Nelfinavir exhibits anti-proliferative effects in breast cancer cell lines.

<p><b>(a)</b> A Cell viability assay was performed in MDA-MB231, MCF-7 and normal breast epithelial cells, treated with indicated concentration of nelfinavir for 24 and 48 hours (* p < 0.05 vs ctrl). <b>(b)</b> Growth curves for 10 μM nelfinavir at indicated times in MDA-MB231, MCF-7 and breast epithelial cells. The data show the mean ± S.D. of three independent biological experiments. Significant differences in cell viability percentage were observed in cell treated with the drug compared to control cells (* p < 0.05 vs ctrl).</p

Nelfinavir causes a dissociation of Akt/HSP90 complex and Akt degradation.

<p><b>(a)</b> MDA-MB231 cells were treated with 10 μM nelfinavir for 24 hours and mRNA expression levels of three Akt isoforms were analyzed by qRT-PCR as indicated in Material and Methods. The values represent means ± S.D. of three independent biological experiments. <b>(b)</b> MDA-MB231 cells were treated with 10 μM nelfinavir for 24 h and incubated with 0,5 μg/mL cycloheximide for the last 1 hour or 6 hours of treatment. Protein lysates were subjected to western blot analysis for Akt and beta-actin. (* p < 0.05 vs cycloheximide 6h) <b>(c)</b> Cells were treated with nelfinavir for 6 hours, then lysed, immunoprecipitated (IP) using Akt antibody and immunoblotted for HSP90 or Akt. <b>(d)</b> Lysates from MDA-MB231 cells co-treated with nelfinavir for 24 hours and proteasomal inhibitor MG132 (10 μM) for the last 8 hours of drug treatment were subjected to western blot for Akt and beta-actin. Akt signals following the indicated treatments were quantified by densitometry and normalized on beta-actin values. The values are representative of three independent biological experiments (* p < 0.05 vs ctrl; # p< 0.05 vs Nelfinavir).</p

Nelfinavir regulates SOD activity and expression in a time-dependent manner.

<p><b>(a)</b> Breast cancer cells were treated with 10 μM nelfinavir at indicated times and SOD activity (relative to activity of untreated cells) was analyzed as indicated in Material and Methods. Values are representative of three independent biological experiments ± S.D., (*p< 0.05 vs ctrl). <b>(b)</b> Protein lysates derived from MDA-MB231, MCF-7, or breast epithelial cells, treated with 10 μM nelfinavir for the indicated time points, were immunoblotted with anti- SOD1, anti-SOD2 and anti-catalase. Beta-actin was used as loading control. <b>(c)</b> MCF-7 cells, treated with 10 μM nelfinavir ± 35 μM tocopherol for 30 minutes or 24 hours, were lysed and subjected to western blot analysis for SOD1. Beta-actin was used as loading control. Densitometric analysis of SOD1 signal relative to beta-actin signal was represented. The values represent the means ± S.D. of three independent biological experiments and are compared to control value (*p< 0.05 vs ctrl).</p

Nelfinavir induces ROS accumulation and lipid peroxidation in breast cancer cells.

<p><b>(a)</b> MDA-MB231, MCF-7 and primary breast epithelial cells were subjected to 10 μM nelfinavir treatment for 30 minutes-72 hours. ROS production was measured by H2DCF-DA staining and fluorescence intensity was expressed as MFI normalized to untreated cell values. Each value is the mean ±S.D. of three different biological experiments (*p< 0.05 vs ctrl). <b>(b)</b> Cells were treated with 10 μM nelfinavir for the indicated time points, and processed as indicated in Material and Methods. Colorimetric analysis revealed the concentration of MDA (nM), a lipid peroxidation marker. The present data derived from three different biological experiments (*p< 0.05 vs ctrl).</p

Nelfinavir induces necrosis and apoptosis in breast cancer cell lines.

