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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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PIK-75 potentiates gemcitabine-induced cytotoxicity in pancreatic cancer cells. (A) AsPC-1 cells, transfected with siRNA (NRF2 vs. control), were treated with gemcitabine for 48 h and the viable cells were determined by MTT assay. Data are presented as mean ± SD performed in triplicate. **P≤0.01 and ***P≤0.001. (B) MIA PaCa-2 and AsPC-1 cells were treated with increasing amount of gemcitabine for 8 h and western blot analysis was performed with indicated antibodies. β-actin was used as a loading control. (C) Cells were treated either gemcitabine, PIK-75 alone or in combination of both drugs for 48 h and viable cells were measured by MTT assay. Data are presented as mean ± SD from two independent experiments performed in triplicate. *P≤0.05; **P≤0.01; and ***P≤0.001. (D) MIA PaCa-2 cells were treated with gemcitabine, PIK-75 or combination of both drugs for 8 h and western blot analysis was performed with indicated antibodies. GSK3β used as a loading control. (E) Cells were treated with gemcitabine, PIK-75 or combination of both drugs for 8 h and western blot analysis was performed with indicated antibodies. β-actin was used as a loading control.
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f5-ijo-44-03-0959: PIK-75 potentiates gemcitabine-induced cytotoxicity in pancreatic cancer cells. (A) AsPC-1 cells, transfected with siRNA (NRF2 vs. control), were treated with gemcitabine for 48 h and the viable cells were determined by MTT assay. Data are presented as mean ± SD performed in triplicate. **P≤0.01 and ***P≤0.001. (B) MIA PaCa-2 and AsPC-1 cells were treated with increasing amount of gemcitabine for 8 h and western blot analysis was performed with indicated antibodies. β-actin was used as a loading control. (C) Cells were treated either gemcitabine, PIK-75 alone or in combination of both drugs for 48 h and viable cells were measured by MTT assay. Data are presented as mean ± SD from two independent experiments performed in triplicate. *P≤0.05; **P≤0.01; and ***P≤0.001. (D) MIA PaCa-2 cells were treated with gemcitabine, PIK-75 or combination of both drugs for 8 h and western blot analysis was performed with indicated antibodies. GSK3β used as a loading control. (E) Cells were treated with gemcitabine, PIK-75 or combination of both drugs for 8 h and western blot analysis was performed with indicated antibodies. β-actin was used as a loading control.

Mentions: Since we found that NRF2 confers resistance of pancreatic cancer cells to various chemotherapeutic agents (20), we assessed the effect of NRF2-KD on the cytotoxicity of gemcitabine in pancreatic cancer cells. AsPC-1 cells were transfected with siRNA (either control or NRF2), then treated with gemcitabine for 48 h and viable cells were determined by MTT assay. Similar to other chemotherapeutic agents (20), the cytotoxic effect of gemcitabine was profound in NRF2-KD cells (Fig. 5A).


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 potentiates gemcitabine-induced cytotoxicity in pancreatic cancer cells. (A) AsPC-1 cells, transfected with siRNA (NRF2 vs. control), were treated with gemcitabine for 48 h and the viable cells were determined by MTT assay. Data are presented as mean ± SD performed in triplicate. **P≤0.01 and ***P≤0.001. (B) MIA PaCa-2 and AsPC-1 cells were treated with increasing amount of gemcitabine for 8 h and western blot analysis was performed with indicated antibodies. β-actin was used as a loading control. (C) Cells were treated either gemcitabine, PIK-75 alone or in combination of both drugs for 48 h and viable cells were measured by MTT assay. Data are presented as mean ± SD from two independent experiments performed in triplicate. *P≤0.05; **P≤0.01; and ***P≤0.001. (D) MIA PaCa-2 cells were treated with gemcitabine, PIK-75 or combination of both drugs for 8 h and western blot analysis was performed with indicated antibodies. GSK3β used as a loading control. (E) Cells were treated with gemcitabine, PIK-75 or combination of both drugs for 8 h and 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

f5-ijo-44-03-0959: PIK-75 potentiates gemcitabine-induced cytotoxicity in pancreatic cancer cells. (A) AsPC-1 cells, transfected with siRNA (NRF2 vs. control), were treated with gemcitabine for 48 h and the viable cells were determined by MTT assay. Data are presented as mean ± SD performed in triplicate. **P≤0.01 and ***P≤0.001. (B) MIA PaCa-2 and AsPC-1 cells were treated with increasing amount of gemcitabine for 8 h and western blot analysis was performed with indicated antibodies. β-actin was used as a loading control. (C) Cells were treated either gemcitabine, PIK-75 alone or in combination of both drugs for 48 h and viable cells were measured by MTT assay. Data are presented as mean ± SD from two independent experiments performed in triplicate. *P≤0.05; **P≤0.01; and ***P≤0.001. (D) MIA PaCa-2 cells were treated with gemcitabine, PIK-75 or combination of both drugs for 8 h and western blot analysis was performed with indicated antibodies. GSK3β used as a loading control. (E) Cells were treated with gemcitabine, PIK-75 or combination of both drugs for 8 h and western blot analysis was performed with indicated antibodies. β-actin was used as a loading control.
Mentions: Since we found that NRF2 confers resistance of pancreatic cancer cells to various chemotherapeutic agents (20), we assessed the effect of NRF2-KD on the cytotoxicity of gemcitabine in pancreatic cancer cells. AsPC-1 cells were transfected with siRNA (either control or NRF2), then treated with gemcitabine for 48 h and viable cells were determined by MTT assay. Similar to other chemotherapeutic agents (20), the cytotoxic effect of gemcitabine was profound in NRF2-KD cells (Fig. 5A).

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