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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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Total body irradiation triggers tumor cell extravasation and lung metastasis in vivo.A: Balb/c mice were irradiated (IR) with 6 Gy. 4 h after total body irradiation (TBI) mRNA expression of cell adhesion molecules was analyzed in large blood vessels from pulmonary and abdominal artery by RT-PCR. GAPDH mRNA expression was measured as internal loading control. As a positive control, isolated blood vessels were incubated with TNFα (10 ng/ml, 30 min, 37 oC). B: Balb/c mice were subjected to TBI (6 Gy). 4 h after radiation (IR), phosphorylation of NF-κB inhibitor IκBα (p-IκBα), which is indicative of activation of NF-κB, was analyzed in liver extracts by Western blot analysis. ERK2 protein expression was determined as internal loading control. C: 2×106 CHO-K1 cells were injected into the tail vein of immunodeficient Rag2−/− Balb/c mice. Immediately after injection, mice were irradiated (4 Gy) (TBI). 3–4 weeks after TBI, the formation of metastases was analyzed in lung and liver. TC, tumor cells injected; TBI, total body irradiation. Shown are representative morphological and histopathological pictures (2–4 animals were analyzed per group). D: Red fluorescent protein overexpressing CHO-K1 cells were used for injection into the tail vein of immunodeficient Rag2−/− Balb/c mice. Formation of lung metastases was analyzed 3–4 weeks after TBI with 4 Gy. Con, non-irradiated control; IR, TBI with 4 Gy. Shown are representative pictures (2–3 animals were analyzed per group). E: Four weeks after i.v. injection of luciferase overexpressing CHO-K1 cells followed by TBI (4 Gy), luciferase activity in the lung was monitored by use of a life imaging instrument as described in Methods. Shown are representative pictures (2–3 animals were analyzed per group). F: 3–4 weeks after i.v. injection of β-galactosidase (β-Gal) overexpressing CHO-K1 cells followed by TBI (4 Gy), relative β-Gal activity in lung extracts of irradiated mice was related to that of non irradiated control mice, which was set to 100%. **p≤0.01 (n = 4–8 mice). G: Formation of lung metastasis was quantitated in the lung of irradiated versus non-irradiated mice by calculating the % tumor area as related to the total lung area. Con, non-irradiated control; IR, TBI with 4 Gy. ***p<0.001 (n = 4–8 mice).
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pone-0026413-g002: Total body irradiation triggers tumor cell extravasation and lung metastasis in vivo.A: Balb/c mice were irradiated (IR) with 6 Gy. 4 h after total body irradiation (TBI) mRNA expression of cell adhesion molecules was analyzed in large blood vessels from pulmonary and abdominal artery by RT-PCR. GAPDH mRNA expression was measured as internal loading control. As a positive control, isolated blood vessels were incubated with TNFα (10 ng/ml, 30 min, 37 oC). B: Balb/c mice were subjected to TBI (6 Gy). 4 h after radiation (IR), phosphorylation of NF-κB inhibitor IκBα (p-IκBα), which is indicative of activation of NF-κB, was analyzed in liver extracts by Western blot analysis. ERK2 protein expression was determined as internal loading control. C: 2×106 CHO-K1 cells were injected into the tail vein of immunodeficient Rag2−/− Balb/c mice. Immediately after injection, mice were irradiated (4 Gy) (TBI). 3–4 weeks after TBI, the formation of metastases was analyzed in lung and liver. TC, tumor cells injected; TBI, total body irradiation. Shown are representative morphological and histopathological pictures (2–4 animals were analyzed per group). D: Red fluorescent protein overexpressing CHO-K1 cells were used for injection into the tail vein of immunodeficient Rag2−/− Balb/c mice. Formation of lung metastases was analyzed 3–4 weeks after TBI with 4 Gy. Con, non-irradiated control; IR, TBI with 4 Gy. Shown are representative pictures (2–3 animals were analyzed per group). E: Four weeks after i.v. injection of luciferase overexpressing CHO-K1 cells followed by TBI (4 Gy), luciferase activity in the lung was monitored by use of a life imaging instrument as described in Methods. Shown are representative pictures (2–3 animals were analyzed per group). F: 3–4 weeks after i.v. injection of β-galactosidase (β-Gal) overexpressing CHO-K1 cells followed by TBI (4 Gy), relative β-Gal activity in lung extracts of irradiated mice was related to that of non irradiated control mice, which was set to 100%. **p≤0.01 (n = 4–8 mice). G: Formation of lung metastasis was quantitated in the lung of irradiated versus non-irradiated mice by calculating the % tumor area as related to the total lung area. Con, non-irradiated control; IR, TBI with 4 Gy. ***p<0.001 (n = 4–8 mice).

