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 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 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

PKA inhibition does not modify PE-induced CaMKII/ERK activation in H9C2 cardiomyoblasts.

<p><b>A:</b> H9C2 cardiomyoblasts were transfected with PKA-I and then stimulated with 100 nm phenylephrine (PE) for 15 min. Total cell extracts were analyzed by western blot for phosphothreonine 286 CaMKII (pCaMKII) and CaMKII with specific antibodies. pCaMKII levels were corrected by CaMKII densitometry. *, <i>P</i> < 0.05 <i>vs</i>. Ctrl. <b>B:</b> H9C2s were stimulated with PE after transfection with PKA-I. Total lysates were analysed by WB for pERK with specific antibody. pERK1/2 levels were corrected by ERK1/2 densitometry. * = p<0.05 vs Ctrl. <b>C:</b> To evaluate that PKA inhibition does not modify CaMKII/ERK interaction, H9C2s were stimulated with 100 nmol/L PE for 15 minutes following 30 min. pretreatment with PKA-I (10 μmol/L). CaMKII was immunoprecipitated from cell lysates using a specific anti- CaMKII antibody, and ERK1/2 was visualized by WB to evaluate its association with CaMKII. ERK1/2 levels were corrected by CaMKII densitometry. * = p<0.05 vs Ctrl. <b>D:</b> To confirm the interaction between CaMKII and ERK in H9C2s, total cell lysates were immunoprecipitated using anti-ERK1/2 antibody, and subjected to western blot using anti-CAMKII antibody. CaMKII levels were corrected by ERK1/2 densitometry. * = p<0.05 vs Ctrl. <b>E:</b> H9C2s were stimulated with PE after trasfection with PKA-I. Subsequently, nuclear extract were prepared from the cells as indicated in the methods section. Nuclear extracts were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were averaged and normalized to histone 3 densitometry. *, <b><i>P</i></b> < 0.05 <b><i>vs</i>.</b> Ctrl. <b>F:</b> To confirm that PE induced ERK nuclear localization was independent from PKA inhibition, H9C2s were transfected with PKA-I and then stimulated with PE. Nuclear extracts were analyzed by WB for total ERK1/2 with specific antibody. ERK1/2 levels were normalized to histone 3 densitometry. *, <b><i>P</i></b> < 0.05 <b><i>vs</i>.</b> Ctrl. Data from all immunoblots were quantified by densitometric analysis. Each data point in all graphs represents the mean±SEM of 3 independent experiments.</p

CaMKII/ERK-dependent regulation of the hypertrophy marker ANF.

<p><b>A:</b> H9C2 cells at ≈ 70% confluence were incubated 1 h at 37°C with 5 mL DMEM containing purified adenovirus at a multiplicity of infection (moi) of 100:1, encoding either the kinase-dead (CaMKII-DN, rCaMKIIdelta, K42M), or the wild type (CaMKII-WT, rCaMKIIdelta) variant of CaMKII or the empty virus as a negative control (Ctr). 48 h after the infection, the cells were stimulated with PE 100 nM for 24 h. Total RNA was isolated from H9C2s using TRIzol reagent, and cDNA was synthesized by means of a Thermo-Script RT-PCR System, following the manufacturer’s instruction. Then ANP gene expression was evaluated by real-time PCR. Results are expressed as mean±SEM from 3 independent experiments. The ratio of fold change was calculated using the Pfaffl method[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130477#pone.0130477.ref036" target="_blank">36</a>]. * = p<0.05 vs Ctrl; # = p<0.05 vs PE. <b>B:</b> The H9C2s infected with adenoviruses encoding wilde type CaMKII (CaMKII-WT) and kinase dead CaMKII (CaMKII-DN) were stimulated with PE 100 nM for 24 h. Total cell lisates were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were corrected by Actin densitometry. Data from the immunoblots were quantified by densitometric analysis.* = p<0.05 vs Ctrl. Each data point in all graphs represents the mean±SEM of 3 independent experiments. <b>C:</b> H9C2 cells were pretreated with CaMK inhibitor KN93 (5 μmol/L), the selective inhibitors AntCaNtide (10 μmol/L) and tat-CN17β (10 μmol/L) and ERK specific inhibitor pathway UO126 (10 μmol/L) for 30 min. and then stimulated with PE (100 nmol/L) for 24 h. cDNA was synthesized from RNA obtained from H9C2s as indicated above. The ANF gene expression was evaluated by real-time PCR. Results are expressed as mean±SEM from 3 independent experiments. The ratio of fold change was calculated using the Pfaffl method[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130477#pone.0130477.ref036" target="_blank">36</a>]. * = p<0.05 vs Ctrl; # = p<0.05 vs PE.</p

The effect of CaMKII selective peptide inhibitors in vivo in SHRs.

