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

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.


Introduction
Natural products with anticancer activities have gained increased attention due to their favorable safety and efficacy profiles as possible therapeutic agents that are not toxic to the surrounding healthy tissue. Of special interest is to determine how these novel compounds affect the endoplasmic reticulum (ER), an essential organelle for proper protein folding. Different physiological and pathological conditions can perturb protein folding in the ER, leading to a condition known as ER stress. ER stress activates the unfolded protein response (UPR), a complex intracellular signal transduction pathway that reestablishes ER homeostasis through adaptive mechanisms involving the stimulation of autophagy. The goal of the UPR is to restore optimal ER function. However, when persistent, ER stress can switch from the cytoprotective functions of UPR and autophagy to promote cell death mechanisms [1].
Flavonoids from naturally rich fruits modulate cell cycles, induce apoptosis, and inhibit extracellular regulated kinase (ERK) phosphorylation; mechanism that are linked to their confirmed anti-carcinogenic, anti-proliferative, co-chemotherapeutic, and estrogenic effects [2]. Numerous reports support the fact that many natural products play a positive role in cancer prevention and treatment by adjusting the oxidative stress response, inhibiting cancer cell proliferation and modulating apoptosis and autophagy; mechanisms that control cellular fate by regulating the turnover of organelles and proteins [3,4]. In general, autophagy blocks the induction of apoptosis, while apoptosis-associated caspase activation shuts off the autophagic process. In spite of its role as a self-digestion mechanism, autophagy is mainly activated to protect against cell death [5]. However, just like in the case of the UPR, stimulation of autophagy can be required to activate the cell death machinery under certain circumstances [6].
Anthocyanin compounds, the main bioactive components found in muscadine grape skin extract (MSKE) from the muscadine grape (Vitis rotundifolia), inhibit prostate cancer cell growth and promote apoptosis in vitro without toxicity to normal prostate epithelial cells [7]. Unlike other grape varieties, the phytochemical constituents of muscadine grapes have a higher concentration of anthocyanin 3,5-diglucosides, ellagic acid, and ellagic acid precursors [8]. In the case of purple skinned muscadine grapes, the anthocyanins are primarily delphinidin-3,5-diglucoside; cyanidin-3,5-diglucoside; and petunidin-3,5-diglucoside [8]. Anthocyanin 3,5 diglucosides are known to inhibit invasion in human hepatoma cells [9], and induce apoptosis and inhibit invasion in colorectal cancer cells [10]. MSKE has been shown to decrease tumor incidence, promote growth inhibition and stimulate cell death in various human cancer cell lines [7]. It can also revert the epithelial mesenchymal transition process [11]. Furthermore, the in vivo therapeutic effects of MSKE against prostate adenocarcinoma (PCa) are currently being investigated in a clinical trial [12].
In this study, proteomics, Western blot, acridine orange, Annexin V and TUNEL staining were used to determine global effects of MSKE on prostate cancer cells using C4-2 cells as a model. Our results revealed that MSKE regulated the expression of proteins important for ER stress response (GRP78, PDIA4, PDIA6, EIF2, EIF4 and Ire-1 alpha) and autophagy (ACIN1, PI4KA, PGK2 and MTDH). Pro-apoptotic markers were up-regulated, while anti-apoptotic protein BCL2 was down-regulated in the presence of MSKE; these effects were antagonized by co-treatment with chloroquine, suggesting that MSKE may promote ER stress-driven autophagic response leading to apoptosis.
Desalination of fractionated samples. A C 18 solid phase extraction SPE column (Thermo Fisher Scientific, Waltham, MA) was used to desalt all collected fractions as previously described [15]. Briefly, the 10 combined fractions were each adjusted to a final volume of 1 ml in a 0.25% trifluoroacetic acid TFA aqueous solution. The C 18 SPE column was preconditioned with 1 ml acetonitrile and eluted in approximately 3 min before it was rinsed with 3 × 1 ml 0.25% TFA. The fractions were loaded on to the top of the SPE cartridge column slowly, and were reloaded once again to decrease lost peptide during column binding. Columns were washed with 4 × 1 ml 0.25% TFA aliquots before the peptides were eluted with 3 × 400 μl 80% acetonitrile/0.1% formic acid (aqueous). The eluted samples were lyophilized prior to the liquid chromatography mass spectrometry LC-MS/MS analysis.
LC-MS/MS analysis. Peptides were analyzed on an LTQ-Orbitrap XL (Thermo Fisher Scientific, Waltham, MA) instrument interfaced with an Ultimate 3000 Dionex LC system (Dionex, Sunnyvale, CA). High mass resolution was utilized for peptide identification and highenergy collision dissociation (HCD) was used for reporter ion quantification as previously described [15]. Briefly, the reverse phase LC system contained a peptide Cap-Trap cartridge (0.5 × 2 mm) (Michrom BioResources, Auburn, CA) and a pre-packed BioBasic C 18 PicoFrit analytical column (75 μm i.d. × 15 cm length, New Objective, Woburn, MA) fitted with a For-tisTip emitter tip. Samples were loaded onto the trap cartridge and washed with mobile phase A (98% H 2 O, 2% acetonitrile, and 0.1% formic acid) for concentration and desalting. Peptides were eluted over 180 min from the analytical column via the trap cartridge by using a linear gradient of 6 to 100% mobile phase B (20% H 2 O, 80% acetonitrile, and 0.1% formic acid) at a flow rate of 0.3 μl/min. The Orbitrap mass spectrometer was operated in a data-dependent mode in which each full MS scan (60,000 resolving power) was followed by six MS/MS scans where the three most abundant molecular ions were dynamically selected and fragmented by collision-induced dissociation with a normalized collision energy of 35% and subsequently scanned by HCD-MS 2 with a collision energy of 45% as previously described [15]. Only the 2+, 3+, and 4+ ions were selected for fragmentation by collision-induced dissociation and HCD.

