sphingosine-1-phosphate and Lung-Neoplasms

Reciprocal interactions between breast cancer cells and the tumor microenvironment (TME) are important for cancer progression and metastasis. We report here that the deletion or inhibition of sphingosine kinase 2 (SphK2), which produces sphingosine-1-phosphate (S1P), markedly suppresses syngeneic breast tumor growth and lung metastasis in mice by creating a hostile microenvironment for tumor growth and invasion. SphK2 deficiency decreased S1P and concomitantly increased ceramides, including C16-ceramide, in stromal fibroblasts. Ceramide accumulation suppressed activation of cancer-associated fibroblasts (CAF) by upregulating stromal p53, which restrained production of tumor-promoting factors to reprogram the TME and to restrict breast cancer establishment. Ablation of p53 in SphK2-deficient fibroblasts reversed these effects, enabled CAF activation and promoted tumor growth and invasion. These data uncovered a novel role of SphK2 in regulating non-cell-autonomous functions of p53 in stromal fibroblasts and their transition to tumor-promoting CAFs, paving the way for the development of a strategy to target the TME and to enhance therapeutic efficacy.. Sphingosine kinase 2 (SphK2) facilitates the activation of stromal fibroblasts to tumor-promoting cancer-associated fibroblasts by suppressing host p53 activity, revealing SphK2 as a potential target to reprogram the TME.

Topics: Animals; Cancer-Associated Fibroblasts; Fibroblasts; Lung Neoplasms; Mammary Neoplasms, Animal; Mice; Phosphotransferases (Alcohol Group Acceptor); Tumor Microenvironment; Tumor Suppressor Protein p53

Metastasis is the leading cause of cancer-related deaths and myeloid cells are critical in the metastatic microenvironment. Here, we explore the implications of reprogramming pre-metastatic niche myeloid cells by inducing trained immunity with whole beta-glucan particle (WGP). WGP-trained macrophages had increased responsiveness not only to lipopolysaccharide but also to tumor-derived factors. WGP in vivo treatment led to a trained immunity phenotype in lung interstitial macrophages, resulting in inhibition of tumor metastasis and survival prolongation in multiple mouse models of metastasis. WGP-induced trained immunity is mediated by the metabolite sphingosine-1-phosphate. Adoptive transfer of WGP-trained bone marrow-derived macrophages reduced tumor lung metastasis. Blockade of sphingosine-1-phosphate synthesis and mitochondrial fission abrogated WGP-induced trained immunity and its inhibition of lung metastases. WGP also induced trained immunity in human monocytes, resulting in antitumor activity. Our study identifies the metabolic sphingolipid-mitochondrial fission pathway for WGP-induced trained immunity and control over metastasis.

Topics: Animals; beta-Glucans; Humans; Lung Neoplasms; Lysophospholipids; Macrophages; Mice; Monocytes; Trained Immunity; Tumor Microenvironment

The stimulator of interferon genes (STING) is a master regulator of innate immunity, involved in several inflammatory diseases. Our previous data showed that sphingosine-1-phosphate (S1P) is released during inflammatory conditions in the lung. The aim of this study was to understand the interplay between S1P and STING during both physiological and pathological conditions. The mRNA levels of ceramidase (ASAH1), S1P precursor enzyme, and STING were inversely correlated in healthy lung tissues, but positively correlated in tumor tissues. The activation of STING induced higher expression of ASAH1 and was accompanied by IFN-β and IL-6 release. ASAH1 and sphingosine kinases (SPHK I/II) blockade significantly reduced IL-6, but not IFNβ, after STING activation. In support of this, taking advantage of a mouse model, we found that inflamed lungs had higher levels of inactive ASAH1 when STING was inhibited. This confirmed the human data, where higher levels of STING promoted the activation of ASAH1. Lung cancer patients positive to STING and ASAH1 mRNA levels had a dismal prognosis in that the overall survival was reduced compared to STING/ASAH1 negative patients. These data highlight that during physiological conditions, STING and the S1P axis do not interfere, whereas in lung cancer patients their interplay is associated to poor prognosis.

