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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

Enhanced ROS and cell death in shNRF2 mammospheres(A)HO-1, AKR1c1, and NQO-1 transcript levels were assessed in the sc and shNRF2 mammospheres by real-time PCR. (B)GCLC, GCLM, GSR, GPx2, and SOD2 transcript levels were monitored in the sc and shNRF2 mammospheres. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared to the sc mammospheres (C) AKR1c1 and HO-1 protein levels were determined in the sc and shNRF2 mammospheres. (D) ARE-driven luciferase activities were monitored in sc and shNRF2 monolayers and mammospheres. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared with the sc monolayers. (E) Intracellular ROS levels in sc and shNRF2 mammospheres were quantified using a fluorogenic dye carboxy-H2DCFDA. (F) To monitor cell death in mammospheres, suspended MCF7 from spheres were stained with an early apoptosis-specific marker Annexin V or a late apoptosis/necrosis-specific marker PI, and confocal microscopic observation was carried out. Annexin V-positive or PI-positive cell numbers are shown in the graphs.
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Figure 7: Enhanced ROS and cell death in shNRF2 mammospheres(A)HO-1, AKR1c1, and NQO-1 transcript levels were assessed in the sc and shNRF2 mammospheres by real-time PCR. (B)GCLC, GCLM, GSR, GPx2, and SOD2 transcript levels were monitored in the sc and shNRF2 mammospheres. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared to the sc mammospheres (C) AKR1c1 and HO-1 protein levels were determined in the sc and shNRF2 mammospheres. (D) ARE-driven luciferase activities were monitored in sc and shNRF2 monolayers and mammospheres. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared with the sc monolayers. (E) Intracellular ROS levels in sc and shNRF2 mammospheres were quantified using a fluorogenic dye carboxy-H2DCFDA. (F) To monitor cell death in mammospheres, suspended MCF7 from spheres were stained with an early apoptosis-specific marker Annexin V or a late apoptosis/necrosis-specific marker PI, and confocal microscopic observation was carried out. Annexin V-positive or PI-positive cell numbers are shown in the graphs.

Mentions: It was apparent that NRF2 stabilization led to elevated detoxifying/antioxidant gene expression in mammospheres. This phenomenon may be an underlying mechanism of delayed mammosphere growth in the NRF2 knockdown group. Indeed, the increase in AKR1c1, NQO1, and HO-1 in mammospheres was suppressed by NRF2 knockdown. AKR1c1 increase in sc mammospheres was 44-fold higher than monolayers, while the value in shNRF2 mammospheres was 19-fold increase (Figure 7A). In addition, mammosphere GSH reductase (GSR) and ROS detoxifying GPx increases were repressed in shNRF2 mammospheres (Figure 7B). Similar patterns were observed in immunoblot analysis using antibodies against HO-1 and AKR1c1 (Figure 7C). Mammosphere ARE luciferase activity was also much lower in NRF2 knockdown cells (Figure 7D).


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

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

Enhanced ROS and cell death in shNRF2 mammospheres(A)HO-1, AKR1c1, and NQO-1 transcript levels were assessed in the sc and shNRF2 mammospheres by real-time PCR. (B)GCLC, GCLM, GSR, GPx2, and SOD2 transcript levels were monitored in the sc and shNRF2 mammospheres. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared to the sc mammospheres (C) AKR1c1 and HO-1 protein levels were determined in the sc and shNRF2 mammospheres. (D) ARE-driven luciferase activities were monitored in sc and shNRF2 monolayers and mammospheres. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared with the sc monolayers. (E) Intracellular ROS levels in sc and shNRF2 mammospheres were quantified using a fluorogenic dye carboxy-H2DCFDA. (F) To monitor cell death in mammospheres, suspended MCF7 from spheres were stained with an early apoptosis-specific marker Annexin V or a late apoptosis/necrosis-specific marker PI, and confocal microscopic observation was carried out. Annexin V-positive or PI-positive cell numbers are shown in the graphs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Enhanced ROS and cell death in shNRF2 mammospheres(A)HO-1, AKR1c1, and NQO-1 transcript levels were assessed in the sc and shNRF2 mammospheres by real-time PCR. (B)GCLC, GCLM, GSR, GPx2, and SOD2 transcript levels were monitored in the sc and shNRF2 mammospheres. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared to the sc mammospheres (C) AKR1c1 and HO-1 protein levels were determined in the sc and shNRF2 mammospheres. (D) ARE-driven luciferase activities were monitored in sc and shNRF2 monolayers and mammospheres. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared with the sc monolayers. (E) Intracellular ROS levels in sc and shNRF2 mammospheres were quantified using a fluorogenic dye carboxy-H2DCFDA. (F) To monitor cell death in mammospheres, suspended MCF7 from spheres were stained with an early apoptosis-specific marker Annexin V or a late apoptosis/necrosis-specific marker PI, and confocal microscopic observation was carried out. Annexin V-positive or PI-positive cell numbers are shown in the graphs.
Mentions: It was apparent that NRF2 stabilization led to elevated detoxifying/antioxidant gene expression in mammospheres. This phenomenon may be an underlying mechanism of delayed mammosphere growth in the NRF2 knockdown group. Indeed, the increase in AKR1c1, NQO1, and HO-1 in mammospheres was suppressed by NRF2 knockdown. AKR1c1 increase in sc mammospheres was 44-fold higher than monolayers, while the value in shNRF2 mammospheres was 19-fold increase (Figure 7A). In addition, mammosphere GSH reductase (GSR) and ROS detoxifying GPx increases were repressed in shNRF2 mammospheres (Figure 7B). Similar patterns were observed in immunoblot analysis using antibodies against HO-1 and AKR1c1 (Figure 7C). Mammosphere ARE luciferase activity was also much lower in NRF2 knockdown cells (Figure 7D).

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