Limits...
MicroRNA-25 regulates chemoresistance-associated autophagy in breast cancer cells, a process modulated by the natural autophagy inducer isoliquiritigenin.

Wang Z, Wang N, Liu P, Chen Q, Situ H, Xie T, Zhang J, Peng C, Lin Y, Chen J - Oncotarget (2014)

Bottom Line: Recent findings have revealed that dysregulated miRNAs contribute significantly to autophagy and chemoresistance.More importantly, miRNA 3.0 array experiments identified miR-25 as the main target of ISL in triggering autophagy flux.Subsequent in vivo experiments showed that ISL had chemosensitizing potency, as revealed by an increase in LC3-II staining, the downregulation of ABCG2, a reduction in miR-25 expression and the activation of the miR-25 target ULK1.

View Article: PubMed Central - PubMed

Affiliation: Department of Mammary Disease, Guangdong Provincial Hospital of Chinese Medicine; School of Chinese Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China; These authors contributed equally to this work.

ABSTRACT
Recent findings have revealed that dysregulated miRNAs contribute significantly to autophagy and chemoresistance. Pharmacologically targeting autophagy-related miRNAs is a novel strategy to reverse drug resistance. Here, we report a novel function of isoliquiritigenin (ISL) as a natural inhibitor of autophagy-related miR-25 in killing drug-resistant breast cancer cells. ISL induced chemosensitization, cell cycle arrest and autophagy, but not apoptosis, in MCF-7/ADR cells. ISL also promoted the degradation of the ATP-binding cassette (ABC) protein ABCG2 primarily via the autophagy-lysosome pathway. More importantly, miRNA 3.0 array experiments identified miR-25 as the main target of ISL in triggering autophagy flux. A mechanistic study validated that miR-25 inhibition led to autophagic cell death by directly increasing ULK1 expression, an early regulator in the autophagy induction phase. miR-25 overexpression was demonstrated to block ISL-induced autophagy and chemosensitization. Subsequent in vivo experiments showed that ISL had chemosensitizing potency, as revealed by an increase in LC3-II staining, the downregulation of ABCG2, a reduction in miR-25 expression and the activation of the miR-25 target ULK1. Overall, our results not only indicate that ISL acts as a natural autophagy inducer to increase breast cancer chemosensitivity, but also reveal that miR-25 functions as a novel regulator of autophagy by targeting ULK1.

Show MeSH

Related in: MedlinePlus

ISL inhibits ABCG2 expression via the autophagy-lysosome pathway(A–B) Treatment with the autophagy inhibitor 3-MA abolished the autophagy-inducing effects of ISL after 24 h of treatment, indicated by decreases in LC3-II expression and AVO formation, whereas adding the lysosome inhibitor CQ enhanced LC3-II accumulation and AVO formation in ADR cells after 24 h, indicating that the ISL-induced enhancement of autophagy markers was due to autophagy induction rather than the inhibition of autophagosome maturation; these results were further validated by a western blotting analysis; (C–D) time point-based immunofluorescence detection was performed to examine the correlation between autophagy induction and ABCG2 inhibition. The results showed that AVO formation and LC3-II accumulation occurred as early as 2 h after ISL treatment, whereas decreased ABCG2 expression was observed at the 5th hour, indicating that autophagy induction occurred prior to the downregulation of the drug resistance marker ABCG2; the results were validated by western blotting; (E–F) 24 h of treatment with the autophagy inhibitors 3-MA and CQ restored the cell viability and ABCG2 expression inhibited by ISL, indicating that ISL may induce cell death primarily by the autophagy pathway (the values represent the means ± SD, n=3, *P<0.05, **P<0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4196180&req=5

Figure 3: ISL inhibits ABCG2 expression via the autophagy-lysosome pathway(A–B) Treatment with the autophagy inhibitor 3-MA abolished the autophagy-inducing effects of ISL after 24 h of treatment, indicated by decreases in LC3-II expression and AVO formation, whereas adding the lysosome inhibitor CQ enhanced LC3-II accumulation and AVO formation in ADR cells after 24 h, indicating that the ISL-induced enhancement of autophagy markers was due to autophagy induction rather than the inhibition of autophagosome maturation; these results were further validated by a western blotting analysis; (C–D) time point-based immunofluorescence detection was performed to examine the correlation between autophagy induction and ABCG2 inhibition. The results showed that AVO formation and LC3-II accumulation occurred as early as 2 h after ISL treatment, whereas decreased ABCG2 expression was observed at the 5th hour, indicating that autophagy induction occurred prior to the downregulation of the drug resistance marker ABCG2; the results were validated by western blotting; (E–F) 24 h of treatment with the autophagy inhibitors 3-MA and CQ restored the cell viability and ABCG2 expression inhibited by ISL, indicating that ISL may induce cell death primarily by the autophagy pathway (the values represent the means ± SD, n=3, *P<0.05, **P<0.01).

Mentions: To confirm that the enhancement of autophagy markers by ISL was due to the induction of autophagy rather than the blockage of autophagosome maturation, the lysosome inhibitor chloroquine (CQ) was added to the culture medium along with ISL. Both the LC3-II level and AVO formation in the cotreatment group were significantly higher than in the group treated with CQ alone (Figure 3A). Western blotting results demonstrated that ISL further induced LC3-II upregulation following CQ treatment (Figure 3B). ISL-induced autophagy was abolished by the autophagy inhibitor 3-methyladenine (3-MA). The addition of 3-MA led to the inhibition of both ISL-induced AVO formation and the upregulation of LC3-II and BECN1 (Figure 3A,B). Because 3-MA is well known as an autophagy inhibitor targeting the upstream PI3K signaling pathway [34], the results indicated that ISL functioned by inducing the early stage of autophagy flux rather than by inhibiting lysosome degradation.


