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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

Celastrol triggers autophagy in prostate cancer cells.Parental LNCaP cells (A, D) or the cells transfected with GFP-LC3 (B, C) were treated with celastrol at 2.0 μM for indicated times. mRNA expressions of autophagy related genes were measured by qPCR (A). RQ, relative quantity. GFP-LC3 transfected cells were stained by DAPI after treatments. GFP-LC3 puncta were observed under confocal microscope (B) and quantified in C. The cells that contained over 5 puncta were selected and fifty cells were analysed for each treatment. D, Protein extracts were immunoblotted with antibodies against LC3, p62 and GAPDH (loading control). Asterisks denote significance compared with control (0 h). *, P <0.05; **, P <0.01.
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pone.0140745.g001: Celastrol triggers autophagy in prostate cancer cells.Parental LNCaP cells (A, D) or the cells transfected with GFP-LC3 (B, C) were treated with celastrol at 2.0 μM for indicated times. mRNA expressions of autophagy related genes were measured by qPCR (A). RQ, relative quantity. GFP-LC3 transfected cells were stained by DAPI after treatments. GFP-LC3 puncta were observed under confocal microscope (B) and quantified in C. The cells that contained over 5 puncta were selected and fifty cells were analysed for each treatment. D, Protein extracts were immunoblotted with antibodies against LC3, p62 and GAPDH (loading control). Asterisks denote significance compared with control (0 h). *, P <0.05; **, P <0.01.

Mentions: Targeting AR with celastrol for prostate cancer treatment has been shown by several groups [3, 5]. Since AR inhibition is associated with autophagy induction, whether celastrol could induce autophagy in human prostate cancer cells was determined. As shown in Fig 1, induction of autophagy-related genes ATG5 and ATG7 was observed at early time points after celastrol treatment in LNCaP prostate cancer cells (Fig 1A). ATG7 acts as the ubiquitin E1-like enzyme while ATG5 acts as the ubiquitination substrate or an E3-like enzyme in the two step ubiquitination process [1]. In accordance with their functions in autophagy induction, ATG7 showed higher expression and earlier response than ATG5 upon celastrol treatment (Fig 1A). Both ATG5 and ATG7 are required for the recruitment of LC3, the microtubule-associated protein which is diffused in the cytoplasm, to autophagosome membrane. To visualize LC3 recruitment, LNCaP cells were transfected with a plasmid encoding GFP-tagged LC3. GFP-LC3 puncta were observed to increase significantly after 6 h treatment with celastrol (Fig 1B and 1C). In accordance, conversion from LC3-I to its lipid form LC3-II, the hallmark of autophagy, was detected after 3–6 h treatments and increased noticeably after 12–24 h treatment (Fig 1D). p62 is a receptor on the autophagosome membrane, which is degraded in the lysosome after autophagosome fusion with it [17]. p62 protein level was reduced after 6–24 h treatment with celastrol (Fig 1D), further confirming that celastrol induced autophagy in LNCaP cells.


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

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

Celastrol triggers autophagy in prostate cancer cells.Parental LNCaP cells (A, D) or the cells transfected with GFP-LC3 (B, C) were treated with celastrol at 2.0 μM for indicated times. mRNA expressions of autophagy related genes were measured by qPCR (A). RQ, relative quantity. GFP-LC3 transfected cells were stained by DAPI after treatments. GFP-LC3 puncta were observed under confocal microscope (B) and quantified in C. The cells that contained over 5 puncta were selected and fifty cells were analysed for each treatment. D, Protein extracts were immunoblotted with antibodies against LC3, p62 and GAPDH (loading control). Asterisks denote significance compared with control (0 h). *, P <0.05; **, P <0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608724&req=5

pone.0140745.g001: Celastrol triggers autophagy in prostate cancer cells.Parental LNCaP cells (A, D) or the cells transfected with GFP-LC3 (B, C) were treated with celastrol at 2.0 μM for indicated times. mRNA expressions of autophagy related genes were measured by qPCR (A). RQ, relative quantity. GFP-LC3 transfected cells were stained by DAPI after treatments. GFP-LC3 puncta were observed under confocal microscope (B) and quantified in C. The cells that contained over 5 puncta were selected and fifty cells were analysed for each treatment. D, Protein extracts were immunoblotted with antibodies against LC3, p62 and GAPDH (loading control). Asterisks denote significance compared with control (0 h). *, P <0.05; **, P <0.01.
Mentions: Targeting AR with celastrol for prostate cancer treatment has been shown by several groups [3, 5]. Since AR inhibition is associated with autophagy induction, whether celastrol could induce autophagy in human prostate cancer cells was determined. As shown in Fig 1, induction of autophagy-related genes ATG5 and ATG7 was observed at early time points after celastrol treatment in LNCaP prostate cancer cells (Fig 1A). ATG7 acts as the ubiquitin E1-like enzyme while ATG5 acts as the ubiquitination substrate or an E3-like enzyme in the two step ubiquitination process [1]. In accordance with their functions in autophagy induction, ATG7 showed higher expression and earlier response than ATG5 upon celastrol treatment (Fig 1A). Both ATG5 and ATG7 are required for the recruitment of LC3, the microtubule-associated protein which is diffused in the cytoplasm, to autophagosome membrane. To visualize LC3 recruitment, LNCaP cells were transfected with a plasmid encoding GFP-tagged LC3. GFP-LC3 puncta were observed to increase significantly after 6 h treatment with celastrol (Fig 1B and 1C). In accordance, conversion from LC3-I to its lipid form LC3-II, the hallmark of autophagy, was detected after 3–6 h treatments and increased noticeably after 12–24 h treatment (Fig 1D). p62 is a receptor on the autophagosome membrane, which is degraded in the lysosome after autophagosome fusion with it [17]. p62 protein level was reduced after 6–24 h treatment with celastrol (Fig 1D), further confirming that celastrol induced autophagy in LNCaP cells.

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