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Lovastatin inhibits Rho-regulated expression of E-selectin by TNF and attenuates tumor cell adhesion¹

TOBIAS NÜBEL, WOLFGANG DIPPOLD^*, HARTMUT KLEINERT, BERND KAINA and GERHARD FRITZ²

University of Mainz, Institute of Toxicology, Division of Applied Toxicology;
^* St. Vincenz and Elisabeth Hospital; and
University of Mainz, Institute of Pharmacology, D-55131 Mainz, Germany

²Correspondence: University of Mainz, Institute of Toxicology, Division of Applied Toxicology, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany. E-mail: fritz{at}mail.uni-mainz.de

SPECIFIC AIMS

E-selectin is involved in metastasis by promoting adhesion of circulating tumor cells to the endothel. We investigated whether small GTPases of the Rho family are essential for E-selectin gene induction by TNF and whether statins, reported to attenuate Rho-regulated functions, inhibit TNF-induced E-selectin gene and protein expression in human umbilical vein endothelial cells (HUVEC). We explored the usefulness of lovastatin for preventing TNF-stimulated tumor cell adhesion and invasion using an in vitro model system.

PRINCIPAL FINDINGS

1. Lovastatin impairs TNF-induced E-selectin gene and protein expression
Treatment of human endothelial cells (HUVEC) with TNF results in a dose-dependent increase in E-selectin protein expression as investigated by an ELISA-based method. Induction of E-selectin expression was confirmed on the level of the mRNA and by reporter gene analysis using a 1.24 kb human E-selectin promoter fragment. We analyzed the effect of HMG-CoA reductase inhibitors (lovastatin and simvastatin) on the expression of E-selectin by TNF. Both types of statin inhibit TNF-stimulated E-selectin expression in a dose-dependent manner (Fig. 1 A). Data obtained by ELISA were confirmed by FACS analysis (Fig. 1B ). A sizable inhibition of TNF-stimulated E-selectin expression by lovastatin was observed on E-selectin mRNA level (Fig. 1C ) and on the level of the E-selectin promoter (Fig. 1D ). Overall, the data show that lovastatin is a powerful inhibitor of TNF-stimulated E-selectin gene and protein expression.

View larger version (33K): [in this window] [in a new window]   Figure 1. TNF-induced E-selectin protein and gene expression are blocked by statins. A, B) Subconfluent HUVEC were pretreated or not overnight with different concentrations of lovastatin or simvastatin. TNF (1 ng/mL) was added and 5 h later E-selectin protein expression was analyzed by an ELISA-based method (A) or FACS analysis (B). Data are mean values ± SD from at least 3 experiments. C) Upon pretreatment of HUVEC with lovastatin (20 µM, overnight), cells were exposed to TNF (1 ng/mL). 3 h later, cells were harvested for analysis of E-selectin mRNA expression by semiquantitative RT-PCR analysis and real-time PCR analysis. Quantitation shown below the autoradiogram is based on real-time PCR analysis. D) 24 h after transfection of HUVEC with E-selectin promoter luciferase construct, cells were treated overnight with lovastatin (20 µM) before TNF stimulation (1 ng/mL). Cells were harvested to determine luciferase activity 8 h later. Data are mean values ± SD from 3 independent experiments performed in duplicate.

2. Physiological relevant concentration of lovastatin impairs E-selectin expression in confluent HUVEC stimulated with low dose of TNF
We examined whether clinically relevant doses of lovastatin (1 µM) are able to inhibit TNF-induced E-selectin expression at nonsaturating concentrations of TNF (10 pg/mL) in confluent HUVEC. As measured by FACS, 0.1–1 µM of lovastatin reduced by 70–80% the level of TNF-stimulated E-selectin expression. The frequency of E-selectin-positive cells after TNF stimulation was not affected by lovastatin. Lovastatin obviously reduces the level of E-selectin expression in all TNF responding cells.