Topics: Animals; Humans; Inflammation; Interleukin-6; Lung; Lung Neoplasms; Lysophospholipids; Mice; Sphingosine

Sex is a biological variable that can reflect clinical outcomes in terms of quality of life, therapy effectiveness, responsiveness and/or toxicity. Sphingosine-1-phosphate (S1P) is a lipidic mediator whose activity can be influenced by sex. To evaluate whether the S1P axis underlies sex 'instructions' in the lung during physiological and oncological lung conditions, sphingosine and S1P were quantified in the blood of healthy (H) volunteers, lung adenocarcinoma (ADK) and squamous cell carcinoma (SCC) patients of both sexes. S1P receptors and their metabolic enzymes were evaluated in the tissues. Circulating levels of S1P were similar among H female and male subjects and female SCC patients. Instead, male and female ADK patients had lower circulating S1P levels. S1P receptor 3 (S1PR3) was physiologically expressed in the lung, but it was overexpressed in male SCC, and female and male ADK, but not in female SCC patients, who showed a significantly reduced ceramide synthase 1 (CERS1) mRNA and an overexpression of the ceramidase (ASAH1) precursor in lung tumor tissues, compared to male SCC and both male and female ADK patients. These findings highlighted sex differences in S1P rheostat in pathological conditions, but not in physiological conditions, identifying S1P as a prognostic mediator depending on lung cancer histotype.

Topics: Ceramidases; Female; Humans; Lung; Lung Neoplasms; Lysophospholipids; Male; Quality of Life; Sex Characteristics; Sphingosine

Sphingosine-1-phosphate (S1P) is involved in inflammatory signaling/s associated with the development of respiratory disorders, including cancer. However, the underlying mechanism/s are still elusive. The aim of this study was to investigate the role of S1P on circulating blood cells obtained from healthy volunteers and non-small cell lung cancer (NSCLC) patients. To pursue our goal, peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with S1P. We found that the administration of S1P did not induce healthy PBMCs to release pro-inflammatory cytokines. In sharp contrast, S1P significantly increased the levels of TNF-α and IL-6 from lung cancer-derived PBMCs. This effect was S1P receptor 3 (S1PR3)-dependent. The pharmacological blockade of ceramidase and sphingosine kinases (SPHKs), key enzymes for S1P synthesis, completely reduced the release of both TNF-α and IL-6 after S1P addition on lung cancer-derived PBMCs. Interestingly, S1P-induced IL-6, but not TNF-α, release from lung cancer-derived PBMCs was mTOR- and K-Ras-dependent, while NF-κB was not involved. These data identify S1P as a bioactive lipid mediator in a chronic inflammation-driven diseases such as NSCLC. In particular, the higher presence of S1P could orchestrate the cytokine milieu in NSCLC, highlighting S1P as a pro-tumor driver.

Topics: Carcinoma, Non-Small-Cell Lung; Cytokines; Humans; Inflammation; Interleukin-6; Leukocytes, Mononuclear; Lung Neoplasms; Lysophospholipids; Pneumonia; Sphingosine; Tumor Necrosis Factor-alpha

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Humans; Lung Neoplasms; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Pre-B-Cell Leukemia Transcription Factor 1; Proto-Oncogene Proteins c-akt; Sphingosine

