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Monitoring the Antioxidant Mediated Chemosensitization and ARE-Signaling in Triple Negative Breast Cancer Therapy.

Foygel K, Sekar TV, Paulmurugan R - PLoS ONE (2015)

Bottom Line: The results identified FLuc2 fusion with N-terminal 100-aa of Nrf2 is sufficient for measuring Nrf2-activation in cancer cells.Tumor xenograft study in mouse demonstrates that combinational treatment by cisplatin/EGCG resulted in tumor growth reduction, compared to cisplatin alone treatment.The results of this study highlight the importance of identifying selective combination of antioxidants/chemotherapeutic agents for customized treatment strategy.

View Article: PubMed Central - PubMed

Affiliation: Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
Chemotherapy often fails due to cellular detoxifying mechanisms, including phase-II enzymes. Activation of Nrf2-Keap1 pathway induces phase-II enzymes expression through ARE-signaling and prevents cancer development. Nrf2-overexpression in cancer cells results in chemo- and/or radioresistance. This necessitates understanding of Nrf2-regulation, and identification of Nrf2 activators/inhibitors sensitizing cancer cells to improve chemotherapy. N-terminal 435-amino acids of Nrf2 are crucial for Keap1 binding during ubiquitination. Identification of a minimum Nrf2-domain required for Keap1 binding without altering endogenous ARE-signaling would be a novel tool to study Nrf2-signaling. Current study developed firefly-luciferase reporter fusion with N-terminal Nrf2-domain of different lengths and examined its response to Nrf2-activators in cells. The results identified FLuc2 fusion with N-terminal 100-aa of Nrf2 is sufficient for measuring Nrf2-activation in cancer cells. We used MDA-MB231 cells expressing this particular construct for studying antioxidant induced Nrf2-activation and chemosensitization in triple-negative breast cancer therapy. While antioxidant EGCG showed chemosensitization of MDA-MB231 cells to cisplatin by activating Nrf2-ARE signaling, PTS, another antioxidant showed chemoprotection. Tumor xenograft study in mouse demonstrates that combinational treatment by cisplatin/EGCG resulted in tumor growth reduction, compared to cisplatin alone treatment. The results of this study highlight the importance of identifying selective combination of antioxidants/chemotherapeutic agents for customized treatment strategy.

No MeSH data available.


Related in: MedlinePlus

Time dependent activation of Nrf2-100-FLuc2 fusions in response to varying anticancer drug (A, RRx-001 and B, cisplatin) concentration in the presence of antioxidant (EGCG) in MDA-MB231 cells, and Nrf2 target genes (GST and NQO1) expression in MDA-MB231-Nrf2-100-FLuc2 cells in response to anticancer drug (RRx-001) in the presence of antioxidant (EGCG).Asterisk (*) denotes statistical significance (p<0.05) of a signal compared to that from the untreated cells (A) and from cells treated with 50 μM EGCG alone (B). C, Immunoblot of cell lysates treated with drug and/or antioxidant for 24 hours (Nrf2, GST and NQO1 expression was normalized to GAPDH expression). Error bars represent standard deviations of triplicate experiments.
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pone.0141913.g006: Time dependent activation of Nrf2-100-FLuc2 fusions in response to varying anticancer drug (A, RRx-001 and B, cisplatin) concentration in the presence of antioxidant (EGCG) in MDA-MB231 cells, and Nrf2 target genes (GST and NQO1) expression in MDA-MB231-Nrf2-100-FLuc2 cells in response to anticancer drug (RRx-001) in the presence of antioxidant (EGCG).Asterisk (*) denotes statistical significance (p<0.05) of a signal compared to that from the untreated cells (A) and from cells treated with 50 μM EGCG alone (B). C, Immunoblot of cell lysates treated with drug and/or antioxidant for 24 hours (Nrf2, GST and NQO1 expression was normalized to GAPDH expression). Error bars represent standard deviations of triplicate experiments.

Mentions: To evaluate the efficiency of Nrf2 activation in response to the treatment of a combination of antioxidant (EGCG) and chemotherapeutic drug combination (RRx-001 or Cisplatin), we used MDA-MB231 cells stably expressing Nrf2-100-FLuc2 reporter protein. The cells were measured for luciferase activity different time points after treatment. We found that even a drug concentration below its IC50 dose showed significant level of luciferase activation when combined with antioxidant. The drug RRx-001 resulted in nearly 12 fold activation at 72 hours post-treatment when combined with EGCG, as compared drug alone treatment and to baseline no treatment control. Similarly, cisplatin showed 3 to 4-fold activation when combined with EGCG (Fig 6A and 6B). The immunoblot analysis of Nrf2 and its downstream target genes found that the GST1 and NQO1 proteins expression were increased in response to RRx-001 and EGCG combination treatment, as compared to control cells (Fig 6C).