3. Rho signaling is essential for E-selectin gene induction by TNF
To characterize the type of isoprenylated protein(s) involved in the regulation of E-selectin gene expression by TNF in more detail, we investigated whether cotreatment with geranylgeranylpyrophosphate (GGPP) or farnesylpyrophosphate (FPP) is able to reverse the inhibitory effect of lovastatin. Supplementation with geranylgeranylpyrophosphate significantly reverted inhibition of E-selectin expression by lovastatin whereas farnesylpyrophosphate did not. In line with this, geranylgeranyltransferase type I inhibitor (GGTI) but not the farnesyltransferase inhibitor (FTI) abrogated induction of E-selectin expression by TNF to a similar extent as lovastatin did. Since Rho GTPases are putative targets of statins and are reported to be involved in the regulation of NF-B, we examined the effect of down-modulation of Rho signaling on basal and TNF-stimulated E-selectin promoter activity. Coexpression of dominant-negative forms of RhoA, RhoB, and Rac did not affect the basal activity of the E-selectin promoter but largely inhibited its TNF-triggered activation. Based on this we suggest that RhoA, RhoB, and Rac-dependent signal mechanisms are essential for TNF-mediated transcriptional activation of the E-selectin gene.

4. Lovastatin attenuates TNF-stimulated adhesion of tumors cells to HUVEC
Stimulation of a confluent layer of human endothelial cells (HUVEC) with TNF causes a dose-dependent increase in the adhesion of human colon carcinoma cells (Fig. 2 A). Already at a concentration as low as 0.1–1 µM, lovastatin significantly blocked cytokine-induced adhesion by 50–60% (Fig. 2B, C ). This is once more illustrated by microscopical analysis shown in Fig. 2C . Expression of P-selectin, which is inducible by TNF in murine endothelial cells, was not induced under our experimental conditions in HUVEC (Fig. 2D ). A low dose of lovastatin (1 µM) did not affect TNF-mediated expression of the adhesion molecule ICAM-1 (Fig. 2D ), showing that the inhibitory effect of lovastatin is specific for E-selectin. Thus, the data strongly supports the conclusion that attenuation of TNF-induced E-selectin expression is a main reason underlying the observed inhibition of tumor cell adhesion by lovastatin.

View larger version (52K): [in this window] [in a new window]   Figure 2. Lovastatin attenuates TNF-induced adhesion of human tumor cells to endothelial cells. A) Confluent HUVEC were left untreated or exposed to increasing concentrations of TNF. After 5 h incubation, 5 x 105 of calcein-labeled human colon carcinoma cells (DLD1) were added and adhesion of DLD1 cells was quantitated 2 h later. Data are mean values ± SD from 3 independent experiments performed in quadruplicate. B) Confluent HUVEC were pretreated or not for 24 h with lovastatin (0.1–20 µM). Cells were exposed to TNF (1 ng/mL). After another incubation of 5 h, adhesion of calcein labeled human tumor cells (DLD1) was assayed. Data are mean values ± SD from at least 2 independent experiments performed in quadruplicate. Identical results were obtained with lower concentration of TNF (10 and 100 pg/mL) (data not shown). C) Illustration of the inhibitory effect of lovastatin (1 µM, overnight) on TNF (1 ng/mL)-induced adhesion of colon carcinoma cells (DLD1) to HUVEC. a) Nonstimulated HUVEC + DLD1; b) Lovastatin pretreated HUVEC + DLD1; c) TNF-stimulated HUVEC + DLD1; d) lovastatin-pretreated, TNF-stimulated HUVEC + DLD1. D) Human endothelial cells (HUVEC) were pretreated or not for 24 h with 0.1 µM or 1.0 µM of lovastatin. Cells were exposed to TNF (10 pg/mL). After TNF exposure, expression of E-selectin, P-selectin, and ICAM-1 was analyzed by an ELISA-based method. Data mean ± SD from 3 independent experiments, each performed in triplicate.

5. Lovastatin attenuates TNF-stimulated tumor cell invasion in vitro
Next we examined the effect of lovastatin on tumor cell invasion using an in vitro transmigration model system. We observed that TNF-stimulated transmigration of colon carcinoma cells through a confluent monolayer of HUVEC was drastically reduced in the presence of low concentration of lovastatin (0.1–1 µM). Thus, lovastatin attenuates both tumor cell adhesion and subsequent invasion in vitro. TNF and lovastatin did not cause any changes in NO production under our experimental conditions. Therefore, the observed effects of lovastatin on cell adhesion and invasion are independent from NO-triggered pathways.

