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Overexpression of RRM2 decreases thrombspondin-1 and increases VEGF production in human cancer cells in vitro and in vivo: implication of RRM2 in angiogenesis.

Zhang K, Hu S, Wu J, Chen L, Lu J, Wang X, Liu X, Zhou B, Yen Y - Mol. Cancer (2009)

Bottom Line: We found that TSP-1 was significantly decreased in both KB-M2-D and KB-HURs cells compared to the parental KB and mock transfected KB-V.In contrast, attenuating RRM2 expression via siRNA resulted in a significant increased TSP-1 expression in both KB and LNCaP cells; while the expression of VEGF by the two cells was significantly decreased under both normoxia and hypoxia.In comparison with KB-V, overexpression of RRM2 had no significant effect on proliferation in vitro, but it dramatically accelerated in vivo subcutaneous growth of KB-M2-D.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical & Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 91010, USA. kzhang@coh.org

ABSTRACT

Background: In addition to its essential role in ribonucleotide reduction, ribonucleotide reductase (RNR) small subunit, RRM2, has been known to play a critical role in determining tumor malignancy. Overexpression of RRM2 significantly enhances the invasive and metastatic potential of tumor. Angiogenesis is critical to tumor malignancy; it plays an essential role in tumor growth and metastasis. It is important to investigate whether the angiogenic potential of tumor is affected by RRM2.

Results: We examined the expression of antiangiogenic thrombospondin-1 (TSP-1) and proangiogenic vascular endothelial growth factor (VEGF) in two RRM2-overexpressing KB cells: KB-M2-D and KB-HURs. We found that TSP-1 was significantly decreased in both KB-M2-D and KB-HURs cells compared to the parental KB and mock transfected KB-V. Simultaneously, RRM2-overexpressing KB cells showed increased production of VEGF mRNA and protein. In contrast, attenuating RRM2 expression via siRNA resulted in a significant increased TSP-1 expression in both KB and LNCaP cells; while the expression of VEGF by the two cells was significantly decreased under both normoxia and hypoxia. In comparison with KB-V, overexpression of RRM2 had no significant effect on proliferation in vitro, but it dramatically accelerated in vivo subcutaneous growth of KB-M2-D. KB-M2-D possessed more angiogenic potential than KB-V, as shown in vitro by its increased chemotaxis for endothelial cells and in vivo by the generation of more vascularized tumor xenografts.

Conclusion: These findings suggest a positive role of RRM2 in tumor angiogenesis and growth through regulation of the expression of TSP-1 and VEGF.

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Overexpression of RRM2 increased VEGF expression in KB-M2-D and KB-HURs cells. (A) VEGF in condition mediums of KB-M2-D and Kb-HURs were increased by 2.4-fold and 2.1-fold respectively. Levels of VEGF in the condition medium were measured with ELISA. Data, presented as ng of VEGF protein/ml of medium and per mg of total protein, was the mean ± SD of 2–3 preparation. (B) q-RT-PCR analysis showed that VEGF mRNA was increased in KB-M2D and KB-HURs by 3~4 fold compared with parental KB and KB-V cells. (C) Compared with KB-V, KB-M2-D secreted more VEGF under both normoxia and hypoxia. (D) q-RT-PCR analysis showed KB-M2D expressed more VEGF mRNA than KB-V under both normoxia and hypoxia, *p < 0.05, **p < 0.01 compared with KB-V.
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Figure 2: Overexpression of RRM2 increased VEGF expression in KB-M2-D and KB-HURs cells. (A) VEGF in condition mediums of KB-M2-D and Kb-HURs were increased by 2.4-fold and 2.1-fold respectively. Levels of VEGF in the condition medium were measured with ELISA. Data, presented as ng of VEGF protein/ml of medium and per mg of total protein, was the mean ± SD of 2–3 preparation. (B) q-RT-PCR analysis showed that VEGF mRNA was increased in KB-M2D and KB-HURs by 3~4 fold compared with parental KB and KB-V cells. (C) Compared with KB-V, KB-M2-D secreted more VEGF under both normoxia and hypoxia. (D) q-RT-PCR analysis showed KB-M2D expressed more VEGF mRNA than KB-V under both normoxia and hypoxia, *p < 0.05, **p < 0.01 compared with KB-V.

Mentions: VEGF is a key mediator of tumor-associated angiogenesis and is thought to support neovascularization by inducing endothelial cell migration and proliferation leading to vascular permeability [18-20]. To answer whether overexpression of RRM2 also effects on VEGF expression in cancer cells, we examined the levels of VEGF in the condition medium of KB cells. By ELISA assay, we found that RRM2-overexpressing KB-M2-D and KB-HURs released about 2.8-fold and 2.2-fold more VEGF than control cells (Figure 2a, P < 0.05). The induction of VEGF expression could be regulated at the level of either mRNA or protein. Measured by Q-RT-PCR, we found that in the two RRM2-overexpressing cells VEGF mRNA was significantly increased (Figure. 2b, P < 0.01). Hypoxia condition in tumor has been shown to be an important stimulus for VEGF gene expression as well as tumor angiogenesis. To determine the effect of RRM2 on VEGF expression under hypoxia, we compared VEGF mRNA and protein in KB-V and KB-M2-D cells. The results showed that hypoxia (1% oxygen for 16h) upregulated VEGF by ~2 fold at protein levels (Figure. 2c, P < 0.05) or ~4-fold at mRNA (Figure. 2d, P < 0.01) in both cells. Compared with that of KB-V, the production of VEGF in KB-M2-D was still significantly higher under both normoxia and hypoxia (Figure. 2c, d, P < 0.05).


