PolymerâKLAK Peptide Conjugates Induce Cancer Cell Death

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Polymer-KLAK Peptide Conjugates Induce Cancer Cell Death through Synergistic Effects of Mitochondria Damage and Autophagy Blockage Zeng-Ying Qiao, Wen-Jia Lai, Yao-Xin Lin, Dan Li, Xiaohui Nan, Yi Wang, Hao Wang, and Qiaojun Fang Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 09 May 2017 Downloaded from http://pubs.acs.org on May 10, 2017

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Bioconjugate Chemistry

Polymer-KLAK Peptide Conjugates Induce Cancer Cell Death through Synergistic Effects of Mitochondria Damage and Autophagy Blockage Zeng-Ying Qiao†,a, Wen-jia Lai†,‡,a, Yao-Xin Lin†,§,a, Dan Li†,§, Xiao-hui Nan†,§, Yi Wang†,§, Hao Wang†,* and Qiao-jun Fang†,‡,§,*

†

CAS Center for Excellence in Nanoscience, Key Laboratory for Biological Effects of

Nanomaterials and Nanosafety of Chinese Academy of Sciences, National Center for Nanoscience and Technology, Beijing 100190, P. R. China. ‡

Beijing Key Laboratory of Ambient Particles Health Effects and Prevention Techniques, National

Center for Nanoscience and Technology, Beijing 100190, P. R. China. §

University of Chinese Academy of Sciences, Beijing, 100049, P. R. China

a

Contributed equally to this work.

* Corresponding author: Qiaojun Fang, Tel: +86(10)82545562, Fax: +86(10)82545643, E-mail: [email protected] Hao Wang, Tel: +86(10) 82545759, E-mail: [email protected]

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ABSTRACT Nanoscaled polymer-peptide conjugates (PPCs) containing both functional peptides and synthetic polymer comprise a new family of biomaterials that can circumvent the limitation of peptides alone. Our previous work showed that PPCs with the therapeutic peptide KLAK, especially PPCs with shorter PEG spacers and a higher degree of polymerization, exhibit enhanced antitumor effects through disrupting mitochondrial membranes. However, as PPCs have a spherical nanostructure (45~60 nm), this may have other effects besides the conjugated therapeutic peptide KLAK itself when they enter cancer cells. In this research, we compared the proteome differences of U87 cells treated with KLAK, polymer, and their conjugates (P-KLAK) through quantitative proteomics technology. The result reveals that proteins involved in oxidative stress response and the Nrf2/ARE pathway were significantly upregulated after P-KLAK treatment. Moreover, the overexpression of sequestosome 1, a protein substrate that is selectively incorporated into the formation of autophagosome and degraded by autophagy, is found in our study, which has not been reported previously in the study of KLAK toxicity. Additional experiments suggest that upon endocytosis, P-KLAK causes lysosome impairment and results in autophagosomes accumulation. Hence, P-KLAK might induce U87 cell death by autophagy blockage due to lysosome impairment as well as mitochondria damage synergistically. KEY WORDS: Polymer-peptide Conjugates, Nrf2, SQSTM1, Lysosome, Autophagy

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INTRODUCTION Peptide drugs have played an increasingly important role in various diseases, ranging from cancer to diabetes and cardiovascular diseases. Nevertheless, a challenge in the application of peptide drugs is their undesirable properties, such as low bioavailability and limited hydrolytic stability or short half-life, which hinder their further therapeutic application.1-3 Thus, many variable approaches have been demonstrated to enhance the bioeffective molecule’s delivery. Polymer-peptide conjugates (PPCs) have a longer circulation time for tumor-targeted delivery and enhanced therapeutic efficacy than free peptides.4,5 Thus, variable approaches have been demonstrated to improve the applications of biofunctional peptides via self-assembled polymer carriers. Due to the self-assembly property of the polymer chains, PPCs usually have nanostructures,6 which not only help peptides to enrich at tumor sites through an enhanced permeability and retention effect (EPR effect) but also facilitate peptides to enter cells through endocytosis, which is a common phenomenon of nanoparticles. Our lab previously established a general strategy for facile synthesis and the in situ rapid screening of a library of functional PPCs. Functional peptides, such as targeting peptides and therapeutic peptides, were copolymerized with a wide variety of spacers to obtain a diverse library of PPCs with different chemical structures, which self-assembled into nanoparticles in the reaction process. After the in situ screening of PPCs, the structure-function relationships of the PPCs were identified. Using the guidelines, we designed PPCs with optimal properties, and the anti-cancer efficacies of PPCs were evaluated in vitro and in vivo, proving their potential application in cancer therapy.7 The 14-amino-acid polycationic peptide (KLAKLAK)2 (abbr. KLAK) is reported as an antibacterial agent that can disrupt anionic prokaryotic cytoplasmic membranes but does not disrupt the zwitterionic plasma membranes of eukaryotic cells because of its difficulty entering these cells.8 3



