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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

Autophagy potentiates the effects of celastrol on inhibition of cell proliferation and induction of apoptosis.Cell viability was measured by MTT assay after treatment with celastrol. A, LNCaP cells were transfected with negative control (NC) or miR-101 mimic at indicated concentrations for 24 h, followed by additional 96 h treatment with DMSO or celastrol at 1.5 μM. **, P<0.01 versus NC with celastrol treatment. B, LNCaP cells were treated with celastrol (1.5 μM) in the presence of miR-101 mimic or negative control (NC) for indicated time points. **, P<0.01 versus NC. C, LNCaP cells were treated with celastrol (CEL, 1.5 μM) or DMSO in the presence or absence of bafilomycin A1 (BAF, 10 nM) for 24 h. #, P <0.05 versus CEL, **, P<0.01 versus other two groups. D, LNCaP cells were treated with celastrol (CEL, 2.0 μM) in the presence or absence of bafilomycin A1 (BAF, 10 nM) or Z-VAD (20 μM) for up to 72 h. Protein extracts were immunoblotted with antibodies against PARP, LC3, p62 and GAPDH (loading control). E, Colony formation assay. LNCaP cells were treated with DMSO or celastrol (CEL, 1.5 μM) in the presence of miR-101 mimic or negative control (NC) or bafilomycin A1 (BAF, 10 nM) for 24 h. Colony formation was performed as described in the Materials and Methods. Colonies with over 50 cells were counted (E) and quantified in F. **, P<0.01 versus NC with celastrol treatment.
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pone.0140745.g007: Autophagy potentiates the effects of celastrol on inhibition of cell proliferation and induction of apoptosis.Cell viability was measured by MTT assay after treatment with celastrol. A, LNCaP cells were transfected with negative control (NC) or miR-101 mimic at indicated concentrations for 24 h, followed by additional 96 h treatment with DMSO or celastrol at 1.5 μM. **, P<0.01 versus NC with celastrol treatment. B, LNCaP cells were treated with celastrol (1.5 μM) in the presence of miR-101 mimic or negative control (NC) for indicated time points. **, P<0.01 versus NC. C, LNCaP cells were treated with celastrol (CEL, 1.5 μM) or DMSO in the presence or absence of bafilomycin A1 (BAF, 10 nM) for 24 h. #, P <0.05 versus CEL, **, P<0.01 versus other two groups. D, LNCaP cells were treated with celastrol (CEL, 2.0 μM) in the presence or absence of bafilomycin A1 (BAF, 10 nM) or Z-VAD (20 μM) for up to 72 h. Protein extracts were immunoblotted with antibodies against PARP, LC3, p62 and GAPDH (loading control). E, Colony formation assay. LNCaP cells were treated with DMSO or celastrol (CEL, 1.5 μM) in the presence of miR-101 mimic or negative control (NC) or bafilomycin A1 (BAF, 10 nM) for 24 h. Colony formation was performed as described in the Materials and Methods. Colonies with over 50 cells were counted (E) and quantified in F. **, P<0.01 versus NC with celastrol treatment.

Mentions: Autophagy plays dual roles in cancer cell survival under stresses, either protects cancer cells from apoptosis or itself leads to cell death, termed as type II programmed cell death. To evaluate the effect of autophagy on cell growth upon celastrol treatment, miR-101 mimic was used to inhibit autophagy and cell proliferation was determined post celastrol treatment. As shown in Fig 7, cell viability after celastrol treatment was significantly decreased by miR-101 mimic compared with the negative control (Fig 7A and 7B). Similarly, treatment with autophagy inhibitor bafilomycin A1 further decreased cell viability in comparison with celastrol treatment alone (Fig 7C). Prolonged treatment (up to 72 h) with celastrol induced apoptosis in LNCaP cells as shown by PARP fragment (p89 kD), a product of caspase-3 cleavage (Fig 7D). When autophagy was blocked with the inhibitor of autophagic flux Bafilomycin A1, the onset of celastrol-induced cell death was shifted to earlier time points. When apoptosis was blocked with the pan-inhibitor of caspase, celastrol-induced autophagy remained largely unaltered (Fig 7D). These results demonstrate that autophagy can delay the onset of apoptosis induced by celastrol in the investigated cell line. To see long term effect of autophagy on cell growth, colony formation was performed. As shown in Fig 7E and 7F, inhibition of autophagy by miR-101 or bafilomycin A1 enhanced celastrol-induced reduction on colonies formation. These results indicate that autophagy serves as a cytoprotective mechanism for prostate cancer cell survival upon celastrol treatment.


