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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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Lovastatin attenuates IR-induced metastasis in vivo.A: Balb/c mice were pretreated with lovastatin (10 mg/kg, p.o.) and irradiated with 6 Gy. 4 h after TBI, large blood vessels (i.e. pulmonary and abdominal artery) were isolated for total mRNA preparation and subsequent semiquantitative RT-PCR. ICAM-1 mRNA expression levels shown in the histogram were obtained by real-time PCR analyses (triplicate determinations). The level of ICAM-1 mRNA was normalized to the levels of GAPDH and β-actin and set to 1.0 in the untreated control (Con). Con, non-irradiated control; IR, irradiation; IR+Lova, IR exposure after lovastatin pre-treatment. B, C: 2×106 CHO-K1 cells were injected into the tail vein of immunodeficient Rag2−/− Balb/c mice which had been pre-treated or not with lovastatin (10 mg/kg, p.o.) for 2 days. Immediately after tumor cell injection, mice were irradiated with 4 Gy (IR). Post-treatment with lovastatin was not performed. 3–4 weeks after TBI, formation of metastasis was analyzed. B, IR, irradiation; IR+Lova, IR exposure after lovastatin pre-treatment. Shown are representative morphological and histopathological pictures (from n = 4–6 animals per group). C, The protective effect of lovastatin on IR-induced extravasation and lung metastasis of CHO-K1 cells was quantitated by determination of β-Gal activity in protein extracts from lung. Relative β-Gal activity in untreated control was set to 100%. See also Figure S3. **p≤0.01 (n = 4 mice). D: 2×106 CHO-K1 cells were injected into the tail vein of immunodeficient Rag2−/− Balb/c mice which had been pretreated or not with lovastatin for 14 days (10 mg/kg, p.o., 3x per week). Immediately after tumor cell injection, mice were irradiated with 4 Gy (IR). 3–4 weeks later, the percent tumor area in lung sections was calculated as described in methods. IR, irradiation; IR+Lova, IR exposure after lovastatin pre-treatment; L-I, lovastatin pre-treatment only; L-II, lovastatin pre- and post-treatment. Post-treatment (10 mg/kg, p.o., 3x per week) was performed for 2 weeks. *** p≤0.001 (n = 4 mice per group).
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pone-0026413-g003: Lovastatin attenuates IR-induced metastasis in vivo.A: Balb/c mice were pretreated with lovastatin (10 mg/kg, p.o.) and irradiated with 6 Gy. 4 h after TBI, large blood vessels (i.e. pulmonary and abdominal artery) were isolated for total mRNA preparation and subsequent semiquantitative RT-PCR. ICAM-1 mRNA expression levels shown in the histogram were obtained by real-time PCR analyses (triplicate determinations). The level of ICAM-1 mRNA was normalized to the levels of GAPDH and β-actin and set to 1.0 in the untreated control (Con). Con, non-irradiated control; IR, irradiation; IR+Lova, IR exposure after lovastatin pre-treatment. B, C: 2×106 CHO-K1 cells were injected into the tail vein of immunodeficient Rag2−/− Balb/c mice which had been pre-treated or not with lovastatin (10 mg/kg, p.o.) for 2 days. Immediately after tumor cell injection, mice were irradiated with 4 Gy (IR). Post-treatment with lovastatin was not performed. 3–4 weeks after TBI, formation of metastasis was analyzed. B, IR, irradiation; IR+Lova, IR exposure after lovastatin pre-treatment. Shown are representative morphological and histopathological pictures (from n = 4–6 animals per group). C, The protective effect of lovastatin on IR-induced extravasation and lung metastasis of CHO-K1 cells was quantitated by determination of β-Gal activity in protein extracts from lung. Relative β-Gal activity in untreated control was set to 100%. See also Figure S3. **p≤0.01 (n = 4 mice). D: 2×106 CHO-K1 cells were injected into the tail vein of immunodeficient Rag2−/− Balb/c mice which had been pretreated or not with lovastatin for 14 days (10 mg/kg, p.o., 3x per week). Immediately after tumor cell injection, mice were irradiated with 4 Gy (IR). 3–4 weeks later, the percent tumor area in lung sections was calculated as described in methods. IR, irradiation; IR+Lova, IR exposure after lovastatin pre-treatment; L-I, lovastatin pre-treatment only; L-II, lovastatin pre- and post-treatment. Post-treatment (10 mg/kg, p.o., 3x per week) was performed for 2 weeks. *** p≤0.001 (n = 4 mice per group).