Monitoring the Antioxidant Mediated Chemosensitization and ARE-Signaling in Triple Negative Breast Cancer Therapy.

Foygel K, Sekar TV, Paulmurugan R - PLoS ONE (2015)

Time dependent activation of Nrf2-100-FLuc2 fusions in response to varying anticancer drug (A, RRx-001 and B, cisplatin) concentration in the presence of antioxidant (EGCG) in MDA-MB231 cells, and Nrf2 target genes (GST and NQO1) expression in MDA-MB231-Nrf2-100-FLuc2 cells in response to anticancer drug (RRx-001) in the presence of antioxidant (EGCG).Asterisk (*) denotes statistical significance (p<0.05) of a signal compared to that from the untreated cells (A) and from cells treated with 50 μM EGCG alone (B). C, Immunoblot of cell lysates treated with drug and/or antioxidant for 24 hours (Nrf2, GST and NQO1 expression was normalized to GAPDH expression). Error bars represent standard deviations of triplicate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4633093&req=5

pone.0141913.g006: Time dependent activation of Nrf2-100-FLuc2 fusions in response to varying anticancer drug (A, RRx-001 and B, cisplatin) concentration in the presence of antioxidant (EGCG) in MDA-MB231 cells, and Nrf2 target genes (GST and NQO1) expression in MDA-MB231-Nrf2-100-FLuc2 cells in response to anticancer drug (RRx-001) in the presence of antioxidant (EGCG).Asterisk (*) denotes statistical significance (p<0.05) of a signal compared to that from the untreated cells (A) and from cells treated with 50 μM EGCG alone (B). C, Immunoblot of cell lysates treated with drug and/or antioxidant for 24 hours (Nrf2, GST and NQO1 expression was normalized to GAPDH expression). Error bars represent standard deviations of triplicate experiments.
Mentions: To evaluate the efficiency of Nrf2 activation in response to the treatment of a combination of antioxidant (EGCG) and chemotherapeutic drug combination (RRx-001 or Cisplatin), we used MDA-MB231 cells stably expressing Nrf2-100-FLuc2 reporter protein. The cells were measured for luciferase activity different time points after treatment. We found that even a drug concentration below its IC50 dose showed significant level of luciferase activation when combined with antioxidant. The drug RRx-001 resulted in nearly 12 fold activation at 72 hours post-treatment when combined with EGCG, as compared drug alone treatment and to baseline no treatment control. Similarly, cisplatin showed 3 to 4-fold activation when combined with EGCG (Fig 6A and 6B). The immunoblot analysis of Nrf2 and its downstream target genes found that the GST1 and NQO1 proteins expression were increased in response to RRx-001 and EGCG combination treatment, as compared to control cells (Fig 6C).

Bottom Line: The results identified FLuc2 fusion with N-terminal 100-aa of Nrf2 is sufficient for measuring Nrf2-activation in cancer cells.Tumor xenograft study in mouse demonstrates that combinational treatment by cisplatin/EGCG resulted in tumor growth reduction, compared to cisplatin alone treatment.The results of this study highlight the importance of identifying selective combination of antioxidants/chemotherapeutic agents for customized treatment strategy.

View Article: PubMed Central - PubMed

Affiliation: Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
Chemotherapy often fails due to cellular detoxifying mechanisms, including phase-II enzymes. Activation of Nrf2-Keap1 pathway induces phase-II enzymes expression through ARE-signaling and prevents cancer development. Nrf2-overexpression in cancer cells results in chemo- and/or radioresistance. This necessitates understanding of Nrf2-regulation, and identification of Nrf2 activators/inhibitors sensitizing cancer cells to improve chemotherapy. N-terminal 435-amino acids of Nrf2 are crucial for Keap1 binding during ubiquitination. Identification of a minimum Nrf2-domain required for Keap1 binding without altering endogenous ARE-signaling would be a novel tool to study Nrf2-signaling. Current study developed firefly-luciferase reporter fusion with N-terminal Nrf2-domain of different lengths and examined its response to Nrf2-activators in cells. The results identified FLuc2 fusion with N-terminal 100-aa of Nrf2 is sufficient for measuring Nrf2-activation in cancer cells. We used MDA-MB231 cells expressing this particular construct for studying antioxidant induced Nrf2-activation and chemosensitization in triple-negative breast cancer therapy. While antioxidant EGCG showed chemosensitization of MDA-MB231 cells to cisplatin by activating Nrf2-ARE signaling, PTS, another antioxidant showed chemoprotection. Tumor xenograft study in mouse demonstrates that combinational treatment by cisplatin/EGCG resulted in tumor growth reduction, compared to cisplatin alone treatment. The results of this study highlight the importance of identifying selective combination of antioxidants/chemotherapeutic agents for customized treatment strategy.

No MeSH data available.


Related in: MedlinePlus