<p><b>A:</b> SHRs were subjected to intramural cardiac injections at the age of 13 week and repeated after 7 and 14 days. Transthoracic echocardiography was performed at each drug administration and once more at the day of euthanasia in untreated WKY rats, AntCaNtide SHRs, tat-CN17β SHRs, and control SHRs (SHR) using a dedicated small-animal high-resolution-ultrasound system (Vevo 770, VisualSonics). ANTCaNtide-SHRs and tat-CN17β-SHRs caused reduction of interventricularseptal (IVS) thickness compared to SHR (*<i>P</i><0.05 vs WKY; #<i>P</i><0.05 vs SHR). <b>B, C:</b> At the end of treatment, rats were weighed and then euthanized. Hearts were immediately removed, rinsed 3 times in cold PBS and blotted dry, weighed and then rapidly frozen. The HW/BW ratio (<b>B</b>) and LVM/BW ratio (<b>C</b>) were measured in ANTCaNtide-SHRs and tat-CN17β-SHRs and compared to WKY and SHR hearts (*<i>P</i><0.05 vs WKY; #<i>P</i><0.05 vs SHR). <b>D:</b> Cardiac Performance at the end of the treatment was assessed by ultrasound. All measurements were averaged on 5 consecutive cardiac cycles and analysed by two experienced investigators blinded to treatment (GS, MC). No differences were observed in LVFS and LVEF in ANTCaNtide and tat-CN17β SHRs when compared with SHR (*P<0.05 vs WKY). <b>E:</b> To evaluate the involvement of BP values in CaMKII–dependent regulation of cardiac hypertrophy, SBP and DBP values were assessed in AntCaNtide- and tat-CN17β- treated and control. The pressure values ​​in the graph represent the measurements made at the end of treatment (*<i>P</i><0.05 vs WKY-SBP; #<i>P</i><0.05 vs WKY-DBP). <b>F:</b> At the end of the treatment the total RNA was isolated from myocardial sample using TRIzol reagent, and cDNA was synthesized by means of a Thermo-Script RT-PCR System, following the manufacturer’s instruction. ANP gene expression was evaluated by real-time PCR in AntCaNtide- and tat-CN17β-SHR rats compared with WKY and SHR (*<i>P</i><0.05 vs WKY). Results are expressed as mean±SEM from 3 independent experiments. The ratio of fold change was calculated using the Pfaffl method[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130477#pone.0130477.ref036" target="_blank">36</a>].</p

CaMKII isoforms.

<p><b>A:</b> To evaluate the expression of CaMKII isoforms in ventricular adult cardiomyocytes from WKY rats, the total cell lysates were immunoprecipitated using anti-CaMKII antibody (lane a) or anti-CaMKII α, β γ or δ specific antibodies (lane b) and together to total samples from WKY cardiomyocytes (lane d) and H9C2 cardiomyoblasts (lane e) were analyzed by western blot with anti-CaMKII α, β γ or δ antibodies as indicated. Total extracts from rat brain (lane f), mouse brain (lane g) and mouse heart (lane h) were used as controls. Total lysates from WKY cardiomyocytes with specific antibody without A/G agarose beads were used as negative control (lane c). <b>B:</b> Total RNA from H9C2 cell line and isolated ventricular cardiomyocytes was extracted with standard methods. RT-PCR for CaMKII α, β, γ, and δ was performed as indicated in methods. The representative graph indicates the relative amounts of transcripts for CaMKII isoforms in H9C2s and ventricular adult myocytes from WKY rats. Cycle threshold (Ct) values from 3 independent experiments were normalized to the internal β-actin control. The ratio of fold change was calculated using the Pfaffl method. * = p<0.05 vs CaMKIIα; # = p<0.05 vs CaMKIIβ.</p