Sphingosine-1-phosphate (S1P) is a membrane-derived bioactive phospholipid involved in many lung physiological and pathological processes. Higher levels of S1P have been registered in a broad range of respiratory diseases, including inflammatory disorders and cancer. The aim of our study was to understand the role of S1P in healthy versus tumor cells after Toll-Like Receptors (TLRs) activation, well-known modulators of sphingolipid metabolism.. Lung adenocarcinoma cells and non-pathological human fibroblasts were stimulated with unmethylated Cytosine phosphate Guanosine (CpG), the TLR9 ligand, and S1P-dependent TNF-α release was evaluated by means of ELISA. Immunofluorescence and LC-MS/MS analysis were performed to evaluate/quantify S1P generation following TLR9 activation.. We found that S1P was involved in TLR9-induced TNF-α release in that the inhibition of both ceramidase and sphingosine kinase I/II (SPHK I/II) significantly reduced the levels of TNF-α after TLR9 triggering in lung adenocarcinoma cells. These results were not observed in healthy fibroblasts, implying that this pathway was mainly involved in pathological conditions. Moreover, the activation of TLR4 by means of LPS did not have similar effects as in the case of CpG-stimulated TLR9. Importantly, the activation of TLR9 induced S1P generation and allowed it to interact on the outside membrane receptor S1P. Our study identifies a novel inflammatory pathway in that TLR9 increases the pro-inflammatory cytokine release, such as TNF-α, via the induction of a ceramide/S1P imbalance in favor of S1P, adding a novel puzzle piece in TLR9-orchestrated inflammatory pathway and shedding more light on the role of the higher levels of S1P during inflammatory conditions.

Topics: A549 Cells; Adenocarcinoma of Lung; Blotting, Western; Fluorescent Antibody Technique; Humans; Inflammation; Lung; Lung Neoplasms; Lysophospholipids; Sphingosine; Tandem Mass Spectrometry; Toll-Like Receptor 9; Tumor Necrosis Factor-alpha

Sphingosine 1-phosphate (S1P), a bioactive lipid, has been shown to mediate cancer processes. Therefore, accurate qualitative and quantitative determination is essential. The current assay method is still cumbersome to be of practical use worldwide and the aim of this study was therefore to develop a fast, accurate, precise and efficient LC-MS/MS method for targeted analyses of S1P in serum samples.. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an established method used for monitoring and analyzing S1P levels in serum. We determined the level of serum S1P in 256 patients with lung cancer and 36 healthy donors, and used Spearman';s rank correlation analysis to evaluate the difference in serum S1P levels between radiotherapy and nonradiotherapy patients.. Our results indicated that this new LC-MS/MS method is rapid, sensitive, specific and reliable for the quantification of S1P levels in serum samples. The level of S1P in serum samples of patients with lung cancer who received radiotherapy was significantly lower than that in patients who did not receive radiotherapy.. An improved method was established to quantify S1P levels in human serum by LC-MS/MS, which enabled the change in serum S1P levels in lung cancer patients to be monitored, in combination with radiotherapy, and their clinical significance to be analyzed.

Topics: Adult; Aged; Biomarkers, Tumor; Chromatography, Liquid; Female; Humans; Lung Neoplasms; Lysophospholipids; Male; Middle Aged; Prognosis; Radiotherapy; Reproducibility of Results; Sphingosine; Tandem Mass Spectrometry; Young Adult

Topics: Animals; Apoptosis; Caspase 3; Cell Proliferation; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Lung Neoplasms; Lysophospholipids; Mice; Mice, Knockout; Mice, SCID; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Telomere; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.

Topics: Animals; Anion Transport Proteins; Cell Line, Tumor; Cell Movement; Disease Models, Animal; Female; Genome; Genomics; Killer Cells, Natural; Lung Neoplasms; Lymphopenia; Lysophospholipids; Male; Mice; Neoplasm Metastasis; Sphingosine; T-Lymphocytes; Tumor Microenvironment

