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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2012 Jun;93(3):202–209. doi: 10.1111/j.1365-2613.2011.00805.x

H-Ras increases release of sphingosine resulting in down-regulation of TSP-1 in non-transformed cells

Wojciech Kalas *, Jacek Rybka †, Ewelina Swiderek *, Ewa Ziolo *, Wojciech Rybka †, Andrzej Gamian †, Janusz Rak ‡, Leon Strzadala *
PMCID: PMC3385918  PMID: 22356213

Summary

Tumour progression is continuously driven by a sequence of genetic events. The presence of mutant or activated Ras proteins represents an interesting paradigm for the investigation of oncogene-dependent induction of tumour angiogenesis. These genes are widely distributed in human cancers. Previously we have shown that cells harbouring mutant H-Ras release soluble unidentified factor(s) associated with lowered expression of an angiogenesis inhibitor – Thrombospondin-1 – (TSP-1) in adjacent normal tissue. In this study, we have addressed the question as to whether or not introduction of the H-ras oncogene leads to increased production of sphingosine. To assess the amount of sphingosine in conditioned media, we developed a technique based on sphingolipid isolation and GC-MSMS detection of specific silylated sphingosine derivatives. Cells harbouring mutant H-Ras, release significant amounts of sphingosine in contrast to normal isogenic cells or premalignant cells. Increased concentration of sphingosine in conditioned media was correlated with their ability to down-regulate the expression of TSP-1. Moreover, medium collected in the presence of U0126, an inhibitor of MAPK kinase (MEK), contained undetectable amounts of sphingosine and had no ability to down-regulate TSP-1 expression. Overall, our studies suggest a H-Ras-dependent mechanism of changing the equilibrium of angiogenic factors in favour of induction of angiogenesis, where a central role is played by sphingosine, a low molecular entity. This represents an example of how a mechanism of translating genetic changes within transformed cells could be amplified into a much larger effect involving the tumour parenchyma and stroma, and this could greatly in turn accelerate local tumour growth and metastasis.

Keywords: cancer, cancer metabolism, Ras, sphingosine, thrombospondin-1

Tumour progression is continuously driven by a sequence of genetic events. The presence of mutant or activated Ras proteins constitutes an interesting paradigm for oncogene-dependent induction of tumour angiogenesis, owing to their widespread presence in human cancers (Downward 2003).

Angiogenesis is a prerequisite for three-dimensional tumour growth, invasion and metastasis and serves as an excellent example of how tumour growth depends on the ability of tumour cells to modulate their own microenvironment (Hanahan & Weinberg 2000; Naumov et al. 2009; Pietras & Ostman 2010). The existing model of induction of angiogenesis suggests that vascular homeostasis is regulated by the local equilibrium of pro- and anti-angiogenic factors (Carmeliet 2005).

Platelet-related sphingolipids may alter the balance of angiogenic factors and this has been shown to involve down-regulation of expression of Thrombospondin-1 (TSP-1) (Kalas et al. 2005a), a potent angiogenesis inhibitor (Lawler 2009). Additionally, we presented the data that even a small number of H-Ras-transformed dermal fibroblasts (528ras1) were able to modulate the expression of TSP-1 in neighbouring cells formatting an proangiogenic environment (Kalas et al. 2005b). This was mediated by a low-molecular weight mediator, in which identity and nature remained unknown.

Sphingolipids are a family of membrane lipids derived from sphingomyelin. Bioactive sphingolipids such as ceramide, sphingosine 1-phosphate, sphingosine and glucosylceramide function as messenger molecules, which are implicated in various aspects of cell physiology and cancer pathogenesis, including regulation of apoptosis, cell proliferation, cell migration, senescence or inflammation (Merrill et al. 1997; Bartke & Hannun 2009; Fyrst & Saba 2010; Ponnusamy et al. 2010). The most abundant sphingoid base in mammalian tissue is d-erythro-sphingosine. Usually, concentrations of free sphingoid in cells are very low. In contrast to sphinganine, free sphingosine present in cells is believed to be primarily derived from turnover of more complex sphingolipids or from dietary sources (Merrill et al. 1997).

Initial interest in sphingolipids as second messengers originates from the observation of Hannun & Bell (1989) that sphingosine can act as an inhibitor of PKC. Further studies, however, revealed numerous other targets for sphingosine, ceramide and last but not least sphingosine-1-phosphate (Merrill et al. 1997).