Database Search and TMT Quantification
The protein search algorithm SEQUEST was used to identify unique protein peptides using the Proteome Discoverer data processing software (version 1.2, Thermo Fisher Scientific, Waltham, MA). The ratios of TMT reporter ion abundances in MS/MS spectra generated by HCD from raw data sets were used For TMT quantification. Fold changes in proteins between control and treatment were calculated as previously described [15].

Western Blot
20-30 μg of proteins extracted with RIPA buffer were analyzed by SDS PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with 5% non-fat dry milk in 1 × TBST buffer (BioRad, Hercules, CA) and incubated with primary antibodies overnight at 4°C. The following primary antibodies were used at a 1:1000 dilution LC3B, cleaved caspase-3, cleaved caspase-7, BAX, and BCL2 (Cell Signaling Technologies Danvers, MA.), anti-caspase-12, Ire-1α, GRP78, DFF45, and PARP (Abcam, Cambridge, MA). Anti-β-actin (1:500, Sigma-Aldrich, St. Louis, MO) were used as a loading control. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma Aldrich, St Louis, MO) were used and protein bands were visualized by a chemiluminescence method using the SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermo Fisher Scientific, Waltham, MA). Images were analyzed with the ImageJ image processing program version 1.50b (National Institutes of Health, Bethesda, MD) to access the differential expression of key ER stress and apoptotic markers.
Immunofluoresence for LC3B 5 × 10 3 cells were plated into Nunc 8-well chamber slides (Bio-Tek Instruments, Winooski, VT). Cells were serum-starved for 4 h and treated with 0 μg/mL or 20 μg/mL of MSKE with or without 20 μM chloroquine co-treatment for 72 h. Fixation was performed with methanol/ethanol (1:1 volume) for 5 min, followed by washes with 1 × PBS. Subsequently, slides were incubated with primary antibody (LC3B) at 1:50dilution in Dako antibody diluent solution for 1 h at room temp. Slides were washed with 1× TBS-T (Dako, Camarillo, CA), then incubated with secondary antibody in the dark for 1 h at room temp. Anti-rabbit Oregon green 488 was utilized as a secondary, and slides were then counterstained with 1 μg/ml DAPI (Santa Cruz Biotechnology, Santa Cruz, CA) before they were fixed with Fluorogel mounting medium (Electron Microscopy Sciences, Hatfield, PA). Fluorescence microscopy was performed at 40 × oil magnification on a Zeiss fluorescence microscope equipped with the AxioVision (release 4.8) imaging software and the ApoTome.2 optical sections system (Carl Zeiss Microscopy GmbH, Jenna, Germany).

