Muscadine Grape Skin Extract Induces an Unfolded Protein Response-Mediated Autophagy in Prostate Cancer Cells: A TMT-Based Quantitative Proteomic Analysis

Abstract

Muscadine grape skin extract (MSKE) is derived from muscadine grape (Vitis rotundifolia), a common red grape used to produce red wine. Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) that serves as a survival mechanism to relieve ER stress and restore ER homeostasis. However, when persistent, ER stress can alter the cytoprotective functions of the UPR to promote autophagy and cell death. Although MSKE has been documented to induce apoptosis, it has not been linked to ER stress/UPR/autophagy. We hypothesized that MSKE may induce a severe ER stress response-mediated autophagy leading to apoptosis. As a model, we treated C4-2 prostate cancer cells with MSKE and performed a quantitative Tandem Mass Tag Isobaric Labeling proteomic analysis. ER stress response, autophagy and apoptosis were analyzed by western blot, acridine orange and TUNEL/Annexin V staining, respectively. Quantitative proteomics analysis indicated that ER stress response proteins, such as GRP78 were greatly elevated following treatment with MSKE. The up-regulation of pro-apoptotic markers PARP, caspase-12, cleaved caspase-3, -7, BAX and down-regulation of anti-apoptotic marker BCL2 was confirmed by Western blot analysis and apoptosis was visualized by increased TUNEL/Annexin V staining upon MSKE treatment. Moreover, increased acridine orange, and LC3B staining was detected in MSKE-treated cells, suggesting an ER stress/autophagy response. Finally, MSKE-mediated autophagy and apoptosis was antagonized by co-treatment with chloroquine, an autophagy inhibitor. Our results indicate that MSKE can elicit an UPR that can eventually lead to apoptosis in prostate cancer cells.

Fig 1. TMT labeled proteins identified by MS and organized according to biological processes.

Fig 1 shows proteins up-regulated or down-regulated at least 1.2 fold in C4-2 cells treated with 20 μg/ml MSKE as identified by the MASCOT database.

Fig 2. Quantitative Western blot of key ER stress markers.

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.

Fig 3. MSKE treatment induces autophagy.

(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 > p value > 0.01, ** means 0.01 > p value > 0.001.

Fig 4. MSKE induces autophagy-mediated apoptosis.

(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 > p value > 0.01, ** means 0.01 > p value > 0.001, and *** means p value < 0.001.

Fig 5. Proposed model highlighting unfolded protein response pathway with pro-apoptotic protein signatures triggered by ER stress in MSKE treated C4-2 prostate cancer cells.

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.