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Role of activated Rac1/Cdc42 in mediating endothelial cell proliferation and tumor angiogenesis in breast cancer.

Ma J, Xue Y, Liu W, Yue C, Bi F, Xu J, Zhang J, Li Y, Zhong C, Chen Y - PLoS ONE (2013)

Bottom Line: However, these effects are reversed after ubiquitin-proteasome breakage.Furthermore, in a xenograft model using nude mice that stably express active Rac1/Cdc42 protein, active Rac1/Cdc42 decreased p53 levels and increased VEGF expression.We conclude that activated Rac1/Cdc42 is a vascular regulator of tumor angiogenesis and that it may reduce stability of the p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shannxi, China.

ABSTRACT
Angiogenesis is a well-established target in anti-cancer therapy. Although vascular endothelial growth factor (VEGF)-mediated angiogenesis apparently requires the Rho GTPases Rac1 and Cdc42, the relevant mechanisms are unclear. Here, we determined that activated Rac1/Cdc42 in MCF-7 breast cancer cells could decrease p53 protein levels and increase VEGF secretion to promote proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). However, these effects are reversed after ubiquitin-proteasome breakage. In exploring potential mechanisms for this relationship, we confirmed that activated Rac1/Cdc42 could enhance p53 protein ubiquitination and weaken p53 protein stability to increase VEGF expression. Furthermore, in a xenograft model using nude mice that stably express active Rac1/Cdc42 protein, active Rac1/Cdc42 decreased p53 levels and increased VEGF expression. Additionally, tumor angiogenesis was inhibited, and p53 protein levels were augmented, by intratumoral injection of the ubiquitin-proteasome inhibitor MG132. Finally in 339 human breast cancer tissues, our analyses indicated that Rac1/Cdc42 expression was related to advanced TNM staging, high proliferation index, ER status, and positive invasive features. In particular, our data suggests that high Rac1/Cdc42 expression is correlated with low wt-p53 and high VEGF expression. We conclude that activated Rac1/Cdc42 is a vascular regulator of tumor angiogenesis and that it may reduce stability of the p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin.

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Effect of MG132 on HUVEC proliferation and tube formation induced by active Rac1/Cdc42.The MTT and tube formation assays were performed as described in Material and Methods. (A) HUVECs were grown to confluence and were then cultured in preconditioned media (derived from GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells pretreated or not with MG132 for 12 h) for 24 h or 48 h; the GST–MCF-7 group was used as a control. (B) HUVECs were plated on Matrigel and incubated with the different preconditioned media for 6 h. Photographs were taken in five random power fields (200 ×). (C) Tube lengths were measured with Image-Pro Plus software. Histograms represent quantification of the HUVEC. The GST–MCF-7 group was used as a control. All data are expressed as mean ± standard deviation (SD) for three independent experiments. Statistical significance was assessed using one-way ANOVA and Student's t-test. *P<0.05, **P<0.01, for the GST–MCF-7 group vs the V12Rac1– or L61Cdc42– MCF-7 group. #P<0.05, ##P<0.01, for the MG132-added group vs the no-MG132 group.
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pone-0066275-g001: Effect of MG132 on HUVEC proliferation and tube formation induced by active Rac1/Cdc42.The MTT and tube formation assays were performed as described in Material and Methods. (A) HUVECs were grown to confluence and were then cultured in preconditioned media (derived from GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells pretreated or not with MG132 for 12 h) for 24 h or 48 h; the GST–MCF-7 group was used as a control. (B) HUVECs were plated on Matrigel and incubated with the different preconditioned media for 6 h. Photographs were taken in five random power fields (200 ×). (C) Tube lengths were measured with Image-Pro Plus software. Histograms represent quantification of the HUVEC. The GST–MCF-7 group was used as a control. All data are expressed as mean ± standard deviation (SD) for three independent experiments. Statistical significance was assessed using one-way ANOVA and Student's t-test. *P<0.05, **P<0.01, for the GST–MCF-7 group vs the V12Rac1– or L61Cdc42– MCF-7 group. #P<0.05, ##P<0.01, for the MG132-added group vs the no-MG132 group.

Mentions: To observe whether Rac1/Cdc42 participates in tumor angiogenesis, the effects of activated Rac1/Cdc42 in MCF-7 cells on HUVEC proliferation and tube formation were examined. The constitutively activated Rac1 plasmid (V12Rac1) and Cdc42 plasmid (L61Cdc42) were used to produce activated Rac1 or Cdc42; pCEFL-GST served as the control plasmid. HUVECs were incubated with conditioned medium from MCF-7 cells stably transfected with one of these plasmids. After 24 h, the changes in HUVEC proliferation were not very obvious. However, after 48 h, the proliferation rates differed among the different groups. Compared to the control group, the medium derived from V12Rac1 or L61Cdc42–MCF-7 cells promoted HUVEC proliferation. Moreover, after treatment with the ubiquitin-proteasome inhibitor MG132, this increased effect was significantly inhibited (Fig. 1A). In the tube formation assay, HUVECs induced by V12Rac1– or L61Cdc42–MCF-7 cells associated with one another and formed more microtubes than did the control group. After MG132 treatment 48 h, however, the number of tubes formed by HUVECs induced by media from V12Rac1– or L61Cdc42–MCF-7 cells was reduced significantly (Fig. 1B and 1C). These results indicated that activated Rac1/Cdc42 in MCF-7 cells can accelerate HUVEC proliferation and tube formation to promote angiogenesis, and that these effects were partly inhibited by MG-132. However, the specific mechanism of angiogenesis induced by activated Rac1/Cdc42 needs to be further investigated.