<p><b>(a)</b> MDA-MB231 and MCF-7 cells were treated with 10 μM nelfinavir for indicated time points and cells were subsequently stained with FITC-conjugated annexin V and PI and analyzed by flow cytometry. <b>(b)</b> Western blot analysis was performed in MDA-MB231 and MCF-7 treated with 10 μM nelfinavir at indicated time points and bak, cytochrome c and pro-caspase 9 proteins were revealed using specific antibodies. Beta-actin immunoblotting was used as loading control. Densitometric analysis of proteins signals relative to beta-actin signal was shown. The present data represent the means ± S.D. of three independent biological experiments and compared to control value (* p< 0.05 vs ctrl). <b>(c)</b> The release of cytochrome c in cytosolic extracts after mitochondria removal was evaluated by western blot in MDA-MB231 and MCF-7 treated with 10 μM nelfinavir at indicated time points and. Beta-actin immunoblotting was used as loading control. Densitometric analysis of proteins signals relative to beta-actin signal was shown. The present data represent the means ± S.D. of three independent biological experiments and compared to control value (* p< 0.05 vs ctrl).</p

The nelfinavir-induced Akt/HSP90 complex disruption and tumor cell-death are ROS-mediated.

<p><b>(a)</b> MDA-MB231, MCF7 and breast epithelial cells were treated with 10 μM nelfinavir and 35 μM tocopherol for 6 hours, and equal amounts of protein lysate were immunoprecipitated using Akt antibody followed by immunoblotting with anti-HSP90 and anti-Akt. <b>(b)</b> MDA-MB231 and MCF-7 cells were treated with 10 μM nelfinavir and 35 μM tocopherol for 24 hours and phospho-Akt, Akt, cyclin D, ERα, beta-actin levels were monitored using the respective antibody by western blot on total lysate. Beta-actin immunoblotting was used as a loading control. <b>(c)</b> MDA-MB231 and MCF-7 cells were treated with nelfinavir (for 72 and 24 hours respectively) in the absence or presence of 35 μM tocopherol. Cell-death profile was examined by cytofluorimetric analysis of annexin V/ PI positivity. The cell percentage were reported in corresponding areas of dot-plot. Three different biological experiments confirmed these cell distributions.</p

Long-term ISA27 treatment induces cell cycle arrest and a persistent increase in p21 mRNA levels in U87MG cells.

<p><i>7A) Flow cytometric cell cycle profiling</i>: the figure shows the percentage of untreated and MDM2 inhibitor-treated U87MG cells in G1-, S- and G2/M-phase. <i>7B) p21 mRNA evaluation</i>: ISA27 treatment resulted in a statistically significant increase in p21 mRNA levels at 24, 48 and 72 h.</p

Effect of ISA27, Temozolomide (TMZ) and their fixed-ratio combinations with regard to growth inhibition of U87MG cells.

<p>Data are presented as IC₅₀±S.E.M. Statistical analysis was performed with Student’s <i>t</i>-test.</p>*<p>P<0.05 <i>vs</i> the respective additive group. γ <1 indicates supra-additivity (synergy).</p

ISA27 induces an increase in PUMA mRNA levels, mitochondrial cytochrome c release, and DNA fragmentation.

<p><i>11A) Relative quantification of PUMA mRNA:</i> ISA27 induced a statistically significant increase in PUMA mRNA levels at 24 and 48 h. Nutlin-3 treatment resulted in a statistically significant increase at 72 h. <i>11B) Evaluation of cytosolic cytochrome c content</i>: ISA27-treated cells showed a 25% increase in cytochrome c levels in the cytosolic fraction. Nutlin-3-treated cells did not give statistically significant results. <i>11C) Evaluation of DNA content</i>: Frequency histograms from a representative experiment are shown. ISA27-treated cells showed a significant increase in the percentage of nuclei with hypodiploid DNA content at 72 h compared with control cells. In contrast, Nutlin-3 did not induce significant nuclear DNA fragmentation.</p

U87MG cells did not recover normal growth upon removal of ISA27.

<p>U87MG cells were cultured in the presence of 2.5 µM ISA27 or 10 µM Nutlin-3. After 4 days of MDM2 inhibitor treatment, cell culture medium was replaced with fresh culture medium without the MDM2 inhibitor. The figure shows the number of viable cells that were counted by Trypan blue exclusion after 4 days of MDM2 inhibitor treatment and 1, 2 and 3 days after MDM2 inhibitor removal. Data represent the means of three independent experiments performed in duplicate; bars, SEM.</p