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Inhibition of NRF2 by PIK-75 augments sensitivity of pancreatic cancer cells to gemcitabine.

Duong HQ, Yi YW, Kang HJ, Hong YB, Tang W, Wang A, Seong YS, Bae I - Int. J. Oncol. (2013)

Bottom Line: The effect of PIK-75 on the level and activity of NRF2 was characterized using various assays including reporter gene, quantitative PCR, DNA-binding and western blot analyses.PIK-75 also reduced the gemcitabine-induced NRF2 levels and the expression of its downstream target MRP5.Co-treatment of PIK-75 augmented the antitumor effect of gemcitabine both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA.

ABSTRACT
We describe the potential benefit of PIK-75 in combination of gemcitabine to treat pancreatic cancer in a preclinical mouse model. The effect of PIK-75 on the level and activity of NRF2 was characterized using various assays including reporter gene, quantitative PCR, DNA-binding and western blot analyses. Additionally, the combinatorial effect of PIK-75 and gemcitabine was evaluated in human pancreatic cancer cell lines and a xenograft model. PIK-75 reduced NRF2 protein levels and activity to regulate its target gene expression through proteasome-mediated degradation of NRF2 in human pancreatic cancer cell lines. PIK-75 also reduced the gemcitabine-induced NRF2 levels and the expression of its downstream target MRP5. Co-treatment of PIK-75 augmented the antitumor effect of gemcitabine both in vitro and in vivo. Our present study provides a strong mechanistic rationale to evaluate NRF2 targeting agents in combination with gemcitabine to treat pancreatic cancers.

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Related in: MedlinePlus

PIK-75 induces the proteasome-mediated degradation of NRF2. (A) AsPC-1 and MIA PaCa-2 cells, treated with 100 μM of tBHQ for 1 h, were further treated with PIK-75 for 4 h in the absence or presence of MG132. (B) AsPC-1 cells, transfected with FLAG-NRF2, were further treated with 0.1 μM of PIK-75 for indicated time in the absence or presence of MG132. (A and B) Western blot analysis was performed with indicated antibodies. β-actin was used as a loading control.
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f3-ijo-44-03-0959: PIK-75 induces the proteasome-mediated degradation of NRF2. (A) AsPC-1 and MIA PaCa-2 cells, treated with 100 μM of tBHQ for 1 h, were further treated with PIK-75 for 4 h in the absence or presence of MG132. (B) AsPC-1 cells, transfected with FLAG-NRF2, were further treated with 0.1 μM of PIK-75 for indicated time in the absence or presence of MG132. (A and B) Western blot analysis was performed with indicated antibodies. β-actin was used as a loading control.

Mentions: NRF2 is actively regulated by proteasomal degradation. Since PIK-75 reduced both endogenous and exogenous NRF2 protein, we further tested the PIK-75-mediated NRF2 downregulation in the presence of proteasome inhibitor. AsPC-1 cells were activated by tBHQ for 1 h followed by treatment of PIK-75 for 4 h in the absence or presence of the proteasome inhibitor MG132. Western blot analysis showed that treatment of MG132 alone induced the level of NRF2 protein similarly to that by tBHQ treatment (Fig. 3A left panel, lanes 2 vs. 4). This indicates that NRF2 is actively degraded by proteasome in this cell line. Indeed, co-treatment of tBHQ and MG132 further increased the NRF protein. Under this condition MG132 treatment was repressed the PIK-75-mediated reduction of NRF2 (Fig. 3A left panel, lanes 3 vs. 6). Inhibition of proteasome by MG132 also recovered by the PIK-75-mediated reduction of NRF2 in MIA PaCa-2 cells (Fig. 3A right panel).


Inhibition of NRF2 by PIK-75 augments sensitivity of pancreatic cancer cells to gemcitabine.

Duong HQ, Yi YW, Kang HJ, Hong YB, Tang W, Wang A, Seong YS, Bae I - Int. J. Oncol. (2013)

PIK-75 induces the proteasome-mediated degradation of NRF2. (A) AsPC-1 and MIA PaCa-2 cells, treated with 100 μM of tBHQ for 1 h, were further treated with PIK-75 for 4 h in the absence or presence of MG132. (B) AsPC-1 cells, transfected with FLAG-NRF2, were further treated with 0.1 μM of PIK-75 for indicated time in the absence or presence of MG132. (A and B) Western blot analysis was performed with indicated antibodies. β-actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928470&req=5

f3-ijo-44-03-0959: PIK-75 induces the proteasome-mediated degradation of NRF2. (A) AsPC-1 and MIA PaCa-2 cells, treated with 100 μM of tBHQ for 1 h, were further treated with PIK-75 for 4 h in the absence or presence of MG132. (B) AsPC-1 cells, transfected with FLAG-NRF2, were further treated with 0.1 μM of PIK-75 for indicated time in the absence or presence of MG132. (A and B) Western blot analysis was performed with indicated antibodies. β-actin was used as a loading control.
Mentions: NRF2 is actively regulated by proteasomal degradation. Since PIK-75 reduced both endogenous and exogenous NRF2 protein, we further tested the PIK-75-mediated NRF2 downregulation in the presence of proteasome inhibitor. AsPC-1 cells were activated by tBHQ for 1 h followed by treatment of PIK-75 for 4 h in the absence or presence of the proteasome inhibitor MG132. Western blot analysis showed that treatment of MG132 alone induced the level of NRF2 protein similarly to that by tBHQ treatment (Fig. 3A left panel, lanes 2 vs. 4). This indicates that NRF2 is actively degraded by proteasome in this cell line. Indeed, co-treatment of tBHQ and MG132 further increased the NRF protein. Under this condition MG132 treatment was repressed the PIK-75-mediated reduction of NRF2 (Fig. 3A left panel, lanes 3 vs. 6). Inhibition of proteasome by MG132 also recovered by the PIK-75-mediated reduction of NRF2 in MIA PaCa-2 cells (Fig. 3A right panel).

Bottom Line: The effect of PIK-75 on the level and activity of NRF2 was characterized using various assays including reporter gene, quantitative PCR, DNA-binding and western blot analyses.PIK-75 also reduced the gemcitabine-induced NRF2 levels and the expression of its downstream target MRP5.Co-treatment of PIK-75 augmented the antitumor effect of gemcitabine both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA.

ABSTRACT
We describe the potential benefit of PIK-75 in combination of gemcitabine to treat pancreatic cancer in a preclinical mouse model. The effect of PIK-75 on the level and activity of NRF2 was characterized using various assays including reporter gene, quantitative PCR, DNA-binding and western blot analyses. Additionally, the combinatorial effect of PIK-75 and gemcitabine was evaluated in human pancreatic cancer cell lines and a xenograft model. PIK-75 reduced NRF2 protein levels and activity to regulate its target gene expression through proteasome-mediated degradation of NRF2 in human pancreatic cancer cell lines. PIK-75 also reduced the gemcitabine-induced NRF2 levels and the expression of its downstream target MRP5. Co-treatment of PIK-75 augmented the antitumor effect of gemcitabine both in vitro and in vivo. Our present study provides a strong mechanistic rationale to evaluate NRF2 targeting agents in combination with gemcitabine to treat pancreatic cancers.

Show MeSH
Related in: MedlinePlus