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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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Anterior gradient 2 (AGR2) binding is required for the CoCl2-induced HIF-1α upregulation. (a,b) Western blot assays were performed on corresponding cell lines. The concentration of CoCl2 is 200 μM and the concentration of doxorubicin is 10 μM. (c) MCF7 cells were treated with 200 μM of CoCl2 for 6 h. The whole-cell lysates were then prepared and used as the input. The lysates were then mixed with a protein G-antibody solution. Western blot was performed with the corresponding antibodies. β-actin was used as a negative control (NC, a mix of the input mentioned above and the direct addition of the G protein). (d) Cells were treated with 200 μM of CoCl2 for 6 h. AGR2 and hypoxia inducible factor-1α (HIF-1α) were detected by immunofluorescence using confocal microscopy. The nuclei were stained with DAPI (blue) as an internal reference. AGR2 and HIF-1α were stained in red and green, respectively. The original magnification was 200× (scale bar: 30 μM). The regions selected in the white frames are amplified, and the overlapping regions (yellow parts) are shown with white arrows. Each experiment was repeated at least three times.
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fig03: Anterior gradient 2 (AGR2) binding is required for the CoCl2-induced HIF-1α upregulation. (a,b) Western blot assays were performed on corresponding cell lines. The concentration of CoCl2 is 200 μM and the concentration of doxorubicin is 10 μM. (c) MCF7 cells were treated with 200 μM of CoCl2 for 6 h. The whole-cell lysates were then prepared and used as the input. The lysates were then mixed with a protein G-antibody solution. Western blot was performed with the corresponding antibodies. β-actin was used as a negative control (NC, a mix of the input mentioned above and the direct addition of the G protein). (d) Cells were treated with 200 μM of CoCl2 for 6 h. AGR2 and hypoxia inducible factor-1α (HIF-1α) were detected by immunofluorescence using confocal microscopy. The nuclei were stained with DAPI (blue) as an internal reference. AGR2 and HIF-1α were stained in red and green, respectively. The original magnification was 200× (scale bar: 30 μM). The regions selected in the white frames are amplified, and the overlapping regions (yellow parts) are shown with white arrows. Each experiment was repeated at least three times.

Mentions: To further elucidate the influence of AGR2 on HIF-1α, we used western blot assays to detect the expression level of HIF-1α in both MCF-7 AGR2-knockdown and MDA-MB-231 AGR2-overexpression cell lines under hypoxic conditions (Fig.3a,b). The results exhibited a stronger HIF-1α upregulation by hypoxia in MDA-MB-231/AGR2 cells, compared with MDA-MB-231/PC cells. By contrast, HIF-1α upregulation was partially blocked in shAGR2 MCF-7 cells, compared with shControl cells. Furthermore, addition of doxorubicin inhibited both hypoxia-induced HIF-1α and AGR2 upregulation. These results suggest that AGR2 is necessary for the full upregulation of HIF-1α by CoCl2.


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)

Anterior gradient 2 (AGR2) binding is required for the CoCl2-induced HIF-1α upregulation. (a,b) Western blot assays were performed on corresponding cell lines. The concentration of CoCl2 is 200 μM and the concentration of doxorubicin is 10 μM. (c) MCF7 cells were treated with 200 μM of CoCl2 for 6 h. The whole-cell lysates were then prepared and used as the input. The lysates were then mixed with a protein G-antibody solution. Western blot was performed with the corresponding antibodies. β-actin was used as a negative control (NC, a mix of the input mentioned above and the direct addition of the G protein). (d) Cells were treated with 200 μM of CoCl2 for 6 h. AGR2 and hypoxia inducible factor-1α (HIF-1α) were detected by immunofluorescence using confocal microscopy. The nuclei were stained with DAPI (blue) as an internal reference. AGR2 and HIF-1α were stained in red and green, respectively. The original magnification was 200× (scale bar: 30 μM). The regions selected in the white frames are amplified, and the overlapping regions (yellow parts) are shown with white arrows. Each experiment was repeated at least three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Anterior gradient 2 (AGR2) binding is required for the CoCl2-induced HIF-1α upregulation. (a,b) Western blot assays were performed on corresponding cell lines. The concentration of CoCl2 is 200 μM and the concentration of doxorubicin is 10 μM. (c) MCF7 cells were treated with 200 μM of CoCl2 for 6 h. The whole-cell lysates were then prepared and used as the input. The lysates were then mixed with a protein G-antibody solution. Western blot was performed with the corresponding antibodies. β-actin was used as a negative control (NC, a mix of the input mentioned above and the direct addition of the G protein). (d) Cells were treated with 200 μM of CoCl2 for 6 h. AGR2 and hypoxia inducible factor-1α (HIF-1α) were detected by immunofluorescence using confocal microscopy. The nuclei were stained with DAPI (blue) as an internal reference. AGR2 and HIF-1α were stained in red and green, respectively. The original magnification was 200× (scale bar: 30 μM). The regions selected in the white frames are amplified, and the overlapping regions (yellow parts) are shown with white arrows. Each experiment was repeated at least three times.
Mentions: To further elucidate the influence of AGR2 on HIF-1α, we used western blot assays to detect the expression level of HIF-1α in both MCF-7 AGR2-knockdown and MDA-MB-231 AGR2-overexpression cell lines under hypoxic conditions (Fig.3a,b). The results exhibited a stronger HIF-1α upregulation by hypoxia in MDA-MB-231/AGR2 cells, compared with MDA-MB-231/PC cells. By contrast, HIF-1α upregulation was partially blocked in shAGR2 MCF-7 cells, compared with shControl cells. Furthermore, addition of doxorubicin inhibited both hypoxia-induced HIF-1α and AGR2 upregulation. These results suggest that AGR2 is necessary for the full upregulation of HIF-1α by CoCl2.

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