Annexin V/Cell Death Apoptosis Kit
Annexin V was also used to detect apoptosis. Briefly C4-2 Cells were serum starved for 4 h and treated with different concentrations of MSKE (0 μg/mL, 5 μg/mL) for 72 h, with or without 20 μM chloroquine. Apoptosis was assessed by Alexa Fluor 488 Annexin V and propidium iodide double staining and flow cytometry was performed using Accuri C6 Flow Cytometer (Accuri Cytometers Inc., Ann Arbor, MI.) according to the manufacterer instructions. Cells that stained positive for Annexin V-Alexa Fluor 488 and negative for PI (Alexa Fluor 488 + / PI -) were considered to be undergoing early apoptosis; Alexa Fluor 488 + /PI + were considered as late apoptosis; Alexa Fluor 488 -/PIconsidered non-apoptotic (viable). Experiments were performed in triplicate in two independent experiments, and a representative result displayed in the form of early/late apoptosis graphs.

Statistical Analysis
Cytotoxicity assays and proteomics analyses were performed in triplicate, and similar results were obtained on at least three separate studies. Statistical analysis was performed using paired or unpaired t-tests, as appropriate, using the GraphPad PRISM v6.03 statistical software (GraphPad Software, La Jolla, CA). A p value of 0.05 was considered statistically significant and all data are presented as means ± standard error and range.

Results
Proteomic Profiling of C4-2 Cells Treated with MSKE with a Gel-Free Isobaric Labeling TMT Quantitative Proteomic Approach The differential expression of proteins between untreated (0.1% ethanol) and treated (20 μg/ml MSKE) C4-2 cells was determined based on a gel-free isobaric labeling TMT quantitative proteomic approach for further validation and identification of novel proteins. Over 1,855 proteins were identified from control and treated C4-2 cell lysates. Among them, 465 significantly differentially expressed proteins contained TMT signals that were used to determine protein expression ratios between treated and control C4-2 cells. Proteins up-regulated or down-regulated at least 1.2 fold were organized according to biological processes (Fig 1). The detailed information including protein ID, gene name, number of amino acids, molecular weight, calculated pI, description, coverage samples, protein expression, change folds, and p-values are shown in S1 Table. Proteins that had a fold change of at least 1.2 and p-values 0.05 were closely examined and are listed in Table 1. MSKE-treated C4-2 cells expressed 254 up-regulated and 211 down-regulated proteins when compared to C4-2 control cells.

MSKE Induces Expression of ER Stress Mediated Pro-Apoptotic Response Proteins
Western blot was used to measure the expression of proteins associated with ER stress response. Key ER stress response markers IRE-1 alpha and GRP78 (Fig 2) were up-regulated when compared to control cells. Apoptotic markers PARP, caspase-12 and DFF45 (Fig 2) were up-regulated in cells exposed to MSKE. These data suggest that MSKE may induce ER stress and UPR-mediated apoptosis.

MSKE Treatment Induces Autophagy
Since MSKE induced expression of ER stress response proteins, we sought to determine if treatment with MSKE induces autophagy. We treated C4-2 cells with 5, 10 or 20 μg/ml MSKE for 72 h and stained with acridine orange. Treatment with higher concentrations of MSKE (10 and 20 μg/mL) showed extensive acridine orange leakage into the cytosol (orange staining), indicating that MSKE induces autophagy (Fig 3A). Since the 20 μg/mL MSKE treatment showed extensive acridine orange staining we decided to see if co-treatment with chloroquine, a known autophagy inhibitor would reverse the effects of MSKE on C4-2 cells. Co-treatment with 20 μg/mL MSKE and 20 μM chloroquine lead to a decrease in acridine orange staining compared to 20 μg/mL MSKE alone, indicating that MSKE may promote autophagy (Fig 3B). To further validate a role for MSKE in autophagy, we also performed immunofluorescence and western blot with the autophagic marker, LC3B. There was increased punctate staining for  LC3B in MSKE plus chloroquine co-treatments, which indicates that chloroquine prevents fusion of autophagosomes to lysosomes, and thus causes an accumulation of autophagosomes (Fig 3C). Similarly, western blot analysis showed increased LC3BII expression in MSKE plus chloroquine treatments indicating that chloroquine had inhbitied MSKE-mediated autophagy and caused accumulation of LC3BII lipidation products (Fig 3D). Of note, we did not observe increased LC3BII with MSKE treatment alone (Fig 3D), possibly due to high autophagic flux that can sometimes not be detected unless one includes an autophagy inhibitor as previously reported [16].