Mentions: In search of drugs counteracting the putative pro-metastatic effect of IR, we explored the HMG-CoA reductase inhibitor lovastatin. The rationale behind this is that statins, which are widely used as lipid-lowering drugs, block the Rac1-regulated and NF-κB-dependent expression of E-selectin following TNFα or IR exposure of endothelial cells in vitro [7], [36]. Lovastatin mitigates TC-EC interaction in vitro (see Fig. 1) and inhibits activation of NF-κB following irradiation in vitro [7] and in vivo [43]. Moreover, statins alleviate normal tissue damage (i.e. pro-inflammatory and pro-fibrotic radiation responses) that results from radiotherapy as acute or delayed side effect [44], [45], [46], [47]. In agreement with our in vitro data, lovastatin impaired the TBI-induced upregulation of cell adhesion molecules in vivo (Fig. 3A). Most importantly, short-time pre-treatment of Rag2−/− Balb/c animals for 2 days with lovastatin clearly attenuated the TBI-induced formation of lung metastases (Fig. 3B). We should note that lovastatin does not increase the sensitivity of CHO-K1 cells to irradiation [48]. Assaying β-galactosidase activity in lung extracts, which was taken as indication of the tumor burden, we calculated that lovastatin reduced the TBI-stimulated formation of lung metastases by about 75% (Fig. 3C). Amazingly, under these experimental conditions of short-time pre-treatment with lovastatin, the statin had a weak prometastatic effect on its own (see Figure S3). Extending the pre-treatment period with lovastatin for 14 days, the prometastatic statin effect was largely vanished and TBI-stimulated lung metastasis was completely blocked (Fig. 3D). Pre- and post-treatment with lovastatin had the same anti-metastatic effect as pre-treatment alone (Fig. 3D), showing that (i) pretreatment with lovastatin is sufficient to block metastasis and (ii) post-treatment with lovastatin for an extended period of time does not result in adverse effects with respect to metastasis.


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)

Lovastatin attenuates IR-induced metastasis in vivo.A: Balb/c mice were pretreated with lovastatin (10 mg/kg, p.o.) and irradiated with 6 Gy. 4 h after TBI, large blood vessels (i.e. pulmonary and abdominal artery) were isolated for total mRNA preparation and subsequent semiquantitative RT-PCR. ICAM-1 mRNA expression levels shown in the histogram were obtained by real-time PCR analyses (triplicate determinations). The level of ICAM-1 mRNA was normalized to the levels of GAPDH and β-actin and set to 1.0 in the untreated control (Con). Con, non-irradiated control; IR, irradiation; IR+Lova, IR exposure after lovastatin pre-treatment. B, C: 2×106 CHO-K1 cells were injected into the tail vein of immunodeficient Rag2−/− Balb/c mice which had been pre-treated or not with lovastatin (10 mg/kg, p.o.) for 2 days. Immediately after tumor cell injection, mice were irradiated with 4 Gy (IR). Post-treatment with lovastatin was not performed. 3–4 weeks after TBI, formation of metastasis was analyzed. B, IR, irradiation; IR+Lova, IR exposure after lovastatin pre-treatment. Shown are representative morphological and histopathological pictures (from n = 4–6 animals per group). C, The protective effect of lovastatin on IR-induced extravasation and lung metastasis of CHO-K1 cells was quantitated by determination of β-Gal activity in protein extracts from lung. Relative β-Gal activity in untreated control was set to 100%. See also Figure S3. **p≤0.01 (n = 4 mice). D: 2×106 CHO-K1 cells were injected into the tail vein of immunodeficient Rag2−/− Balb/c mice which had been pretreated or not with lovastatin for 14 days (10 mg/kg, p.o., 3x per week). Immediately after tumor cell injection, mice were irradiated with 4 Gy (IR). 3–4 weeks later, the percent tumor area in lung sections was calculated as described in methods. IR, irradiation; IR+Lova, IR exposure after lovastatin pre-treatment; L-I, lovastatin pre-treatment only; L-II, lovastatin pre- and post-treatment. Post-treatment (10 mg/kg, p.o., 3x per week) was performed for 2 weeks. *** p≤0.001 (n = 4 mice per group).