Recent studies reveal that chemotherapy can enhance metastasis due to host responses, such as augmented expression of adhesion molecules in endothelial cells and increased populations of myeloid cells. However, it is still unclear how tumour cells contribute to this process. Here, we observed that paclitaxel and carboplatin accelerated lung metastasis in tumour-bearing mice, while doxorubicin and fluorouracil did not. Mechanistically, paclitaxel and carboplatin induced similar changes in cytokine and angiogenic factors. Increased levels of CXCR2, CXCR4, S1P/S1PR1, PlGF and PDGF-BB were identified in the serum or primary tumour tissues of tumour-bearing mice treated by paclitaxel. The serum levels of CXCL1 and PDGF-BB and the tissue level of CXCR4 were also elevated by carboplatin. On the other hand, doxorubicin and fluorouracil did not induce such changes. The chemotherapy-induced cytokine and angiogenic factor changes were also confirmed in gene expression datasets from human patients following chemotherapy treatment. These chemotherapy-enhanced cytokines and angiogenic factors further induced angiogenesis, destabilized vascular integrity, recruited BMDCs to metastatic organs and mediated the proliferation, migration and epithelial-to-mesenchymal transition of tumour cells. Interestingly, inhibitors of these factors counteracted chemotherapy-enhanced metastasis in both tumour-bearing mice and normal mice injected intravenously with B16F10-GFP cells. In particular, blockade of the SDF-1α-CXCR4 or S1P-S1PR1 axes not only compromised chemotherapy-induced metastasis but also prolonged the median survival time by 33.9% and 40.3%, respectively. The current study delineates the mechanism of chemotherapy-induced metastasis and provides novel therapeutic strategies to counterbalance pro-metastatic effects of chemo-drugs via combination treatment with anti-cytokine/anti-angiogenic therapy.

Topics: Angiogenesis Inhibitors; Angiogenic Proteins; Animals; Antineoplastic Agents; Becaplermin; Carboplatin; Cell Line, Tumor; Cell Movement; Cell Survival; Cytokines; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Female; Humans; Lung Neoplasms; Lysophospholipids; Melanoma, Experimental; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Paclitaxel; Placenta Growth Factor; Pregnancy Proteins; Proto-Oncogene Proteins c-sis; Receptors, CXCR4; Receptors, Interleukin-8B; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Stromal Cells; Time Factors; Up-Regulation; Xenograft Model Antitumor Assays

The sphingosine-1-phosphate (S1P) transporter Spns2 regulates myocardial precursor migration in zebrafish and lymphocyte trafficking in mice. However, its function in cancer has not been investigated. We show here that ectopic Spns2 expression induced apoptosis and its knockdown enhanced cell migration in non-small cell lung cancer (NSCLC) cells. Metabolically, Spns2 expression increased the extracellular S1P level while its knockdown the intracellular. Pharmacological inhibition of S1P synthesis abolished the augmented cell migration mediated by Spns2 knockdown, indicating that intracellular S1P plays a key role in this process. Cell signaling studies indicated that Spns2 expression impaired GSK-3β and Stat3 mediated pro-survival pathways. Conversely, these pathways were activated by Spns2 knockdown, which explains the increased cell migration since they are also crucial for migration. Alterations of Spns2 were found to affect several enzymes involved in S1P metabolism, including sphingosine kinases, S1P phosphatases, and S1P lyase 1. Genetically, Spns2 mRNA level was found to be reduced in advanced lung cancer (LC) patients as quantified by using a small scale qPCR array. These data show for the first time that Spns2 plays key roles in regulating the cellular functions in NSCLC cells, and that its down-regulation is a potential risk factor for LC.

Topics: Animals; Anion Transport Proteins; Apoptosis; Biological Transport; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Survival; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Intracellular Space; Lung Neoplasms; Lysophospholipids; Mice; Receptors, Lysosphingolipid; RNA, Messenger; Sphingosine

Sphingosine-1-phosphate (S1P) and ceramides are bioactive signaling sphingolipids that regulate pathways that are central to cancer pathogenesis.. A nested case-control study was implemented to test whether prediagnostic circulating concentrations of S1P and ceramides were associated with future lung cancer risk. In the community-based CLUE II cohort study in Washington County, Maryland, the study consisted of 100 incident lung cancer cases, each matched to two cancer-free controls on age, sex, race, and cigarette smoking status. Plasma stored at -70°C at the beginning of follow-up in 1989 was assayed for sphingolipids using liquid chromatography/tandem mass spectrometry methodology (LC/MS-MS).. Compared with controls, geometric mean plasma concentrations of S1P and total ceramides were 2.9% (P = 0.10) and 5.1% (P = 0.02), respectively, greater in lung cancer cases. For S1P, the ORs and 95% confidence intervals (CI) for lung cancer risk were 2.7 (1.2-5.9), 2.7 (1.1-6.4), and 1.9 (0.8-4.5) for the second, third, and highest fourth, respectively, compared with the lowest fourth (overall P = 0.006). Compared with those with total ceramide concentrations in the lowest fourth, the ORs (and 95% CI) for lung cancer risk were 1.6 (0.7-3.3), 1.5 (0.7-3.4), and 2.1 (0.9-4.7) for the second, third, and highest fourth, respectively (P(trend) = 0.01).. Higher concentrations of S1P and total ceramide in plasma were associated with increased future risk of lung cancer.. These novel findings suggest that perturbation of sphingolipid metabolism and S1P generation may either contribute to the etiology of lung cancer or be a marker of latent lung cancer.