MSKE Promotes Autophagy-Mediated Apoptosis
To confirm that MSKE causes apoptosis, we treated the C4-2 cells with 5, 10, or 20 μg/ml MSKE and performed a TUNEL assay. Increased TUNEL staining (green) was observed with higher doses of MSKE (Fig 4A). These findings correlate with the Western blot analysis where up-regulation of pro-apoptotic markers PARP and caspase-12 was observed (Fig 2). These results support the hypothesis that MSKE induces apoptosis in C4-2 prostate cancer cells. To support the findings in the TUNEL assay and show that there was a correlation between autophagy and apoptosis, we performed quantitative apoptosis assay using flow cytometry via the Alexa Fluor 488 Annexin V in the presence or absence of chloroquine (autophagy inhibitor). Graphical representation of the apoptosis assay showed that treatment with MSKE led to an increase of apoptosis which was abrogated by treatment with chloroquine ( Fig 4B). Western blot analysis confirmed these findings by showing that MSKE-mediated increase in pro-apoptotic proteins BAX, cleaved caspase-3 and -7, and decrease in anti-apoptotic protein BCL2 was abrogated by co-treatment with chloroquine (Fig 4C and 4D). Therefore, MSKE-mediated autophagy leads to apoptosis.

Discussion
We investigated the impact of MSKE on the C4-2 proteome using a quantitative TMT isobaric labeling approach with subsequent protein identification by mass spectrometry. Changes in C4-2 cells were also examined by Western blot. Autophagy and apoptosis were determined by acridine orange, TUNEL/Annexin V analyses, respectively. The results indicated that MSKE significantly regulated components of the UPR stress inducing pathways that include chaperones, ER stress antioxidant enzymes, proteolytic enzymes, cytoskeletal proteins, as well as enzymes involved in autophagy or apoptosis. Autophagy is a dynamic process, whereby cytoplasmic proteins and cellular organelles are enveloped in autophagosomes and degraded by fusion with lysosomes for amino acid and energy recycling [17]. There is evidence that autophagy can play a critical role in cellular survival [18] but, it is frequently activated in tumor cells following anticancer therapies such as drug treatment and gamma-irradiation [19]. Apoptosis is defined as an active, fixed-pathway process of cell death characterized by cell shrinking, cytoplasmic condensation, ladder DNA degradation, and nuclear fragmentation resulting in the formation of apoptotic bodies. Initial studies on the mechanism of action of MSKE, suggest the ability to induce apoptosis via activation of the caspase cascade. The increase of apoptosis after MSKE treatment was confirmed with the TUNEL and Annexin V assays, in agreement with previous studies that have shown that MSKE promotes apoptosis in prostate cancer cells [7]. Our current study identified several proteins associated with apoptosis triggered by MSKE treatment such as STEAP2, PDCD6, MTDH, RAB5B, and cytochrome C oxidase (Table 1). STEAP2, also known as 6-transmembrane protein of the prostate 1 (STAMP1) is overexpressed in several types of human cancers, namely prostate, bladder, colon, pancreas, ovary, testis, breast, and cervix, but its clinical significance and role in cancer cells is still unclear [20]. STEAP2 participates in a wide range of biological processes, including molecular trafficking in the endocytic and exocytic pathways. It also controls cell proliferation and apoptosis. STEAP2 was down-regulated in C4-2 cells. It has been shown that STEAP2 blockage has a 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. pro-apoptotic role that causes mitochondrial damage, and decreases cell proliferation and glucose uptake [20]. MTDH was up-regulated by 1.91 fold upon MSKE treatment in C4-2 cells. MTDH has been proposed to promote tumor progression through the integration of multiple signaling pathways including ras, myc, Wnt, PI3K/AKT, and NF-κB in various types of cancer [21][22][23][24].
The nuclear protein Apoptotic Chromatin Condensation Inducer 1 (ACIN1) is known to function in DNA fragmentation and is activated during apoptosis by caspase-3. It also plays an active role in spliceosome assembly because it is a critical subunit of an apoptosis and splicingassociated protein (ASAP) complex [25,26]. ACIN1 was down-regulated in our study by -1.60 fold and we speculate that MSKE induced an anti-apoptotic/survival response in C4-2 cells by down-regulating ACIN1. Taxilin protein (TXLNA), a binding partner of the syntaxin family that functions as a central player in the intracellular vesicle traffic, and is a marker of testicular injury [27] was also upregulated upon MSKE treatment.
