IMP-1 inhibits renal cell carcinoma 786-O cell growth by targeting EphrinB2 signaling pathway

EphrinB2 plays a critical role in tumor growth. In this study, we studied the antitumor activity of imperatorin derivative IMP-1 in renal cell carcinoma (RCC) by regulating EphrinB2 pathway.. Results showed that IMP-1 inhibited the proliferation of 786-O cells in a dose- and time-dependent manner. More importantly, knockdown and transfection of EphrinB2 altered the inhibitory effect of IMP-1 on the activity of 786-O cells. IMP-1 arrested 786-O cell cycle at G0/G1 phase by decreasing the expression of cyclin D1 and cyclin E. Moreover, IMP-1 regulated Bcl-2 family proteins’ expression, thus inducing apoptosis of 786-O cells. IMP-1 down-regulated the expression of EphrinB2, Syntenin1 and PICK1. Then, IMP-1 decreased the phosphorylation of Erk1/2 and AKT. In all, IMP-1 could regulate the EphrinB2 pathway in order to inhibit 786-O cell growth by arresting the cell cycle at G0/G1 phase and inducing cell apoptosis. Thus, IMP-1 may present as a potential strategy for RCC treatment.

Keywords:
Renal cell carcinoma; IMP-1; EphrinB2; Cell cycle; Cell apoptosis.

Authorship

Juan Liu

Department of Oncology, The Weapons Industry of institutes of health, Xi’an, Shaanxi, ChinaThe Weapons Industry of institutes of healthChinaXi’an, Shaanxi, ChinaDepartment of Oncology, The Weapons Industry of institutes of health, Xi’an, Shaanxi, China

Hong Shi ^**Correspondence: H. Shi. Department of Oncology, Tangdu Hospital. The Airforce Military Medical University. Xi’an, Shaanxi, 710038, China. E-mail: blogmm202010@163.com Phone: +86-29-82656788. Fax: +86-29-82655451.

Department of Oncology, Tangdu Hospital, The Airforce Military Medical University, Xi’an, Shaanxi, ChinaThe Airforce Military Medical UniversityChinaXi’an, Shaanxi, ChinaDepartment of Oncology, Tangdu Hospital, The Airforce Military Medical University, Xi’an, Shaanxi, China

http://orcid.org/0000-0002-3491-6127

*Correspondence: H. Shi. Department of Oncology, Tangdu Hospital. The Airforce Military Medical University. Xi’an, Shaanxi, 710038, China. E-mail: blogmm202010@163.com Phone: +86-29-82656788. Fax: +86-29-82655451.

CONFLICT OF INTEREST The authors declare that they have no conflict of interest.

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Figures

Figures (6)

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FIGURE 1
The effect of IMP-1 on renal carcinoma cell growth. (a) The chemical structure of IMP-1. (b) Effect of IMP-1 on renal carcinoma cell growth at 48 h. (c) Effect of IMP-1 on 786-O cell growth at different time point. Cells were treated with various concentrations of IMP-1 for 24, 48 and 72 h and measured by MTT assay. Five wells were treated in each experiment and the data are presented as the mean ± standard error of the mean from three repeated experiments.

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FIGURE 2
Effect of IMP-1 on cell cycle distribution. (a) 786-O cells were treated with IMP-1 (0, 0.78, 1.56 and 3.12 μM) for 48 h followed by staining with propidium iodide for flow cytometric analysis. (b) Expression levels of CDC2, CDK2, Cyclin A2, Cyclin B1, Cyclin D1 and Cyclin E in 786-O cells treated with IMP-1 (0, 0.78, 1.56and 3.12 μM) for 48 h were examined by western blot assay and the results were quantified by densitometry analysis of the bands and normalization to GAPDH. Data are presented as the mean standard error of the mean (n=3).

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FIGURE 3
Effect of IMP-1 on cell apoptosis. (a) IMP-1-induced apoptosis in 789-O cells were characterized by nuclear condensation or nuclear fragmentation after Hoechst staining. (b) Flow cytometric analysis of IMP-1-induced apoptosis in 789-O cells. The flow cytometry profile presents Annexin V FITC (x axis) and PI staining (y axis). The values represent the percentage of cells in each of the four quadrants (lower left quadrant, viable cells; upper left quadrant, necrotic or dead cells; lower right quadrant, early stage apoptotic cells; and upper right quadrant, late stage apoptotic cells).

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FIGURE 4
The mechanism of IMP-1 on cell apoptosis. (a) The mitochondrial membrane potential (Δψm) were assessed using flow cytometry following treatment of the 786-O cells with IMP-1 (0.78, 1.56 or 3.12 μM) for 48 h. (b) Expression levels of Bad, Bak, Bax, Bcl-2 and Mcl-1 in 786-O cells treated with IMP-1 (0, 0.78, 1.56 and 3.12 μM) for 48 h were examined by western blot assay and the results were quantified by densitometry analysis of the bands and normalization to GAPDH. Data are presented as the mean standard error of the mean (n=3).