Topics: Aged; Biomarkers, Tumor; Case-Control Studies; Ceramides; Cohort Studies; Female; Humans; Lung Neoplasms; Lysophospholipids; Male; Maryland; Middle Aged; Sphingolipids; Sphingosine

Macrophages frequently infiltrate tumors and can enhance cancer growth, yet the origins of the macrophage response are not well understood. Here we address molecular mechanisms of macrophage production in a conditional mouse model of lung adenocarcinoma. We report that overproduction of the peptide hormone Angiotensin II (AngII) in tumor-bearing mice amplifies self-renewing hematopoietic stem cells (HSCs) and macrophage progenitors. The process occurred in the spleen but not the bone marrow, and was independent of hemodynamic changes. The effects of AngII required direct hormone ligation on HSCs, depended on S1P(1) signaling, and allowed the extramedullary tissue to supply new tumor-associated macrophages throughout cancer progression. Conversely, blocking AngII production prevented cancer-induced HSC and macrophage progenitor amplification and thus restrained the macrophage response at its source. These findings indicate that AngII acts upstream of a potent macrophage amplification program and that tumors can remotely exploit the hormone's pathway to stimulate cancer-promoting immunity.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Angiotensin II; Animals; Carcinoma, Non-Small-Cell Lung; Cell Communication; Cell Movement; Cell Proliferation; Gene Expression; Hematopoietic Stem Cells; Humans; Lung Neoplasms; Lysophospholipids; Macrophages; Mice; Mice, Transgenic; Signal Transduction; Sphingosine; Spleen; Tumor Burden

Sphingosine-1-phosphate (S1P) regulates a wide array of biological functions. However, the role of S1P signaling in tumorigenesis remains to be elucidated. In this study, we show that S1P receptor subtype 3 (S1P₃) is markedly up-regulated in a subset of lung adenocarcinoma cells compared to normal lung epithelial cells. Specific knockdown of S1P₃ receptors inhibits proliferation and anchorage-independent growth of lung adenocarcinoma cells. Mechanistically, we demonstrate that S1P₃ signaling increases epidermal growth factor receptor (EGFR) expression via the Rho kinase (ROCK) pathway in lung adenocarcinoma cells. Nuclear run-off analysis indicates that S1P/S1P₃ signaling transcriptionally increases EGFR expression. Knockdown of S1P₃ receptors diminishes the S1P-stimulated EGFR expression in lung adenocarcinoma cells. Moreover, S1P treatment greatly enhances EGF-stimulated colony formation, proliferation and invasion of lung adenocarcinoma cells. Together, these results suggest that the enhanced S1P₃-EGFR signaling axis may contribute to the tumorigenesis or progression of lung adenocarcinomas.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Carcinoma, Lewis Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lysophospholipids; Mice; Neoplasm Invasiveness; Receptors, Lysosphingolipid; rho-Associated Kinases; RNA Interference; RNA, Messenger; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Time Factors; Transcriptional Activation; Transfection; Up-Regulation