In this work, using newly developed technique based on GC-MSMS detection of specific silylated derivative of sphingosine, we directly address the question whether introduction of H-Ras oncogene leads to increased release of sphingosine, which serves as mediator of down-regulation of TSP-1 expression in non-transformed cells.

Materials and methods

Cells and reagents

The immortalized, non-tumorigenic, polyclonal mouse dermal fibroblastic cell line MDFB6 was derived from dermal explants of C57Bl/6 mice (Kalas et al. 2005b). These cells and their polyclonal variant harbouring mutant H-Ras oncogene B6ras and CLS-1 and B1v cells were cultured in Dulbecco Modified Eagle’s Medium (HyClone, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Invitrogen Corp., Carlsbad, CA, USA). T/G HA-VSMCs, a human aortic smooth muscle cell line established by telomerase transfection, were cultured in F12K medium supplemented with 2 mM l-glutamate (Gibco) and 0.1 mg/ml heparin (Sigma-Aldrich, St. Louis, MO, USA), 0.03 mg/ml Endothelial Cell Growth Supplement (ECGS; Sigma) and 10% fetal calf serum (Gibco). The human cancer cell line A549 was cultured in a 1:1 mixture of OptiMEM (Gibco) and RPMI (IITD, Wroclaw, Poland) supplemented with 5% FBS (PAA Laboratories GmbH, Pasching, Austria). The rat intestinal epithelium–derived cell line MT-Ras was maintained in minimal essential medium (aMEM; Gibco), supplemented with 5% FBS, 4 mM l-glutamine, 20 mM glucose and 10 μg/ml insulin (Sigma). In order to activate the metallothionein (MT) promoter driving H-Ras expression in MT-Ras cells, the growth medium was supplemented with 2 μM CdCl2 (Sigma) as described earlier (Filmus et al. 1993, 1994; Kalas et al. 2003). All cultures were supplemented with antibiotic and antimycotic solution (Sigma). Culture plates and flasks were purchased from Nunc (Thermo Fisher Scientific Inc.). U0124 and U0126 inhibitors were purchased from Calbiochem (Merck, Darmstadt, Germany), commercial standard of the d-erythro-sphingosine from Avanti Polar Lipids (Alabaster, AL, USA), and chloroform, methanol and hexane from Sigma-Aldrich.

Conditioned medium preparation

Indicated cells were grown (37 °C, 5% CO2) in 50-ml flasks in the presence of DMEM medium (HyClone) supplemented with 0.5% FCS (Gibco). After 72-h incubation, medium was collected and centrifuged (4000 g, 15 min).

Preparation of lipid fraction of conditioned medium

Lipid and sphingolipid fractions of conditioned media were prepared using the modified protocol described by Bodennec et al. (2000). Collected medium was lyophilized and resuspended in chloroform and washed twice with chloroform/methanol/water 3:48:47 (v/v/v). The lower chloroform phase was dried under nitrogen and dissolved in 1 ml of chloroform and applied to hexane preconditioned LC-NH2 SPE column (Supelco, Ballefonte, PA, USA). Column was subsequently washed with hexane and acetone/methanol 9:1.35 (v/v) and again with hexane (all Sigma-Aldrich). The sphingolipid fraction was eluted by solvent system consisting of chloroform/methanol 2:1 (v/v). The aluted fraction was dried under nitrogen for subsequent analysis.

Derivatization with BSTFA

A 2-ml sample of chloroform cell extract was dried under a stream of N2. 100 μl of silylation reagent – BSTFA [trimethylsilyl-2, 2, 2-trifluoro-n-(trimethylsilyl acetimidate); Sigma-Aldrich] – and 100 μl of dry pyridine were added to the dried sample. The reaction was carried out for 30 min in the temperature of 80 °C. After the reaction, the sample was diluted with 200 μl of hexane and analysed on gas–liquid chromatography connected with tandem mass spectrometry (GLC-MSMS).

GC-MSMS analysis

Analysis was carried out on the instrument Focus GC connected with ion trap tandem mass detector ITQ 700 (Thermo-Fisher Scientific Inc., Waltham, MA, USA). Temperature of ion source was 250 °C and ionization current was 250 μA. The spectrometric method was based on the selection of the primary ion m/z = 338 from the primary mass spectrum of silylated sphingosine derivative (M = 443) and its secondary fragmentation. The product ion with the m/z = 264 was used as the marker ion. The amount of sphingosine in the sample was estimated by determining the area of the peak representing sphingosine derivative on the gas–liquid chromatography in comparison with the standard curve obtained using commercial standard of the d-erythro-sphingosine. The coefficient of variation (CV) of the sample pretreatment and MSMS analysis ranged, depending on the sphingosine concentration in the sample, from 35% (for low concentrations) to 11% (for high concentrations). Higher variations, shown on graphs, were obtained for the repetitions of the biological experiments.

