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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 inhibition on celastrol-induced autophagy is related with miR-101 transactivation.To see whether miR-101 could affect AR suppression on celastrol-induced autophagy, AR positive LNCaP cells were transfected with miR-101 mimic or negative control (NC) for 24 h (A), followed by additional 24 h treatment with celastrol (2.0 μM, CEL). MiR-101 levels were determined by RT-PCR. #, P<0.05 versus NC transfection without celastrol treament. **, P<0.01 between miR-101 and NC transfections with celastrol treatment. The protein levels of LC3 and p62 as well as AR were determined by Western blotting using GAPDH as a loading control. B, AR negative DU145 cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) in the presence of miR-101 inhibitor (miR-101 Inh) or negative control (NC). Cells were incubated in celastrol (2.0 μM, CEL) for an additional 24 h. MiR-101 levels were determined by RT-PCR. #, P<0.05 versus EGFP-V plus NC transfections. *, P<0.05 between EGFP-V and EGFP-AR in the presence of miR-101 inhibitor. The protein levels of AR, LC3and p62 were detected by Western blotting. C, DU145 cells were transfected with pGL3-B-miR-101-W (with wild type AR binding site) or pGL3-B-miR-101-M (with mutant AR binding site), along with AR expression vector (EGFP-AR) or empty vector (EGFP-V) for 24 h, then treated with DMSO or celastrol (CEL, 2.0 μM) for additional 24 h. MiR-101 levels were determined by RT-PCR. #, P<0.05 versus EGFP-V plus pGL3-B-miR-101-M transfections. *, P<0.05 between pGL3-B-miR-101-M and pGL3-B-miR-101-W transfections. The protein levels of AR, LC3 and p62 were detected by Western blotting using GAPDH as a loading control.
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pone.0140745.g005: AR inhibition on celastrol-induced autophagy is related with miR-101 transactivation.To see whether miR-101 could affect AR suppression on celastrol-induced autophagy, AR positive LNCaP cells were transfected with miR-101 mimic or negative control (NC) for 24 h (A), followed by additional 24 h treatment with celastrol (2.0 μM, CEL). MiR-101 levels were determined by RT-PCR. #, P<0.05 versus NC transfection without celastrol treament. **, P<0.01 between miR-101 and NC transfections with celastrol treatment. The protein levels of LC3 and p62 as well as AR were determined by Western blotting using GAPDH as a loading control. B, AR negative DU145 cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) in the presence of miR-101 inhibitor (miR-101 Inh) or negative control (NC). Cells were incubated in celastrol (2.0 μM, CEL) for an additional 24 h. MiR-101 levels were determined by RT-PCR. #, P<0.05 versus EGFP-V plus NC transfections. *, P<0.05 between EGFP-V and EGFP-AR in the presence of miR-101 inhibitor. The protein levels of AR, LC3and p62 were detected by Western blotting. C, DU145 cells were transfected with pGL3-B-miR-101-W (with wild type AR binding site) or pGL3-B-miR-101-M (with mutant AR binding site), along with AR expression vector (EGFP-AR) or empty vector (EGFP-V) for 24 h, then treated with DMSO or celastrol (CEL, 2.0 μM) for additional 24 h. MiR-101 levels were determined by RT-PCR. #, P<0.05 versus EGFP-V plus pGL3-B-miR-101-M transfections. *, P<0.05 between pGL3-B-miR-101-M and pGL3-B-miR-101-W transfections. The protein levels of AR, LC3 and p62 were detected by Western blotting using GAPDH as a loading control.

Mentions: Next, we determined if AR negatively regulates celastrol-induced autophagy through inhibition of miR-101 expression in prostate cancer cells. MiR-101 mimic was used to increase the miR-101 level in LNCaP cells. With miR-101 upregulation by addition of miR-101 mimic (Fig 5A, upper), basal level autophagy was inhibited (LC3-II/LC3-I ratio decreased by half) (Fig 5A, bottom). Although AR reduction by celastrol was favorable for autophagy induction, addition of miR-101 mimic resulted in autophagy inhibition as shown by the half-fold decrease of LC3-II/LC3-I ratio and increase of p62 level (Fig 5A, bottom). In DU145 cells, stable expression of exogenous AR could upregulate miR-101 expression and could suppress autophagy triggered by celastrol. When miR-101 was inhibited by addition of miR-101 inhibitor, as determined by qPCR (Fig 5B, upper), autophagy was rescued regardless of AR overexpression (Fig 5B, bottom). These data suggest that the negative role AR played in the regulation of autophagy depends on its downstream targets. Furthermore, a pair of miR-101 expression constructs, pGL3-B-miR-101-W that contains the wild type ARE, and pGL3-B-miR-101-M that has mutant ARE were generated. pGL3-B-miR-101-W significantly enhanced miR-101 expression than pGL3-B-miR-101-M in co-transfection with exogenous AR in DU145 cells with or without celastrol treatment (Fig 5C). In accordance with the miR-101 levels, co-transfection with pGL3-B-miR-101-W that can be transactivated by AR showed more suppression on autophagy at the basal level or celastrol induced compared with pGL3-B-miR-101-M that could not be recognized by AR (Fig 5C), indicating that AR inhibits celastrol-induced autophagy via transactivation of miR-101.