MicroRNA-25 regulates chemoresistance-associated autophagy in breast cancer cells, a process modulated by the natural autophagy inducer isoliquiritigenin.

Wang Z, Wang N, Liu P, Chen Q, Situ H, Xie T, Zhang J, Peng C, Lin Y, Chen J - Oncotarget (2014)

ISL inhibits ABCG2 expression via the autophagy-lysosome pathway(A–B) Treatment with the autophagy inhibitor 3-MA abolished the autophagy-inducing effects of ISL after 24 h of treatment, indicated by decreases in LC3-II expression and AVO formation, whereas adding the lysosome inhibitor CQ enhanced LC3-II accumulation and AVO formation in ADR cells after 24 h, indicating that the ISL-induced enhancement of autophagy markers was due to autophagy induction rather than the inhibition of autophagosome maturation; these results were further validated by a western blotting analysis; (C–D) time point-based immunofluorescence detection was performed to examine the correlation between autophagy induction and ABCG2 inhibition. The results showed that AVO formation and LC3-II accumulation occurred as early as 2 h after ISL treatment, whereas decreased ABCG2 expression was observed at the 5th hour, indicating that autophagy induction occurred prior to the downregulation of the drug resistance marker ABCG2; the results were validated by western blotting; (E–F) 24 h of treatment with the autophagy inhibitors 3-MA and CQ restored the cell viability and ABCG2 expression inhibited by ISL, indicating that ISL may induce cell death primarily by the autophagy pathway (the values represent the means ± SD, n=3, *P<0.05, **P<0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196180&req=5

Figure 3: ISL inhibits ABCG2 expression via the autophagy-lysosome pathway(A–B) Treatment with the autophagy inhibitor 3-MA abolished the autophagy-inducing effects of ISL after 24 h of treatment, indicated by decreases in LC3-II expression and AVO formation, whereas adding the lysosome inhibitor CQ enhanced LC3-II accumulation and AVO formation in ADR cells after 24 h, indicating that the ISL-induced enhancement of autophagy markers was due to autophagy induction rather than the inhibition of autophagosome maturation; these results were further validated by a western blotting analysis; (C–D) time point-based immunofluorescence detection was performed to examine the correlation between autophagy induction and ABCG2 inhibition. The results showed that AVO formation and LC3-II accumulation occurred as early as 2 h after ISL treatment, whereas decreased ABCG2 expression was observed at the 5th hour, indicating that autophagy induction occurred prior to the downregulation of the drug resistance marker ABCG2; the results were validated by western blotting; (E–F) 24 h of treatment with the autophagy inhibitors 3-MA and CQ restored the cell viability and ABCG2 expression inhibited by ISL, indicating that ISL may induce cell death primarily by the autophagy pathway (the values represent the means ± SD, n=3, *P<0.05, **P<0.01).
Mentions: To confirm that the enhancement of autophagy markers by ISL was due to the induction of autophagy rather than the blockage of autophagosome maturation, the lysosome inhibitor chloroquine (CQ) was added to the culture medium along with ISL. Both the LC3-II level and AVO formation in the cotreatment group were significantly higher than in the group treated with CQ alone (Figure 3A). Western blotting results demonstrated that ISL further induced LC3-II upregulation following CQ treatment (Figure 3B). ISL-induced autophagy was abolished by the autophagy inhibitor 3-methyladenine (3-MA). The addition of 3-MA led to the inhibition of both ISL-induced AVO formation and the upregulation of LC3-II and BECN1 (Figure 3A,B). Because 3-MA is well known as an autophagy inhibitor targeting the upstream PI3K signaling pathway [34], the results indicated that ISL functioned by inducing the early stage of autophagy flux rather than by inhibiting lysosome degradation.

Bottom Line: Recent findings have revealed that dysregulated miRNAs contribute significantly to autophagy and chemoresistance.More importantly, miRNA 3.0 array experiments identified miR-25 as the main target of ISL in triggering autophagy flux.Subsequent in vivo experiments showed that ISL had chemosensitizing potency, as revealed by an increase in LC3-II staining, the downregulation of ABCG2, a reduction in miR-25 expression and the activation of the miR-25 target ULK1.

View Article: PubMed Central - PubMed

Affiliation: Department of Mammary Disease, Guangdong Provincial Hospital of Chinese Medicine; School of Chinese Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China; These authors contributed equally to this work.

ABSTRACT
Recent findings have revealed that dysregulated miRNAs contribute significantly to autophagy and chemoresistance. Pharmacologically targeting autophagy-related miRNAs is a novel strategy to reverse drug resistance. Here, we report a novel function of isoliquiritigenin (ISL) as a natural inhibitor of autophagy-related miR-25 in killing drug-resistant breast cancer cells. ISL induced chemosensitization, cell cycle arrest and autophagy, but not apoptosis, in MCF-7/ADR cells. ISL also promoted the degradation of the ATP-binding cassette (ABC) protein ABCG2 primarily via the autophagy-lysosome pathway. More importantly, miRNA 3.0 array experiments identified miR-25 as the main target of ISL in triggering autophagy flux. A mechanistic study validated that miR-25 inhibition led to autophagic cell death by directly increasing ULK1 expression, an early regulator in the autophagy induction phase. miR-25 overexpression was demonstrated to block ISL-induced autophagy and chemosensitization. Subsequent in vivo experiments showed that ISL had chemosensitizing potency, as revealed by an increase in LC3-II staining, the downregulation of ABCG2, a reduction in miR-25 expression and the activation of the miR-25 target ULK1. Overall, our results not only indicate that ISL acts as a natural autophagy inducer to increase breast cancer chemosensitivity, but also reveal that miR-25 functions as a novel regulator of autophagy by targeting ULK1.

Show MeSH
Related in: MedlinePlus