Role of activated Rac1/Cdc42 in mediating endothelial cell proliferation and tumor angiogenesis in breast cancer.

Ma J, Xue Y, Liu W, Yue C, Bi F, Xu J, Zhang J, Li Y, Zhong C, Chen Y - PLoS ONE (2013)

Effect of MG132 on HUVEC proliferation and tube formation induced by active Rac1/Cdc42.The MTT and tube formation assays were performed as described in Material and Methods. (A) HUVECs were grown to confluence and were then cultured in preconditioned media (derived from GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells pretreated or not with MG132 for 12 h) for 24 h or 48 h; the GST–MCF-7 group was used as a control. (B) HUVECs were plated on Matrigel and incubated with the different preconditioned media for 6 h. Photographs were taken in five random power fields (200 ×). (C) Tube lengths were measured with Image-Pro Plus software. Histograms represent quantification of the HUVEC. The GST–MCF-7 group was used as a control. All data are expressed as mean ± standard deviation (SD) for three independent experiments. Statistical significance was assessed using one-way ANOVA and Student's t-test. *P<0.05, **P<0.01, for the GST–MCF-7 group vs the V12Rac1– or L61Cdc42– MCF-7 group. #P<0.05, ##P<0.01, for the MG132-added group vs the no-MG132 group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672132&req=5

pone-0066275-g001: Effect of MG132 on HUVEC proliferation and tube formation induced by active Rac1/Cdc42.The MTT and tube formation assays were performed as described in Material and Methods. (A) HUVECs were grown to confluence and were then cultured in preconditioned media (derived from GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells pretreated or not with MG132 for 12 h) for 24 h or 48 h; the GST–MCF-7 group was used as a control. (B) HUVECs were plated on Matrigel and incubated with the different preconditioned media for 6 h. Photographs were taken in five random power fields (200 ×). (C) Tube lengths were measured with Image-Pro Plus software. Histograms represent quantification of the HUVEC. The GST–MCF-7 group was used as a control. All data are expressed as mean ± standard deviation (SD) for three independent experiments. Statistical significance was assessed using one-way ANOVA and Student's t-test. *P<0.05, **P<0.01, for the GST–MCF-7 group vs the V12Rac1– or L61Cdc42– MCF-7 group. #P<0.05, ##P<0.01, for the MG132-added group vs the no-MG132 group.
Mentions: To observe whether Rac1/Cdc42 participates in tumor angiogenesis, the effects of activated Rac1/Cdc42 in MCF-7 cells on HUVEC proliferation and tube formation were examined. The constitutively activated Rac1 plasmid (V12Rac1) and Cdc42 plasmid (L61Cdc42) were used to produce activated Rac1 or Cdc42; pCEFL-GST served as the control plasmid. HUVECs were incubated with conditioned medium from MCF-7 cells stably transfected with one of these plasmids. After 24 h, the changes in HUVEC proliferation were not very obvious. However, after 48 h, the proliferation rates differed among the different groups. Compared to the control group, the medium derived from V12Rac1 or L61Cdc42–MCF-7 cells promoted HUVEC proliferation. Moreover, after treatment with the ubiquitin-proteasome inhibitor MG132, this increased effect was significantly inhibited (Fig. 1A). In the tube formation assay, HUVECs induced by V12Rac1– or L61Cdc42–MCF-7 cells associated with one another and formed more microtubes than did the control group. After MG132 treatment 48 h, however, the number of tubes formed by HUVECs induced by media from V12Rac1– or L61Cdc42–MCF-7 cells was reduced significantly (Fig. 1B and 1C). These results indicated that activated Rac1/Cdc42 in MCF-7 cells can accelerate HUVEC proliferation and tube formation to promote angiogenesis, and that these effects were partly inhibited by MG-132. However, the specific mechanism of angiogenesis induced by activated Rac1/Cdc42 needs to be further investigated.

Bottom Line: However, these effects are reversed after ubiquitin-proteasome breakage.Furthermore, in a xenograft model using nude mice that stably express active Rac1/Cdc42 protein, active Rac1/Cdc42 decreased p53 levels and increased VEGF expression.We conclude that activated Rac1/Cdc42 is a vascular regulator of tumor angiogenesis and that it may reduce stability of the p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shannxi, China.

ABSTRACT
Angiogenesis is a well-established target in anti-cancer therapy. Although vascular endothelial growth factor (VEGF)-mediated angiogenesis apparently requires the Rho GTPases Rac1 and Cdc42, the relevant mechanisms are unclear. Here, we determined that activated Rac1/Cdc42 in MCF-7 breast cancer cells could decrease p53 protein levels and increase VEGF secretion to promote proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). However, these effects are reversed after ubiquitin-proteasome breakage. In exploring potential mechanisms for this relationship, we confirmed that activated Rac1/Cdc42 could enhance p53 protein ubiquitination and weaken p53 protein stability to increase VEGF expression. Furthermore, in a xenograft model using nude mice that stably express active Rac1/Cdc42 protein, active Rac1/Cdc42 decreased p53 levels and increased VEGF expression. Additionally, tumor angiogenesis was inhibited, and p53 protein levels were augmented, by intratumoral injection of the ubiquitin-proteasome inhibitor MG132. Finally in 339 human breast cancer tissues, our analyses indicated that Rac1/Cdc42 expression was related to advanced TNM staging, high proliferation index, ER status, and positive invasive features. In particular, our data suggests that high Rac1/Cdc42 expression is correlated with low wt-p53 and high VEGF expression. We conclude that activated Rac1/Cdc42 is a vascular regulator of tumor angiogenesis and that it may reduce stability of the p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin.

Show MeSH
Related in: MedlinePlus