TSP-1 promoter activity assays

The pMTSP-1 vector containing 2800 bp of the mouse TSP-1 (mTSP-1) promoter upstream of firefly luciferase pGL2 reporter, a gift from Dr. Paul Bornstein, has been described in detail elsewhere (Shingu & Bornstein 1994). MDFB6 detector cells were transfected using Lipofectamine2000 (Invitrogen, Life Technologies, Carlsbad, CA), and after 12 h, they were pooled into 24-well plates for treatment, which mostly lasted for 18–24 h. Cells were treated with indicated conditioned media or with control treatments, and then the cells were lysed in Cell Culture Lysis Reagent (Promega, Madison, WI, USA), supplemented with a protease inhibitor mix (Sigma-Aldrich) and stored at −80 °C until used. mTSP-1 promoter activity was measured in cell lysates using Luciferase Assay Reagent (Promega) and Luminometer TD20/20 (Turner Design, Sunnyvale, CA, USA). Results were normalized to protein content, as determined by Bradford Assay (Bio-Rad, Hercules, CA, USA).

Quantitative polymerase chain reaction (Real-Time PCR)

Total RNA was extracted from the cells using a Total RNA Minipreps Super kit (Bio Basic, Ontario, Canada) followed by DNase I (Fermentas, Ontario, Canada) treatment. RNA was reverse-transcribed with oligo(dT) primers (Sigma-Aldrich) using SuperScript III Reverse Transcriptase (Invitrogen). Quantitative PCR was performed with a 7300 Real-Time PCR system using reagents from Applied Biosystems (Foster City, CA, USA). The expression levels of TSP-1 were evaluated using the primer/probe set Mm01335418_m1. GAPDH was used as an endogenous control.

Western blotting

Cells were lysed in RIPA buffer supplemented with a protease inhibitor mix (Sigma-Aldrich). Protein content was quantified using Bradford Assay (Bio-Rad), and 50 or 30 μg/lane of protein was resolved by SDS–PAGE (8% or 12%) gel and transferred to Immobilon-P membranes (Millipore Corp., Billerica, MA, USA). Membranes were probed with anti-Ras (Pierce Biotechnology, Rockford, IL, USA) or rabbit anti-ERK2 0.3 μg/ml (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The signal was visualized using HRP-conjugated anti-mouse secondary antibody, 1/2000 (DAKO, Glostrup, Denmark), followed by incubation with the ECL reagent (Pierce).

Statistical analysis

P-value (statistical significance) was computed using unpaired Student’s t-test. Statistically significant differences were distinguished with asterisk.

Results

Presence of H-Ras oncogene results in increased secretion of sphingosine

For assessment of the amount of sphingosine present in conditioned media, a new method, based on GC-MSMS tandem chromatography, was developed. To enrich the lipid fraction, a known volume of conditioned media was reduced by lyophilization and then the sphingolipid fraction of the media was isolated using the extraction method described by Bodennec et al. (2000).

For the GC-MSMS analysis, samples were silylated using BSTFA. The spectrometric method was based on isolation of the primary ion of m/z = 338 from the whole primary mass spectrum of silylated sphingosine derivative (Figure 1a; M = 443) and its second fragmentation. The product ion with the m/z = 264 was used as the marker ion (Figure 1b). The amount of sphingosine in the sample was established by the assessment of area of the peak representing sphingosine derivative on the gas–liquid chromatogram in comparison with the standard curve obtained using commercial standard of the d-erythro-sphingosine.

Figure 1.

Figure 1

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Principles of sphingosine detection. (a) Structure of TMS derivative of sphingosine and ions used for its detection in tandem mass spectrometry. (b) Primary mass spectrum of silylated derivative of sphingosine (MS1) and the secondary mass spectrum of the fragmentation of the primary ion of m/z = 338 with its product ion m/z = 264 used for the quantification of sphingosine (MS2).

To study whether introduction of H-Ras oncogene leads to increased production of sphingosine, we have used a previously developed model of mouse dermal fibroblasts MDFB6 and their isogenic counterparts B6ras (Kalas et al. 2005b), stably transformed with H-Ras oncogene (Figure 2b). We estimated the concentration of sphingosine in the conditioned media of MDFB6 and B6ras cells, and we found that the amounts of sphingosine observed in media of B6ras cells were significantly higher than in media of MDFB6 (Figure 2a).