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FIGURE 5
Effect of IMP-1 on EphrinB2. (a) Expression level of EphrinB2 mRNA in 786-O cells treated with IMP-1 (0, 0.78, 1.56 and 3.12 μM) for 48 h were examined by PCR. (b) Protein level of EphrinB2 in 786-O cells treated with IMP-1 (0, 0.78, 1.56 and 3.12 μM) for 48 h were examined by western blot assay. (c) The expression of EphrinB2 on 786-O cells and 786-O-KO cells. (d) The effect of IMP-1 in inhibiting the growth of 786-O cells and 786-O-KO cells. *p< 0.05 versus the 786-O group. (e) The expression of EphrinB2 on 786- O cells and 786-O/EphrinB2 transfection cells. (f) The effect of ND-17 in inhibiting the growth of 786-O cells and 786-O/EphrinB2 transfection cells. *p< 0.05 versus the 786-O group. Data are presented as the mean standard error of the mean (n=3).

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FIGURE 6
Effect of IMP-1 on EphrinB2 signaling pathway. (a) Protein levels of Syntenin1 and PICK1 in 786-O cells treated with IMP-1 (0, 0.78, 1.56 and 3.12 μM) for 48 h were examined by western blot assay. (b) The phosphorylation of AKT and Erk1/2 in 786-O cells treated with IMP-1 (0, 0.78, 1.56 and 3.12 μM) for 48 h were examined by western blot assay. Data are presented as the mean standard error of the mean (n=3).

FIGURE 1 The effect of IMP-1 on renal carcinoma cell growth. (a) The chemical structure of IMP-1. (b) Effect of IMP-1 on renal carcinoma cell growth at 48 h. (c) Effect of IMP-1 on 786-O cell growth at different time point. Cells were treated with various concentrations of IMP-1 for 24, 48 and 72 h and measured by MTT assay. Five wells were treated in each experiment and the data are presented as the mean ± standard error of the mean from three repeated experiments.

FIGURE 2 Effect of IMP-1 on cell cycle distribution. (a) 786-O cells were treated with IMP-1 (0, 0.78, 1.56 and 3.12 μM) for 48 h followed by staining with propidium iodide for flow cytometric analysis. (b) Expression levels of CDC2, CDK2, Cyclin A2, Cyclin B1, Cyclin D1 and Cyclin E in 786-O cells treated with IMP-1 (0, 0.78, 1.56and 3.12 μM) for 48 h were examined by western blot assay and the results were quantified by densitometry analysis of the bands and normalization to GAPDH. Data are presented as the mean standard error of the mean (n=3).

FIGURE 3 Effect of IMP-1 on cell apoptosis. (a) IMP-1-induced apoptosis in 789-O cells were characterized by nuclear condensation or nuclear fragmentation after Hoechst staining. (b) Flow cytometric analysis of IMP-1-induced apoptosis in 789-O cells. The flow cytometry profile presents Annexin V FITC (x axis) and PI staining (y axis). The values represent the percentage of cells in each of the four quadrants (lower left quadrant, viable cells; upper left quadrant, necrotic or dead cells; lower right quadrant, early stage apoptotic cells; and upper right quadrant, late stage apoptotic cells).

FIGURE 4 The mechanism of IMP-1 on cell apoptosis. (a) The mitochondrial membrane potential (Δψm) were assessed using flow cytometry following treatment of the 786-O cells with IMP-1 (0.78, 1.56 or 3.12 μM) for 48 h. (b) Expression levels of Bad, Bak, Bax, Bcl-2 and Mcl-1 in 786-O cells treated with IMP-1 (0, 0.78, 1.56 and 3.12 μM) for 48 h were examined by western blot assay and the results were quantified by densitometry analysis of the bands and normalization to GAPDH. Data are presented as the mean standard error of the mean (n=3).

FIGURE 5 Effect of IMP-1 on EphrinB2. (a) Expression level of EphrinB2 mRNA in 786-O cells treated with IMP-1 (0, 0.78, 1.56 and 3.12 μM) for 48 h were examined by PCR. (b) Protein level of EphrinB2 in 786-O cells treated with IMP-1 (0, 0.78, 1.56 and 3.12 μM) for 48 h were examined by western blot assay. (c) The expression of EphrinB2 on 786-O cells and 786-O-KO cells. (d) The effect of IMP-1 in inhibiting the growth of 786-O cells and 786-O-KO cells. p< 0.05 versus the 786-O group. (e) The expression of EphrinB2 on 786- O cells and 786-O/EphrinB2 transfection cells. (f) The effect of ND-17 in inhibiting the growth of 786-O cells and 786-O/EphrinB2 transfection cells. p< 0.05 versus the 786-O group. Data are presented as the mean standard error of the mean (n=3).

FIGURE 6 Effect of IMP-1 on EphrinB2 signaling pathway. (a) Protein levels of Syntenin1 and PICK1 in 786-O cells treated with IMP-1 (0, 0.78, 1.56 and 3.12 μM) for 48 h were examined by western blot assay. (b) The phosphorylation of AKT and Erk1/2 in 786-O cells treated with IMP-1 (0, 0.78, 1.56 and 3.12 μM) for 48 h were examined by western blot assay. Data are presented as the mean standard error of the mean (n=3).

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IMP-1 inhibits renal cell carcinoma 786-O cell growth by targeting EphrinB2 signaling pathway

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[1] *Correspondence: H. Shi. Department of Oncology, Tangdu Hospital. The Airforce Military Medical University. Xi’an, Shaanxi, 710038, China. E-mail: blogmm202010@163.com Phone: +86-29-82656788. Fax: +86-29-82655451.