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Anterior gradient 2 is a binding stabilizer of hypoxia inducible factor-1α that enhances CoCl2 -induced doxorubicin resistance in breast cancer cells.

Li Z, Zhu Q, Hu L, Chen H, Wu Z, Li D - Cancer Sci. (2015)

Bottom Line: Our results show that knockdown of AGR2 in MCF-7 cells leads to the suppression of HIF-1α-induced doxorubicin resistance, whereas elevated levels of AGR2 in MDA-MB-231 cells enhance HIF-1α-induced doxorubicin resistance.By specific binding to HIF-1α, the increased level of intracellular AGR2 stabilizes HIF-1α and delays its proteasomal degradation.Finally, we found that AGR2-stabilized HIF-1α escalates multiple drug resistance protein 1 (MDR1) mRNA levels and limits doxorubicin intake of MCF-7 cells, whereas MCF-7/ADR, a doxorubicin resistant cell line with deficient AGR2 and HIF-1α, acquires wild-type MDR1 overexpression.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.

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Intracellular but not extracellular anterior gradient 2 (AGR2) stabilizes CoCl2-induced hypoxia inducible factor-1α (HIF-1α). (a,b) Western blot was performed on corresponding cell lines with treatment of 200 μM CoCl2, 20 μM MG-132 and their combination. β-actin was used as a loading control. (c,d) Cells were pre-treated with or without 200 μM CoCl2 for 6 h, and subjected to 20 μM of cycloheximide (CHX). The external AGR2 protein concentration was 1 μg/mL when added with or without CoCl2. Whole-cell lysates were prepared 15, 30, 45, 60 and 90 min after treatment. Western blots were performed with specific antibodies. The HIF-1α protein levels, compared to the β-actin levels at the corresponding time points, are displayed in the line chart in (e) and (f). (g,h) The same treatments were assigned to MCF-7 and MDA-MB-231 cell lines. The HIF-1α protein level at 0 and 1 h after CHX treatment is shown based on results of the immunofluorescence assay. The original magnification was 200× (scale bar: 30 μM). Each experiment was repeated at least three times. *P < 0.05; **P < 0.01
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fig04: Intracellular but not extracellular anterior gradient 2 (AGR2) stabilizes CoCl2-induced hypoxia inducible factor-1α (HIF-1α). (a,b) Western blot was performed on corresponding cell lines with treatment of 200 μM CoCl2, 20 μM MG-132 and their combination. β-actin was used as a loading control. (c,d) Cells were pre-treated with or without 200 μM CoCl2 for 6 h, and subjected to 20 μM of cycloheximide (CHX). The external AGR2 protein concentration was 1 μg/mL when added with or without CoCl2. Whole-cell lysates were prepared 15, 30, 45, 60 and 90 min after treatment. Western blots were performed with specific antibodies. The HIF-1α protein levels, compared to the β-actin levels at the corresponding time points, are displayed in the line chart in (e) and (f). (g,h) The same treatments were assigned to MCF-7 and MDA-MB-231 cell lines. The HIF-1α protein level at 0 and 1 h after CHX treatment is shown based on results of the immunofluorescence assay. The original magnification was 200× (scale bar: 30 μM). Each experiment was repeated at least three times. *P < 0.05; **P < 0.01

Mentions: Previous researches have uncovered that hypoxia increases HIF-1α levels by delaying rapid proteasomal degradation of HIF-1α. In the present study, proteasome inhibitor MG-132 was used to treat MCF-7 and MDA-MB-231 cells with or without CoCl2, to investigate whether AGR2 enhances CoCl2-induced HIF-1α upregulation by interfering with HIF-1α proteasomal degradation (Fig.4a,b). Western blot analysis confirmed that the higher AGR2 levels resulted in the stronger HIF-1α upregulation under CoCl2 treatment in both cell lines. However, combined treatment with MG-132 and CoCl2 caused an equivalent level of HIF-1α upregulation independent of AGR2 levels, which implicates that AGR2 stabilizes CoCl2-induced HIF-1α by affecting the proteasomal degradation process.


