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Activation of NRF2 by p62 and proteasome reduction in sphere-forming breast carcinoma cells.

Ryoo IG, Choi BH, Kwak MK - Oncotarget (2015)

Bottom Line: The MCF7 mammospheres expressed significantly higher levels of the NRF2 protein and target gene expression compared to the monolayer.Moreover, unlike the control mammospheres, NRF2-knockdown mammospheres did not develop anticancer drug resistance.Collectively, these results indicated that altered proteasome function and p62 expression caused NRF2 activation in CSC-enriched mammospheres.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, The Catholic University of Korea, Bucheon, Gyeonggi-do, Republic of Korea.

ABSTRACT
Cancer stem cells (CSCs) express high levels of drug efflux transporters and antioxidant genes, and are therefore believed to be responsible for cancer recurrence following chemo/radiotherapy intervention. In this study, we investigated the role of NF-E2-related factor 2 (NRF2), a master regulator of antioxidant gene expression, in the growth and stress resistance of CSC-enriched mammosphere. The MCF7 mammospheres expressed significantly higher levels of the NRF2 protein and target gene expression compared to the monolayer. As underlying mechanisms, we observed that proteolytic activity and expression of the proteasome catalytic subunits were decreased in the mammospheres. Additionally, mammospheres retained a high level of p62 and the silencing of p62 was observed to attenuate NRF2 activation. NRF2 increase was confirmed in sphere-cultures of the colon and ovarian cancer cells. The functional implication of NRF2 was demonstrated in NRF2-knockdown mammospheres. NRF2-silenced mammospheres demonstrated increased cell death and retarded sphere growth as a result of target gene repression. Moreover, unlike the control mammospheres, NRF2-knockdown mammospheres did not develop anticancer drug resistance. Collectively, these results indicated that altered proteasome function and p62 expression caused NRF2 activation in CSC-enriched mammospheres. In addition, NRF2 appeared to play a role in CSC survival and anticancer drug resistance.

No MeSH data available.


Related in: MedlinePlus

NRF2 knockdown-mediated sensitization of mammospheres to anticancer drugs(A)BCRP, MRP2, MRP3, and MRP5 transcript levels were assessed in the sc and shNRF2 mammospheres by real-time PCR. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared to the sc mammospheres. (B) BCRP and MRP2 protein levels were evaluated in the sc and shNRF2 mammospheres by western blot analysis. (C–D) The sc or shNRF2 mammosphere were incubated with Hoechst 33342 (H342, C) or doxorubicin (Dox, D) and fluorescent intensity was evaluated using a confocal microscopy. (E–F) Cell viability was monitored in the sc and shNRF2 mammospheres after doxorubicin (Dox, E) or 5-FU (F) treatment for 72 h. Values represent the mean ± SE from six to eight sampled wells. aP < 0.05 compared to sc control. bP < 0.05 compared to sc monolayers.
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Figure 8: NRF2 knockdown-mediated sensitization of mammospheres to anticancer drugs(A)BCRP, MRP2, MRP3, and MRP5 transcript levels were assessed in the sc and shNRF2 mammospheres by real-time PCR. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared to the sc mammospheres. (B) BCRP and MRP2 protein levels were evaluated in the sc and shNRF2 mammospheres by western blot analysis. (C–D) The sc or shNRF2 mammosphere were incubated with Hoechst 33342 (H342, C) or doxorubicin (Dox, D) and fluorescent intensity was evaluated using a confocal microscopy. (E–F) Cell viability was monitored in the sc and shNRF2 mammospheres after doxorubicin (Dox, E) or 5-FU (F) treatment for 72 h. Values represent the mean ± SE from six to eight sampled wells. aP < 0.05 compared to sc control. bP < 0.05 compared to sc monolayers.

Mentions: Mammosphere chemoresistance can be related with NRF2 activation and consequent drug efflux. In NRF2 knockdown mammospheres, the increase in efflux transporters such as BCRP and MRP2 were significantly attenuated when compared to the sc control mammospheres (Figure 8A). Repressed expression of efflux transporters was confirmed by immunoblot analysis using MRP2 and BCRP antibodies (Figure 8B). Accordingly, Hoechst 33342-resistant cells, which represent a side population of CSC, were decreased in NRF2 knockdown mammospheres (Figure 8C). Differential transporter increases can lead to sensitization to anticancer drug treatment. Indeed, NRF2 knockdown mammospheres accumulated higher amount of doxorubicin than sc control mammospheres (Figure 8D). In addition, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) analysis after a 72 h-doxorubicin incubation revealed that shNRF2 mammospheres did not develop doxorubicin resistance in contrast to the sc control mammospheres (Figure 8E). Similarly, the sc control MCF7 acquired 5-fluorouracil (5-FU) resistance after sphere formation, whereas shNRF2 mammospheres showed a similar response to 5-FU cytotoxicity compared to monolayers (Figure 8F). Collectively, these data suggested that mammosphere anticancer resistance, which is one of CSC characteristics, is largely mediated by NRF2 activation.


