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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

ERp29 expression reduces DNMT1 to increase MGMT promoter demethylation in MDA-MB-231 cells.(a) ERp29 expression decreased the level of DNMT1 whereas ERp29 knockdown upregulated the expression of DNMT1. The expression of DNMT3A or 3B was not markedly affected by ERp29. *p < 0.05, **p < 0.01, relative mock-transfected control or controL siRNA. (b) Reduction of DNMT1 by siRNA upregulated MGMT expression in MDA-MB-231 cells. MDA-MB-231 cells were transiently transfected with control siRNA or DNMT1 siRNA (#1) for 48hours and the expression of DNMT1 and MGMT was examined. (c) MGMT promoter methylation/demethylation. Genomic DNA was extracted from the MDA-MB-231 cells transfected with control siRNA or DNMT1 siRNA and the MS-PCR was done as described in “Materials and Methods”. **p < 0.01, ***p < 0.001, versus control.
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f3: ERp29 expression reduces DNMT1 to increase MGMT promoter demethylation in MDA-MB-231 cells.(a) ERp29 expression decreased the level of DNMT1 whereas ERp29 knockdown upregulated the expression of DNMT1. The expression of DNMT3A or 3B was not markedly affected by ERp29. *p < 0.05, **p < 0.01, relative mock-transfected control or controL siRNA. (b) Reduction of DNMT1 by siRNA upregulated MGMT expression in MDA-MB-231 cells. MDA-MB-231 cells were transiently transfected with control siRNA or DNMT1 siRNA (#1) for 48hours and the expression of DNMT1 and MGMT was examined. (c) MGMT promoter methylation/demethylation. Genomic DNA was extracted from the MDA-MB-231 cells transfected with control siRNA or DNMT1 siRNA and the MS-PCR was done as described in “Materials and Methods”. **p < 0.01, ***p < 0.001, versus control.

Mentions: Since DNA methyltransferase is responsible for increase of DNA methylation, the expression of DNMT1, DNMT3A and DNMT3B was analysed in mock-transfected control cells and MB-231/ERp29 cells. As indicated in Fig. 3a, relative to control cells, ERp29 overexpression in MDA-MB-231 cells significantly inhibited the expression of DNMT1, rather than the expression of DNMT3A or 3B. The role of DNMT1 in epigenetic regulation of MGMT expression was further supported by the fact that DNMT1 knockdown by siRNA in MDA-MB-231 cells (Suppl. Fig. 1C) led to an increase of MGMT expression compared to the cells treated with non-targeted control siRNA (Fig. 3b). MS-PCR analysis showed that DNMT1 knockdown in MDA-MB-231 cells enhanced demethylation and reduced methylation of MGMT promoter relative to the cells treated with control siRNA (Fig. 3c). These data indicate a critical role of DNMT1 in ERp29-mediated inhibition of MGMT promoter methylation.


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)

ERp29 expression reduces DNMT1 to increase MGMT promoter demethylation in MDA-MB-231 cells.(a) ERp29 expression decreased the level of DNMT1 whereas ERp29 knockdown upregulated the expression of DNMT1. The expression of DNMT3A or 3B was not markedly affected by ERp29. *p < 0.05, **p < 0.01, relative mock-transfected control or controL siRNA. (b) Reduction of DNMT1 by siRNA upregulated MGMT expression in MDA-MB-231 cells. MDA-MB-231 cells were transiently transfected with control siRNA or DNMT1 siRNA (#1) for 48hours and the expression of DNMT1 and MGMT was examined. (c) MGMT promoter methylation/demethylation. Genomic DNA was extracted from the MDA-MB-231 cells transfected with control siRNA or DNMT1 siRNA and the MS-PCR was done as described in “Materials and Methods”. **p < 0.01, ***p < 0.001, versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: ERp29 expression reduces DNMT1 to increase MGMT promoter demethylation in MDA-MB-231 cells.(a) ERp29 expression decreased the level of DNMT1 whereas ERp29 knockdown upregulated the expression of DNMT1. The expression of DNMT3A or 3B was not markedly affected by ERp29. *p < 0.05, **p < 0.01, relative mock-transfected control or controL siRNA. (b) Reduction of DNMT1 by siRNA upregulated MGMT expression in MDA-MB-231 cells. MDA-MB-231 cells were transiently transfected with control siRNA or DNMT1 siRNA (#1) for 48hours and the expression of DNMT1 and MGMT was examined. (c) MGMT promoter methylation/demethylation. Genomic DNA was extracted from the MDA-MB-231 cells transfected with control siRNA or DNMT1 siRNA and the MS-PCR was done as described in “Materials and Methods”. **p < 0.01, ***p < 0.001, versus control.
Mentions: Since DNA methyltransferase is responsible for increase of DNA methylation, the expression of DNMT1, DNMT3A and DNMT3B was analysed in mock-transfected control cells and MB-231/ERp29 cells. As indicated in Fig. 3a, relative to control cells, ERp29 overexpression in MDA-MB-231 cells significantly inhibited the expression of DNMT1, rather than the expression of DNMT3A or 3B. The role of DNMT1 in epigenetic regulation of MGMT expression was further supported by the fact that DNMT1 knockdown by siRNA in MDA-MB-231 cells (Suppl. Fig. 1C) led to an increase of MGMT expression compared to the cells treated with non-targeted control siRNA (Fig. 3b). MS-PCR analysis showed that DNMT1 knockdown in MDA-MB-231 cells enhanced demethylation and reduced methylation of MGMT promoter relative to the cells treated with control siRNA (Fig. 3c). These data indicate a critical role of DNMT1 in ERp29-mediated inhibition of MGMT promoter methylation.

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