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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 depletion by siRNA stimulates radiation-induced cell apoptosis in ERp29-transfected MDA-MB-231 cells.(a) Reduction of MGMT in ERp29-overexpressed clone B cells increased irradiation-induced expression of cleaved caspase 3 compared to the control siRNA-treated cells. Expression of cleaved caspase 3 was highly induced by irradiation in the vector-transfected MDA-MB-231 cells (column 2 vs. 1). In the ERp29-overespressed clone B cells, depletion of MGMT alone did not activate expression of cleaved caspase 3 (column 3 vs. 1). However, these cells treated with MGMT siRNA (#3) markedly increased the expression of cleaved caspase 3 after irradiation (column 5 vs. 4). (b) Cell viability. Irradiation treatment caused significant reduction of cell viability in vector-transfected MDA-MB-231 cells (column 2 vs. 1). Combinatory treatment of irradiation/MGMT siRNA (column 5) in the ERp29-overexpressed clone B cells significantly reduced cell viability compared to the cells treated with MGMT siRNA alone (column 3) or the irradiation/control siRNA (column 4). **p < 0.01, ***p < 0.001, relative to control.
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f7: MGMT depletion by siRNA stimulates radiation-induced cell apoptosis in ERp29-transfected MDA-MB-231 cells.(a) Reduction of MGMT in ERp29-overexpressed clone B cells increased irradiation-induced expression of cleaved caspase 3 compared to the control siRNA-treated cells. Expression of cleaved caspase 3 was highly induced by irradiation in the vector-transfected MDA-MB-231 cells (column 2 vs. 1). In the ERp29-overespressed clone B cells, depletion of MGMT alone did not activate expression of cleaved caspase 3 (column 3 vs. 1). However, these cells treated with MGMT siRNA (#3) markedly increased the expression of cleaved caspase 3 after irradiation (column 5 vs. 4). (b) Cell viability. Irradiation treatment caused significant reduction of cell viability in vector-transfected MDA-MB-231 cells (column 2 vs. 1). Combinatory treatment of irradiation/MGMT siRNA (column 5) in the ERp29-overexpressed clone B cells significantly reduced cell viability compared to the cells treated with MGMT siRNA alone (column 3) or the irradiation/control siRNA (column 4). **p < 0.01, ***p < 0.001, relative to control.

Mentions: To examine whether the ERp29-induced activation of MGMT is responsible for antagonizing radiation-induced apoptosis, the MB-231/ERp29 cells were treated with MGMT siRNA or control siRNA for 48 hours and exposed to radiation treatment (4 Gy). After 48 hours post-incubation, these cells were used for examining the expression of cleaved caspase 3 and cell viability. In parallel, the vector-transfected control cells were also exposed to radiation treatment (4 Gy). As shown in Fig. 7a, radiation treatment significantly increased the expression of cleaved caspase-3 (column 2 vs. 1) in the vector-transfected control cells. Interestingly, MGMT depletion alone in MB-231/ERp29 cells could not activate the expression of cleaved caspase 3 (column 3). However, compared to MB-231/ERp29 cells pre-treated with control siRNA, knockdown of the ERp29-induced MGMT significantly increased the expression of cleaved caspase-3 after exposure to irradiation (column 5 vs. 4). Hence, MGMT depletion facilitates the radiation-induced caspase activation 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 depletion by siRNA stimulates radiation-induced cell apoptosis in ERp29-transfected MDA-MB-231 cells.(a) Reduction of MGMT in ERp29-overexpressed clone B cells increased irradiation-induced expression of cleaved caspase 3 compared to the control siRNA-treated cells. Expression of cleaved caspase 3 was highly induced by irradiation in the vector-transfected MDA-MB-231 cells (column 2 vs. 1). In the ERp29-overespressed clone B cells, depletion of MGMT alone did not activate expression of cleaved caspase 3 (column 3 vs. 1). However, these cells treated with MGMT siRNA (#3) markedly increased the expression of cleaved caspase 3 after irradiation (column 5 vs. 4). (b) Cell viability. Irradiation treatment caused significant reduction of cell viability in vector-transfected MDA-MB-231 cells (column 2 vs. 1). Combinatory treatment of irradiation/MGMT siRNA (column 5) in the ERp29-overexpressed clone B cells significantly reduced cell viability compared to the cells treated with MGMT siRNA alone (column 3) or the irradiation/control siRNA (column 4). **p < 0.01, ***p < 0.001, relative to control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4588584&req=5

f7: MGMT depletion by siRNA stimulates radiation-induced cell apoptosis in ERp29-transfected MDA-MB-231 cells.(a) Reduction of MGMT in ERp29-overexpressed clone B cells increased irradiation-induced expression of cleaved caspase 3 compared to the control siRNA-treated cells. Expression of cleaved caspase 3 was highly induced by irradiation in the vector-transfected MDA-MB-231 cells (column 2 vs. 1). In the ERp29-overespressed clone B cells, depletion of MGMT alone did not activate expression of cleaved caspase 3 (column 3 vs. 1). However, these cells treated with MGMT siRNA (#3) markedly increased the expression of cleaved caspase 3 after irradiation (column 5 vs. 4). (b) Cell viability. Irradiation treatment caused significant reduction of cell viability in vector-transfected MDA-MB-231 cells (column 2 vs. 1). Combinatory treatment of irradiation/MGMT siRNA (column 5) in the ERp29-overexpressed clone B cells significantly reduced cell viability compared to the cells treated with MGMT siRNA alone (column 3) or the irradiation/control siRNA (column 4). **p < 0.01, ***p < 0.001, relative to control.
Mentions: To examine whether the ERp29-induced activation of MGMT is responsible for antagonizing radiation-induced apoptosis, the MB-231/ERp29 cells were treated with MGMT siRNA or control siRNA for 48 hours and exposed to radiation treatment (4 Gy). After 48 hours post-incubation, these cells were used for examining the expression of cleaved caspase 3 and cell viability. In parallel, the vector-transfected control cells were also exposed to radiation treatment (4 Gy). As shown in Fig. 7a, radiation treatment significantly increased the expression of cleaved caspase-3 (column 2 vs. 1) in the vector-transfected control cells. Interestingly, MGMT depletion alone in MB-231/ERp29 cells could not activate the expression of cleaved caspase 3 (column 3). However, compared to MB-231/ERp29 cells pre-treated with control siRNA, knockdown of the ERp29-induced MGMT significantly increased the expression of cleaved caspase-3 after exposure to irradiation (column 5 vs. 4). Hence, MGMT depletion facilitates the radiation-induced caspase activation 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