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Endoplasmic reticulum protein 29 (ERp29) confers radioresistance through the DNA repair gene, O(6)-methylguanine DNA-methyltransferase, in breast cancer cells.

Chen S, Zhang Y, Zhang D - Sci Rep (2015)

Bottom Line: Our previous studies have demonstrated a significant role of ERp29 in breast cancer cell survival against doxorubicin-induced genotoxic stress.We here reported that ERp29 expression in the triple negative MDA-MB-231 breast cancer cells significantly increased cell survival against ionizing radiation.In addition, radiation treatment in the MGMT-knockdown cells elevated phosphorylation of γ-H2AX and cleavage of caspase 3, indicating that depletion of MGMT facilitates DNA double strands breaks and increases cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.

ABSTRACT
Resistance of cancer cells to radiotherapy is a major clinical problem in cancer treatment. Therefore, understanding the molecular basis of cellular resistance to radiotherapy and identification of novel targets are essential for improving treatment efficacy for cancer patients. Our previous studies have demonstrated a significant role of ERp29 in breast cancer cell survival against doxorubicin-induced genotoxic stress. We here reported that ERp29 expression in the triple negative MDA-MB-231 breast cancer cells significantly increased cell survival against ionizing radiation. Methylation PCR array analysis identified that ERp29 expression increased promoter hypomethylation of the DNA repair gene, O(6)-methylguanine DNA-methyltransferase (MGMT), by downregulating DNA methyltransferase 1. Knockdown of MGMT in the ERp29-transfected cancer cells increased radiosensitivity, leading to a decreased post-irradiation survival. In addition, radiation treatment in the MGMT-knockdown cells elevated phosphorylation of γ-H2AX and cleavage of caspase 3, indicating that depletion of MGMT facilitates DNA double strands breaks and increases cell apoptosis. Hence, our studies prove a novel function of ERp29\MGMT in cancer cell survival against radiation. Targeting ERp29\MGMT axis may be useful for providing better treatment efficacy in combination with radiotherapy in breast cancer.

No MeSH data available.


Related in: MedlinePlus

MGMT mediates ERp29-induced post-irradiation survival rate. and facilitates DNA damage in ERp29-transfected MDA-MB-231 cells.(a) Repression of MGMT by siRNA (#3) reduced the ERp29-enhanced post-irradiation survival rate. Cells were treated with control or MGMT siRNA (#3) for 48 hours and then exposed to irradiation at the indicated doses. Expression of MGMT was efficiently repressed by siRNA in the ERp29-overexpressed clone B cells. (b) Depletion of endogenous MGMT by siRNA in MCF-7 cells sensitized to radiation treatment. MCF-7 cells were treated with control or MGMT siRNA (#3) for 48 hours and then exposed to irradiation at the indicated doses. The expression of MGMT in MCF-7 cells was efficiently reduced by siRNA. (c) Re-expression of MGMT in the MB-231/ERp29 siRNA cells restores radioresistance. Cells were transfected with pcDNA-MGMT or pcDNA for 24 hours and exposed to radiation treatment. MGMT expressed was examined by immunoblot. Post-radiation survival rate was assessed by clonogenic assay as described in Fig. 1
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f5: MGMT mediates ERp29-induced post-irradiation survival rate. and facilitates DNA damage in ERp29-transfected MDA-MB-231 cells.(a) Repression of MGMT by siRNA (#3) reduced the ERp29-enhanced post-irradiation survival rate. Cells were treated with control or MGMT siRNA (#3) for 48 hours and then exposed to irradiation at the indicated doses. Expression of MGMT was efficiently repressed by siRNA in the ERp29-overexpressed clone B cells. (b) Depletion of endogenous MGMT by siRNA in MCF-7 cells sensitized to radiation treatment. MCF-7 cells were treated with control or MGMT siRNA (#3) for 48 hours and then exposed to irradiation at the indicated doses. The expression of MGMT in MCF-7 cells was efficiently reduced by siRNA. (c) Re-expression of MGMT in the MB-231/ERp29 siRNA cells restores radioresistance. Cells were transfected with pcDNA-MGMT or pcDNA for 24 hours and exposed to radiation treatment. MGMT expressed was examined by immunoblot. Post-radiation survival rate was assessed by clonogenic assay as described in Fig. 1

Mentions: We showed that ERp29 overexpression resulted in radioresistance in MDA-MB-231 cells (Fig. 1). To understand whether the upregulated MGMT by ERp29 is involved in radioresistance, the MB-231/ERp29 cells (clone B) were treated for 48 hours with MGMT siRNA to reduce MGMT expression or with control siRNA. These cells were then irradiated (0–6 Gy) and the post-irradiation survival assessed using the clonogenic assay. Relative to MB-231/ERp29 cells treated with control siRNA (D37 = 2.72 ± 0.09 Gy), MGMT knockdown in these cells caused a significantly decrease of D37 (1.82 ± 0.13 Gy, p < 0.05) (Fig. 5a). These data indicate that MGMT depletion re-sensitizes to radiation in MB-231/ERp29 cells.


