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Celastrol Induces Autophagy by Targeting AR/miR-101 in Prostate Cancer Cells.

Guo J, Huang X, Wang H, Yang H - PLoS ONE (2015)

Bottom Line: In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy.Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR.Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Technology, Harbin Institute of Technology, Harbin, 150001, China.

ABSTRACT
Autophagy is an evolutionarily conserved process responsible for the degradation and recycling of cytoplasmic components through autolysosomes. Targeting AR axis is a standard strategy for prostate cancer treatment; however, the role of AR in autophagic processes is still not fully understood. In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy. Knockdown of AR in AR-positive prostate cancer cells resulted in enhanced autophagy. Ectopic expression of AR in AR-negative prostate cancer cells, or gain of function of the AR signaling in AR-positive cells, led to suppression of autophagy. Since miR-101 is an inhibitor of autophagy and its expression was decreased along with AR in the process of celastrol-induced autophagy, we hypothesize that AR inhibits autophagy through transactivation of miR-101. AR binding site was defined in the upstream of miR-101 gene by luciferase reporter and ChIP assays. MiR-101 expression correlated with AR status in prostate cancer cell lines. The inhibition of celastrol-induced autophagy by AR was compromised by blocking miR-101; while transfection of miR-101 led to inhibition of celastrol-induced autophagy in spite of AR depletion. Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR. Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells. Our results demonstrate that AR inhibits autophagy via transactivation of miR-101, thus combination of miR-101 mimics with celastrol may represent a promising therapeutic approach for treating prostate cancer.

No MeSH data available.


Related in: MedlinePlus

AR modulates miR-101 levels without affecting cell death.A and B, LNCaP cells were treated with MDV3100 at 5 μM for indicated times. Protein extracts were immunoblotted with antibodies against PARP, AR, LC3, p62 and GAPDH (loading control) (A). Mature miR-101 levels were determined by qPCR (B). C and D, LNCaP cells were subjected to androgen starvation as described in "Materials and Methods", followed by 1 nM of R1881 treatment for indicated times. PARP, AR, LC3, p62 and GAPDH (loading control) were detected by Western blotting (C). Mature miR-101 levels were determined by qPCR (D). Asterisks denote significance compared with control (0 h). *, P <0.05.
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pone.0140745.g006: AR modulates miR-101 levels without affecting cell death.A and B, LNCaP cells were treated with MDV3100 at 5 μM for indicated times. Protein extracts were immunoblotted with antibodies against PARP, AR, LC3, p62 and GAPDH (loading control) (A). Mature miR-101 levels were determined by qPCR (B). C and D, LNCaP cells were subjected to androgen starvation as described in "Materials and Methods", followed by 1 nM of R1881 treatment for indicated times. PARP, AR, LC3, p62 and GAPDH (loading control) were detected by Western blotting (C). Mature miR-101 levels were determined by qPCR (D). Asterisks denote significance compared with control (0 h). *, P <0.05.

Mentions: Since AR reduction also results in reduced cell viability, whether the observed miR-101 suppression and autophagy induction were due to cell death was determined. LNCaP cells were treated with AR antagonist MDV3100 (Enzalutamide) for different times. As expected, AR protein was decreased (Fig 6A). Along with AR reduction, miR-101 expression was decreased while autophagy was induced, as shown by decreased p62 levels and increased LC3-II/LC3-I ratios. In the same treated samples, PARP was kept intact (Fig 6A and 6B), indicating that cell viability was not affected post MDV3100 treatment at current conditions. Reversely, AR was activated by R1881 (Fig 6C). With AR activation, miR-101 expression was increased (Fig 6D) along with autophagy inhibition (Fig 6C and 6D). Again, cell death was not observed post R1881 treatment (Fig 6C). These results indicate that miR-101 reduction was due to AR suppression, but not cell death.


Celastrol Induces Autophagy by Targeting AR/miR-101 in Prostate Cancer Cells.

Guo J, Huang X, Wang H, Yang H - PLoS ONE (2015)

AR modulates miR-101 levels without affecting cell death.A and B, LNCaP cells were treated with MDV3100 at 5 μM for indicated times. Protein extracts were immunoblotted with antibodies against PARP, AR, LC3, p62 and GAPDH (loading control) (A). Mature miR-101 levels were determined by qPCR (B). C and D, LNCaP cells were subjected to androgen starvation as described in "Materials and Methods", followed by 1 nM of R1881 treatment for indicated times. PARP, AR, LC3, p62 and GAPDH (loading control) were detected by Western blotting (C). Mature miR-101 levels were determined by qPCR (D). Asterisks denote significance compared with control (0 h). *, P <0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608724&req=5

pone.0140745.g006: AR modulates miR-101 levels without affecting cell death.A and B, LNCaP cells were treated with MDV3100 at 5 μM for indicated times. Protein extracts were immunoblotted with antibodies against PARP, AR, LC3, p62 and GAPDH (loading control) (A). Mature miR-101 levels were determined by qPCR (B). C and D, LNCaP cells were subjected to androgen starvation as described in "Materials and Methods", followed by 1 nM of R1881 treatment for indicated times. PARP, AR, LC3, p62 and GAPDH (loading control) were detected by Western blotting (C). Mature miR-101 levels were determined by qPCR (D). Asterisks denote significance compared with control (0 h). *, P <0.05.
Mentions: Since AR reduction also results in reduced cell viability, whether the observed miR-101 suppression and autophagy induction were due to cell death was determined. LNCaP cells were treated with AR antagonist MDV3100 (Enzalutamide) for different times. As expected, AR protein was decreased (Fig 6A). Along with AR reduction, miR-101 expression was decreased while autophagy was induced, as shown by decreased p62 levels and increased LC3-II/LC3-I ratios. In the same treated samples, PARP was kept intact (Fig 6A and 6B), indicating that cell viability was not affected post MDV3100 treatment at current conditions. Reversely, AR was activated by R1881 (Fig 6C). With AR activation, miR-101 expression was increased (Fig 6D) along with autophagy inhibition (Fig 6C and 6D). Again, cell death was not observed post R1881 treatment (Fig 6C). These results indicate that miR-101 reduction was due to AR suppression, but not cell death.

Bottom Line: In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy.Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR.Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Technology, Harbin Institute of Technology, Harbin, 150001, China.

ABSTRACT
Autophagy is an evolutionarily conserved process responsible for the degradation and recycling of cytoplasmic components through autolysosomes. Targeting AR axis is a standard strategy for prostate cancer treatment; however, the role of AR in autophagic processes is still not fully understood. In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy. Knockdown of AR in AR-positive prostate cancer cells resulted in enhanced autophagy. Ectopic expression of AR in AR-negative prostate cancer cells, or gain of function of the AR signaling in AR-positive cells, led to suppression of autophagy. Since miR-101 is an inhibitor of autophagy and its expression was decreased along with AR in the process of celastrol-induced autophagy, we hypothesize that AR inhibits autophagy through transactivation of miR-101. AR binding site was defined in the upstream of miR-101 gene by luciferase reporter and ChIP assays. MiR-101 expression correlated with AR status in prostate cancer cell lines. The inhibition of celastrol-induced autophagy by AR was compromised by blocking miR-101; while transfection of miR-101 led to inhibition of celastrol-induced autophagy in spite of AR depletion. Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR. Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells. Our results demonstrate that AR inhibits autophagy via transactivation of miR-101, thus combination of miR-101 mimics with celastrol may represent a promising therapeutic approach for treating prostate cancer.

No MeSH data available.


Related in: MedlinePlus