Limits...
Nrf2 Expression and Apoptosis in Quercetin-treated Malignant Mesothelioma Cells.

Lee YJ, Lee DM, Lee SH - Mol. Cells (2015)

Bottom Line: Effective reversal of apoptosis was observed following pretreatment with the pan-caspase inhibitor Z-VAD.Moreover, Nrf2 knockdown exhibited increased sensitivity to the anticancer drug, cisplatin, presumably by potentiating the oxidative stress induced by cisplatin.Collectively, our data demonstrate the importance of Nrf2 in cytoprotection, survival, and drug resistance with implications for the potential significance of targeting Nrf2 as a promising strategy for overcoming resistance to chemotherapeutics in MM.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Cancer Research, Soonchunhyang Medical Research Institute, Soonchunhyang University Cheonan Hospital, Cheonan, Korea.

ABSTRACT
NF-E2-related factor 2 (Nrf2), a basic leucine zipper transcription factor, has recently received a great deal of attention as an important molecule that enhances antioxidative defenses and induces resistance to chemotherapy or radiotherapy. In this study, we investigated the apoptosis-inducing and Nrf2-upregulating effects of quercetin on malignant mesothelioma (MM) MSTO-211H and H2452 cells. Quercetin treatment inhibited cell growth and led to upregulation of Nrf2 at both the mRNA and protein levels without altering the ubiquitination and extending the half-life of the Nrf2 protein. Following treatment with quercetin, analyses of the nuclear level of Nrf2, Nrf2 antioxidant response element-binding assay, Nrf2 promoter-luc assay, and RT-PCR toward the Nrf2-regulated gene, heme oxygenase-1, demonstrated that the induced Nrf2 is transcriptionally active. Knockdown of Nrf2 expression with siRNA enhanced cytotoxicity due to the induction of apoptosis, as evidenced by an increase in the level of proapoptotic Bax, a decrease in the level of antiapoptotic Bcl-2 with enhanced cleavage of caspase-3 and PARP proteins, the appearance of a sub-G0/G1 peak in the flow cytometric assay, and increased percentage of apoptotic propensities in the annexin V binding assay. Effective reversal of apoptosis was observed following pretreatment with the pan-caspase inhibitor Z-VAD. Moreover, Nrf2 knockdown exhibited increased sensitivity to the anticancer drug, cisplatin, presumably by potentiating the oxidative stress induced by cisplatin. Collectively, our data demonstrate the importance of Nrf2 in cytoprotection, survival, and drug resistance with implications for the potential significance of targeting Nrf2 as a promising strategy for overcoming resistance to chemotherapeutics in MM.

No MeSH data available.


Related in: MedlinePlus

Effects of quercetin treatment on Nrf2 expression in MM cells. Cells were incubated with the indicated concentrations of quercetin for 48 h before the extraction of cell lysates and total RNA for Western blot (A) and RT-PCR (B) analyses, respectively. (C) Cells were treated with quercetin (20 μM) for 48 h before immunoprecipitation of Nrf2 or ubiquitin from cell lysate (500 μg), after which immunoprecipitates were analyzed by Western blotting with the anti-ubiquitin or anti-Nrf2 antibody, respectively. (D) Cells were pretreated with 0.1 μM CHX for 2 h and followed by treatment with or without 20 μM quercetin for varying intervals as indicated. Immunodetection was carried out by using antibodies against Nrf2 and β-actin. Normalized intensity of Nrf2 versus β-actin was presented as the mean value from two independent experiments. Q, quercetin; Ub, ubiquitnated; IP, immunoprecipitation; WB, Western blotting; CHX, cycloheximide.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4443283&req=5

f2-molce-38-5-416: Effects of quercetin treatment on Nrf2 expression in MM cells. Cells were incubated with the indicated concentrations of quercetin for 48 h before the extraction of cell lysates and total RNA for Western blot (A) and RT-PCR (B) analyses, respectively. (C) Cells were treated with quercetin (20 μM) for 48 h before immunoprecipitation of Nrf2 or ubiquitin from cell lysate (500 μg), after which immunoprecipitates were analyzed by Western blotting with the anti-ubiquitin or anti-Nrf2 antibody, respectively. (D) Cells were pretreated with 0.1 μM CHX for 2 h and followed by treatment with or without 20 μM quercetin for varying intervals as indicated. Immunodetection was carried out by using antibodies against Nrf2 and β-actin. Normalized intensity of Nrf2 versus β-actin was presented as the mean value from two independent experiments. Q, quercetin; Ub, ubiquitnated; IP, immunoprecipitation; WB, Western blotting; CHX, cycloheximide.