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pone-0026413-g003: Lovastatin attenuates IR-induced metastasis in vivo.A: Balb/c mice were pretreated with lovastatin (10 mg/kg, p.o.) and irradiated with 6 Gy. 4 h after TBI, large blood vessels (i.e. pulmonary and abdominal artery) were isolated for total mRNA preparation and subsequent semiquantitative RT-PCR. ICAM-1 mRNA expression levels shown in the histogram were obtained by real-time PCR analyses (triplicate determinations). The level of ICAM-1 mRNA was normalized to the levels of GAPDH and β-actin and set to 1.0 in the untreated control (Con). Con, non-irradiated control; IR, irradiation; IR+Lova, IR exposure after lovastatin pre-treatment. B, C: 2×106 CHO-K1 cells were injected into the tail vein of immunodeficient Rag2−/− Balb/c mice which had been pre-treated or not with lovastatin (10 mg/kg, p.o.) for 2 days. Immediately after tumor cell injection, mice were irradiated with 4 Gy (IR). Post-treatment with lovastatin was not performed. 3–4 weeks after TBI, formation of metastasis was analyzed. B, IR, irradiation; IR+Lova, IR exposure after lovastatin pre-treatment. Shown are representative morphological and histopathological pictures (from n = 4–6 animals per group). C, The protective effect of lovastatin on IR-induced extravasation and lung metastasis of CHO-K1 cells was quantitated by determination of β-Gal activity in protein extracts from lung. Relative β-Gal activity in untreated control was set to 100%. See also Figure S3. **p≤0.01 (n = 4 mice). D: 2×106 CHO-K1 cells were injected into the tail vein of immunodeficient Rag2−/− Balb/c mice which had been pretreated or not with lovastatin for 14 days (10 mg/kg, p.o., 3x per week). Immediately after tumor cell injection, mice were irradiated with 4 Gy (IR). 3–4 weeks later, the percent tumor area in lung sections was calculated as described in methods. IR, irradiation; IR+Lova, IR exposure after lovastatin pre-treatment; L-I, lovastatin pre-treatment only; L-II, lovastatin pre- and post-treatment. Post-treatment (10 mg/kg, p.o., 3x per week) was performed for 2 weeks. *** p≤0.001 (n = 4 mice per group).
Mentions: In search of drugs counteracting the putative pro-metastatic effect of IR, we explored the HMG-CoA reductase inhibitor lovastatin. The rationale behind this is that statins, which are widely used as lipid-lowering drugs, block the Rac1-regulated and NF-κB-dependent expression of E-selectin following TNFα or IR exposure of endothelial cells in vitro [7], [36]. Lovastatin mitigates TC-EC interaction in vitro (see Fig. 1) and inhibits activation of NF-κB following irradiation in vitro [7] and in vivo [43]. Moreover, statins alleviate normal tissue damage (i.e. pro-inflammatory and pro-fibrotic radiation responses) that results from radiotherapy as acute or delayed side effect [44], [45], [46], [47]. In agreement with our in vitro data, lovastatin impaired the TBI-induced upregulation of cell adhesion molecules in vivo (Fig. 3A). Most importantly, short-time pre-treatment of Rag2−/− Balb/c animals for 2 days with lovastatin clearly attenuated the TBI-induced formation of lung metastases (Fig. 3B). We should note that lovastatin does not increase the sensitivity of CHO-K1 cells to irradiation [48]. Assaying β-galactosidase activity in lung extracts, which was taken as indication of the tumor burden, we calculated that lovastatin reduced the TBI-stimulated formation of lung metastases by about 75% (Fig. 3C). Amazingly, under these experimental conditions of short-time pre-treatment with lovastatin, the statin had a weak prometastatic effect on its own (see Figure S3). Extending the pre-treatment period with lovastatin for 14 days, the prometastatic statin effect was largely vanished and TBI-stimulated lung metastasis was completely blocked (Fig. 3D). Pre- and post-treatment with lovastatin had the same anti-metastatic effect as pre-treatment alone (Fig. 3D), showing that (i) pretreatment with lovastatin is sufficient to block metastasis and (ii) post-treatment with lovastatin for an extended period of time does not result in adverse effects with respect to metastasis.

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