Mechanisms by which cancer cells communicate with the host organism to regulate lung colonization/metastasis are unclear. We show that this communication occurs via sphingosine 1-phosphate (S1P) generated systemically by sphingosine kinase 1 (SK1), rather than via tumour-derived S1P. Modulation of systemic, but not tumour SK1, prevented S1P elevation, and inhibited TRAMP-induced prostate cancer growth in TRAMP(+/+) SK1(-/-) mice, or lung metastasis of multiple cancer cells in SK1(-/-) animals. Genetic loss of SK1 activated a master metastasis suppressor, Brms1 (breast carcinoma metastasis suppressor 1), via modulation of S1P receptor 2 (S1PR2) in cancer cells. Alterations of S1PR2 using pharmacologic and genetic tools enhanced Brms1. Moreover, Brms1 in S1PR2(-/-) MEFs was modulated by serum S1P alterations. Accordingly, ectopic Brms1 in MB49 bladder cancer cells suppressed lung metastasis, and stable knockdown of Brms1 prevented this process. Importantly, inhibition of systemic S1P signalling using a novel anti-S1P monoclonal antibody (mAb), Sphingomab, attenuated lung metastasis, which was prevented by Brms1 knockdown in MB49 cells. Thus, these data suggest that systemic SK1/S1P regulates metastatic potential via regulation of tumour S1PR2/Brms1 axis.

Topics: Animals; Disease Models, Animal; Humans; Lung Neoplasms; Lysophospholipids; Male; Mice; Mice, Knockout; Neoplasm Metastasis; Phosphotransferases (Alcohol Group Acceptor); Prostatic Neoplasms; Receptors, Lysosphingolipid; Repressor Proteins; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Urinary Bladder Neoplasms

The lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are small lipid molecules with a variety of physiological roles. Additionally, their involvement in the initiation and progression of malignant tumors has been increasingly recognized in recent years. However, the data on the expression of S1P and LPA receptors on different cancer cells are very few. Real-time polymerase chain reaction was used for the analysis of mRNA expression of five S1P((1-5)) and three LPA((1-3)) receptors on a large panel of 13 colon, breast, melanoma, and lung cancer cell lines. Furthermore, the modulation of S1P and LPA receptor mRNA expression was studied upon xenotransplantation of tumor cells into severe combined immunodeficient (SCID) mice. The S1P and LPA receptors were expressed to a variable degree on all tumor cell lines tested (with exception of colon cancer SW480). Most notably, tumor cell lines in vitro expressed S1P(2) mRNA that was down-regulated upon xenotransplantation, whereas LPA(2) receptor mRNA was strongly expressed both in vitro and in vivo (except by breast cancer cells). The latter was especially distinctive for small cell lung tumor cells. The S1P and LPA receptors are differentially expressed on tumor cell lines in vitro. Their expression is modulated upon xenografting into SCID mice in vivo.

Topics: Animals; Breast Neoplasms; Colonic Neoplasms; Endothelium, Vascular; Female; Humans; Immunoenzyme Techniques; Lung Neoplasms; Lysophospholipids; Melanoma; Mice; Mice, SCID; Neoplasm Transplantation; Receptors, Lysophosphatidic Acid; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine; Transplantation, Heterologous; Tumor Cells, Cultured; Umbilical Cord

Urethane is a chemical carcinogen which causes lung tumorigenesis in mice with similarities to human adenocarcinoma (AC). The sphingosine 1-phosphate agonist FTY720 administered to mice in doses above 5 mg/kg/day has been able to prevent hepatocellular carcinoma and bladder cancer. We used BALB/c mice in urethane-induced lung cancer model to investigate the effects of a lower dose of FTY720 (1 mg/kg/day). The benefits of FTY720 were associated with the time point of the compound administration. FTY720 30 Group presented lower incidence and smaller area of lung nodules, decreased PCNA and increased Caspase-3 expressions. The findings in FTY720 0 Group (nodule multiplicity and area, PCNA expression) were similar to Urethane Group suggesting that the administration of the compound at early time point did not affect lung tumor development. FTY720 90 Group presented the biggest nodule area which was associated with increased PCNA and decreased Caspase-3 expressions. FTY720 (30 days and 90 days) administration decreased CD4 + splenocytes and blood lymphocytes which caused opposite effects in lung tumor development - impairment and improvement respectively.In conclusion, FTY720 in low dose did not provide lung tumor inhibition in mice but its administration 30 days after the chemical carcinogen (Urethane) injection was associated with impaired tumor development.