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

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

AR inhibition on celastrol-induced autophagy is related with miR-101 transactivation.To see whether miR-101 could affect AR suppression on celastrol-induced autophagy, AR positive LNCaP cells were transfected with miR-101 mimic or negative control (NC) for 24 h (A), followed by additional 24 h treatment with celastrol (2.0 μM, CEL). MiR-101 levels were determined by RT-PCR. #, P<0.05 versus NC transfection without celastrol treament. **, P<0.01 between miR-101 and NC transfections with celastrol treatment. The protein levels of LC3 and p62 as well as AR were determined by Western blotting using GAPDH as a loading control. B, AR negative DU145 cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) in the presence of miR-101 inhibitor (miR-101 Inh) or negative control (NC). Cells were incubated in celastrol (2.0 μM, CEL) for an additional 24 h. MiR-101 levels were determined by RT-PCR. #, P<0.05 versus EGFP-V plus NC transfections. *, P<0.05 between EGFP-V and EGFP-AR in the presence of miR-101 inhibitor. The protein levels of AR, LC3and p62 were detected by Western blotting. C, DU145 cells were transfected with pGL3-B-miR-101-W (with wild type AR binding site) or pGL3-B-miR-101-M (with mutant AR binding site), along with AR expression vector (EGFP-AR) or empty vector (EGFP-V) for 24 h, then treated with DMSO or celastrol (CEL, 2.0 μM) for additional 24 h. MiR-101 levels were determined by RT-PCR. #, P<0.05 versus EGFP-V plus pGL3-B-miR-101-M transfections. *, P<0.05 between pGL3-B-miR-101-M and pGL3-B-miR-101-W transfections. The protein levels of AR, LC3 and p62 were detected by Western blotting using GAPDH as a loading control.
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pone.0140745.g005: AR inhibition on celastrol-induced autophagy is related with miR-101 transactivation.To see whether miR-101 could affect AR suppression on celastrol-induced autophagy, AR positive LNCaP cells were transfected with miR-101 mimic or negative control (NC) for 24 h (A), followed by additional 24 h treatment with celastrol (2.0 μM, CEL). MiR-101 levels were determined by RT-PCR. #, P<0.05 versus NC transfection without celastrol treament. **, P<0.01 between miR-101 and NC transfections with celastrol treatment. The protein levels of LC3 and p62 as well as AR were determined by Western blotting using GAPDH as a loading control. B, AR negative DU145 cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) in the presence of miR-101 inhibitor (miR-101 Inh) or negative control (NC). Cells were incubated in celastrol (2.0 μM, CEL) for an additional 24 h. MiR-101 levels were determined by RT-PCR. #, P<0.05 versus EGFP-V plus NC transfections. *, P<0.05 between EGFP-V and EGFP-AR in the presence of miR-101 inhibitor. The protein levels of AR, LC3and p62 were detected by Western blotting. C, DU145 cells were transfected with pGL3-B-miR-101-W (with wild type AR binding site) or pGL3-B-miR-101-M (with mutant AR binding site), along with AR expression vector (EGFP-AR) or empty vector (EGFP-V) for 24 h, then treated with DMSO or celastrol (CEL, 2.0 μM) for additional 24 h. MiR-101 levels were determined by RT-PCR. #, P<0.05 versus EGFP-V plus pGL3-B-miR-101-M transfections. *, P<0.05 between pGL3-B-miR-101-M and pGL3-B-miR-101-W transfections. The protein levels of AR, LC3 and p62 were detected by Western blotting using GAPDH as a loading control.
Mentions: Next, we determined if AR negatively regulates celastrol-induced autophagy through inhibition of miR-101 expression in prostate cancer cells. MiR-101 mimic was used to increase the miR-101 level in LNCaP cells. With miR-101 upregulation by addition of miR-101 mimic (Fig 5A, upper), basal level autophagy was inhibited (LC3-II/LC3-I ratio decreased by half) (Fig 5A, bottom). Although AR reduction by celastrol was favorable for autophagy induction, addition of miR-101 mimic resulted in autophagy inhibition as shown by the half-fold decrease of LC3-II/LC3-I ratio and increase of p62 level (Fig 5A, bottom). In DU145 cells, stable expression of exogenous AR could upregulate miR-101 expression and could suppress autophagy triggered by celastrol. When miR-101 was inhibited by addition of miR-101 inhibitor, as determined by qPCR (Fig 5B, upper), autophagy was rescued regardless of AR overexpression (Fig 5B, bottom). These data suggest that the negative role AR played in the regulation of autophagy depends on its downstream targets. Furthermore, a pair of miR-101 expression constructs, pGL3-B-miR-101-W that contains the wild type ARE, and pGL3-B-miR-101-M that has mutant ARE were generated. pGL3-B-miR-101-W significantly enhanced miR-101 expression than pGL3-B-miR-101-M in co-transfection with exogenous AR in DU145 cells with or without celastrol treatment (Fig 5C). In accordance with the miR-101 levels, co-transfection with pGL3-B-miR-101-W that can be transactivated by AR showed more suppression on autophagy at the basal level or celastrol induced compared with pGL3-B-miR-101-M that could not be recognized by AR (Fig 5C), indicating that AR inhibits celastrol-induced autophagy via transactivation of miR-101.

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