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Rac1-regulated endothelial radiation response stimulates extravasation and metastasis that can be blocked by HMG-CoA reductase inhibitors.

Hamalukic M, Huelsenbeck J, Schad A, Wirtz S, Kaina B, Fritz G - PLoS ONE (2011)

Bottom Line: IR-stimulated TC-EC adhesion was blocked by the HMG-CoA reductase inhibitor lovastatin.Glycyrrhizic acid from liquorice root, which acts as a Sialyl-Lewis X mimetic drug, and the Rac1 inhibitor NSC23766 also reduced TC-EC adhesion.To examine the in vivo relevance of these findings, tumorigenic cells were injected into the tail vein of immunodeficient mice followed by total body irradiation (TBI).

View Article: PubMed Central - PubMed

Affiliation: Institute of Toxicology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.

ABSTRACT
Radiotherapy (RT) plays a key role in cancer treatment. Although the benefit of ionizing radiation (IR) is well established, some findings raise the possibility that irradiation of the primary tumor not only triggers a killing response but also increases the metastatic potential of surviving tumor cells. Here we addressed the question of whether irradiation of normal cells outside of the primary tumor augments metastasis by stimulating the extravasation of circulating tumor cells. We show that IR exposure of human endothelial cells (EC), tumor cells (TC) or both increases TC-EC adhesion in vitro. IR-stimulated TC-EC adhesion was blocked by the HMG-CoA reductase inhibitor lovastatin. Glycyrrhizic acid from liquorice root, which acts as a Sialyl-Lewis X mimetic drug, and the Rac1 inhibitor NSC23766 also reduced TC-EC adhesion. To examine the in vivo relevance of these findings, tumorigenic cells were injected into the tail vein of immunodeficient mice followed by total body irradiation (TBI). The data obtained show that TBI dramatically enhances tumor cell extravasation and lung metastasis. This pro-metastatic radiation effect was blocked by pre-treating mice with lovastatin, glycyrrhizic acid or NSC23766. TBI of mice prior to tumor cell transplantation also stimulated metastasis, which was again blocked by lovastatin. The data point to a pro-metastatic trans-effect of RT, which likely rests on the endothelial radiation response promoting the extravasation of circulating tumor cells. Administration of the widely used lipid-lowering drug lovastatin prior to irradiation counteracts this process, likely by suppressing Rac1-regulated E-selectin expression following irradiation. The data support the concern that radiation exposure might increase the extravasation of circulating tumor cells and recommend co-administration of lipid-lowering drugs to avoid this adverse effect of ionizing radiation.

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Irradiation of both endothelial and tumor cells stimulates TC-EC adhesion that is attenuated by lovastatin.A: Human colon carcinoma cells (HT29) were irradiated with 2–10 Gy. 4 h later, adhesion to primary human endothelial cells (HUVEC) was analyzed as described in Methods. Relative cell adhesion in untreated control was set to 1.0. *p≤0.05, **p≤0.01 (n = 15). B: Human colon carcinoma cells (HT29) or endothelial cells (HUVEC) were irradiated with 5 Gy. 4 h later, TC-EC adhesion was analyzed as described in Methods. **p≤0.01 (n = 8). C: Human colon carcinoma (HT29) and/or endothelial cells (HUVEC) were left untreated or were pretreated overnight with lovastatin (10 µM) (Lova). Afterwards, cells were irradiated (10 Gy) (IR) and TC-EC interactions were assayed after further incubation period of 4 h as described in Methods. See also Figure S1 and S2. ***p≤0.001; **p≤0.01 (n = 8). D: Logarithmically growing human endothelial (HUVEC) or tumor cells (HT29) were left untreated or were pretreated overnight with lovastatin (Lova). Afterwards cells were irradiated (IR) with 10 Gy and harvested 2 h or 4 h later for analysis of mRNA expression of various cell adhesion molecules. GAPDH mRNA expression was determined as internal loading control.
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pone-0026413-g001: Irradiation of both endothelial and tumor cells stimulates TC-EC adhesion that is attenuated by lovastatin.A: Human colon carcinoma cells (HT29) were irradiated with 2–10 Gy. 4 h later, adhesion to primary human endothelial cells (HUVEC) was analyzed as described in Methods. Relative cell adhesion in untreated control was set to 1.0. *p≤0.05, **p≤0.01 (n = 15). B: Human colon carcinoma cells (HT29) or endothelial cells (HUVEC) were irradiated with 5 Gy. 4 h later, TC-EC adhesion was analyzed as described in Methods. **p≤0.01 (n = 8). C: Human colon carcinoma (HT29) and/or endothelial cells (HUVEC) were left untreated or were pretreated overnight with lovastatin (10 µM) (Lova). Afterwards, cells were irradiated (10 Gy) (IR) and TC-EC interactions were assayed after further incubation period of 4 h as described in Methods. See also Figure S1 and S2. ***p≤0.001; **p≤0.01 (n = 8). D: Logarithmically growing human endothelial (HUVEC) or tumor cells (HT29) were left untreated or were pretreated overnight with lovastatin (Lova). Afterwards cells were irradiated (IR) with 10 Gy and harvested 2 h or 4 h later for analysis of mRNA expression of various cell adhesion molecules. GAPDH mRNA expression was determined as internal loading control.