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Various modifications of KLAK have been made to allow it to pass through cell membranes to disrupt mitochondrial function and induce apoptosis in cancer cells.9-11 When internalized, KLAK can target the negatively charged mitochondrial membrane and trigger mitochondrial permeabilization, resulting in the generation of relatively high levels of reactive oxygen species (ROS) and the induction of mitochondria-dependent apoptosis.12,13 However, as nanoparticles (NPs), PPCs may gain the additional biological effects and mechanisms of actions of other NPs, such as the regulation of cell-membrane penetration,14,15 the degeneration of cytoskeletons,16,17 or the interference of cell autophagy.18-20 A recent study has shown that possible different drug resistance mechanisms exist for NPs drug compared to its single molecular form, indicating that besides those caused by a single molecule, other biological effects originating from nanoparticles may involve and act synergistically.21 And, though many PPCs have been reported, the bioeffects mechanism of nanoscaled PPCs, except the conjugated peptide itself, is less clear. Herein, we focus on the mechanism study of nanoscaled P-KLAK, one of the most efficient antitumor PPCs in our previous study,7 in the molecular level to understand its effects as nanoparticle beyond the conjugated peptide KLAK when they entered cancer cells through quantitative proteomics technology. Subsequent data suggested that a synergistic mechanism of autophagosome accumulation induced by lysosome impairment and oxidative stress response caused by mitochondrial damage is responsible for cell death upon P-KLAK stimulated U87 cells. These findings indicate that the cells’ response to PPCs is more complex than that of free peptides and polymers, which might have important implications for developing self-assembled PPCs NPs for biomedical applications.

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RESULTS AND DISCUSSION P-KLAK cytotoxicity and cellular location. In a recent paper, we reported that KLAK side chains were conjugated onto polymer backbones, which self-assembled into PPC nanoparticles with high anti-cancer activities and significant tumor suppression effects.7 In the present work, incubation with free KLAK alone did not significantly affect the human glioblastoma cell line U87 cells’ viability or cause apoptosis (Figure 1A and B). This observation is consistent with previous studies showing that the KLAK peptide has very low efficiency in passing through the cell membrane and is nontoxic outside of the cell.12,22 It was also found that the polymer carrier P was nontoxic toward cells. However, P-KLAK has a relative high cytotoxicity on U87 cells, as evaluated by CCK8 and annexin V-FITC/propidium iodide double staining (Figure 1A and 1B). To understand the mechanism of how P-KLAK causes cell death, we first examined the cellular locations of P-KLAK after entering cells using FITC labeled P and P-KLAK. As shown in Figure 1C and Figure S1, after 12 h of treatment, most of the green fluorescence of FITC-P remained on the cell surface, but not inside the cells, suggesting that no FITC-P enters the cells. Even after 24 h, only a few FITC-P co-localized with lysosomes, while the remaining large number was still surrounding the cells (Figure S1B). This suggests that, without linking to KLAK, the polymer itself tends to accumulate outside the cell membrane because of its low cell-penetrating ability. This is possibly due to the fact that the polymer is dispersed in an aqueous solution with random coil morphology owing to the lack of hydrophobic microdomains, which makes it difficult for the polymer to enter cells by cell endocytosis. Conversely, exposing cells with FITC-P-KLAK at the same concentration showed that the green fluorescence overlapped with the red, which was the label for lysosomes (Figure 1C), indicating that FITC labeled self-assembled P-KLAK conjugates can enter cells through endocytosis and then co-localize with lysosomes. Notably, free KLAK was 5



