Western blot <i>in vivo</i> analysis.

<p>Female nude mice bearing MDA-MB-231 tumors were treated with either vehicle (control) or the compounds AuD6 and AuD8 at 1 mg kg⁻¹ d⁻¹. Tumors were collected and the corresponding tissues prepared for Western blot analysis after either 13-day treatment (<b>A</b>) or 27-day treatment (<b>B</b>) [I and II denote distinct experiments]. Tissues were also prepared for the assays of caspase-3 activity (<b>C</b>) and of proteasomal CT-like activity (<b>D</b>) after 27 days of treatment.</p

Antitumor activity <i>in vivo</i> on MDA-MB-231 xenografts.

<p>Female nude mice bearing MDA-MB-231 tumors were treated with either vehicle (control) or the compounds AuD6 and AuD8 at 1 mg kg⁻¹ d⁻¹. <b>A,</b> Inhibition of xenograft growth by both complexes. Tumor volumes were measured every other day using a caliper. Points represent the mean ± SD (bars) of seven mice per group. The insert depicts representative tumors from each treatment group; * = p<0.05. <b>B</b>, If only the most responsive mice are considered, the xenograft growth inhibition is greater. The insert shows average weights of mice over time; ** = p<0.01. <b>C</b>, Immunohistochemical p27 and TUNEL staining of tumor samples indicates proteasome inhibition and apoptosis as a result of both compounds. Stronger p27 staining is observed following AuD8 treatment, and more TUNEL positive cells are observed following AuD6 treatment. Brown colored cells are considered positive.</p

B-DIM activates the AMPK pathway, resulting in inhibition of AR and PSA expression and induction of apoptosis in prostate cancer cells.

<p>Human prostate cancer C4-2B (A) or LNCaP (B) cells were treated with indicated concentrations of B-DIM for 3 hours to measure protein levels of phosphor-AMPKα, AMPKα, phosphor-Raptor, phosphor-ACC, phosphor-mTOR, or for 24 hours to measure protein levels of AR, PSA or PARP cleavage by Western blot analysis. Measurement of β-actin served as loading controls. The numbers underneath the Western results of phosphor-AMPKα indicate quantified normalized phosphor-AMPKα and β-actin ratios.</p

Western blot and morphological analysis (concentration-dependent study).

<p><b>A</b>, Western blot analysis of breast cancer MDA-MB-231 cell extracts. Cells were treated with the complexes AuD6 and AuD8 at the indicated concentrations for 24 h. <b>B</b>, Western blot analysis of the p27 protein amount in MDA-MB-231 cell extract after treatment with the compound AuD8 at the indicated concentrations for 24 h. The solvent DMSO was used as a control while GAPDH as a loading control. <b>C</b>, Apoptotic morphological changes of MDA-MB-231 cells after treatment with AuD6 and AuD8 at the indicated concentrations for 24 h (phase contrast imaging, 100× magnification).</p

<i>In vitro</i> inhibition of proteasome.

<p>IC₅₀ values (µM±SD) obtained for the three proteasomal activities on the purified 20S proteasome and on MDA-MB-231 cell extract after 2 h incubation.</p

Annexin–V FITC/PI assay.

<p>MDA-MB-231 cells were treated with the complexes AuD6 and AuD8 (20 µM) for 16 and 24 h. Then, cells were labeled with Annexin–V FITC and PI and analyzed by flow cytometry in order to evaluate the percentage of apoptotic cells. Apoptotic cells at early stage occur in the lower right quadrant while apoptotic cells at late stage set in the up-right part. The percentage in the lower left quadrant is due to viable cells whereas the upper left part to non-apoptotic cell death.</p

Chemical structures of investigated gold(III)-based compounds AuD6 (A) and AuD8 (B).

<p>Chemical structures of investigated gold(III)-based compounds AuD6 (A) and AuD8 (B).</p

B-DIM activates AMPK signaling and inhibits AR expression in prostate tumor xenograft tissues.

<p>ICR SCID mice were implanted with C4-2B cells and treated with 5 mg/mouse of B-DIM by oral gavages daily for 4 wks. Tumor tissues were analyzed by immunohistochemistry using anti-phosphor-AMPKα (T172), phosphor-ACC (S79) or AR antibodies. The stained sections were visualized under the microscope (400×amplification). Unlike solvent-treated control, mice treated with B-DIM presented with activation of AMPK and significant loss of AR in tumor sections.</p

Treatment with B-DIM or metformin inhibits prostasphere-forming ability.

<p>C4-2B cells were treated with indicated doses of B-DIM (A, B) for 6 days. Treatment with different concentrations of metformin served as controls (C, D). After 6 days, prostasphere numbers were counted under the microscope and the proportion of sphere-generating cells was calculated by dividing the number of cells seeded by the number of prostaspheres (A, C). Prostaspheres from C4-2B cells treated with B-DIM (B) or metformin (D) were photographed and the results showed that 10 and 25 µM of B-DIM significantly reduced size and numbers of prostaspheres. n = 6, * P<0.05, ** P<0.01.</p

AMPK inhibitor Compound C can block AMPK activation by B-DIM in prostate cancer cells.

<p>Prostate cancer C4-2B and LNCaP cells were pre-treated with 20 µM of AMPK inhibitor Compound C (CC) for 6 hours, followed by co-treatment with indicated concentrations of B-DIM for 3 hours. Cell extracts of the treated cells were immunoblotted for anti-phosphor-AMPKα, phosphor-ACC or β-actin antibodies.</p