Mentions: As an initial approach in determining effective doses for the treatment of MM cells with quercetin, a dose- and time-response study was carried out using the MTT assay. The results revealed a concentration-dependent decrease in cell viability due to treatment with quercetin. At concentrations ≥ 20 μM, quercetin significantly decreased cell viability of both MSTO-211H and H2452 cells (Fig. 1A). Western blot analysis showed that an increase in the Nrf2 level was first observed at 2 h incubation with 20 μM quercetin and remained upregulated at longer incubation (Fig. 1B). However, the treatment of cells with quercetin at toxic doses ≥ 60 μM did not influence on the Nrf2 levels compared to untreated controls (Fig. 1C). Based on this observation, 40 μM was regarded as a subtoxic dose at which quercetin caused mild cytotoxicity in MM cells. Concentrations below that were then selected for further study to examine the efficacy of quercetin as an activator of Nrf2. Besides the total levels of Nrf2, the Nrf2-regulated gene product HO-1 was dose-dependently increased in cultures treated with ≤30 μM quercetin (Fig. 2A). To determine whether the upregulation of Nrf2 protein is due to gene expression and if it involves transcriptional activation of its downstream target genes, the levels of Nrf2 and HO-1 transcripts were analyzed by RT-PCR. As shown in Fig. 2B, treatment with quercetin increased the mRNA levels of Nrf2 and its transcription target HO-1 in both types of cells, consistent with the results obtained for the proteins. To evaluate whether quercetin has an effect on Nrf2 stability, the level of Nrf2 polyubiquitination was investigated by immunoprecipitation assay using the anti-Nrf2 antibody followed by Western blotting with anti-ubiquitin antibody, and vice versa. As shown in Fig. 2C, immunoprecipitation revealed approximately 100 kDa band, which remained unchanged among cells cultured with or without quercetin. Next, Nrf2 protein turnover was analyzed via cycloheximide (CHX) chase experiments. As shown in Fig. 2D, the level of Nrf2 protein rapidly declined over 160 min of treatment with CHX alone, or in combination with quercetin. For the decay curve, the half-lives of endogenous Nrf2 were approximately 20.1 min in MSTO-211H and 27.4 min in H2452 cells, and treatment with quercetin caused no delay compared to the cells treated with CHX alone. Collectively, the data suggest that that the quercetin-induced upregulation of Nrf2 in MM cells was accomplished largely at the transcriptional level rather than by the prolongation of protein stability.


Nrf2 Expression and Apoptosis in Quercetin-treated Malignant Mesothelioma Cells.

Lee YJ, Lee DM, Lee SH - Mol. Cells (2015)

Effects of quercetin treatment on Nrf2 expression in MM cells. Cells were incubated with the indicated concentrations of quercetin for 48 h before the extraction of cell lysates and total RNA for Western blot (A) and RT-PCR (B) analyses, respectively. (C) Cells were treated with quercetin (20 μM) for 48 h before immunoprecipitation of Nrf2 or ubiquitin from cell lysate (500 μg), after which immunoprecipitates were analyzed by Western blotting with the anti-ubiquitin or anti-Nrf2 antibody, respectively. (D) Cells were pretreated with 0.1 μM CHX for 2 h and followed by treatment with or without 20 μM quercetin for varying intervals as indicated. Immunodetection was carried out by using antibodies against Nrf2 and β-actin. Normalized intensity of Nrf2 versus β-actin was presented as the mean value from two independent experiments. Q, quercetin; Ub, ubiquitnated; IP, immunoprecipitation; WB, Western blotting; CHX, cycloheximide.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4443283&req=5