Figure 2.

Figure 2

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Presence of oncogenic Ras results in increased release of sphingosine to conditioned media (CM). (a) Concentration of sphingosine in CM. Averages ± SD from three experiments are shown. Asterisks indicate statistically significant difference, P < 0.05, n = 4. (b) B6ras cells express increased amounts of Ras protein assayed by Western blotting. The signal was quantified by densitometry and normalized to the ERK2 signal, which served as a loading control. Representative blots are shown. (c, d) Sphingosine concentration (c) in CM of CLS-1, B1v, H/A VSMC and A549 cell lines correlates with Ras expression (d). Averages ± SD from two experiments are shown. Asterisks indicate statistically significant difference, P < 0.05, n = 4–6. VSMC, vascular smooth muscle cells.

Next, we examined the amounts of sphingosine present in conditioned media of premalignant cell lines derived from mouse mammary gland (CLS-1) or intestine (B1v) or immortalized with telomerase normal human vascular smooth muscle cells (H/A VSMCs) with relatively low expression of Ras (Figure 2d) and carcinomic human alveolar basal epithelial cells (A549), expressing significant amounts of Ras protein. We found that the concentration of sphingosine in media of A549 cells was the highest and about threefold higher than in premalignant cell lines (Figure 2c).

For further studies of role of H-Ras oncogene in sphingosine release, we used the model of MT-Ras cells (Filmus et al. 1993, 1994) where H-Ras is placed under the control of heavy metal inducible promoter. Presence of heavy metal ions (delivered as cadmium chloride) induced at least threefold increase in Ras expression (Figure 3a). Conditioned media of non-induced MT-Ras cells contained relatively low amounts of sphingosine (Figure 3b). Induction of Ras expression resulted in fivefold increase in sphingosine concentration in conditioned media. Next, to reverse the effect of Ras induction, we used UO126, a selective inhibitor of MEK1 and MEK2, key kinases of Ras signalling pathway, which completely diminished the release of sphingosine by Ras-expressing cells. In the same circumstances, the inactive structural analogue of UO126 – UO124 – did not decrease the release of sphingosine, showing that the Ras presence and activity is crucial for raised release of sphingosine.

Figure 3.

Figure 3

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Presence of sphingosine in conditioned media is correlated with TSP-1 down-regulation. (a) Induction of the expression of Ras in MT-Ras cells by treatment with CdCl2 assayed by Western blotting. The signal was quantified by densitometry and normalized to the ERK2 signal, which served as a loading control. Representative blots are shown. (b) Release of sphingosine depends on Ras-pathway activity. The amount of sphingosine in conditioned media (CM) of MT-Ras cells treated with (to induce Ras expression) and/or MEK inhibitor UO126 or its inactive structural analogue UO124. Significant amounts of sphingosine were observed only in media collected in the presence of CdCl2 and/or inactive compound UO124. Media collected from cells where Ras was not induced (control) or activity of Ras pathway was down-regulated by UO126 contained undetectable amounts of sphingosine. Averages ± SD from three experiments are shown. Asterisks indicate statistically significant difference, P < 0.005, n = 3–6.

Sphingosine or sphingosine-rich media down-regulate TSP-1 expression in MDFB6 fibroblasts

We have previously shown that cells stably transfected with H-Ras oncogene release the unknown non-protein factor with the activity of diminishing expression of angiogenesis inhibitor – TSP-1 (Kalas et al. 2005b).

To test whether activation of Ras signalling, crucial for raised release of sphingosine, is also correlated with the ability to down-regulate expression of TSP-1, we used a MT-Ras cell model. TSP-1 expression was assayed by Luciferase Reporter assay in MDFB6 detector cells treated with conditioned media of MT-Ras cells (Figure 4a,b). Conditioned media of non-induced MT-Ras cells (endogenous Ras only) did not affect TSP-1 expression, but when Ras expression and sphingosine production was induced (Figure 3), then conditioned media down-regulated the expression of the TSP-1 mRNA (Figure 4b) and the activity of TSP-1 promoter by 60% (Figure 4a). Moreover, further addition of MEK inhibitor UO126, which diminished the release of sphingosine completely, resulted on regaining activity of the TSP-1 promoter. At the same time, the presence of then analogue but inactive compound UO124 did not restore the activity of TSP-1 promoter as it did not decrease. sphingosine release (Figure 4a). It is worth mentioning that cadmium chloride alone or used in combination with any of the aforementioned compound (control treatments) had no significant influence on the activity of TSP-1 reporter in MDFB6 detector cells.