Activation of NRF2 by p62 and proteasome reduction in sphere-forming breast carcinoma cells.

Ryoo IG, Choi BH, Kwak MK - Oncotarget (2015)

NRF2 knockdown-mediated sensitization of mammospheres to anticancer drugs(A)BCRP, MRP2, MRP3, and MRP5 transcript levels were assessed in the sc and shNRF2 mammospheres by real-time PCR. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared to the sc mammospheres. (B) BCRP and MRP2 protein levels were evaluated in the sc and shNRF2 mammospheres by western blot analysis. (C–D) The sc or shNRF2 mammosphere were incubated with Hoechst 33342 (H342, C) or doxorubicin (Dox, D) and fluorescent intensity was evaluated using a confocal microscopy. (E–F) Cell viability was monitored in the sc and shNRF2 mammospheres after doxorubicin (Dox, E) or 5-FU (F) treatment for 72 h. Values represent the mean ± SE from six to eight sampled wells. aP < 0.05 compared to sc control. bP < 0.05 compared to sc monolayers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480743&req=5

Figure 8: NRF2 knockdown-mediated sensitization of mammospheres to anticancer drugs(A)BCRP, MRP2, MRP3, and MRP5 transcript levels were assessed in the sc and shNRF2 mammospheres by real-time PCR. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared to the sc mammospheres. (B) BCRP and MRP2 protein levels were evaluated in the sc and shNRF2 mammospheres by western blot analysis. (C–D) The sc or shNRF2 mammosphere were incubated with Hoechst 33342 (H342, C) or doxorubicin (Dox, D) and fluorescent intensity was evaluated using a confocal microscopy. (E–F) Cell viability was monitored in the sc and shNRF2 mammospheres after doxorubicin (Dox, E) or 5-FU (F) treatment for 72 h. Values represent the mean ± SE from six to eight sampled wells. aP < 0.05 compared to sc control. bP < 0.05 compared to sc monolayers.
Mentions: Mammosphere chemoresistance can be related with NRF2 activation and consequent drug efflux. In NRF2 knockdown mammospheres, the increase in efflux transporters such as BCRP and MRP2 were significantly attenuated when compared to the sc control mammospheres (Figure 8A). Repressed expression of efflux transporters was confirmed by immunoblot analysis using MRP2 and BCRP antibodies (Figure 8B). Accordingly, Hoechst 33342-resistant cells, which represent a side population of CSC, were decreased in NRF2 knockdown mammospheres (Figure 8C). Differential transporter increases can lead to sensitization to anticancer drug treatment. Indeed, NRF2 knockdown mammospheres accumulated higher amount of doxorubicin than sc control mammospheres (Figure 8D). In addition, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) analysis after a 72 h-doxorubicin incubation revealed that shNRF2 mammospheres did not develop doxorubicin resistance in contrast to the sc control mammospheres (Figure 8E). Similarly, the sc control MCF7 acquired 5-fluorouracil (5-FU) resistance after sphere formation, whereas shNRF2 mammospheres showed a similar response to 5-FU cytotoxicity compared to monolayers (Figure 8F). Collectively, these data suggested that mammosphere anticancer resistance, which is one of CSC characteristics, is largely mediated by NRF2 activation.

Bottom Line: The MCF7 mammospheres expressed significantly higher levels of the NRF2 protein and target gene expression compared to the monolayer.Moreover, unlike the control mammospheres, NRF2-knockdown mammospheres did not develop anticancer drug resistance.Collectively, these results indicated that altered proteasome function and p62 expression caused NRF2 activation in CSC-enriched mammospheres.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, The Catholic University of Korea, Bucheon, Gyeonggi-do, Republic of Korea.

ABSTRACT
Cancer stem cells (CSCs) express high levels of drug efflux transporters and antioxidant genes, and are therefore believed to be responsible for cancer recurrence following chemo/radiotherapy intervention. In this study, we investigated the role of NF-E2-related factor 2 (NRF2), a master regulator of antioxidant gene expression, in the growth and stress resistance of CSC-enriched mammosphere. The MCF7 mammospheres expressed significantly higher levels of the NRF2 protein and target gene expression compared to the monolayer. As underlying mechanisms, we observed that proteolytic activity and expression of the proteasome catalytic subunits were decreased in the mammospheres. Additionally, mammospheres retained a high level of p62 and the silencing of p62 was observed to attenuate NRF2 activation. NRF2 increase was confirmed in sphere-cultures of the colon and ovarian cancer cells. The functional implication of NRF2 was demonstrated in NRF2-knockdown mammospheres. NRF2-silenced mammospheres demonstrated increased cell death and retarded sphere growth as a result of target gene repression. Moreover, unlike the control mammospheres, NRF2-knockdown mammospheres did not develop anticancer drug resistance. Collectively, these results indicated that altered proteasome function and p62 expression caused NRF2 activation in CSC-enriched mammospheres. In addition, NRF2 appeared to play a role in CSC survival and anticancer drug resistance.

No MeSH data available.


Related in: MedlinePlus