Topics: Adenoma; Animals; Apoptosis; Caspase 3; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Fingolimod Hydrochloride; Lung; Lung Neoplasms; Lysophospholipids; Male; Mice; Mice, Inbred BALB C; Proliferating Cell Nuclear Antigen; Propylene Glycols; Sphingosine; Urethane

Hypoxia and signaling via hypoxia-inducible factor-1 (HIF-1) is a key feature of solid tumors and is related to tumor progression as well as treatment failure. Although it is generally accepted that HIF-1 provokes tumor cell survival and induces chemoresistance under hypoxia, HIF-1-independent mechanisms operate as well. We present evidence that conditioned medium obtained from A549 cells, incubated for 24 h under hypoxia, protected naive A549 cells from etoposide-induced cell death. Lipid extracts generated from hypoxia-conditioned medium still rescued cells from apoptosis induced by etoposide. Specifically, the bioactive lipid sphingosine-1-phosphate (S1P) not only was essential for cell viability of A549 cells but also protected cells from apoptosis. We noticed an increase in sphingosine kinase 2 (SphK2) protein level and enzymatic activity under hypoxia, which correlated with the release of S1P into the medium. Knockdown of SphK2 using specific small interfering RNA relieved chemoresistance of A549 cells under hypoxia and conditioned medium obtained from SphK2 knockdown cells was only partially protective. Coincubations of conditioned medium with VPC23019, a S1P(1)/S1P(3) antagonist, reduced protection of conditioned medium, with the further notion that p42/44 mitogen-activated protein kinase transmits autocrine or paracrine survival signaling downstream of S1P(1)/S1P(3) receptors. Our data suggest that hypoxia activates SphK2 to promote the synthesis and release of S1P, which in turn binds to S1P(1)/S1P(3) receptors, thus activating p42/44 mitogen-activated protein kinase to convey autocrine or paracrine protection of A549 cells.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Hypoxia; Cell Line, Tumor; Culture Media; Drug Resistance, Neoplasm; Etoposide; Humans; Lung Neoplasms; Lysophospholipids; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Small Interfering; Sphingosine; Transfection

Ceramide, a sphingolipid, is an important signaling molecule in the inflammatory response. Mediators of acute lung injury such as TNF-alpha, platelet-activating factor, and Fas/Apo ligand stimulate sphingomyelin hydrolysis to increase intracellular ceramide levels. Surfactant protein B (SP-B), a hydrophobic protein of pulmonary surfactant, is essential for surfactant function and lung stability. In this study we investigated the effects of ceramide on SP-B gene expression in H441 lung epithelial cells. Ceramide decreased SP-B mRNA levels in control and dexamethasone-treated cells after 24-h incubation and inhibition of SP-B mRNA was associated with inhibition of immunoreactive SP-B. In transient transfections assays, ceramide inhibited SP-B promoter activity, indicating that the inhibitory effects are exerted at the transcriptional level. Deletion mapping experiments showed that the ceramide-responsive region is located within the -233/-80-bp region of human SP-B promoter. Electrophoretic mobility shift and reporter assays showed that ceramide reduced the DNA binding activity and transactivation capability of thyroid transcription factor 1 (TTF-1/Nkx2.1), a key factor for SP-B promoter activity. Collectively these data showed that ceramide inhibits SP-B gene expression by reducing the DNA biding activity of TTF-1/Nkx2.1 transcription factor. Protein kinase C inhibitor bisindolylmaleimide and the protein tyrosine kinase inhibitor genistein partially reversed ceramide inhibition, indicating that protein kinases play important roles in the ceramide inhibition of SP-B gene expression. Chemical inhibitors of de novo ceramide synthesis and sphingomyelin hydrolysis had no effect on TNF-alpha inhibition of SP-B promoter activity and mRNA levels, suggesting that ceramide does not play a role in the inhibition.