Figure 4.

Figure 4

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H-Ras-dependent release of sphingosine down-regulates TSP-1 expression. MDFB6 detector cells transfected with mTSP-1 LucFL reporter vector were treated with specified conditioned medium (CM) or control treatment. (a) Media collected from cells treated with CdCl2 (to induce Ras expression) down-regulated the expression of TSP-1, measured in Luciferase Reporter Assay. When besides CdCl2, UO126 MEK inhibitor (but not its inactive structural analogue – UO124) was used, then media lost their ability to down-regulate the expression of TSP-1. Control treatments of MDFB6 cells with CdCl2 and/or UO126, UO124, did not down-regulate TSP-1 reporter activity in MDFB6 cells. Averages ± SD from three experiments are shown. Asterisks indicate statistically significant difference against cells treated with CM of untreated MT-Ras cells (control; P < 0.005), while hash indicates statistically significant difference against control treatment of MDFB6 (P < 0.005) n = 6–12. (b) Media collected from MT-Ras cells treated with CdCl2 (to induce Ras expression) down-regulated the expression of TSP-1 mRNA, measured by Real-Time PCR. Untreated MDFB6 cells served as control (RQ = 1). Averages and data ranges are shown. n = 3. (c) B6ras conditioned media lowered TSP-1 reporter activity to 70%. Averages ± SD from three experiments are shown. Asterisks indicate statistically significant difference, P < 0.005, n = 8–16. (d) Conditioned media of premalignant cell lines CLS-1, B1v or VSMCs increased the activity of TSP-1 reporter in contrast to A549 conditioned media which lowered TSP-1 reporter activity to 70%. Averages ± SD from three experiments are shown. Asterisks indicate statistically significant difference, P < 0.005, n = 8. VSMC, vascular smooth muscle cells.

The effect of sphingosine-rich conditioned media on TSP-1 expression can be recapitulated by the addition of sphingosine to sphingosine-low media of MT-Ras cells (Figure 3b). Sphingosine, but not sphingosine-1-phosphate or sphinganine, down-regulated the expression of TSP-1 in MDFB6 cells assayed by Real-Time PCR or TSP-1 promoter assay (Figure 5).

Figure 5.

Figure 5

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Sphingosine, but not sphingosine-1-phosphate, down-regulates TSP-1 expression. (a) MDFB6 detector cells transfected with mTSP-1 LucFL reporter vector were treated with sphingosine-low conditioned medium (CM) of MT-Ras cells supplemented with sphingosine, sphingosine-1-phosphate (Sp-1-P) and sphinganine. Averages ± SD from three experiments are shown. Asterisks indicate statistically significant difference, P < 0.005, n = 12–18. (b) TSP-1 mRNA expression in MDFB6 cells treated with 1 μM of sphingosine (Sph), sphingosine-1-phosphate (Sp-1-P) and sphinganine (Spn) assayed by Real-Time-PCR. Averages and data ranges are shown. n = 3.

The down-regulation of TSP-1 promoter activity was observed in cells treated with conditioned media of B6ras fibroblasts (Figure 4c) or lung carcinoma A549 (Figure 4d). Both media containing increased amounts of sphingosine significantly down-regulated the activity of the TSP-1 promoter. In contrast, sphingosine-low conditioned media from normal fibroblasts MDFB6 or premalignant cells CLS-1, B1v and H/A VSMCs did not decrease the activity of TSP-1 promoter in MDFB6. It suggests that the ability to down-regulate TSP-1 expression is exerted only by media produced by Ras-transformed cells, containing high amounts of sphingosine.

Discussion

It is now widely accepted that oncogenic mutation leading to the development of tumour results in perturbation of homeostasis of the whole organism (Rak & Klement 2000). For example, in tumours with HER-2 or EGFR mutations, increased expression of tissue factor was found, manifesting as Trousseau syndrome (Milsom & Rak 2008; Yu et al. 2010). The other example is the data presented by Hirsch et al. showing that surprisingly the transcriptional signature of cancer is linked tp lipid metabolism, in particular to the expression of the oxidized LDL receptor (OLR1) gene as well as to other genes related to lipid metabolism and this correlated with the progression of breast and prostate cancers (Hirsch et al. 2010). Interestingly, metabolism of LDL was shown to be regulated by sphingosine and PKC (Rodriguez-Lafrasse et al. 1997), which suggests a potential role of sphingosine in cancer progression.