Mentions: Previously, we reported a pro-adhesive IR response of human endothelial cells (EC), which is due to the upregulation of endothelial adhesion molecules, in particular E-selectin, via NF-κB [7]. Here, we expand on this observation showing that irradiation of HT29 human colon carcinoma cells (TC) increases their binding to human umbilical vein endothelial cells (HUVEC) in vitro. Tumor cell-endothelial cell (TC-EC) adhesion following irradiation of tumor cells occurred in a dose dependent manner (Fig. 1A). Irradiation of both tumor and endothelial cells exerted additive effects (Fig. 1B). Similar pro-adhesive effects were obtained after irradiation of tumorigenic rodent cells (i.e. CHO-K1 cells; see Figure S1). Whereas pre-treatment of HUVEC with the lipid lowering drug lovastatin attenuated TC-EC adhesion, pre-treatment of tumor cells with lovastatin was ineffective (Fig. 1C). This indicates that the anti-adhesive effect of lovastatin mainly rests on a reduced upregulation of endothelial adhesion factors following radiation exposure. Similar to IR, treatment of HT29 cells with the pro-inflammatory cytokine TNFα, which is another prototypical NF-κB activating agent, also augmented TC-EC adhesion (see Figure S2A). We also show that TC-EC adhesion following TNFα treatment of HUVEC [7], was further enhanced when the colon carcinoma cells were irradiated (see Figure S2B and S2C). Whether treatment of either HUVEC or HT29 cells with TNFα plus IR ameliorates TC-EC adhesion as compared to a single treatment remains to be tested. Regarding HUVEC, such additivity might be expected since we observed TNFα and IR to have additive effects on the expression level of E-selectin protein [7]. Overall, the data indicate that activation of the transcription factor NF-κB, either by TNFα or ionizing radiation, is of relevance for TC-EC adhesion.


Rac1-regulated endothelial radiation response stimulates extravasation and metastasis that can be blocked by HMG-CoA reductase inhibitors.

Hamalukic M, Huelsenbeck J, Schad A, Wirtz S, Kaina B, Fritz G - PLoS ONE (2011)

Irradiation of both endothelial and tumor cells stimulates TC-EC adhesion that is attenuated by lovastatin.A: Human colon carcinoma cells (HT29) were irradiated with 2–10 Gy. 4 h later, adhesion to primary human endothelial cells (HUVEC) was analyzed as described in Methods. Relative cell adhesion in untreated control was set to 1.0. *p≤0.05, **p≤0.01 (n = 15). B: Human colon carcinoma cells (HT29) or endothelial cells (HUVEC) were irradiated with 5 Gy. 4 h later, TC-EC adhesion was analyzed as described in Methods. **p≤0.01 (n = 8). C: Human colon carcinoma (HT29) and/or endothelial cells (HUVEC) were left untreated or were pretreated overnight with lovastatin (10 µM) (Lova). Afterwards, cells were irradiated (10 Gy) (IR) and TC-EC interactions were assayed after further incubation period of 4 h as described in Methods. See also Figure S1 and S2. ***p≤0.001; **p≤0.01 (n = 8). D: Logarithmically growing human endothelial (HUVEC) or tumor cells (HT29) were left untreated or were pretreated overnight with lovastatin (Lova). Afterwards cells were irradiated (IR) with 10 Gy and harvested 2 h or 4 h later for analysis of mRNA expression of various cell adhesion molecules. GAPDH mRNA expression was determined as internal loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198428&req=5