Several results support the induction of apoptosis by MSKE via autophagy. The acridine orange staining showed that induced autophagy increased with MSKE treatment in a dosedependent manner. Additionally, ER stress was significantly induced upon MSKE treatment in C4-2 cells as seen in the up-regulation of several stress-related proteins (e.g., GRP78, PDIA4, PDIA6, eIF2B, eIF4EBP1). We also identified COL4A3BP whose down-regulation sensitizes cancer cells to multiple cytotoxic agents, potentiating ER stress [28]. Lysosomal inhibitors such as chloroquine that inhibit acidification inside the lysosome or inhibit autophagosome-lysosome fusion and can block the degradation of LC3BII, leading to the accumulation of LC3BII, are good indicators for autophagy [29]. Co-treatment with MSKE plus chloroquine in C4-2 cells increased LC3B punctate staining as shown by immunofluorescence, and increased LC3-BII expression as shown by western blot analysis, proving that MSKE promotes autophagy which is blocked by chloroquine leading to accumulation of LC3BII. We speculated, however, that at 72 h we failed to see increased LC3BII with MSKE treatment alone due to high autophagic flux. This problem that sometimes arises when LC3BII expression does not increase when assayed at a certain time point as expected due to high autophagic flux is discussed in great detail with the conclusion that it is better to gauge autophagy by examining autophagosome accumulation in the presence of chloroquine [16]. We also demonstrated that chloroquine was able to reverse the extensive acridine orange leakage into the cytosol.
Degradation of cytosolic proteins in lysosomes is a hallmark of autophagy [30]. The absence of chromatin condensation is another characteristic of autophagy and indicative of apoptosis [31]. Recent studies have shown that a variety of anticancer therapies (including those that stimulate ER stress) activate autophagy in tumor cells, which has been proposed to either enhance cancer cell death or act as a mechanism of resistance to chemotherapy [32]. Autophagy works as a tumor suppression mechanism by removing damaged organelles/proteins, limiting cell growth and minimizing genomic instability [33]. Phosphatidylinositol 4-Kinase (PI4KA), a protein involved in both nonselective and selective types of autophagy [34], was upregulated in MSKE-treated C4-2 cells. The expression of metadherin (MTDH), also known as astrocyte-elevated gene-1, a protein that may protect normal cells from serum starvationinduced death through protective autophagy [35], increased 1.91 fold in the presence of MSKE. In addition, phosphoglycerate kinase (PGK2), a cytosolic and glycolytic marker, was downregulated by MSKE in C4-2 cells, suggesting that it may have been taken up into autophagic bodies [36].
ER stress was evident by the up-regulation of key ER stress markers like GRP78 and PDIA4, A6. Proper protein folding, maturation, and stabilization of the nascent protein in the ER requires a highly oxidizing ER environment, which is essential for the diverse post-translational and co-translational modifications (e.g., glycosylation, disulfide bridge formation) to which proteins are subjected after entering the ER. These processes are assisted and monitored by several resident chaperones and binding proteins, including glucose-regulated proteins like GRP78, which was up-regulated in the presence of MSKE. Folding enzymes, such as the thioredoxin-like protein (PDI), oxidize cysteine residues in nascent proteins (i.e., oxidative folding) resulting in the formation of intra-and intermolecular disulfide bonds. MSKE resulted in ER stress pathway activation and subsequent translation initiation component activation (i.e., eIF4EPB, eIF2). Moreover, the upregulation of pro-apoptotic markers MTDH, caspase 12, Cytochrome C oxidase, PDCD6 and NQO1, along with the increased apoptosis visualized by TUNEL and Annexin V staining, suggest that the ER stress response might eventually trigger apoptosis.
Overexpression of MTDH, also known as LYRIC, is observed in a variety of cancers and is involved in cancer initiation, proliferation, invasion, metastasis and chemoresistance [37]. MTDH also activates the PI3K/Akt pro-apoptotic pathway in cancer [38] and its inhibition induces apoptosis in prostate cancer cells [39]. Hudson et al. have also shown that treatment with MSKE activates the PI3K/Akt pro-apoptotic pathway in prostate cancer cells [7].