Endoplasmic reticulum protein 29 (ERp29) confers radioresistance through the DNA repair gene, O(6)-methylguanine DNA-methyltransferase, in breast cancer cells.

Chen S, Zhang Y, Zhang D - Sci Rep (2015)

MGMT mediates ERp29-induced post-irradiation survival rate. and facilitates DNA damage in ERp29-transfected MDA-MB-231 cells.(a) Repression of MGMT by siRNA (#3) reduced the ERp29-enhanced post-irradiation survival rate. Cells were treated with control or MGMT siRNA (#3) for 48 hours and then exposed to irradiation at the indicated doses. Expression of MGMT was efficiently repressed by siRNA in the ERp29-overexpressed clone B cells. (b) Depletion of endogenous MGMT by siRNA in MCF-7 cells sensitized to radiation treatment. MCF-7 cells were treated with control or MGMT siRNA (#3) for 48 hours and then exposed to irradiation at the indicated doses. The expression of MGMT in MCF-7 cells was efficiently reduced by siRNA. (c) Re-expression of MGMT in the MB-231/ERp29 siRNA cells restores radioresistance. Cells were transfected with pcDNA-MGMT or pcDNA for 24 hours and exposed to radiation treatment. MGMT expressed was examined by immunoblot. Post-radiation survival rate was assessed by clonogenic assay as described in Fig. 1
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5: MGMT mediates ERp29-induced post-irradiation survival rate. and facilitates DNA damage in ERp29-transfected MDA-MB-231 cells.(a) Repression of MGMT by siRNA (#3) reduced the ERp29-enhanced post-irradiation survival rate. Cells were treated with control or MGMT siRNA (#3) for 48 hours and then exposed to irradiation at the indicated doses. Expression of MGMT was efficiently repressed by siRNA in the ERp29-overexpressed clone B cells. (b) Depletion of endogenous MGMT by siRNA in MCF-7 cells sensitized to radiation treatment. MCF-7 cells were treated with control or MGMT siRNA (#3) for 48 hours and then exposed to irradiation at the indicated doses. The expression of MGMT in MCF-7 cells was efficiently reduced by siRNA. (c) Re-expression of MGMT in the MB-231/ERp29 siRNA cells restores radioresistance. Cells were transfected with pcDNA-MGMT or pcDNA for 24 hours and exposed to radiation treatment. MGMT expressed was examined by immunoblot. Post-radiation survival rate was assessed by clonogenic assay as described in Fig. 1
Mentions: We showed that ERp29 overexpression resulted in radioresistance in MDA-MB-231 cells (Fig. 1). To understand whether the upregulated MGMT by ERp29 is involved in radioresistance, the MB-231/ERp29 cells (clone B) were treated for 48 hours with MGMT siRNA to reduce MGMT expression or with control siRNA. These cells were then irradiated (0–6 Gy) and the post-irradiation survival assessed using the clonogenic assay. Relative to MB-231/ERp29 cells treated with control siRNA (D37 = 2.72 ± 0.09 Gy), MGMT knockdown in these cells caused a significantly decrease of D37 (1.82 ± 0.13 Gy, p < 0.05) (Fig. 5a). These data indicate that MGMT depletion re-sensitizes to radiation in MB-231/ERp29 cells.

Bottom Line: Our previous studies have demonstrated a significant role of ERp29 in breast cancer cell survival against doxorubicin-induced genotoxic stress.We here reported that ERp29 expression in the triple negative MDA-MB-231 breast cancer cells significantly increased cell survival against ionizing radiation.In addition, radiation treatment in the MGMT-knockdown cells elevated phosphorylation of γ-H2AX and cleavage of caspase 3, indicating that depletion of MGMT facilitates DNA double strands breaks and increases cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.

ABSTRACT
Resistance of cancer cells to radiotherapy is a major clinical problem in cancer treatment. Therefore, understanding the molecular basis of cellular resistance to radiotherapy and identification of novel targets are essential for improving treatment efficacy for cancer patients. Our previous studies have demonstrated a significant role of ERp29 in breast cancer cell survival against doxorubicin-induced genotoxic stress. We here reported that ERp29 expression in the triple negative MDA-MB-231 breast cancer cells significantly increased cell survival against ionizing radiation. Methylation PCR array analysis identified that ERp29 expression increased promoter hypomethylation of the DNA repair gene, O(6)-methylguanine DNA-methyltransferase (MGMT), by downregulating DNA methyltransferase 1. Knockdown of MGMT in the ERp29-transfected cancer cells increased radiosensitivity, leading to a decreased post-irradiation survival. In addition, radiation treatment in the MGMT-knockdown cells elevated phosphorylation of γ-H2AX and cleavage of caspase 3, indicating that depletion of MGMT facilitates DNA double strands breaks and increases cell apoptosis. Hence, our studies prove a novel function of ERp29\MGMT in cancer cell survival against radiation. Targeting ERp29\MGMT axis may be useful for providing better treatment efficacy in combination with radiotherapy in breast cancer.

No MeSH data available.


Related in: MedlinePlus