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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 intratumoral injections of MG132 on vascularization induced by active Rac1/Cdc42 in MCF-7 cell xenografts.These experiments were described in the Materials and Methods section. Stably transfected cells (MCF-7, GST–MCF-7, V12Rac1–MCF-7, L61Cdc42–MCF-7) were used for xenografts in nude mice. When the tumors reached a volume of 200 mm3, the mice were treated with intratumoral injections of a specific dose of MG132 (10 mg/kg) or PBS. (A, B) Intratumoral vascularization was assessed by VEGF and p53 immunolabeling (400 × power) on paraffin-embedded MCF-7 cell tumor sections. Representative images are shown. Integrated optical density (IOD) values of VEGF and p53 protein expression were evaluated. ImagePro Plus software was used to analyze the IOD values of the positive areas of immunohistochemical staining. The resulting histograms are presented here. A statistical analysis was performed using a one-way ANOVA. The results are presented as mean ± SD for six mice. *P<0.05; **P<0.01.
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pone-0066275-g004: Effect of intratumoral injections of MG132 on vascularization induced by active Rac1/Cdc42 in MCF-7 cell xenografts.These experiments were described in the Materials and Methods section. Stably transfected cells (MCF-7, GST–MCF-7, V12Rac1–MCF-7, L61Cdc42–MCF-7) were used for xenografts in nude mice. When the tumors reached a volume of 200 mm3, the mice were treated with intratumoral injections of a specific dose of MG132 (10 mg/kg) or PBS. (A, B) Intratumoral vascularization was assessed by VEGF and p53 immunolabeling (400 × power) on paraffin-embedded MCF-7 cell tumor sections. Representative images are shown. Integrated optical density (IOD) values of VEGF and p53 protein expression were evaluated. ImagePro Plus software was used to analyze the IOD values of the positive areas of immunohistochemical staining. The resulting histograms are presented here. A statistical analysis was performed using a one-way ANOVA. The results are presented as mean ± SD for six mice. *P<0.05; **P<0.01.

Mentions: To further evaluate the effects of MG132 on tumor angiogenesis induced by V12Rac1/LCdc42, we injected 10 mg/kg MG132 or PBS every 2 days into pre-established, MCF-7 breast tumors (approximately 200 mm3) grown in nude mice. Immunolabeling for VEGF was much higher in tumors excised from mice in the V12Rac1 and L61Cdc42 groups than in those from the control group (Fig. 4). Moreover, p53 expression was lower in the V12Rac1 and L61Cdc42 groups than in the blank or control groups. However, MG312 injections dramatically inhibited the increase in VEGF expression and enhanced the decrease in p53 expression in the V12Rac1 and L61Cdc42 groups compared with the control group. There was no significant difference between the blank and control groups. These data revealed that in vivo, MG132 could reverse the effects of activated Rac1/Cdc42 on p53 and VEGF expression to suppress tumor angiogenesis.


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 intratumoral injections of MG132 on vascularization induced by active Rac1/Cdc42 in MCF-7 cell xenografts.These experiments were described in the Materials and Methods section. Stably transfected cells (MCF-7, GST–MCF-7, V12Rac1–MCF-7, L61Cdc42–MCF-7) were used for xenografts in nude mice. When the tumors reached a volume of 200 mm3, the mice were treated with intratumoral injections of a specific dose of MG132 (10 mg/kg) or PBS. (A, B) Intratumoral vascularization was assessed by VEGF and p53 immunolabeling (400 × power) on paraffin-embedded MCF-7 cell tumor sections. Representative images are shown. Integrated optical density (IOD) values of VEGF and p53 protein expression were evaluated. ImagePro Plus software was used to analyze the IOD values of the positive areas of immunohistochemical staining. The resulting histograms are presented here. A statistical analysis was performed using a one-way ANOVA. The results are presented as mean ± SD for six mice. *P<0.05; **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672132&req=5

pone-0066275-g004: Effect of intratumoral injections of MG132 on vascularization induced by active Rac1/Cdc42 in MCF-7 cell xenografts.These experiments were described in the Materials and Methods section. Stably transfected cells (MCF-7, GST–MCF-7, V12Rac1–MCF-7, L61Cdc42–MCF-7) were used for xenografts in nude mice. When the tumors reached a volume of 200 mm3, the mice were treated with intratumoral injections of a specific dose of MG132 (10 mg/kg) or PBS. (A, B) Intratumoral vascularization was assessed by VEGF and p53 immunolabeling (400 × power) on paraffin-embedded MCF-7 cell tumor sections. Representative images are shown. Integrated optical density (IOD) values of VEGF and p53 protein expression were evaluated. ImagePro Plus software was used to analyze the IOD values of the positive areas of immunohistochemical staining. The resulting histograms are presented here. A statistical analysis was performed using a one-way ANOVA. The results are presented as mean ± SD for six mice. *P<0.05; **P<0.01.
Mentions: To further evaluate the effects of MG132 on tumor angiogenesis induced by V12Rac1/LCdc42, we injected 10 mg/kg MG132 or PBS every 2 days into pre-established, MCF-7 breast tumors (approximately 200 mm3) grown in nude mice. Immunolabeling for VEGF was much higher in tumors excised from mice in the V12Rac1 and L61Cdc42 groups than in those from the control group (Fig. 4). Moreover, p53 expression was lower in the V12Rac1 and L61Cdc42 groups than in the blank or control groups. However, MG312 injections dramatically inhibited the increase in VEGF expression and enhanced the decrease in p53 expression in the V12Rac1 and L61Cdc42 groups compared with the control group. There was no significant difference between the blank and control groups. These data revealed that in vivo, MG132 could reverse the effects of activated Rac1/Cdc42 on p53 and VEGF expression to suppress tumor angiogenesis.

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