Article Title: The andean anticancer herbal product BIRM causes destabilization of androgen receptor and induces caspase-8 mediated-apoptosis in prostate cancer
Figure Lengend Snippet: BIRM induced decrease in AR is due to increased proteasomal degradation of AR ( A ) Total RNA was purified from LAPC-4 treated with 10 μl/ml BIRM with or without 1 nM DHT for 48 h and semi-quantitative RT-PCR (i) was performed. GAPDH was amplified as an internal control. (ii) Quantitative real time PCR was performed on LAPC-4 cells treated with BIRM with 1nM DHT, the graph shows a relative AR mRNA expression normalized against a β-actin mRNA. ( B ) LAPC-4 and LNCaP cells incubated with or without BIRM (20 μl/ml) for 48 h, were pretreated with 10 μM LY294002 (PI-3Kinase inhibitor) for 1 h, or co-treatment with MG132 (proteasome inhibitor) for 4 h at the end of treatment. Decrease AR levels were monitored by Immunoblotting. ( C ) LAPC-4 cells were treated with indicated doses of BIRM for 48 h with or without MG132 treatment as described above. Immunoprecipitation was performed using anti-AR antibody and Western blotting was performed with anti-Ubiquitin antibody. Emodin treatment was performed for positive control of AR ubiquitination. ( D ) LAPC-4 cells were treated with various doses of BIRM for 48 h or 5 μg ml geldanamycin (GA) for 18 h and harvested. Cell lysate was used for Immunoprecipitation with a-Hsp90 antibody, separated by SDS-PAGE and probed with a-AR or a-acetyl lysine antibody. GA treated samples was used as a positive control of decrease interaction between AR and Hsp90. Due to high background in control band (0 μl/ml BIRM) in lane1, accurate densitometry of band intensity was difficult. However, there was > 50% decrease in the Hsp90-bound AR at 5 μl/ml BIRM. BIRM induced strong acetylation of Hsp90 at 5μl/ml. The disintegrated band at higher BIRM exposure might be associated with cells undergoing apoptosis. GA: Geldanamycin treated cell lysates, IgG: normal mouse IgG as negative control in IP.
Article Snippet: LAPC-4 cells were treated with 5 μl/ml BIRM for 24 h and AR levels in cytosol and nucleus were determined by first isolating the two fractions as described in methods or using a kit (NE-PER Nuclear and Cytoplasmic extraction reagents, Thermo-Scientific, Cat#78833) then by western blotting of respective fractions.
Techniques: Purification, Quantitative RT-PCR, Amplification, Real-time Polymerase Chain Reaction, Expressing, Incubation, Immunoprecipitation, Western Blot, Positive Control, SDS Page, Negative Control