The upregulation of cytoplasmic C oxidase and Glyoxalase I (GLO1) suggests that MSKE also interferes with glycolysis and mitochondrial metabolism in C4-2 cells. GLO1 is a ubiquitous cellular defense enzyme involved in detoxification. GLO1 expression may protect cells against methylglyoxal-dependent protein adduction and cellular damage associated with diabetes, cancer, and chronological aging. GLO1 upregulation has been shown to play a pivotal role in glycolytic adaptations of cancer cells [40]. Phosphoglycerate kinase, a protein encoded by the PGK1 and PGK2 genes, is a glycolytic protein activated by MSKE. Phosphoglycerate kinase converts 1,3-diphosphoglycerate into 3-phosphoglycerate in the glycolysis pathway. PGK1 is located on the X chromosome and is ubiquitously expressed whereas PGK2, whose differential expression was induced by MSKE in C4-2 cells, is a retrotransposed copy of PGK1 located on chromosome 6 that shows a testis specific expression pattern [41,42]. Our analyses demonstrated overexpression of HYOU1, CTTN and DPYSL5, and down-regulation of ABCA5 proteins in C4-2 cells. These proteins have been implicated in other cancer cells and tumor types and are involved in relevant cell mechanisms for apoptosis evasion, increased tumor invasiveness, tumor hypoxia, cellular respiration, and mitochondrial fragmentation. ABCA5 is a member of the ATP-binding cassette transporter 1 subfamily of genes whose mutations are linked to several human genetic disorders including cystic fibrosis, neurological disease, retinal degeneration, cholesterol and bile transport defects, anemia, and drug response phenotypes [43].
MSKE also activates oxidative stress and ROS pathways through aldehyde dehydrogenases (ALD), sideroflexin, sulfiredoxin and DHRS1. ALDH belongs to a group of NAD(P) + -dependent enzymes involved in oxidation of a large number of aldehydes into their weak carboxylic acids [44]. ALDH is important for drug resistance, cell proliferation, differentiation, and response to oxidative stress in prostate cancer and its activity is used to distinguish between normal cells and their malignant counterparts. In a previous study, high ALDH activity was used to isolate human prostate cancer cells with significantly enhanced clonogenic and migratory properties both in vitro and in vivo. As seen in other cancer tissues, the percentage of ALDH cells in prostate cancer cell lines are also related to tumorigenicity and metastatic behavior [45].
MSKE induced apoptosis via the up-regulation of ER stress-driven caspase-3,-7 and -12. Treatment with chloroquine blocked the effects of MSKE on apoptosis by up-regulating BCL-2, decreasing BAX, preventing the cleavage of caspase-3 and -7 and antagonizing MSKE-mediated increase in early and late apoptosis. This further supports the findings that MSKE-mediated autophagy leads to apoptosis. MSKE also prompted the down-regulation of anti-apoptotic and survival proteins like Annexin A4 (ANXA4), a member of the Ca 2+ -regulated and phospholipid-binding annexin superfamily. ANXA4 expression is increased in many cancer types, including cancers of renal, gastric, colonic, ovarian, and cervical origins [46][47][48][49][50]. Its expression has been linked to loss of cell-to-cell adhesion, increased metastasis, and chemo-resistance, and it is considered a potential cancer diagnostic and therapeutic target [51,52]. In vitro studies suggest that ANXA4 exhibits an anti-apoptotic effect by activating NF-κB transcriptional activity [53,54]. Our data suggests that MSKE may induce apoptosis by decreasing ANXA4, and increasing MTDH.
Recently, the cross-talk between autophagy and apoptosis has been considered as a key factor in the development and treatment of cancer [55]. The two pathways share molecular regulators and, in some cases, are activated by the same stimulus. Taken together, the results of this study suggest that MSKE induces apoptosis through signaling pathways that modulate ER stress, autophagy, cytoskeletal changes, cell-matrix, and cell-cell adhesion, as well as glycolysis and mitochondrial metabolism ( Fig 5); opening the door to novel therapeutic and clinical exploitations.
Supporting Information S1 Table. Differentially expressed proteins in MSKE-treated and control cultures. Detailed information including protein ID, gene name, number of amino acids, molecular weight, calculated pI, description, coverage samples, protein expression, change folds, and p-values. (XLSX) Acknowledgments C4-2 human prostate cancer cells were a gift from Dr. Leland Chung (Cedars Sinai Medical Center, Los Angeles, CA). Dr. Tamaro Hudson (Howard University, Washington, DC) kindly provided the MSKE. The authors thank Dr. Lycely del C. Sepúlveda-Torres for editorial assistance.