Limits...
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 suppression on celastrol induced autophagy.A, LNCaP cells were treated with celastrol at 2.0 μM for indicated times, the protein level of AR was revealed by Western blotting using GAPDH as a loading control. B, LNCaP cells were transfected with AR siRNA or control siRNA for 24 h, the effects on AR knockdown and autophagy induction were verified by Western blotting using AR, LC3, p62 and GAPDH (loading control) antibodies. C, LNCaP cells were subjected to androgen starvation as described in "Materials and Methods", followed by 1 nM of R1881 treatment for 24 h. AR and its target PSA, as well as autophagic makers LC3 and p62 were detected by Western blotting using GAPDH as a loading control. LNCaP (D) or DU145 (E) cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V). D, After transfection, LNCaP cells were cultured in the medium containing R1881 (1 nM) and treated with or without celastrol (CEL) at 2.0 μM for 24 h (D). E, DU145 stably transfected cells were pretreated with R1881 (1 nM) for 24 h before celastrol treatment as D. LC3 was detected by Western blotting using GAPDH as a loading control.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608724&req=5

pone.0140745.g002: AR suppression on celastrol induced autophagy.A, LNCaP cells were treated with celastrol at 2.0 μM for indicated times, the protein level of AR was revealed by Western blotting using GAPDH as a loading control. B, LNCaP cells were transfected with AR siRNA or control siRNA for 24 h, the effects on AR knockdown and autophagy induction were verified by Western blotting using AR, LC3, p62 and GAPDH (loading control) antibodies. C, LNCaP cells were subjected to androgen starvation as described in "Materials and Methods", followed by 1 nM of R1881 treatment for 24 h. AR and its target PSA, as well as autophagic makers LC3 and p62 were detected by Western blotting using GAPDH as a loading control. LNCaP (D) or DU145 (E) cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V). D, After transfection, LNCaP cells were cultured in the medium containing R1881 (1 nM) and treated with or without celastrol (CEL) at 2.0 μM for 24 h (D). E, DU145 stably transfected cells were pretreated with R1881 (1 nM) for 24 h before celastrol treatment as D. LC3 was detected by Western blotting using GAPDH as a loading control.

Mentions: In the same samples from above, AR protein was decreased by celastrol during autophagy inducing processes. Celastrol treatment caused a substantial decrease in AR expression levels at 3–6 h time points and an almost complete depletion at 12–24 h time points (Fig 2A). In line with the above findings, siRNA knockdown of AR in AR positive LNCaP cells led to ~2-fold increase of LC3-I to LC3-II conversion (Fig 2B). Compared with the control, p62 protein was decreased by AR siRNA (Fig 2B), indicating that AR negatively regulated autophagy. Synthetic androgen R1881 was used to activate endogenous AR signaling in LNCaP cells, which was evidenced by the increased protein levels of AR and its target PSA (Fig 2C). With AR signaling activation, autophagy was inhibited as shown by the increase of p62 level and half-fold decrease of LC3-II/LC3-I ratio (Fig 2C), further confirming that AR plays a negative role in autophagy regulation. In addition, pEGFP-AR was transfected into LNCaP cells. After celastrol treatment, decreased conversion of LC3-I to LC3-II was detected in pEGFP-AR transfected cells compared with the mock transfection (Fig 2D), indicating that forced AR overexpression inhibited autophagy triggered by celastrol. Celastrol also induced autophagy in AR negative DU145 cells, which could be suppressed by exogenous AR (Fig 2E).


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

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

AR suppression on celastrol induced autophagy.A, LNCaP cells were treated with celastrol at 2.0 μM for indicated times, the protein level of AR was revealed by Western blotting using GAPDH as a loading control. B, LNCaP cells were transfected with AR siRNA or control siRNA for 24 h, the effects on AR knockdown and autophagy induction were verified by Western blotting using AR, LC3, p62 and GAPDH (loading control) antibodies. C, LNCaP cells were subjected to androgen starvation as described in "Materials and Methods", followed by 1 nM of R1881 treatment for 24 h. AR and its target PSA, as well as autophagic makers LC3 and p62 were detected by Western blotting using GAPDH as a loading control. LNCaP (D) or DU145 (E) cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V). D, After transfection, LNCaP cells were cultured in the medium containing R1881 (1 nM) and treated with or without celastrol (CEL) at 2.0 μM for 24 h (D). E, DU145 stably transfected cells were pretreated with R1881 (1 nM) for 24 h before celastrol treatment as D. LC3 was detected by Western blotting using GAPDH as a loading control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608724&req=5

pone.0140745.g002: AR suppression on celastrol induced autophagy.A, LNCaP cells were treated with celastrol at 2.0 μM for indicated times, the protein level of AR was revealed by Western blotting using GAPDH as a loading control. B, LNCaP cells were transfected with AR siRNA or control siRNA for 24 h, the effects on AR knockdown and autophagy induction were verified by Western blotting using AR, LC3, p62 and GAPDH (loading control) antibodies. C, LNCaP cells were subjected to androgen starvation as described in "Materials and Methods", followed by 1 nM of R1881 treatment for 24 h. AR and its target PSA, as well as autophagic makers LC3 and p62 were detected by Western blotting using GAPDH as a loading control. LNCaP (D) or DU145 (E) cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V). D, After transfection, LNCaP cells were cultured in the medium containing R1881 (1 nM) and treated with or without celastrol (CEL) at 2.0 μM for 24 h (D). E, DU145 stably transfected cells were pretreated with R1881 (1 nM) for 24 h before celastrol treatment as D. LC3 was detected by Western blotting using GAPDH as a loading control.
Mentions: In the same samples from above, AR protein was decreased by celastrol during autophagy inducing processes. Celastrol treatment caused a substantial decrease in AR expression levels at 3–6 h time points and an almost complete depletion at 12–24 h time points (Fig 2A). In line with the above findings, siRNA knockdown of AR in AR positive LNCaP cells led to ~2-fold increase of LC3-I to LC3-II conversion (Fig 2B). Compared with the control, p62 protein was decreased by AR siRNA (Fig 2B), indicating that AR negatively regulated autophagy. Synthetic androgen R1881 was used to activate endogenous AR signaling in LNCaP cells, which was evidenced by the increased protein levels of AR and its target PSA (Fig 2C). With AR signaling activation, autophagy was inhibited as shown by the increase of p62 level and half-fold decrease of LC3-II/LC3-I ratio (Fig 2C), further confirming that AR plays a negative role in autophagy regulation. In addition, pEGFP-AR was transfected into LNCaP cells. After celastrol treatment, decreased conversion of LC3-I to LC3-II was detected in pEGFP-AR transfected cells compared with the mock transfection (Fig 2D), indicating that forced AR overexpression inhibited autophagy triggered by celastrol. Celastrol also induced autophagy in AR negative DU145 cells, which could be suppressed by exogenous AR (Fig 2E).

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