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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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Knockdown of RRM2 increased TSP-1 and decreased VEGF expression in LNCaP Cells. (A) q-RT-PCR analysis confirmed that RRM2 mRNA decreased by ~80%, and TSP-1 mRNA increased by ~8 folds in LNCaP cells at 48 h post-transfection with RRM2 siRNA. (B) Western blot analysis showed that TSP-1 protein was significantly increased by ~3.4-fold (Ln-Wt: LNCaP cell, Sc-siR: scramble siRNA transfected LNCaP, R2-siR: RRM2 siRNA transfected LNCaP). (C) q-RT-PCR analysis confirmed that RRM2 mRNA was significantly decreased in LNCaP at 48 h post-transfection under both normoxia and hypoxia. (D) q-RT-PCR analysis showed VEGF mRNA in LNCaP cells was decreased after RRM2 knockdown under both normoxia and hypoxia. (E) VEGF in condition medium of LNCaP was significantly decreased after RRM2 knockdown under both normoxia and hypoxia. Data was presented as the mean ± SD of 2–3 independent experiments, *p < 0.05, **p < 0.01, compared with scramble siRNA transefected.
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Figure 4: Knockdown of RRM2 increased TSP-1 and decreased VEGF expression in LNCaP Cells. (A) q-RT-PCR analysis confirmed that RRM2 mRNA decreased by ~80%, and TSP-1 mRNA increased by ~8 folds in LNCaP cells at 48 h post-transfection with RRM2 siRNA. (B) Western blot analysis showed that TSP-1 protein was significantly increased by ~3.4-fold (Ln-Wt: LNCaP cell, Sc-siR: scramble siRNA transfected LNCaP, R2-siR: RRM2 siRNA transfected LNCaP). (C) q-RT-PCR analysis confirmed that RRM2 mRNA was significantly decreased in LNCaP at 48 h post-transfection under both normoxia and hypoxia. (D) q-RT-PCR analysis showed VEGF mRNA in LNCaP cells was decreased after RRM2 knockdown under both normoxia and hypoxia. (E) VEGF in condition medium of LNCaP was significantly decreased after RRM2 knockdown under both normoxia and hypoxia. Data was presented as the mean ± SD of 2–3 independent experiments, *p < 0.05, **p < 0.01, compared with scramble siRNA transefected.

Mentions: It is important to address whether the regulation of RRM2 on TSP-1 and VEGF expression is a cell specific response or not. To answer this question, we then measured the effect of the knockdown of RRM2 on VEGF and TSP-1 expression in human prostate cancer cell: LNCaP, which also contains a wild-type p53. Similar to the change in KB cells, knockdown of RRM2 also significantly increased TSP-1 mRNA and protein (Figure 4a, b, P < 0.05). Knockdown of RRM2 in LNCaP cells (Figure 4c, P < 0.01) significantly decreased VEGF mRNA and protein under both normoxia and hypoxia conditions (Figure 4d, e, P < 0.05). The decreased VEGF in LNCaP was more pronounced under hypoxia condition. The data indicates that the regulation of RRM2 on TSP-1 and VEGF may not be a cell type specific but a common event; and a common mechanism may responsible for this regulation.


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)

Knockdown of RRM2 increased TSP-1 and decreased VEGF expression in LNCaP Cells. (A) q-RT-PCR analysis confirmed that RRM2 mRNA decreased by ~80%, and TSP-1 mRNA increased by ~8 folds in LNCaP cells at 48 h post-transfection with RRM2 siRNA. (B) Western blot analysis showed that TSP-1 protein was significantly increased by ~3.4-fold (Ln-Wt: LNCaP cell, Sc-siR: scramble siRNA transfected LNCaP, R2-siR: RRM2 siRNA transfected LNCaP). (C) q-RT-PCR analysis confirmed that RRM2 mRNA was significantly decreased in LNCaP at 48 h post-transfection under both normoxia and hypoxia. (D) q-RT-PCR analysis showed VEGF mRNA in LNCaP cells was decreased after RRM2 knockdown under both normoxia and hypoxia. (E) VEGF in condition medium of LNCaP was significantly decreased after RRM2 knockdown under both normoxia and hypoxia. Data was presented as the mean ± SD of 2–3 independent experiments, *p < 0.05, **p < 0.01, compared with scramble siRNA transefected.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662784&req=5

Figure 4: Knockdown of RRM2 increased TSP-1 and decreased VEGF expression in LNCaP Cells. (A) q-RT-PCR analysis confirmed that RRM2 mRNA decreased by ~80%, and TSP-1 mRNA increased by ~8 folds in LNCaP cells at 48 h post-transfection with RRM2 siRNA. (B) Western blot analysis showed that TSP-1 protein was significantly increased by ~3.4-fold (Ln-Wt: LNCaP cell, Sc-siR: scramble siRNA transfected LNCaP, R2-siR: RRM2 siRNA transfected LNCaP). (C) q-RT-PCR analysis confirmed that RRM2 mRNA was significantly decreased in LNCaP at 48 h post-transfection under both normoxia and hypoxia. (D) q-RT-PCR analysis showed VEGF mRNA in LNCaP cells was decreased after RRM2 knockdown under both normoxia and hypoxia. (E) VEGF in condition medium of LNCaP was significantly decreased after RRM2 knockdown under both normoxia and hypoxia. Data was presented as the mean ± SD of 2–3 independent experiments, *p < 0.05, **p < 0.01, compared with scramble siRNA transefected.
Mentions: It is important to address whether the regulation of RRM2 on TSP-1 and VEGF expression is a cell specific response or not. To answer this question, we then measured the effect of the knockdown of RRM2 on VEGF and TSP-1 expression in human prostate cancer cell: LNCaP, which also contains a wild-type p53. Similar to the change in KB cells, knockdown of RRM2 also significantly increased TSP-1 mRNA and protein (Figure 4a, b, P < 0.05). Knockdown of RRM2 in LNCaP cells (Figure 4c, P < 0.01) significantly decreased VEGF mRNA and protein under both normoxia and hypoxia conditions (Figure 4d, e, P < 0.05). The decreased VEGF in LNCaP was more pronounced under hypoxia condition. The data indicates that the regulation of RRM2 on TSP-1 and VEGF may not be a cell type specific but a common event; and a common mechanism may responsible for this regulation.

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