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

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

Autophagy potentiates the effects of celastrol on inhibition of cell proliferation and induction of apoptosis.Cell viability was measured by MTT assay after treatment with celastrol. A, LNCaP cells were transfected with negative control (NC) or miR-101 mimic at indicated concentrations for 24 h, followed by additional 96 h treatment with DMSO or celastrol at 1.5 μM. **, P<0.01 versus NC with celastrol treatment. B, LNCaP cells were treated with celastrol (1.5 μM) in the presence of miR-101 mimic or negative control (NC) for indicated time points. **, P<0.01 versus NC. C, LNCaP cells were treated with celastrol (CEL, 1.5 μM) or DMSO in the presence or absence of bafilomycin A1 (BAF, 10 nM) for 24 h. #, P <0.05 versus CEL, **, P<0.01 versus other two groups. D, LNCaP cells were treated with celastrol (CEL, 2.0 μM) in the presence or absence of bafilomycin A1 (BAF, 10 nM) or Z-VAD (20 μM) for up to 72 h. Protein extracts were immunoblotted with antibodies against PARP, LC3, p62 and GAPDH (loading control). E, Colony formation assay. LNCaP cells were treated with DMSO or celastrol (CEL, 1.5 μM) in the presence of miR-101 mimic or negative control (NC) or bafilomycin A1 (BAF, 10 nM) for 24 h. Colony formation was performed as described in the Materials and Methods. Colonies with over 50 cells were counted (E) and quantified in F. **, P<0.01 versus NC with celastrol treatment.
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pone.0140745.g007: Autophagy potentiates the effects of celastrol on inhibition of cell proliferation and induction of apoptosis.Cell viability was measured by MTT assay after treatment with celastrol. A, LNCaP cells were transfected with negative control (NC) or miR-101 mimic at indicated concentrations for 24 h, followed by additional 96 h treatment with DMSO or celastrol at 1.5 μM. **, P<0.01 versus NC with celastrol treatment. B, LNCaP cells were treated with celastrol (1.5 μM) in the presence of miR-101 mimic or negative control (NC) for indicated time points. **, P<0.01 versus NC. C, LNCaP cells were treated with celastrol (CEL, 1.5 μM) or DMSO in the presence or absence of bafilomycin A1 (BAF, 10 nM) for 24 h. #, P <0.05 versus CEL, **, P<0.01 versus other two groups. D, LNCaP cells were treated with celastrol (CEL, 2.0 μM) in the presence or absence of bafilomycin A1 (BAF, 10 nM) or Z-VAD (20 μM) for up to 72 h. Protein extracts were immunoblotted with antibodies against PARP, LC3, p62 and GAPDH (loading control). E, Colony formation assay. LNCaP cells were treated with DMSO or celastrol (CEL, 1.5 μM) in the presence of miR-101 mimic or negative control (NC) or bafilomycin A1 (BAF, 10 nM) for 24 h. Colony formation was performed as described in the Materials and Methods. Colonies with over 50 cells were counted (E) and quantified in F. **, P<0.01 versus NC with celastrol treatment.
Mentions: Autophagy plays dual roles in cancer cell survival under stresses, either protects cancer cells from apoptosis or itself leads to cell death, termed as type II programmed cell death. To evaluate the effect of autophagy on cell growth upon celastrol treatment, miR-101 mimic was used to inhibit autophagy and cell proliferation was determined post celastrol treatment. As shown in Fig 7, cell viability after celastrol treatment was significantly decreased by miR-101 mimic compared with the negative control (Fig 7A and 7B). Similarly, treatment with autophagy inhibitor bafilomycin A1 further decreased cell viability in comparison with celastrol treatment alone (Fig 7C). Prolonged treatment (up to 72 h) with celastrol induced apoptosis in LNCaP cells as shown by PARP fragment (p89 kD), a product of caspase-3 cleavage (Fig 7D). When autophagy was blocked with the inhibitor of autophagic flux Bafilomycin A1, the onset of celastrol-induced cell death was shifted to earlier time points. When apoptosis was blocked with the pan-inhibitor of caspase, celastrol-induced autophagy remained largely unaltered (Fig 7D). These results demonstrate that autophagy can delay the onset of apoptosis induced by celastrol in the investigated cell line. To see long term effect of autophagy on cell growth, colony formation was performed. As shown in Fig 7E and 7F, inhibition of autophagy by miR-101 or bafilomycin A1 enhanced celastrol-induced reduction on colonies formation. These results indicate that autophagy serves as a cytoprotective mechanism for prostate cancer cell survival upon celastrol treatment.

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