CONCLUSIONS AND SIGNIFICANCE

Modulation of expression of the cell adhesion molecule E-selectin can be regarded as a promising strategy for improving tumor therapy. Since NF-B is essential for TNF-stimulated E-selectin expression and statins inhibit stress-induced activation of NF-B, we investigated whether lovastatin is pharmacologically useful for down-modulation of E-selectin expression. We included FACS- and ELISA-based protein and mRNA and promoter analyses in our study, providing convincing evidence that lovastatin abrogates the TNF-stimulated E-selectin gene and protein expression in HUVEC. Our findings show that inhibition of gene expression is the major mechanism by which lovastatin causes a reduction in TNF-induced E-selectin protein expression.

We observed inhibition of E-selectin expression by lovastatin at very low concentrations, suggesting the clinical relevance of the findings. If nongrowing HUVEC were stimulated with nonsaturating doses (10 pg/ml) of TNF, the lovastatin concentration inhibited E-selectin expression by 50% (ID₅₀) was <100 nM. This concentration is in the range of that achieved in vivo in patients. The TNF-triggered increase in the adhesion of human colon carcinoma cells to endothelial cells was attenuated by lovastatin at a physiological dose of 0.1–1 µM. Bearing in mind that E-selectin-mediated adhesion of tumor cells to the endothelium is considered to promote extravasation of tumor cells, our data support the view that statins might be useful as anti-metastatic drugs, particularly under conditions of increased TNF production (e.g., during inflammation, often observed in large necrotic tumors). This hypothesis gains support by our observation that low doses of lovastatin (0.1–1 µM) reduced tumor cell invasion in vitro. A suggested model to explain the lovastatin-mediated inhibition of E-selectin expression and tumor cell adhesion is presented in Fig. 3 .

View larger version (60K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating statin-mediated inhibition of E-selectin expression and tumor cell adhesion. Statins decrease the intracellular concentration of isoprene precursor moieties required for carboxyl-terminal isoprenylation of Rho proteins by corresponding isoprene transferases. Isoprenylation is essential for correct intracellular localization and function of Rho GTPases. By inhibiting Rho-dependent activation of NF-B, statins attenuate TNF-induced E-selectin gene expression and E-selectin protein induction. Under blockage of E-selectin protein expression, a putative inhibitory effect of lovastatin on receptor clustering is no longer relevant. Since E-selectin acts as a ligand for tumor-specific expressed Sialyl-Lewis X and Sialyl-Lewis A glycoproteins, it promotes adhesion of tumor cells to the endothel. By blocking TNF-induced Rho-dependent E-selectin expression, lovastatin causes inhibition of the adhesion of tumor cells to endothelial cells.

Overexpression of Rac or Cdc42 was shown to potentiate TNF-driven E-selectin expression. We speculated that inhibition of Rho/Rac signaling might be responsible for inhibition of E-selectin induction by lovastatin. This hypothesis is supported by the observation that coexpression of dominant-negative RhoA, RhoB, and Rac abrogated transcriptional activation of the E-selectin promoter by TNF. Obviously, Rho/Rac signaling is essential for stimulation of E-selectin gene expression by TNF. Thus, E-selectin-promoted adhesion of tumor cells to the endothel might contribute to the frequently reported prometastatic potency of Rho proteins. We suggest that inhibition of Rho by lovastatin leads to a block of TNF-stimulated activation of NF-B, thereby reducing E-selectin gene expression and thus attenuating E-selectin-promoted tumor cell adhesion (Fig. 3) . Tumor cell invasion was also strongly attenuated by lovastatin, supporting the view of an anti-metastatic effect of statins. Since this class of drugs is well established and frequently used, our finding of inhibition of cytokine-induced E-selectin expression and tumor cell adhesion and invasion in vitro might have clinical impact.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0261fje

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