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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 KB cells. (A) q-RT-PCR analysis showed that RRM2 mRNA was decreased by ~80%, and TSP-1 mRNA was increased by ~5 folds in KB cells at 48 h post-transfection with RRM2 siRNA. (B) Western blot analysis showed that TSP-1 protein was significantly increased by ~2.6-fold (KB-W: KB cell, Sc-siR: scramble siRNA transfected KB, R2-siR: RRM2 siRNA transfected KB). (C) q-RT-PCR analysis confirmed RRM2 mRNA was significantly decreased in KB cells at 48 h post-transfection under both normoxia and hypoxia. (D) q-RT-PCR analysis showed VEGF mRNA in KB cells was decreased after RRM2 knockdown under both normoxia and hypoxia. (E) VEGF in condition medium of KB 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 transfected.
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Figure 3: Knockdown of RRM2 increased TSP-1 and decreased VEGF expression in KB cells. (A) q-RT-PCR analysis showed that RRM2 mRNA was decreased by ~80%, and TSP-1 mRNA was increased by ~5 folds in KB cells at 48 h post-transfection with RRM2 siRNA. (B) Western blot analysis showed that TSP-1 protein was significantly increased by ~2.6-fold (KB-W: KB cell, Sc-siR: scramble siRNA transfected KB, R2-siR: RRM2 siRNA transfected KB). (C) q-RT-PCR analysis confirmed RRM2 mRNA was significantly decreased in KB cells at 48 h post-transfection under both normoxia and hypoxia. (D) q-RT-PCR analysis showed VEGF mRNA in KB cells was decreased after RRM2 knockdown under both normoxia and hypoxia. (E) VEGF in condition medium of KB 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 transfected.

Mentions: Overexpression of RRM2 significantly decreased TSP-1, simultaneously increased VEGF expression in KB cells. To further confirm the implication of RRM2 in the regulation of TSP-1 and VEGF expression, we measured TSP-1 and VEGF expression level, after RRM2 in cancer cells was knocked down by its specific siRNA. Q-RT-PCR and Western blot analysis showed that RRM2 mRNA and protein in KB cells was significantly decreased at 48 h post-transfection of siRNA (Figure 3a, b). RRM2 level at 24 h and 72 h post-transfection of siRNA was also significantly decreased (data not shown). Simultaneously, we measured TSP-1 expression in KB cells, we found TSP-1 mRNA and protein was significantly upregulated at all three points (Figure 3a, b, P < 0.05). By using of q-RT-PCR analysis, we confirmed under both normoxia and hypoxia RRM2 mRNA in KB cells was significantly decreased by siRNA transfection (Figure 3c, P < 0.01). And knockdown of RRM2 in KB cells moderately decreased VEGF mRNA and protein expression under normoxia, significantly decreased VEGF expression under hypoxia (Figure 3d and 3e, 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)

Knockdown of RRM2 increased TSP-1 and decreased VEGF expression in KB cells. (A) q-RT-PCR analysis showed that RRM2 mRNA was decreased by ~80%, and TSP-1 mRNA was increased by ~5 folds in KB cells at 48 h post-transfection with RRM2 siRNA. (B) Western blot analysis showed that TSP-1 protein was significantly increased by ~2.6-fold (KB-W: KB cell, Sc-siR: scramble siRNA transfected KB, R2-siR: RRM2 siRNA transfected KB). (C) q-RT-PCR analysis confirmed RRM2 mRNA was significantly decreased in KB cells at 48 h post-transfection under both normoxia and hypoxia. (D) q-RT-PCR analysis showed VEGF mRNA in KB cells was decreased after RRM2 knockdown under both normoxia and hypoxia. (E) VEGF in condition medium of KB 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 transfected.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Knockdown of RRM2 increased TSP-1 and decreased VEGF expression in KB cells. (A) q-RT-PCR analysis showed that RRM2 mRNA was decreased by ~80%, and TSP-1 mRNA was increased by ~5 folds in KB cells at 48 h post-transfection with RRM2 siRNA. (B) Western blot analysis showed that TSP-1 protein was significantly increased by ~2.6-fold (KB-W: KB cell, Sc-siR: scramble siRNA transfected KB, R2-siR: RRM2 siRNA transfected KB). (C) q-RT-PCR analysis confirmed RRM2 mRNA was significantly decreased in KB cells at 48 h post-transfection under both normoxia and hypoxia. (D) q-RT-PCR analysis showed VEGF mRNA in KB cells was decreased after RRM2 knockdown under both normoxia and hypoxia. (E) VEGF in condition medium of KB 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 transfected.
Mentions: Overexpression of RRM2 significantly decreased TSP-1, simultaneously increased VEGF expression in KB cells. To further confirm the implication of RRM2 in the regulation of TSP-1 and VEGF expression, we measured TSP-1 and VEGF expression level, after RRM2 in cancer cells was knocked down by its specific siRNA. Q-RT-PCR and Western blot analysis showed that RRM2 mRNA and protein in KB cells was significantly decreased at 48 h post-transfection of siRNA (Figure 3a, b). RRM2 level at 24 h and 72 h post-transfection of siRNA was also significantly decreased (data not shown). Simultaneously, we measured TSP-1 expression in KB cells, we found TSP-1 mRNA and protein was significantly upregulated at all three points (Figure 3a, b, P < 0.05). By using of q-RT-PCR analysis, we confirmed under both normoxia and hypoxia RRM2 mRNA in KB cells was significantly decreased by siRNA transfection (Figure 3c, P < 0.01). And knockdown of RRM2 in KB cells moderately decreased VEGF mRNA and protein expression under normoxia, significantly decreased VEGF expression under hypoxia (Figure 3d and 3e, 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