lapcs  (Thermo Fisher)

Journal: Oncotarget

Article Title: The andean anticancer herbal product BIRM causes destabilization of androgen receptor and induces caspase-8 mediated-apoptosis in prostate cancer

doi: 10.18632/oncotarget.12393

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

Journal: Oncotarget

Article Title: The andean anticancer herbal product BIRM causes destabilization of androgen receptor and induces caspase-8 mediated-apoptosis in prostate cancer

doi: 10.18632/oncotarget.12393

Figure Lengend Snippet: Effect of BIRM in phosphorylation of Akt ( A ) LNCaP, LAPC-4, PC-3 and DU145 cells were treated with various doses of BIRM for 48 h and p-Akt levels were monitored by Immunoblotting. Relative levels of p-Akt against t-Akt were calculated compared to untreated samples. ( B ) LAPC-4 cells were transiently transfected with either KDAkt or P PCDNA3 (control). Then, cells were treated with 10 μl/ml BIRM for 24 h. KDAkt: Kinase-Dead domain containing AKT transfectants.

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: Transfection

Journal: Oncotarget

Article Title: The andean anticancer herbal product BIRM causes destabilization of androgen receptor and induces caspase-8 mediated-apoptosis in prostate cancer

doi: 10.18632/oncotarget.12393

Figure Lengend Snippet: BIRM induces apoptosis by extrinsic (DISC) pathway ( A ) FADD, Cleaved caspase-8 and cleaved PARP levels were monitored in LAPC-4 and LNCaP cells treated with indicated doses of BIRM for 48 h (left panel), and DU145 and PC-3 cells treated with 10 μl/ml BIRM for indicated time points (right panel) by Immunoblotting. ( B ) DU145 cells were treated with BIRM for 48 h or staurosporine (STS) for 18 h and indicated protein levels were monitored by Immunoblotting. ( C ) LAPC-4 cells were pretreated with 50 μM caspase-8 inhibitor (Ac-IETD-CHO) for 1 h, followed by 50 μl/ml BIRM for 48 h. Then, cells were harvested for Immunoblotting to detect cleaved caspase-8 and PARP. ( D ) DU145 cells were pretreated with 1 μM CHO for 1 h, followed by BIRM for 48 h. Cytotoxicity of BIRM was monitored by MTT assay. The data presented as means ± SEM from two or more independent experiments in triplicate. FADD: Fas-Associated via Death Domain. PARP: Poly-ADP-Ribose Polymerase; C-PARP: Cleaved PARP; UT: Untreated; C-Casp 8: Cleaved (activated) caspase 8; C-casp9: Cleaved (activated) caspase 9; STS: Staurosporin. CHO: Inhibitor of Caspase 8 activation.

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: MTT Assay, Activation Assay

Journal: Oncotarget

Article Title: The andean anticancer herbal product BIRM causes destabilization of androgen receptor and induces caspase-8 mediated-apoptosis in prostate cancer

doi: 10.18632/oncotarget.12393

Figure Lengend Snippet: BIRM mediated AR down regulation ( A ) DNA synthesis inhibition in LAPC-4 and LNCaP cells treated with indicated dose of BIRM for 48 h with or without DHT were measured by [ 3 H] Thymidine incorporation assay. ( B ) LAPC-4 cells were transiently transfected with PSA-luc and TK-Renilla. 24 h later, cells were exposed to 10 μl/ml BIRM in medium with 1 nM DHT for indicated time points and promoter activity was monitored by Dual-luciferase assay. Luciferase activity was normalized by Renilla activity and% promoter activity was measured compared to untreated (left panel). LAPC-4 cells were treated with 10 μl/ml BIRM in medium with or without DHT for 48 h and PSA production was monitored by ELISA (Inset). *p = 0.002; ( C ) Androgen dependent LAPC-4 and LNCaP cells cultured in growth medium were exposed to 10 μl/ml BIRM for indicated time point (left panel) or treated with various concentrations of BIRM for 24 h. Cells were harvested after incubation time and AR levels were analyzed by Immunoblotting. Actin was used as loading control. ( D ) LAPC-4 cells were treated with 10 μl/ml BIRM for 24 h and cytosol and nuclear fractions were prepared for immunoblotting to examine the effect of BIRM on AR localization. C: cytoplasmic AR, N: Nuclear AR in (A) and (C), the data presented as means ± SEM from two or more independent experiments in triplicate.

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: DNA Synthesis, Inhibition, Thymidine Incorporation Assay, Transfection, Activity Assay, Luciferase, Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation

Journal: Molecular Therapy

Article Title: 6BIO Enhances Oligonucleotide Activity in Cells: A Potential Combinatorial Anti-androgen Receptor Therapy in Prostate Cancer Cells

doi: 10.1016/j.ymthe.2016.10.017

Figure Lengend Snippet: 6BIO Targets the AR and Inhibits AR Signaling in Prostate Cancer Cells (A–C) Relative AR, PSA, and TMPRSS3 gene expression as determined by RT-PCR from total RNA derived from (A) LNCaP, (B) LAPC-4, and (C) 22Rv1 cells treated with 1 μM 6BIO for 24 hr. Values were normalized to β-actin mRNA expression and expressed as the mean ± SD. n = 3. ***p

Article Snippet: Each siRNA was delivered to LAPC-4 cells, to make a final concentration of 50 nM, 4 hr after seeding, using Lipofectamine 3000 (Thermo Fisher Scientific) as recommended by the manufacturer.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: EJNMMI Research

Article Title: Evaluation of androgen-induced effects on the uptake of [18F]FDG, [11C]choline and [11C]acetate in an androgen-sensitive and androgen-independent prostate cancer xenograft model

doi: 10.1186/2191-219X-3-31

Figure Lengend Snippet: Percentage changed SUV max and SUV meanTH values of tracer uptake in control and castrated LAPC-4 tumour-xenografted mice. Percentage changed SUV values of ( a ) [ 18 F]FDG, ( b ) [ 11 C]choline and ( c ) [ 11 C]acetate in control ( n = 6) and castrated ( n = 7) LAPC-4 tumour xenografted mice. Data are presented as mean ± SEM. Significant differences between both groups are indicated with p

Article Snippet: LAPC-4 cells were grown in Iscove’s modified Dulbecco’s medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (FBS) (Invitrogen, Life Technologies, Carlsbad, CA, USA), 2 mM l -glutamine (Invitrogen), and 100 μg/ml streptomycin and 100 U/ml penicillin (Invitrogen).

Techniques: Mouse Assay

Journal: EJNMMI Research

Article Title: Evaluation of androgen-induced effects on the uptake of [18F]FDG, [11C]choline and [11C]acetate in an androgen-sensitive and androgen-independent prostate cancer xenograft model

doi: 10.1186/2191-219X-3-31

Figure Lengend Snippet: H E staining of representative LAPC-4 and 22Rv1 tumour tissues grown in control and castrated animals. Original magnification is ×100. AD did not affect tumour cell morphology, and necrosis occurred equally in both experimental groups of the animal models. N, necrosis.

Article Snippet: LAPC-4 cells were grown in Iscove’s modified Dulbecco’s medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (FBS) (Invitrogen, Life Technologies, Carlsbad, CA, USA), 2 mM l -glutamine (Invitrogen), and 100 μg/ml streptomycin and 100 U/ml penicillin (Invitrogen).

Techniques: Staining