Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of MG132 on VEGF and p53 expression induced by active Rac1/Cdc42.(A) GST–MCF-7, V12Rac1–MCF-7, and L61Cdc42–MCF-7 cells were incubated, and MG132 was added to select samples for 12 and 24 h, respectively. After incubation, the media were analyzed for VEGF levels using ELISA assay; cell numbers were counted. Data are expressed as the mean ± standard deviation (SD) for three independent experiments. Statistical significance was assessed with one-way ANOVA and Student's t-test. *P<0.05, **P<0.01, for the GST–MCF-7 group vs V12Rac1– or L61Cdc42–MCF-7 group. #P<0.05, ##P<0.01, for the MG132-added group vs the no-MG132-added group. (B, C) MCF-7, GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells were incubated, and MG132 was added to select samples for 24 h. After incubation, the cells were analyzed by western blot assay. p53, p21 and β-actin expression was examined. β-actin protein levels were used as a loading control. (D) The immunoprecipitation assay was performed as described in Material and Methods. Total protein was extracted from MCF-7, GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells and subjected to an immunoprecipitation assay. p53 protein expression was examined. β-actin protein levels were used as a loading control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3672132&req=5

pone-0066275-g003: Effect of MG132 on VEGF and p53 expression induced by active Rac1/Cdc42.(A) GST–MCF-7, V12Rac1–MCF-7, and L61Cdc42–MCF-7 cells were incubated, and MG132 was added to select samples for 12 and 24 h, respectively. After incubation, the media were analyzed for VEGF levels using ELISA assay; cell numbers were counted. Data are expressed as the mean ± standard deviation (SD) for three independent experiments. Statistical significance was assessed with one-way ANOVA and Student's t-test. *P<0.05, **P<0.01, for the GST–MCF-7 group vs V12Rac1– or L61Cdc42–MCF-7 group. #P<0.05, ##P<0.01, for the MG132-added group vs the no-MG132-added group. (B, C) MCF-7, GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells were incubated, and MG132 was added to select samples for 24 h. After incubation, the cells were analyzed by western blot assay. p53, p21 and β-actin expression was examined. β-actin protein levels were used as a loading control. (D) The immunoprecipitation assay was performed as described in Material and Methods. Total protein was extracted from MCF-7, GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells and subjected to an immunoprecipitation assay. p53 protein expression was examined. β-actin protein levels were used as a loading control.

Mentions: Given that the amount of p53 protein could be modified by changes in the rate of synthesis or degradation [22], we hypothesized that Rac1/Cdc42 affected degradation of p53. To test this hypothesis, the ubiquitin-proteasome inhibitor MG-132 was utilized to analyze p53 protein levels. Western blot results revealed that p53 protein was inhibited by V12Rac1/Cdc42, but a large accumulation of p53 protein was observed in the presence of V12Rac1/Cdc42, together with MG-132 (Fig. 3A). Additionally, immunoprecipitation data showed that V12Rac1/Cdc42 could reduce p53 protein levels and increase the amount of ubiquitinated p53 (Fig. 3B). The VEGF ELISA assay also showed that in MCF-7 cells, V12Rac1/LCdc42 increased VEGF secretion after 24 h and 48 h, compared to the control group. However, VEGF expression induced by V12Rac1 or L61Cdc42 decreased after treatment with MG132 (Fig. 3C). These results confirmed that active Rac1/Cdc42 affected and promoted ubiquitin-mediated degradation of p53 to increase VEGF production.


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 VEGF and p53 expression induced by active Rac1/Cdc42.(A) GST–MCF-7, V12Rac1–MCF-7, and L61Cdc42–MCF-7 cells were incubated, and MG132 was added to select samples for 12 and 24 h, respectively. After incubation, the media were analyzed for VEGF levels using ELISA assay; cell numbers were counted. Data are expressed as the mean ± standard deviation (SD) for three independent experiments. Statistical significance was assessed with one-way ANOVA and Student's t-test. *P<0.05, **P<0.01, for the GST–MCF-7 group vs V12Rac1– or L61Cdc42–MCF-7 group. #P<0.05, ##P<0.01, for the MG132-added group vs the no-MG132-added group. (B, C) MCF-7, GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells were incubated, and MG132 was added to select samples for 24 h. After incubation, the cells were analyzed by western blot assay. p53, p21 and β-actin expression was examined. β-actin protein levels were used as a loading control. (D) The immunoprecipitation assay was performed as described in Material and Methods. Total protein was extracted from MCF-7, GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells and subjected to an immunoprecipitation assay. p53 protein expression was examined. β-actin protein levels were used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0066275-g003: Effect of MG132 on VEGF and p53 expression induced by active Rac1/Cdc42.(A) GST–MCF-7, V12Rac1–MCF-7, and L61Cdc42–MCF-7 cells were incubated, and MG132 was added to select samples for 12 and 24 h, respectively. After incubation, the media were analyzed for VEGF levels using ELISA assay; cell numbers were counted. Data are expressed as the mean ± standard deviation (SD) for three independent experiments. Statistical significance was assessed with one-way ANOVA and Student's t-test. *P<0.05, **P<0.01, for the GST–MCF-7 group vs V12Rac1– or L61Cdc42–MCF-7 group. #P<0.05, ##P<0.01, for the MG132-added group vs the no-MG132-added group. (B, C) MCF-7, GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells were incubated, and MG132 was added to select samples for 24 h. After incubation, the cells were analyzed by western blot assay. p53, p21 and β-actin expression was examined. β-actin protein levels were used as a loading control. (D) The immunoprecipitation assay was performed as described in Material and Methods. Total protein was extracted from MCF-7, GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells and subjected to an immunoprecipitation assay. p53 protein expression was examined. β-actin protein levels were used as a loading control.
Mentions: Given that the amount of p53 protein could be modified by changes in the rate of synthesis or degradation [22], we hypothesized that Rac1/Cdc42 affected degradation of p53. To test this hypothesis, the ubiquitin-proteasome inhibitor MG-132 was utilized to analyze p53 protein levels. Western blot results revealed that p53 protein was inhibited by V12Rac1/Cdc42, but a large accumulation of p53 protein was observed in the presence of V12Rac1/Cdc42, together with MG-132 (Fig. 3A). Additionally, immunoprecipitation data showed that V12Rac1/Cdc42 could reduce p53 protein levels and increase the amount of ubiquitinated p53 (Fig. 3B). The VEGF ELISA assay also showed that in MCF-7 cells, V12Rac1/LCdc42 increased VEGF secretion after 24 h and 48 h, compared to the control group. However, VEGF expression induced by V12Rac1 or L61Cdc42 decreased after treatment with MG132 (Fig. 3C). These results confirmed that active Rac1/Cdc42 affected and promoted ubiquitin-mediated degradation of p53 to increase VEGF production.

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