Overexpression of RRM2 decreases thrombspondin-1 and increases VEGF production in human cancer cells in vitro and in vivo: implication of RRM2 in angiogenesis.

Zhang K, Hu S, Wu J, Chen L, Lu J, Wang X, Liu X, Zhou B, Yen Y - Mol. Cancer (2009)

Overexpression of RRM2 increased VEGF expression in KB-M2-D and KB-HURs cells. (A) VEGF in condition mediums of KB-M2-D and Kb-HURs were increased by 2.4-fold and 2.1-fold respectively. Levels of VEGF in the condition medium were measured with ELISA. Data, presented as ng of VEGF protein/ml of medium and per mg of total protein, was the mean ± SD of 2–3 preparation. (B) q-RT-PCR analysis showed that VEGF mRNA was increased in KB-M2D and KB-HURs by 3~4 fold compared with parental KB and KB-V cells. (C) Compared with KB-V, KB-M2-D secreted more VEGF under both normoxia and hypoxia. (D) q-RT-PCR analysis showed KB-M2D expressed more VEGF mRNA than KB-V under both normoxia and hypoxia, *p < 0.05, **p < 0.01 compared with KB-V.
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Figure 2: Overexpression of RRM2 increased VEGF expression in KB-M2-D and KB-HURs cells. (A) VEGF in condition mediums of KB-M2-D and Kb-HURs were increased by 2.4-fold and 2.1-fold respectively. Levels of VEGF in the condition medium were measured with ELISA. Data, presented as ng of VEGF protein/ml of medium and per mg of total protein, was the mean ± SD of 2–3 preparation. (B) q-RT-PCR analysis showed that VEGF mRNA was increased in KB-M2D and KB-HURs by 3~4 fold compared with parental KB and KB-V cells. (C) Compared with KB-V, KB-M2-D secreted more VEGF under both normoxia and hypoxia. (D) q-RT-PCR analysis showed KB-M2D expressed more VEGF mRNA than KB-V under both normoxia and hypoxia, *p < 0.05, **p < 0.01 compared with KB-V.
Mentions: VEGF is a key mediator of tumor-associated angiogenesis and is thought to support neovascularization by inducing endothelial cell migration and proliferation leading to vascular permeability [18-20]. To answer whether overexpression of RRM2 also effects on VEGF expression in cancer cells, we examined the levels of VEGF in the condition medium of KB cells. By ELISA assay, we found that RRM2-overexpressing KB-M2-D and KB-HURs released about 2.8-fold and 2.2-fold more VEGF than control cells (Figure 2a, P < 0.05). The induction of VEGF expression could be regulated at the level of either mRNA or protein. Measured by Q-RT-PCR, we found that in the two RRM2-overexpressing cells VEGF mRNA was significantly increased (Figure. 2b, P < 0.01). Hypoxia condition in tumor has been shown to be an important stimulus for VEGF gene expression as well as tumor angiogenesis. To determine the effect of RRM2 on VEGF expression under hypoxia, we compared VEGF mRNA and protein in KB-V and KB-M2-D cells. The results showed that hypoxia (1% oxygen for 16h) upregulated VEGF by ~2 fold at protein levels (Figure. 2c, P < 0.05) or ~4-fold at mRNA (Figure. 2d, P < 0.01) in both cells. Compared with that of KB-V, the production of VEGF in KB-M2-D was still significantly higher under both normoxia and hypoxia (Figure. 2c, d, P < 0.05).

Bottom Line: We found that TSP-1 was significantly decreased in both KB-M2-D and KB-HURs cells compared to the parental KB and mock transfected KB-V.In contrast, attenuating RRM2 expression via siRNA resulted in a significant increased TSP-1 expression in both KB and LNCaP cells; while the expression of VEGF by the two cells was significantly decreased under both normoxia and hypoxia.In comparison with KB-V, overexpression of RRM2 had no significant effect on proliferation in vitro, but it dramatically accelerated in vivo subcutaneous growth of KB-M2-D.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical & Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 91010, USA. kzhang@coh.org

ABSTRACT

Background: In addition to its essential role in ribonucleotide reduction, ribonucleotide reductase (RNR) small subunit, RRM2, has been known to play a critical role in determining tumor malignancy. Overexpression of RRM2 significantly enhances the invasive and metastatic potential of tumor. Angiogenesis is critical to tumor malignancy; it plays an essential role in tumor growth and metastasis. It is important to investigate whether the angiogenic potential of tumor is affected by RRM2.

Results: We examined the expression of antiangiogenic thrombospondin-1 (TSP-1) and proangiogenic vascular endothelial growth factor (VEGF) in two RRM2-overexpressing KB cells: KB-M2-D and KB-HURs. We found that TSP-1 was significantly decreased in both KB-M2-D and KB-HURs cells compared to the parental KB and mock transfected KB-V. Simultaneously, RRM2-overexpressing KB cells showed increased production of VEGF mRNA and protein. In contrast, attenuating RRM2 expression via siRNA resulted in a significant increased TSP-1 expression in both KB and LNCaP cells; while the expression of VEGF by the two cells was significantly decreased under both normoxia and hypoxia. In comparison with KB-V, overexpression of RRM2 had no significant effect on proliferation in vitro, but it dramatically accelerated in vivo subcutaneous growth of KB-M2-D. KB-M2-D possessed more angiogenic potential than KB-V, as shown in vitro by its increased chemotaxis for endothelial cells and in vivo by the generation of more vascularized tumor xenografts.

Conclusion: These findings suggest a positive role of RRM2 in tumor angiogenesis and growth through regulation of the expression of TSP-1 and VEGF.

Show MeSH
Related in: MedlinePlus