Egr-1 Enhances Drug Resistance of Breast Cancer Cells by MDR1 Dependence

Paclitaxel (PTX) is one of the most effective drugs used in the treatment of breast cancer. Nonetheless, the appearance of MDR1 (multidrug resistance 1) in tumor cells has become a significant hindrance for efficacious chemotherapy. In this study, we show that the expression level of Egr-1 (early growth response gene-1) in cancer tissues (from paclitaxel chemotherapy failure patients) and MCF-7/PTX cells (the breast cancer cell line that was resistant to paclitaxel) was increased. Cell proliferation assay and apoptosis assay revealed that Egr-1 could promote cell growth and inhibit apoptosis in MCF-7/PTX. Mechanistic studies indicated that Egr-1 could bind to the proximal MDR1 promoter and enhance MDR1 transcription. These findings indicate that paclitaxel induced Egr-1 accumulation and upregulated the expression of MDR1, thereby inducing the drug resistance in MCF-7/PTX. Our results suggest a novel pathway by which paclitaxel induces MDR1 expression, possibly illuminating a potential target pathway for the prevention of MDR1-mediated drug resistance.

Key words:
Egr-1; Breast cancer; MDR1; Drug resistance.

Authorship

Pei-yao Gong ^**Correspondence: P. Gong. Department of Pathology. Xuzhou Health Hospital of Women and Children. 46 He-ping Road, Xuzhou, Jiangsu. 221004, P.R.China. mail: jsxzgpy1@126.com.

Department of Pathology, Xuzhou Health Hospital of Women and Children, Xuzhou, P. R. ChinaXuzhou Health Hospital of Women and ChildrenChinaXuzhou, ChinaDepartment of Pathology, Xuzhou Health Hospital of Women and Children, Xuzhou, P. R. China

http://orcid.org/0000-0002-1355-5879

*Correspondence: P. Gong. Department of Pathology. Xuzhou Health Hospital of Women and Children. 46 He-ping Road, Xuzhou, Jiangsu. 221004, P.R.China. mail: jsxzgpy1@126.com.

CONFLICT OF INTERESTS

The authors declare that they have no conflict of interest.

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (4)

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FIGURE 1
Egr-1 was overexpressed in drug resistance BC. A. The expression levels of Egr-1 in cancerous tissues and paracancerous tissues of PTX chemotherapy failure BC by Western blot. Comparison of Egr-1 in PTX chemotherapy failure BC cancerous tissues and paracancerous tissues, *P < 0.05, **P < 0.01, ***P < 0.001. B. The expression of Egr-1 in PTX chemotherapy failure BC cancerous tissues and paracancerous tissues of BC by immunohistochemistry. (Upper figure 10x, lower figure 40x, n = 30). C. The expression of Egr-1 in MCF-7 and in MCF-7/PTX (paclitaxel at 5 nmol/L concentration) by Western blot, *P < 0.05, n = 3.

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FIGURE 2
Downregulating Egr-1 inhibited the cell proliferation of MCF-7/PTX. A. Western blot of the expression of Egr-1 protein in BC cell lines MCF-7/PTX (paclitaxel at 5 nmol/L concentration) transfected with siRNA of Egr-1 (siEgr-1) and siRNA of negative control (siNC). CCK8 analysis was performed to examine the cell proliferation of MCF-7/PTX cells transfected with siRNA of Egr-1 (siEgr-1) and siRNA of negative control (siNC). The cell proliferation absorbance was detected in 24 h, 48 h, 72 h and 96 h, *P < 0.05, **P < 0.01, n = 3. B. Knocking-down Egr-1 suppressed clone formation in MCF-7/PTX (paclitaxel at 5 nmol/L concentration), **P < 0.01, n = 3.

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FIGURE 3
Downregulating Egr-1 promoted the apoptosis of MCF-7/PTX. A. Annexin V-FITC binding assay was used to observe apoptotic cells by fluorescence microscope in MCF-7/PTX cells (paclitaxel at 5 nmol/L concentration) transfected with siRNA of Egr-1 (siEgr-1) and siRNA of negative control (siNC). Prophase apoptotic cells were recognized by binding with FITC on the membrane (cell membrane displays green). Anaphase apoptotic cells were recognized by binding with FITC and PI on the nuclei (nuclei displays red). Data shown were from a typical experiment performed in triplicate. B. Western blot analyzed the expression of Egr-1 and cleaved caspase 3 protein in BC cell lines MCF-7/PTX (paclitaxel at 5 nmol/L concentration) transfected with siRNA of Egr-1 (siEgr-1) and siRNA of negative control (siNC). *P < 0.05, n = 3.