Mentions: Binding of circulating tumor cells to the endothelium is a prerequisite for their extravasation in vivo. Hence, the in vitro data are alarming because they implicate that radiotherapy might promote metastasis in vivo. To scrutinize this hypothesis we performed experiments using different mouse models. We showed that total body irradiation (TBI) of Balb/c mice results in the upregulation of both E-selectin and ICAM-1 mRNA levels (Fig. 2A), which was accompanied by activation of NF-κB (Fig. 2B). This is fully in line with other experimental systems demonstrating activation of NF-κB and subsequent expression of E-selectin and ICAM mRNA and protein by IR [20], [34], [35], [41]. Next, we determined whether TBI promotes extravasation and metastasis. To this end, tumorigenic Chinese hamster (CHO-K1) cells, which were shown to have significant metastatic potential [42], were injected into the tail vein of immunodeficient mice (Rag2−/− Balb/c strain). In our hands, CHO-K1 cells turned out to be the most appropriate cell line in this mouse model. Other cell lines tested were not metastatic at all or showed too high basal level of metastasis (see Figure S4). Immediately after injection, mice were subjected to TBI with a single dose of 4 Gy and colonization of the lung with metastases was analyzed 3–4 weeks later in comparison with non-irradiated animals. As shown in Fig. 2C, TBI caused a dramatic increase in the formation of lung metastases and, to a lesser extent, also increased the formation of liver metastases. Apparently, TBI increases the likelihood of extravasation of circulating tumor cells into the lung vasculature and subsequent formation of lung metastases. The pro-metastatic effect of TBI was further illustrated using CHO-K1 cells for transplantation that overexpress the red fluorescent protein (Fig. 2D) or the luciferase protein (Fig. 2E). Lung metastasis was also quantitated after intravenous injection of β-Gal overexpressing CHO-K1 cells followed by the analysis of β-galactosidase activity in protein extracts prepared from the lung of irradiated versus non irradiated mice. These studies revealed that TBI following transplantation of tumorigenic cells stimulated lung metastasis by about 4–5-fold (Fig. 2F). The pro-metastatic effect of TBI was also demonstrated by calculating the tumor area in lung sections, which was significantly enhanced as compared to non-irradiated mice (Fig. 2G). The data clearly show that TBI of mice harbouring circulating tumor cells leads to a massive colonization of the lung with metastases. Since extravasation of circulating tumorigenic cells is a prerequisite for the formation of lung metastases and, as shown above, TC-EC adhesion is stimulated by IR, we conclude that TBI promotes the process of tumor cell extravasation. With respect to the clinical situation, the data point to the possibility that the well established and highly appreciated therapeutic effect of radiotherapy (i.e. killing of tumor cells) might be influenced by an increased probability of circulating tumor cells, which have survived irradiation, to extravasate and develop tumors at secondary sites.


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)

Total body irradiation triggers tumor cell extravasation and lung metastasis in vivo.A: Balb/c mice were irradiated (IR) with 6 Gy. 4 h after total body irradiation (TBI) mRNA expression of cell adhesion molecules was analyzed in large blood vessels from pulmonary and abdominal artery by RT-PCR. GAPDH mRNA expression was measured as internal loading control. As a positive control, isolated blood vessels were incubated with TNFα (10 ng/ml, 30 min, 37 oC). B: Balb/c mice were subjected to TBI (6 Gy). 4 h after radiation (IR), phosphorylation of NF-κB inhibitor IκBα (p-IκBα), which is indicative of activation of NF-κB, was analyzed in liver extracts by Western blot analysis. ERK2 protein expression was determined as internal loading control. C: 2×106 CHO-K1 cells were injected into the tail vein of immunodeficient Rag2−/− Balb/c mice. Immediately after injection, mice were irradiated (4 Gy) (TBI). 3–4 weeks after TBI, the formation of metastases was analyzed in lung and liver. TC, tumor cells injected; TBI, total body irradiation. Shown are representative morphological and histopathological pictures (2–4 animals were analyzed per group). D: Red fluorescent protein overexpressing CHO-K1 cells were used for injection into the tail vein of immunodeficient Rag2−/− Balb/c mice. Formation of lung metastases was analyzed 3–4 weeks after TBI with 4 Gy. Con, non-irradiated control; IR, TBI with 4 Gy. Shown are representative pictures (2–3 animals were analyzed per group). E: Four weeks after i.v. injection of luciferase overexpressing CHO-K1 cells followed by TBI (4 Gy), luciferase activity in the lung was monitored by use of a life imaging instrument as described in Methods. Shown are representative pictures (2–3 animals were analyzed per group). F: 3–4 weeks after i.v. injection of β-galactosidase (β-Gal) overexpressing CHO-K1 cells followed by TBI (4 Gy), relative β-Gal activity in lung extracts of irradiated mice was related to that of non irradiated control mice, which was set to 100%. **p≤0.01 (n = 4–8 mice). G: Formation of lung metastasis was quantitated in the lung of irradiated versus non-irradiated mice by calculating the % tumor area as related to the total lung area. Con, non-irradiated control; IR, TBI with 4 Gy. ***p<0.001 (n = 4–8 mice).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198428&req=5