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reported as a mitochondrial target peptide once in the cytoplasm.10,23 The different cellular location of P-KLAK is not surprising, since studies have shown that many NPs (e.g., iron oxide, polystyrene, Au, or Ag) uptaken by cells are

Figure 1. P-KLAK toxicity and lysosome location in U87 cell. A) U87 cell viability of P, K, and P-KLAK with different concentration at 24 h was measured by CCK-8 assay (Mean ± SD). B) Apoptosis of P, K, and P-KLAK (12 µM) in U87 cells was analyzed using Annexin V-FITC/propidium iodide double staining after 24 h treatments. C) CLSM observation of cellular uptake of fluorescein labeled P and P-KLAK in U87 cell lines. U87 cells were incubated with KLAK, FITC labeled P-KLAK, and FITC labeled P for 24 h. Nuclei were labeled with Hoechst 33342, and lysosomes were labeled with LysoTracker Red. Yellow spots in the merged pictures denote the 6


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co-localization of the P-KLAK within lysosome compartments. Bar as 20 µm.

present in lysosomes.24-27 The size of polymeric nanoparticles with a polydispersity of 0.168 was measured by dynamic light scattering (DLS), and TEM images revealed that P-KLAK had a spherical nanostructure of 45~60 nm, which was concentration-independent (Figure S2). In addition, P-KLAK showed a positive Zeta potential around 17.3 mV, which could be attributed to the positive charge of the peptides and can facilitate the entrance of the nanoparticles into the cells.7 Hence, it is possible that the cytotoxicity mechanism of P-KLAK may differ from that of free KLAK.

Proteomic analyses of U87 cells with treatment of KLAK, P, and P-KLAK. To gain further insights into the molecular basis of the higher anti-cancer activity of P-KLAK as compared to P and KLAK, proteomics analysis was utilized for investigating the biological effects of PPCs (P-KLAK) on cells. Experiments with stable isotope labeling by amino acids in cell culture (SILAC) were carried out by labeling the U87 cell line and then treating it with KLAK, P, and P-KLAK (12 µM for peptides).28 Both forward and reverse labeling experiments were designed as described in the supporting information (Figure S3). Liquid chromatography mass spectrometry (LC-MS/MS) was employed to detect protein changes. Altogether, about 4,000 proteins were identified; detailed quantitation information of the proteins is listed in Table S1. An overview of scatter plots showed that for both forward and reverse labeling experiments, compared to the control, the log2 ratios of proteins for cells treated with P-KLAK had greater change than those of cells treated with P and KLAK (Figure 2A). In fact, when the ratios from both forward and reverse labeling experiments were plotted together, no consistent protein changes were observed for P and KLAK, indicating that these two compounds had no significant biological effect on U87 in proteomics analysis. However, 24 proteins were constantly upregulated, and 13 proteins were downregulated in cells treated with P-KLAK, shown in red and blue dots on the scatter plots (Figure 2A). In addition, these 7



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differentially expressed proteins were not changed in the P and KLAK groups (Figure 2B and Table S2). Our proteomics results were consistent with the cellular toxicity and locations mentioned previously, which showed very few P and KLAK entered the cells at the same concentration, resulting in no detectable protein changes in our analysis.

Figure 2. Overview of the identified proteins in three groups. A) Scatterplots of forward and reverse labeling experiments of U87 cells treated with KLAK, P, and P-KLAK compared to the control. SILAC-based ratios (normalized to median logarithm ratio of zero by MaxQuant) of KLAK, P, and P-KLAK present as heavy to light. Blue and red dots represent the decreased and increased proteins in 24 h P-KLAK experiment group, respectively. 8


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B) Heat map showing logarithm of averaged expression ratios of decreased and increased proteins (in gene names) in 24 h P-KLAK experiment and their relative ratios in KLAK and P groups.