Some connection between presence of oncogenic H-Ras and sphingolipid metabolism was found in the studies showing increased sphingosine kinase activity and subsequent sphingosine-1-phosphate accumulation in Ras-transformed cells (Xia et al. 2000). Similarly Knapp et al. 2010 presented the data showing the accumulation of sphinganine, sphingosine, ceramide and other sphingolipids in the endometrial cancer tissue. But the mechanism of alternation of sphingolipids metabolism observed in H-ras-transformed cells still remains unclear. Here, using multiple models, we provide the direct evidence that introduction of oncogenic H-Ras alters the sphingolipid metabolism in the manner that leads to increased release of sphingosine. The metabolic origin of sphingosine produced and released by Ras-transformed cells is unknown. Sphingosine may be generated in two ways: by deacylation of ceramide mediated by CDases or dephosphorylation of sphingosine-1-phosphate (Gault et al. 2010). The results of Knapp et al. show increased amounts of sphingosine, as well as sphinganine and ceramide (which could be converted to sphingosine), suggesting up-regulation in the synthesis of sphingolipids in endometrial cancer tissue, while the data of Xia et al. indicate the another possibility that sphingosine could be generated from sphingosine-1-phosphate by S1P phosphatases acting inside or outside the cell. Nevertheless, the origin of sphingosine expressed by Ras-transformed cells remains to be determined.

Aforementioned studies of impact of impaired sphingolipid metabolism were limited to the transformed cells itself. In the previous studies, we have shown that even a small number of H-Ras-transformed dermal fibroblasts (528ras1) were able to down-regulate TSP-1 expression in the surrounding cells, formatting a proangiogenic environment. It was suggested and presented that H-Ras-transformed cells release the unidentified low-molecular weight and non-protein factor with the activity of diminishing the expression of angiogenesis inhibitor – TSP-1 (Kalas et al. 2005b). We have also shown that platelets and their related sphingolipids have the ability to down-regulate the expression of TSP-1 (Kalas et al. 2005a), suggesting its role in maintaining the balance of angiogenic factors.

Here, we show that sphingosine and sphingosine-rich media, but not sphinganine or sphingosine-1-phosphate, down-regulate the expression of TSP-1 in normal cells in each model we have used. This strongly suggests that H-Ras decreases the expression of angiogenesis inhibitor TSP-1 in the surrounding normal cells using the low-molecular weight, non-protein mediator – sphingosine. Thus, this finding allows to explain the previously published data and reveals novel features about the nature of proangiogenic field formation (Kalas et al. 2005b).

It is not clear why down-regulation of TSP-1 by sphingosine depends on sphingosine kinase activity, but interestingly at the same time, it is not diminished by the inhibition of G-coupled receptors of sphingosine-1-phosphate and cannot be recapitulated by extracellular sphingosine-1-phosphate (Kalas et al. 2005a,b). Some explanation may be linked with different transport of free sphingosine and sphingosine-1-phosphate. Sphingosine in contrast to sphingosine-1-phosphate easily passes through the cellular membrane into the cell (Garmy et al. 2005). In the cytoplasm, sphingosine is rapidly converted into sphingosine-1-phosphate by sphingosine kinase (Zhao et al. 2007). Interestingly, the intracellular sphingosine-1-phosphate was shown to act differently than extracellular (Kim et al. 2009). Even more its action was implicated in the down-regulation of angiogenesis-related genes (Limaye et al. 2005, 2009). Similar action of sphingosine was demonstrated on growth-arrested Swiss 3T3 cells (Olivera et al. 1992). Sphingosine was shown to be rapidly taken up by these cells, phosphorylated by sphingosine kinase and eventually induced proliferation (Zhang et al. 1991). Presented data suggest that the impact of altered sphingolipid metabolism extends beyond the single tumour cell or tumour but may also affect genetically normal cells surrounding tumour tissue. Overall, our studies demonstrate a H-Ras-dependent mechanism of changing the equilibrium of angiogenic factors in favour of angiogenesis induction, where a central role is played by the low molecular entity: sphingosine. This represents a mechanism of translating genetic changes within transformed cells into much larger, amplified effect involving tumour parenchyma and stroma, which could greatly accelerate local tumour growth and metastasis.

Acknowledgments

This work was supported by grant no. N301 104 31/3087 and 1243/B/P01/2007/33 from the Polish State Committee for Scientific Research.

Conflict of interest

All authors declare that they have no competing interests.

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