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[FIGURE 4
Egr-1 was responsible for the activation of MDR1. A. Effects of knocking down Egr-1 on protein expression of MDR1 in MCF-7/PTX cells (paclitaxel at 5 nmol/L concentration) by Western blot, *P < 0.05, **P < 0.01, n = 3. B. MCF-7/PTX cells (paclitaxel at 5 nmol/L concentration) were transfected with siRNA of Egr-1 (siEgr-1) and siRNA of negative control (siNC). Lysates were immunoprecipitated with anti-Egr-1 antibody and immunoblotted with anti-MDR1 and anti-Egr-1 antibody. Chromatin immunoprecipitation (ChIP) analysis in MCF-7/PTX cells. The results verified that Egr-1 could bind with the gene promoter region of MDR1. The input DNA sample was 10% of the sample used for ChIP and anti-IgG was used for negative control. C. MCF-7/PTX cells (paclitaxel at 5 nmol/L concentration) incubated with 20 mM rhodamine-123 for 90 min. MCF-7/PTX cells were transfected with siRNA of Egr-1 (siEgr-1) and siRNA of negative control (siNC) for 48 h. Rhodamine-123 florescence in cell lysates was measured at excitation and emission wavelengths of 485 and 528 nm, respectively. The fluorescence values were normalized to the total protein content of each sample. *P < 0.05, n = 3.

FIGURE 1 Egr-1 was overexpressed in drug resistance BC. A. The expression levels of Egr-1 in cancerous tissues and paracancerous tissues of PTX chemotherapy failure BC by Western blot. Comparison of Egr-1 in PTX chemotherapy failure BC cancerous tissues and paracancerous tissues, P < 0.05, P < 0.01, P < 0.001. B. The expression of Egr-1 in PTX chemotherapy failure BC cancerous tissues and paracancerous tissues of BC by immunohistochemistry. (Upper figure 10x, lower figure 40x, n = 30). C. The expression of Egr-1 in MCF-7 and in MCF-7/PTX (paclitaxel at 5 nmol/L concentration) by Western blot, *P < 0.05, n = 3.

FIGURE 2 Downregulating Egr-1 inhibited the cell proliferation of MCF-7/PTX. A. Western blot of the expression of Egr-1 protein in BC cell lines MCF-7/PTX (paclitaxel at 5 nmol/L concentration) transfected with siRNA of Egr-1 (siEgr-1) and siRNA of negative control (siNC). CCK8 analysis was performed to examine the cell proliferation of MCF-7/PTX cells transfected with siRNA of Egr-1 (siEgr-1) and siRNA of negative control (siNC). The cell proliferation absorbance was detected in 24 h, 48 h, 72 h and 96 h, *P < 0.05, P < 0.01, n = 3. B. Knocking-down Egr-1 suppressed clone formation in MCF-7/PTX (paclitaxel at 5 nmol/L concentration), P < 0.01, n = 3.

FIGURE 3 Downregulating Egr-1 promoted the apoptosis of MCF-7/PTX. A. Annexin V-FITC binding assay was used to observe apoptotic cells by fluorescence microscope in MCF-7/PTX cells (paclitaxel at 5 nmol/L concentration) transfected with siRNA of Egr-1 (siEgr-1) and siRNA of negative control (siNC). Prophase apoptotic cells were recognized by binding with FITC on the membrane (cell membrane displays green). Anaphase apoptotic cells were recognized by binding with FITC and PI on the nuclei (nuclei displays red). Data shown were from a typical experiment performed in triplicate. B. Western blot analyzed the expression of Egr-1 and cleaved caspase 3 protein in BC cell lines MCF-7/PTX (paclitaxel at 5 nmol/L concentration) transfected with siRNA of Egr-1 (siEgr-1) and siRNA of negative control (siNC). *P < 0.05, n = 3.

[FIGURE 4 Egr-1 was responsible for the activation of MDR1. A. Effects of knocking down Egr-1 on protein expression of MDR1 in MCF-7/PTX cells (paclitaxel at 5 nmol/L concentration) by Western blot, *P < 0.05, **P < 0.01, n = 3. B. MCF-7/PTX cells (paclitaxel at 5 nmol/L concentration) were transfected with siRNA of Egr-1 (siEgr-1) and siRNA of negative control (siNC). Lysates were immunoprecipitated with anti-Egr-1 antibody and immunoblotted with anti-MDR1 and anti-Egr-1 antibody. Chromatin immunoprecipitation (ChIP) analysis in MCF-7/PTX cells. The results verified that Egr-1 could bind with the gene promoter region of MDR1. The input DNA sample was 10% of the sample used for ChIP and anti-IgG was used for negative control. C. MCF-7/PTX cells (paclitaxel at 5 nmol/L concentration) incubated with 20 mM rhodamine-123 for 90 min. MCF-7/PTX cells were transfected with siRNA of Egr-1 (siEgr-1) and siRNA of negative control (siNC) for 48 h. Rhodamine-123 florescence in cell lysates was measured at excitation and emission wavelengths of 485 and 528 nm, respectively. The fluorescence values were normalized to the total protein content of each sample. *P < 0.05, n = 3.

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Egr-1 Enhances Drug Resistance of Breast Cancer Cells by MDR1 Dependence

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[1] *Correspondence: P. Gong. Department of Pathology. Xuzhou Health Hospital of Women and Children. 46 He-ping Road, Xuzhou, Jiangsu. 221004, P.R.China. mail: jsxzgpy1@126.com.