Topics: DNA; Down-Regulation; Gene Expression Regulation; Hepatocyte Nuclear Factor 3-alpha; Humans; Lung Neoplasms; Lysophospholipids; Nuclear Proteins; Promoter Regions, Genetic; Protein Kinases; Pulmonary Surfactant-Associated Protein B; RNA, Messenger; Signal Transduction; Sphingosine; Thyroid Nuclear Factor 1; Transcription Factors; Tumor Cells, Cultured

Phospholipase D (PLD) is involved in the signaling by many extracellular ligands, and its regulation appears to be quite complex. We investigated the signaling pathways initiated by bradykinin (BK) or sphingosine 1-phosphate (S1P) in A549 cells to define molecular mechanisms responsible for their additive effects on PLD activity. BK and S1P each elicited a sustained increase in phosphatidic acid content through a rapid and transient activation of PLD. The two pathways demonstrated rapid homologous downregulation, but heterologous desensitization was not observed. Action of both agonists required protein kinase C (PKC) activation and Ca(2+) influx but was mediated by different heterotrimeric G proteins. In membranes, inhibition of PKCdelta by rottlerin enhanced BK activation of PLD but inhibited that by S1P. Rottlerin inhibited activation of PLD in nuclei by both BK and S1P. By in situ immunofluorescence or cell fractionation followed by immunoblotting, PLD1 was concentrated primarily in nuclei, whereas the membrane fraction contained PLD2 and PLD1. Moreover, PKCdelta specifically phosphorylated recombinant PLD2, but not PLD1. BK and S1P similarly enhanced RhoA translocation to nuclei, whereas BK was less efficacious than S1P on RhoA relocalization to membranes. Effects of both agonists on the nuclear fraction, which contains only PLD1, are compatible with a RhoA- and PKCdelta-dependent process. In membranes, which contain both PLD1 and PLD2, the stimulatory effect of S1P on PLD activity can best be explained by RhoA- and PKCdelta-dependent activation of PLD1; in contrast, the effects of BK on RhoA translocation and enhancement of BK-stimulated PLD activity by PKC inhibition are both consistent with PLD2 serving as its primary target.

Topics: Adenocarcinoma; Bradykinin; Calcium; Enzyme Activation; Glycerophospholipids; Heterotrimeric GTP-Binding Proteins; Humans; Lung Neoplasms; Lysophospholipids; Phosphatidic Acids; Phospholipase D; Protein Kinase C; Protein Kinase C-delta; Protein Transport; rhoA GTP-Binding Protein; Signal Transduction; Sphingosine; Subcellular Fractions; Tritium; Tumor Cells, Cultured

In the present paper, the effect of sphingosine 1-phosphate (Sph-1-P) on arachidonic acid mobilization in A549 human lung adenocarcinoma cells was investigated. Sph-1-P provoked a rapid and relevant release of arachidonic acid which was similar to that elicited by bradykinin, well-known pro-inflammatory agonist. The Sph-1-P-induced release of arachidonic acid involved Ca(2+)-independent phospholipase A(2) (iPLA2) activity, as suggested by the dose-dependent inhibition exerted by the rather specific inhibitor bromoenol lactone. The Sph-1-P-induced release of arachidonic acid was pertussis toxin-sensitive, pointing at a receptor-mediated mechanism, which involves heterotrimeric Gi proteins. The action of Sph-1-P was totally dependent on protein kinase C (PKC) catalytic activity and seemed to involve agonist-stimulated phospholipase D (PLD) activity. This study represents the first evidence for Sph-1-P-induced release of arachidonic acid which occurs through a specific signaling pathway involving Gi protein-coupled receptor(s), PKC, PLD and iPLA2 activities.

Topics: Adenocarcinoma; Arachidonic Acid; Enzyme Inhibitors; Humans; Lipid Mobilization; Lung Neoplasms; Lysophospholipids; Phosphatidic Acids; Phospholipase D; Phospholipases A; Protein Kinase C; Sphingosine; Tritium; Tumor Cells, Cultured

Topics: Bradykinin; Enzyme Activation; Epithelial Cells; Humans; Lung Neoplasms; Lysophospholipids; Phospholipase D; Pulmonary Alveoli; Signal Transduction; Sphingosine; Tumor Cells, Cultured