pone-0026413-g002: Total body irradiation triggers tumor cell extravasation and lung metastasis in vivo.A: Balb/c mice were irradiated (IR) with 6 Gy. 4 h after total body irradiation (TBI) mRNA expression of cell adhesion molecules was analyzed in large blood vessels from pulmonary and abdominal artery by RT-PCR. GAPDH mRNA expression was measured as internal loading control. As a positive control, isolated blood vessels were incubated with TNFα (10 ng/ml, 30 min, 37 oC). B: Balb/c mice were subjected to TBI (6 Gy). 4 h after radiation (IR), phosphorylation of NF-κB inhibitor IκBα (p-IκBα), which is indicative of activation of NF-κB, was analyzed in liver extracts by Western blot analysis. ERK2 protein expression was determined as internal loading control. C: 2×106 CHO-K1 cells were injected into the tail vein of immunodeficient Rag2−/− Balb/c mice. Immediately after injection, mice were irradiated (4 Gy) (TBI). 3–4 weeks after TBI, the formation of metastases was analyzed in lung and liver. TC, tumor cells injected; TBI, total body irradiation. Shown are representative morphological and histopathological pictures (2–4 animals were analyzed per group). D: Red fluorescent protein overexpressing CHO-K1 cells were used for injection into the tail vein of immunodeficient Rag2−/− Balb/c mice. Formation of lung metastases was analyzed 3–4 weeks after TBI with 4 Gy. Con, non-irradiated control; IR, TBI with 4 Gy. Shown are representative pictures (2–3 animals were analyzed per group). E: Four weeks after i.v. injection of luciferase overexpressing CHO-K1 cells followed by TBI (4 Gy), luciferase activity in the lung was monitored by use of a life imaging instrument as described in Methods. Shown are representative pictures (2–3 animals were analyzed per group). F: 3–4 weeks after i.v. injection of β-galactosidase (β-Gal) overexpressing CHO-K1 cells followed by TBI (4 Gy), relative β-Gal activity in lung extracts of irradiated mice was related to that of non irradiated control mice, which was set to 100%. **p≤0.01 (n = 4–8 mice). G: Formation of lung metastasis was quantitated in the lung of irradiated versus non-irradiated mice by calculating the % tumor area as related to the total lung area. Con, non-irradiated control; IR, TBI with 4 Gy. ***p<0.001 (n = 4–8 mice).
Mentions: Binding of circulating tumor cells to the endothelium is a prerequisite for their extravasation in vivo. Hence, the in vitro data are alarming because they implicate that radiotherapy might promote metastasis in vivo. To scrutinize this hypothesis we performed experiments using different mouse models. We showed that total body irradiation (TBI) of Balb/c mice results in the upregulation of both E-selectin and ICAM-1 mRNA levels (Fig. 2A), which was accompanied by activation of NF-κB (Fig. 2B). This is fully in line with other experimental systems demonstrating activation of NF-κB and subsequent expression of E-selectin and ICAM mRNA and protein by IR [20], [34], [35], [41]. Next, we determined whether TBI promotes extravasation and metastasis. To this end, tumorigenic Chinese hamster (CHO-K1) cells, which were shown to have significant metastatic potential [42], were injected into the tail vein of immunodeficient mice (Rag2−/− Balb/c strain). In our hands, CHO-K1 cells turned out to be the most appropriate cell line in this mouse model. Other cell lines tested were not metastatic at all or showed too high basal level of metastasis (see Figure S4). Immediately after injection, mice were subjected to TBI with a single dose of 4 Gy and colonization of the lung with metastases was analyzed 3–4 weeks later in comparison with non-irradiated animals. As shown in Fig. 2C, TBI caused a dramatic increase in the formation of lung metastases and, to a lesser extent, also increased the formation of liver metastases. Apparently, TBI increases the likelihood of extravasation of circulating tumor cells into the lung vasculature and subsequent formation of lung metastases. The pro-metastatic effect of TBI was further illustrated using CHO-K1 cells for transplantation that overexpress the red fluorescent protein (Fig. 2D) or the luciferase protein (Fig. 2E). Lung metastasis was also quantitated after intravenous injection of β-Gal overexpressing CHO-K1 cells followed by the analysis of β-galactosidase activity in protein extracts prepared from the lung of irradiated versus non irradiated mice. These studies revealed that TBI following transplantation of tumorigenic cells stimulated lung metastasis by about 4–5-fold (Fig. 2F). The pro-metastatic effect of TBI was also demonstrated by calculating the tumor area in lung sections, which was significantly enhanced as compared to non-irradiated mice (Fig. 2G). The data clearly show that TBI of mice harbouring circulating tumor cells leads to a massive colonization of the lung with metastases. Since extravasation of circulating tumorigenic cells is a prerequisite for the formation of lung metastases and, as shown above, TC-EC adhesion is stimulated by IR, we conclude that TBI promotes the process of tumor cell extravasation. With respect to the clinical situation, the data point to the possibility that the well established and highly appreciated therapeutic effect of radiotherapy (i.e. killing of tumor cells) might be influenced by an increased probability of circulating tumor cells, which have survived irradiation, to extravasate and develop tumors at secondary sites.

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