Biological process and pathway analysis. Biological process and pathway analysis were performed to study the differentially expressed proteins observed in P-KLAK stimulated cells to understand the mechanisms of P-KLAK on cells and compare it with reported free KLAK peptides. Functional classification and enrichment analysis were performed using the Cytoscape platform (see Methods) for differentially expressed proteins. Basically, statistical tests were performed to assess the over-represented categories with reference to the databases of KEGG pathways, Gene Ontology (GO), WikiPathways, and REACTOME. Moreover, protein-protein interactions were analyzed using the STRING database. Figure 3A summarizes the enriched bioprocess or pathways with p < 0.05 using the above four databases for proteins regulated in P-KLAK treatment (details in Figure S4 and Table S3). Many of these were related to oxidative stress—for example, “response to reactive oxygen species,” “response to oxidative stress,” and “response to hyperoxia.” This indicated that increased ROS production occurs after P-KLAK treatment. Other processes such as the “organic substance catabolic process” or “response to extracellular stimulus” suggested that changes in the constitutional expression of the proteins participated in cell response and catabolism. Furthermore, the GO molecular function analysis (Figure 3B, details in Figure S5 and Table S3) also showed that the significant enriched terms were associated with oxidative stress (proteins had an “oxidoreductase activity” function). An overview of the proteomic data presents a general picture of cells under the stress caused by P-KLAK. In the following study, molecular and biochemical experiments will be performed to explain and compare the mechanisms of P-KLAK and free KLAK.

Oxidative stress response and ROS production in U87 cells. The KLAK peptide is an 9



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amphipathic α helix peptide known to be toxic after cellular internalization12 and functions as a proapoptotic peptide to disrupt negatively charged mitochondrial membranes and induce mitochondria-dependent apoptosis.12,29 The classic mitochondria-dependent apoptosis involves the persistent opening of the permeability transition pore, membrane potential change, the release of

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Figure 3. Enrichment of differentially expressed proteins of P-KLAK treated U87 cells. A) Enriched process or pathway terms (p < 0.05) from 4 databases were shown in distinct colors (the left panel). The p value of every enriched term was rainbow-colored and sorted from high on the top to low in the bottom; the bar’s length represented the number of differentially expressed genes included in each term (the right panel). B) GO molecular functions with p < 0.01 were shown. Each protein was represented by a certain gene name; surrounding color shows the expression ratio. Red: up-regulated genes; blue: down-regulated. C) Summary of overexpressed proteins in response to oxidative stress process and Nrf2/ARE pathway. Nrf2 harbors two Keap1 binding motifs, DLG and ETGE.30 Stress conditions or accumulation of disruptor proteins, such as p62 (SQSTM1) and p21 (CDKN1A), can disturb Nrf2-Keap1 binding and result in an increase in nuclear Nrf2. Consequently, Nrf2 bind to the antioxidant response element (ARE) and drive the expression of Nrf2 target genes.

cytochrome c, and the activation of caspase proteins. Since mitochondria produces most of the cellular ROS, its damage along with the electron transport chain’s deficiency will cause the generation of large amounts of O2-, which further convert to other ROS and release into cytoplasma.31 Our previous work proved that P-KLAK can cause mitochondrial membrane potential depolarization7 and may lead to oxidative stress response. Furthermore, we performed the co-localization experiment of P-KLAK and mitochondria (Figure S6). P-KLAK were labeled by Cy5 (red signal on KLAK part), and mitochondria were labeled by Mito Tracker Green (green signal). The overlap of red and green signals proved that the P-KLAK could co-localize with the mitochondria. In the proteomic result, out of 24 increased proteins, 10 (CAT, PRDX6, SRXN1, NQO1, KDM2A, BLVRB, HMOX1, GCLM, CDKN1A and CBR3) belong to significantly enriched (p < 0.01) the oxidative stress and ROS pathway (Figure 4A). Specifically, peroxiredoxin-6 (PRDX6), catalase (CAT), and sulfiredoxin-1 (SRXN1) can reduce H2O2 to protect against H2O2-mediated cell toxicity.32-35 NAD(P)H dehydrogenase [quinone] 1 (NQO1) is a member of the NAD(P)H dehydrogenase (quinone) family and encodes a cytoplasmic 2-electron reductase that prevents the 11



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one electron reduction resulting in semi-quinone radicals and therefore mediates a detoxification reaction.36 Heme oxygenase 1 (HMOX1) is an isoform of heme oxygenase that cleaves the heme ring at the alpha methene bridge to form biliverdin, which is subsequently converted to bilirubin by a biliverdin reductase such as the flavin reductase [NADPH] (BLVRB).37 By changing heme as a potent pro-oxidant to bilirubin as an equipotent anti-oxidant, HMOX1 plays an important role against oxidant stress.38 The glutamate-cysteine ligase regulatory subunit (GCLM) is one subunit of glutamate-cysteine ligase (gamma-glutamylcysteine synthetase) and is the first-rate limiting enzyme in glutathione (GSH) synthesis. Increased levels of GCLM and the glutamate-cysteine