Anterior gradient 2 is a binding stabilizer of hypoxia inducible factor-1α that enhances CoCl2 -induced doxorubicin resistance in breast cancer cells.

Li Z, Zhu Q, Hu L, Chen H, Wu Z, Li D - Cancer Sci. (2015)

Intracellular but not extracellular anterior gradient 2 (AGR2) stabilizes CoCl2-induced hypoxia inducible factor-1α (HIF-1α). (a,b) Western blot was performed on corresponding cell lines with treatment of 200 μM CoCl2, 20 μM MG-132 and their combination. β-actin was used as a loading control. (c,d) Cells were pre-treated with or without 200 μM CoCl2 for 6 h, and subjected to 20 μM of cycloheximide (CHX). The external AGR2 protein concentration was 1 μg/mL when added with or without CoCl2. Whole-cell lysates were prepared 15, 30, 45, 60 and 90 min after treatment. Western blots were performed with specific antibodies. The HIF-1α protein levels, compared to the β-actin levels at the corresponding time points, are displayed in the line chart in (e) and (f). (g,h) The same treatments were assigned to MCF-7 and MDA-MB-231 cell lines. The HIF-1α protein level at 0 and 1 h after CHX treatment is shown based on results of the immunofluorescence assay. The original magnification was 200× (scale bar: 30 μM). Each experiment was repeated at least three times. *P < 0.05; **P < 0.01
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556394&req=5

fig04: Intracellular but not extracellular anterior gradient 2 (AGR2) stabilizes CoCl2-induced hypoxia inducible factor-1α (HIF-1α). (a,b) Western blot was performed on corresponding cell lines with treatment of 200 μM CoCl2, 20 μM MG-132 and their combination. β-actin was used as a loading control. (c,d) Cells were pre-treated with or without 200 μM CoCl2 for 6 h, and subjected to 20 μM of cycloheximide (CHX). The external AGR2 protein concentration was 1 μg/mL when added with or without CoCl2. Whole-cell lysates were prepared 15, 30, 45, 60 and 90 min after treatment. Western blots were performed with specific antibodies. The HIF-1α protein levels, compared to the β-actin levels at the corresponding time points, are displayed in the line chart in (e) and (f). (g,h) The same treatments were assigned to MCF-7 and MDA-MB-231 cell lines. The HIF-1α protein level at 0 and 1 h after CHX treatment is shown based on results of the immunofluorescence assay. The original magnification was 200× (scale bar: 30 μM). Each experiment was repeated at least three times. *P < 0.05; **P < 0.01
Mentions: Previous researches have uncovered that hypoxia increases HIF-1α levels by delaying rapid proteasomal degradation of HIF-1α. In the present study, proteasome inhibitor MG-132 was used to treat MCF-7 and MDA-MB-231 cells with or without CoCl2, to investigate whether AGR2 enhances CoCl2-induced HIF-1α upregulation by interfering with HIF-1α proteasomal degradation (Fig.4a,b). Western blot analysis confirmed that the higher AGR2 levels resulted in the stronger HIF-1α upregulation under CoCl2 treatment in both cell lines. However, combined treatment with MG-132 and CoCl2 caused an equivalent level of HIF-1α upregulation independent of AGR2 levels, which implicates that AGR2 stabilizes CoCl2-induced HIF-1α by affecting the proteasomal degradation process.

Bottom Line: Our results show that knockdown of AGR2 in MCF-7 cells leads to the suppression of HIF-1α-induced doxorubicin resistance, whereas elevated levels of AGR2 in MDA-MB-231 cells enhance HIF-1α-induced doxorubicin resistance.By specific binding to HIF-1α, the increased level of intracellular AGR2 stabilizes HIF-1α and delays its proteasomal degradation.Finally, we found that AGR2-stabilized HIF-1α escalates multiple drug resistance protein 1 (MDR1) mRNA levels and limits doxorubicin intake of MCF-7 cells, whereas MCF-7/ADR, a doxorubicin resistant cell line with deficient AGR2 and HIF-1α, acquires wild-type MDR1 overexpression.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.

Show MeSH
Related in: MedlinePlus