Differentially expressed proteins in MSKE-treated and control cultures.

<p>Differentially expressed proteins in MSKE-treated and control cultures.</p

MSKE treatment induces autophagy.

<p>(A) C4-2 prostate cancer cells were treated with MSKE (0 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL) for 72 h. Fixation was performed with methanol/ethanol 1:1 volume followed by washes with 1× PBS. The cells were then exposed to acridine orange (5 μg/ml) for 15 min at 37°C, followed by washes with 1× PBS, prior to counterstaining with DAPI. We observed that treatment with higher concentrations of MSKE (10 and 20μg/ml) showed extensive acridine orange leakage into the cytosol, producing a diffuse yellow color and an increase in lysosomes indicating that MSKE induces cell death via autophagy compared to control. (B) C4-2 cells were treated with MSKE, with and without 20 μM chloroquine, for 72 h. The cells were then exposed to acridine orange (5 μg/ml) for 15 min at at 37°C, followed by washes with 1× PBS, prior to counterstaining with DAPI. Chloroquine treatment reversed the effects of MSKE. (C) Immunofluorescence staining for LC3B was performed on C4-2 cells treated with MSKE plus or minus chloroquine for 72 h. (D) Western blot analysis for LC3B was performed on C4-2 cells treated with MSKE plus/minus chloroquine. Image J analysis was performed to plot the ratios of LC3BI and LC3BII relative to actin loading control. The results are representative of experiments that have been performed in triplicate at least two times independently. Graphical data represents three independent experiments; * means 0.05 > <i>p</i> value > 0.01, ** means 0.01 > <i>p</i> value > 0.001.</p

Proposed model highlighting unfolded protein response pathway with pro-apoptotic protein signatures triggered by ER stress in MSKE treated C4-2 prostate cancer cells.

<p>MSKE treatment of C4-2 cells promoted an unfolded protein response (UPR) pathway in a mitochondria-specific stress response (UPRmt) with pro- and anti-apoptotic protein signature triggered by ER stress. Strong ER stress and activation of the UPR initiate apoptosis. In contrast, mild UPR activation induces a beneficial ER homeotic response by reducing the load of misfolded proteins and by activating cellular protective mechanisms like autophagy. PERK mediates phosphorylation of eIF2α and ATF4-dependent transcriptional activation of autophagy proteins. The model highlights insufficient folding or degradation capacity in the mitochondria, contributing to apoptosis.</p

MSKE induces autophagy-mediated apoptosis.

<p>(A) C4-2 prostate cancer cells were treated with increasing concentrations of 0μ g/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL for 72 h. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% sodium citrate and 0.1% Triton X. DNA fragmentation was determined by TdT-mediated dUTP nick end labeling (TUNEL). TUNEL assay (green channel). DAPI (blue channel) is used to locate the nuclei of the cells. (B) Cells were stained with Annexin V- Alexa Fluor 488 and PI and analyzed by flow cytometry following treatment with 5 μg/mL MSKE with or without 20 μM chloroquine. Percent of cells in the lower right quadrant that represent Annexin V ⁺/PI^- or early apoptotis and cells in the upper right quadrant that represent Annexin V ⁺/PI⁺ or late apoptotis was graphed. (C) Western blot analysis was performed to examine pro-apoptotic markers (Bax, cleaved caspase-3 and -7) and anti-apoptotic marker (BCL2) following treatment with MSKE in the presence or absence of 20 μM chloroquine. Actin was utilized as a loading control. (D) Results of western blot were analyzed using Image J and graphed. The experiments were performed in triplicate at least two times independently. Graphical data represents three independent experiments; * means 0.05 > <i>p</i> value > 0.01, ** means 0.01 > <i>p</i> value > 0.001, and *** means <i>p</i> value < 0.001.</p

Quantitative Western blot of key ER stress markers.

<p>Western blot analysis in C4-2 cells treated with 20 μg/ml MSKE as compared to ethanol-treated controls. As a loading control total protein from Ponceau S staining was assessed. Expression of ER stress markers IRE1-alpha and GRP78 and pro-apoptotic markers DFF45, PARP and caspase-12 was analyzed and quantification of western blot analysis performed using Image J, NIH. The standard deviation was used to assess data dispersion.</p