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Figure 4. P-KLAK induced oxidative stress response and Nrf2 translocation in U87 cell. A) Overview of regulated proteins in U87 cell treated with P-KLAK and enriched process with p value < 0.01. Each protein was represented by certain gene name; surrounding color shows the expression ratio. Red: up-regulated genes; blue: down-regulated. B) Western blot of Nrf2 in cell nucleus component and whole cell lysis of U87 cells treated with

KLAK, P, and P-KLAK for 24 h (peptide concentration: 12 µM). Laminin B and β-actin serve as loading controls. C) Western blot of NQO1, HMOX1, and GCLM in U87 cells treated with KLAK, P, and P-KLAK for 24 h (peptide concentration: 12 µM). β-actin serves as a loading control.

ligase catalytic subunit (also upregulated after 48 h P-KLAK treatment, data not shown) are accompanied by a successive increase in GSH levels,39 and GSH is an important antioxidant preventing the damage of cellular components caused by ROS.40 Carbonyl reductase [NADPH] 3 (CBR3) catalyzes the two electron reduction of carbonyl compounds, which is a central metabolic process that controls the level of key regulatory molecules as well as xenobiotics.41 Recently, the expression of CBR3 has been suggested to be modulated by nuclear factor-erythroid 2-related factor 2 (Nrf2).42 Moreover, SRXN1, NQO1, HMOX1, and GCLM are also reported as target genes of the nuclear factor-erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE).43,44 This pathway is considered to be the major player in cellular protection against insults coming from the heightened production of ROS or electrophilic metabolites of xenobiotics/carcinogens.45 In response to these insults, Keap1 (Kelch ECH associating protein 1), a repressor protein that binds to Nrf2 and promotes its degradation by the ubiquitin proteasome pathway, will undergo conformation change and disassociate with Nrf2, which makes Nrf2 translocate into the nucleus. In addition, cyclin-dependent kinase inhibitor 1 (CDKN1A or p21) and sequestosome-1 (SQSTM1 or p62) can also regulate the Nrf2/ARE pathway through disturbing Nrf2-Keap1 binding, resulting in an increase in nuclear Nrf2.46,47 Once in the nucleus, Nrf2 complexes will bind to ARE, a cis-acting DNA promoter sequence, and drive the transcription of a large battery of cytoprotective genes, 13



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including those that defend against electrophilic stressors and oxidative insults.48 The upregulation of NQO1, HMOX1 GCLM, and p62 as well as the Nrf2 translocation into the nucleus after adding P-KLAK into U87 cells were validated by western blots (Figure 4B, C and Figure 5B). Therefore, our results indicated that under the survival pressures from P-KLAK, cells upregulated proteins in the Nrf2/ARE pathway against oxidative stress and xenobiotic P-KLAK itself (Figure 3C and Figure S7). Consistently, high levels of ROS and increased amounts of cytochrome c (Cyt c) and caspase-3 cleavage were detected in U87 cells under P-KLAK stimulation (Figure 5A and Figure S8), confirming that intracellular P-KLAK could cause mitochondrial dysfunctions and remarkably increased Cyt c and ROS production, contributing to caspase activation and cell apoptosis.

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Figure 5. P-KLAK induced ROS and lysosome impairment in U87 cells. A) The ROS expression in U87 cells treated with various concentrations of P-KLAK for 12 h. B) Western blot of cytosolic P62, CSTD, and Cyt c in U87 cells treated with P-KLAK (10 µM, 20 µM, and 30 µM) for 24 h. β-actin served as a loading control. C) Flow cytometry analysis of U87 cells stained with DCFH-DA or BODIPY FL-Pepstatin A after 12 h or 24 h treatment of

P, KLAK, P-KLAK (peptide concentration: 12 µM).

Our results suggest P-KLAK induced oxidative stress response and excess ROS production in U87 cells. Moreover, we have identified the downstream molecules in response to ROS. The final death of cells suggests that P-KLAK at a certain concentration can overwhelm the Nrf2/ARE pathway mediated detoxication of ROS as well as xenobiotic. On the basis of the result, we suspect that blocking the Nrf2/ARE pathway may enhance the antitumor effect of P-KLAK and that a further design of PPCs with a similar function may consider including Nrf2 targeted siRNAs or 15



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pathway inhibitors49 to improve the efficiency.