f2-molce-38-5-416: Effects of quercetin treatment on Nrf2 expression in MM cells. Cells were incubated with the indicated concentrations of quercetin for 48 h before the extraction of cell lysates and total RNA for Western blot (A) and RT-PCR (B) analyses, respectively. (C) Cells were treated with quercetin (20 μM) for 48 h before immunoprecipitation of Nrf2 or ubiquitin from cell lysate (500 μg), after which immunoprecipitates were analyzed by Western blotting with the anti-ubiquitin or anti-Nrf2 antibody, respectively. (D) Cells were pretreated with 0.1 μM CHX for 2 h and followed by treatment with or without 20 μM quercetin for varying intervals as indicated. Immunodetection was carried out by using antibodies against Nrf2 and β-actin. Normalized intensity of Nrf2 versus β-actin was presented as the mean value from two independent experiments. Q, quercetin; Ub, ubiquitnated; IP, immunoprecipitation; WB, Western blotting; CHX, cycloheximide.
Mentions: As an initial approach in determining effective doses for the treatment of MM cells with quercetin, a dose- and time-response study was carried out using the MTT assay. The results revealed a concentration-dependent decrease in cell viability due to treatment with quercetin. At concentrations ≥ 20 μM, quercetin significantly decreased cell viability of both MSTO-211H and H2452 cells (Fig. 1A). Western blot analysis showed that an increase in the Nrf2 level was first observed at 2 h incubation with 20 μM quercetin and remained upregulated at longer incubation (Fig. 1B). However, the treatment of cells with quercetin at toxic doses ≥ 60 μM did not influence on the Nrf2 levels compared to untreated controls (Fig. 1C). Based on this observation, 40 μM was regarded as a subtoxic dose at which quercetin caused mild cytotoxicity in MM cells. Concentrations below that were then selected for further study to examine the efficacy of quercetin as an activator of Nrf2. Besides the total levels of Nrf2, the Nrf2-regulated gene product HO-1 was dose-dependently increased in cultures treated with ≤30 μM quercetin (Fig. 2A). To determine whether the upregulation of Nrf2 protein is due to gene expression and if it involves transcriptional activation of its downstream target genes, the levels of Nrf2 and HO-1 transcripts were analyzed by RT-PCR. As shown in Fig. 2B, treatment with quercetin increased the mRNA levels of Nrf2 and its transcription target HO-1 in both types of cells, consistent with the results obtained for the proteins. To evaluate whether quercetin has an effect on Nrf2 stability, the level of Nrf2 polyubiquitination was investigated by immunoprecipitation assay using the anti-Nrf2 antibody followed by Western blotting with anti-ubiquitin antibody, and vice versa. As shown in Fig. 2C, immunoprecipitation revealed approximately 100 kDa band, which remained unchanged among cells cultured with or without quercetin. Next, Nrf2 protein turnover was analyzed via cycloheximide (CHX) chase experiments. As shown in Fig. 2D, the level of Nrf2 protein rapidly declined over 160 min of treatment with CHX alone, or in combination with quercetin. For the decay curve, the half-lives of endogenous Nrf2 were approximately 20.1 min in MSTO-211H and 27.4 min in H2452 cells, and treatment with quercetin caused no delay compared to the cells treated with CHX alone. Collectively, the data suggest that that the quercetin-induced upregulation of Nrf2 in MM cells was accomplished largely at the transcriptional level rather than by the prolongation of protein stability.

Bottom Line: Effective reversal of apoptosis was observed following pretreatment with the pan-caspase inhibitor Z-VAD.Moreover, Nrf2 knockdown exhibited increased sensitivity to the anticancer drug, cisplatin, presumably by potentiating the oxidative stress induced by cisplatin.Collectively, our data demonstrate the importance of Nrf2 in cytoprotection, survival, and drug resistance with implications for the potential significance of targeting Nrf2 as a promising strategy for overcoming resistance to chemotherapeutics in MM.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Cancer Research, Soonchunhyang Medical Research Institute, Soonchunhyang University Cheonan Hospital, Cheonan, Korea.

ABSTRACT
NF-E2-related factor 2 (Nrf2), a basic leucine zipper transcription factor, has recently received a great deal of attention as an important molecule that enhances antioxidative defenses and induces resistance to chemotherapy or radiotherapy. In this study, we investigated the apoptosis-inducing and Nrf2-upregulating effects of quercetin on malignant mesothelioma (MM) MSTO-211H and H2452 cells. Quercetin treatment inhibited cell growth and led to upregulation of Nrf2 at both the mRNA and protein levels without altering the ubiquitination and extending the half-life of the Nrf2 protein. Following treatment with quercetin, analyses of the nuclear level of Nrf2, Nrf2 antioxidant response element-binding assay, Nrf2 promoter-luc assay, and RT-PCR toward the Nrf2-regulated gene, heme oxygenase-1, demonstrated that the induced Nrf2 is transcriptionally active. Knockdown of Nrf2 expression with siRNA enhanced cytotoxicity due to the induction of apoptosis, as evidenced by an increase in the level of proapoptotic Bax, a decrease in the level of antiapoptotic Bcl-2 with enhanced cleavage of caspase-3 and PARP proteins, the appearance of a sub-G0/G1 peak in the flow cytometric assay, and increased percentage of apoptotic propensities in the annexin V binding assay. Effective reversal of apoptosis was observed following pretreatment with the pan-caspase inhibitor Z-VAD. Moreover, Nrf2 knockdown exhibited increased sensitivity to the anticancer drug, cisplatin, presumably by potentiating the oxidative stress induced by cisplatin. Collectively, our data demonstrate the importance of Nrf2 in cytoprotection, survival, and drug resistance with implications for the potential significance of targeting Nrf2 as a promising strategy for overcoming resistance to chemotherapeutics in MM.

No MeSH data available.


Related in: MedlinePlus