and IL-6 formation by TNF-α treatment were enhanced as compared to those of wild type. Furthermore, human fibroblasts from Gaucher's disease patients also displayed increases in p38 activation and IL-6 formation as comparison to healthy counterpart. These results raise the potential that proinflammatory responses such as p38 activation and IL-6 formation are augmented in Gaucher's disease.

Role of Sphingolipids and Metabolizing Enzymes in Hematological Malignancies.

Sphingolipids such as ceramide, sphingosine-1-phosphate and sphingomyelin have been emerging as bioactive lipids since ceramide was reported to play a role in human leukemia HL-60 cell differentiation and death. Recently, it is well-known that ceramide acts as an inducer of cell death, that sphingomyelin works as a regulator for microdomain function of the cell membrane, and that sphingosine-1-phosphate plays a role in cell survival/proliferation. The lipids are metabolized by the specific enzymes, and each metabolite could be again returned to the original form by the reverse action of the different enzyme or after a long journey of many metabolizing/synthesizing pathways. In addition, the metabolites may serve as reciprocal bio-modulators like the rheostat between ceramide and sphingosine-1-phosphate. Therefore, the change of lipid amount in the cells, the subcellular localization and the downstream signal in a specific subcellular organelle should be clarified to understand the pathobiological significance of sphingolipids when extracellular stimulation induces a diverse of cell functions such as cell death, proliferation and migration. In this review, we focus on how sphingolipids and their metabolizing enzymes cooperatively exert their function in proliferation, migration, autophagy and death of hematopoetic cells, and discuss the way developing a novel therapeutic device through the regulation of sphingolipids for effectively inhibiting cell proliferation and inducing cell death in hematological malignancies such as leukemia, malignant lymphoma and multiple myeloma.

Inhibition of plasminogen activator inhibitor-1 is a potential therapeutic strategy in ovarian cancer.

Plasminogen activator inhibitor (PAI)-1 is predictive of poor outcome in several types of cancer. The present study investigated the biological role for PAI-1 in ovarian cancer and potential of targeted pharmacotherapeutics. In patients with ovarian cancer, PAI-1 mRNA expression in tumor tissues was positively correlated with poor prognosis. To determine the role of PAI-1 in cell proliferation in ovarian cancer, the effects of PAI-1 inhibition were examined in PAI-1-expressing ovarian cancer cells. PAI-1 knockdown by small interfering RNA resulted in significant suppression of cell growth accompanied with G2/M cell cycle arrest and intrinsic apoptosis. Similarly, treatment with the small molecule PAI-1 inhibitor TM5275 effectively blocked cell proliferation of ovarian cancer cells that highly express PAI-1. Together these results suggest that PAI-1 promotes cell growth in ovarian cancer. Interestingly, expression of PAI-1 was increased in ovarian clear cell carcinoma compared with that in serous tumors. Our results suggest that PAI-1 inhibition promotes cell cycle arrest and apoptosis in ovarian cancer and that PAI-1 inhibitors potentially represent a novel class of anti-tumor agents.

Dietary glucosylceramides suppress tumor growth in a mouse xenograft model of head and neck squamous cell carcinoma by the inhibition of angiogenesis through an increase in ceramide.

BACKGROUND: We previously reported that dietary glucosylceramides show cancer-prevention activity in a mouse xenograft model of human head and neck cancer cells (SCCKN). However, the mechanism was unclear. Ceramides, metabolites of glucosylceramides, induce apoptotic cell death in various malignancies. Here, we investigated the inhibitory effects of dietary glucosylceramides on tumor growth in vivo and in vitro. METHODS: SCCKN were subcutaneously inoculated into the right flanks of NOD/SCID mice. Mice were treated with or without dietary glucosylceramides (300 mg/kg) daily for 14 consecutive days after confirmation of tumor progression. Microvessel areas around the tumor were assessed by hematoxylin-eosin staining and immunohistochemistry of CD31, and, as markers for angiogenesis, protein levels of VEGF, VEGF receptor-2, and HIF-1α were assessed by Western blotting. Mass spectrometry was performed to measure the levels of sphingolipids in mouse serum after treatment with dietary glucosylceramides. RESULTS: Oral administration of glucosylceramides significantly decreased SCCKN growth in the xenograft model with inhibition of angioinvasion. In tumor-invasive areas, VEGF and HIF-1α in the tumor cells, and VEGF receptor-2 in endothelial cells decreased after treatment with dietary glucosylceramides. Dietary glucosylceramides increased serum levels of sphingosine-based ceramides as compared to the control. In SCCKN and UV♀2 cells, C6-ceramide suppressed the expressions of VEGF, VEGF receptor-2, and HIF-1α in vitro. CONCLUSION: These results suggest that dietary glucosylceramides trigger the de novo pathway of ceramide synthesis, indicating that sphingosine-based ceramide suppresses the growth of head and neck tumors through the inhibition of pro-angiogenic signals such as VEGF, VEGF receptor-2, and HIF-1α.

Defining a role for acid sphingomyelinase in the p38/interleukin-6 pathway.

Acid sphingomyelinase (ASM) is one of the key enzymes involved in regulating the metabolism of the bioactive sphingolipid ceramide in the sphingolipid salvage pathway, yet defining signaling pathways by which ASM exerts its effects has proven difficult. Previous literature has implicated sphingolipids in the regulation of cytokines such as interleukin-6 (IL-6), but the specific sphingolipid pathways and mechanisms involved in inflammatory signaling need to be further elucidated. In this work, we sought to define the role of ASM in IL-6 production because our previous work showed that a parallel pathway of ceramide metabolism, acid ß-glucosidase 1, negatively regulates IL-6. First, silencing ASM with siRNA abrogated IL-6 production in response to the tumor promoter, 4ß-phorbol 12-myristate 13-acetate (PMA), in MCF-7 cells, in distinction to acid ß-glucosidase 1 and acid ceramidase, suggesting specialization of the pathways. Moreover, treating cells with siRNA to ASM or with the indirect pharmacologic inhibitor desipramine resulted in significant inhibition of TNFα- and PMA-induced IL-6 production in MDA-MB-231 and HeLa cells. Knockdown of ASM was found to significantly inhibit PMA-dependent IL-6 induction at the mRNA level, probably ruling out mechanisms of translation or secretion of IL-6. Further, ASM knockdown or desipramine blunted p38 MAPK activation in response to TNFα, revealing a key role for ASM in activating p38, a signaling pathway known to regulate IL-6 induction. Last, knockdown of ASM dramatically blunted invasion of HeLa and MDA-MB-231 cells through Matrigel. Taken together, these results demonstrate that ASM plays a critical role in p38 signaling and IL-6 synthesis with implications for tumor pathobiology.