pone-0026413-g001: Irradiation of both endothelial and tumor cells stimulates TC-EC adhesion that is attenuated by lovastatin.A: Human colon carcinoma cells (HT29) were irradiated with 2–10 Gy. 4 h later, adhesion to primary human endothelial cells (HUVEC) was analyzed as described in Methods. Relative cell adhesion in untreated control was set to 1.0. *p≤0.05, **p≤0.01 (n = 15). B: Human colon carcinoma cells (HT29) or endothelial cells (HUVEC) were irradiated with 5 Gy. 4 h later, TC-EC adhesion was analyzed as described in Methods. **p≤0.01 (n = 8). C: Human colon carcinoma (HT29) and/or endothelial cells (HUVEC) were left untreated or were pretreated overnight with lovastatin (10 µM) (Lova). Afterwards, cells were irradiated (10 Gy) (IR) and TC-EC interactions were assayed after further incubation period of 4 h as described in Methods. See also Figure S1 and S2. ***p≤0.001; **p≤0.01 (n = 8). D: Logarithmically growing human endothelial (HUVEC) or tumor cells (HT29) were left untreated or were pretreated overnight with lovastatin (Lova). Afterwards cells were irradiated (IR) with 10 Gy and harvested 2 h or 4 h later for analysis of mRNA expression of various cell adhesion molecules. GAPDH mRNA expression was determined as internal loading control.
Mentions: Previously, we reported a pro-adhesive IR response of human endothelial cells (EC), which is due to the upregulation of endothelial adhesion molecules, in particular E-selectin, via NF-κB [7]. Here, we expand on this observation showing that irradiation of HT29 human colon carcinoma cells (TC) increases their binding to human umbilical vein endothelial cells (HUVEC) in vitro. Tumor cell-endothelial cell (TC-EC) adhesion following irradiation of tumor cells occurred in a dose dependent manner (Fig. 1A). Irradiation of both tumor and endothelial cells exerted additive effects (Fig. 1B). Similar pro-adhesive effects were obtained after irradiation of tumorigenic rodent cells (i.e. CHO-K1 cells; see Figure S1). Whereas pre-treatment of HUVEC with the lipid lowering drug lovastatin attenuated TC-EC adhesion, pre-treatment of tumor cells with lovastatin was ineffective (Fig. 1C). This indicates that the anti-adhesive effect of lovastatin mainly rests on a reduced upregulation of endothelial adhesion factors following radiation exposure. Similar to IR, treatment of HT29 cells with the pro-inflammatory cytokine TNFα, which is another prototypical NF-κB activating agent, also augmented TC-EC adhesion (see Figure S2A). We also show that TC-EC adhesion following TNFα treatment of HUVEC [7], was further enhanced when the colon carcinoma cells were irradiated (see Figure S2B and S2C). Whether treatment of either HUVEC or HT29 cells with TNFα plus IR ameliorates TC-EC adhesion as compared to a single treatment remains to be tested. Regarding HUVEC, such additivity might be expected since we observed TNFα and IR to have additive effects on the expression level of E-selectin protein [7]. Overall, the data indicate that activation of the transcription factor NF-κB, either by TNFα or ionizing radiation, is of relevance for TC-EC adhesion.

Bottom Line: IR-stimulated TC-EC adhesion was blocked by the HMG-CoA reductase inhibitor lovastatin.Glycyrrhizic acid from liquorice root, which acts as a Sialyl-Lewis X mimetic drug, and the Rac1 inhibitor NSC23766 also reduced TC-EC adhesion.To examine the in vivo relevance of these findings, tumorigenic cells were injected into the tail vein of immunodeficient mice followed by total body irradiation (TBI).

View Article: PubMed Central - PubMed

Affiliation: Institute of Toxicology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.

ABSTRACT
Radiotherapy (RT) plays a key role in cancer treatment. Although the benefit of ionizing radiation (IR) is well established, some findings raise the possibility that irradiation of the primary tumor not only triggers a killing response but also increases the metastatic potential of surviving tumor cells. Here we addressed the question of whether irradiation of normal cells outside of the primary tumor augments metastasis by stimulating the extravasation of circulating tumor cells. We show that IR exposure of human endothelial cells (EC), tumor cells (TC) or both increases TC-EC adhesion in vitro. IR-stimulated TC-EC adhesion was blocked by the HMG-CoA reductase inhibitor lovastatin. Glycyrrhizic acid from liquorice root, which acts as a Sialyl-Lewis X mimetic drug, and the Rac1 inhibitor NSC23766 also reduced TC-EC adhesion. To examine the in vivo relevance of these findings, tumorigenic cells were injected into the tail vein of immunodeficient mice followed by total body irradiation (TBI). The data obtained show that TBI dramatically enhances tumor cell extravasation and lung metastasis. This pro-metastatic radiation effect was blocked by pre-treating mice with lovastatin, glycyrrhizic acid or NSC23766. TBI of mice prior to tumor cell transplantation also stimulated metastasis, which was again blocked by lovastatin. The data point to a pro-metastatic trans-effect of RT, which likely rests on the endothelial radiation response promoting the extravasation of circulating tumor cells. Administration of the widely used lipid-lowering drug lovastatin prior to irradiation counteracts this process, likely by suppressing Rac1-regulated E-selectin expression following irradiation. The data support the concern that radiation exposure might increase the extravasation of circulating tumor cells and recommend co-administration of lipid-lowering drugs to avoid this adverse effect of ionizing radiation.

Show MeSH
Related in: MedlinePlus