Autophagosome accumulation. The proteomic result showed that p62/SQSTM1 was remarkably increased in the P-KLAK group by an average of 2.5 fold (Table S2 and Figure 2B). The multi-functional adapter protein p62 is not only an activator and target gene of the ARE response in the Nrf2/ARE pathway under environmental stress,50 but more importantly, p62 is also the selective autophagy substrate.51 When autophagosomes form, the cytosolic microtubule-associated protein 1 light chain 3 (LC3) is recruited to the membrane of nascent autophagosomes to control autophagosomes expansion.52 LC3 is the most widely known autophagy related protein used for monitoring autophagosomes, and p62 localizes onto the autophagosomes via binding to ubiquitylated LC3 and is constantly being degraded by the autophagy-lysosome system.53 Studies have suggested that both the perturbation of lysosomal function and blockade of autophagic flux will lead to marked accumulation of p62.52,54 Our western blot analysis showed that the p62/β-actin ratio became higher as the concentration of P-KLAK increased (Figure 5B). The increased level of p62 upon P-KLAK treatment suggested autophagosomes accumulation in U87 cells. To test this, acridine orange (AO), which can mark acidic vesicular organelles (red signal) in cells, was used to estimate the level of autophagy. Autophagy is the process of sequestering cytoplasmic proteins into the lytic component and is characterized by the formation and promotion of acidic vesicular organelles.24,54 CLSM images revealed there were more red signals in the P-KLAK treated cells than that in the P and KLAK treated cells (Figure 6A). Furthermore, LC3 was also examined using U87 cells expressing green fluorescent protein (GFP)-tagged LC3 (GFP-LC3). Normally, the GFP-LC3 protein is diffused in the cytoplasm but accumulates in the autophagosome membrane and thus appears as green punctate dots in the cell upon autophagy.55 CLSM observation showed that P-KLAK (peptide concentration: 20 µM) can induce the accumulation of GFP-LC3 positive dots and 16


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that there were many more GFP-LC3 dots in the P-KLAK treated cells than in the P and KLAK treated cells (Figure 6B and C). Moreover, the conversion of LC3-I to LC3-II is widely used to

Figure 6. P-KLAK induced autophagosome accumulation in U87 cells. A) Acridine orange (AO) staining of cells treated with KLAK, P, and P-KLAK for 24 h (peptide concentration: 20 µM). B) CLSM images of GFP-LC3 positive dots in U87/GFP-LC3 cells treated with KLAK, P, and P-KLAK for 24 h (peptide concentration: 20 µM). C) Quantified results of B). D) LC3 western blot of U87 cells treated with KLAK, P, and P-KLAK for 24 h (peptide concentration: 20 µM). E) The quantified band intensity of LC3-II relative to that of β-actin in D).

monitor autophagosome formation, because LC3-II is recruited to the autophagosome membrane 17



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during autophagy.56 As shown in Figure 6D and E, after treatment with P-KLAK (peptide concentration: 20 µM) for 24 h, the LC3-II/β-actin ratio increased dramatically and was much larger than that of P and KLAK. All these analyses confirmed autophagosome accumulation in P-KLAK treated cells.

P-KLAK treatment causes lysosome impairment. Autophagosome accumulation can result from the blockade of autophagy flux as well as autophagy induction.56 The blockade of autophagy flux is usually caused by the destabilization of lysosomes, which fuse with autophagosomes to form autolysosomes for degradation.57 Previous studies have suggested that the perturbation of lysosomal function will lead to the blockade of autophagic flux and cause an accumulation of p62.18,20,24,52 In our results, p62 elevated in a concentration-dependent way (Figure 5B), suggesting that the normal autophagosome degradation is blocked. We have shown that self-assembled nanoscaled P-KLAK entered cells by endocytosis and co-localized with lysosomes (Figure 1C). Next, lysosomal function was tested by measuring cathepsin D (CSTD). CSTD is one of the most abundantly expressed aspartic endopeptidases in the lysosomal compartment. It degrades proteins at low pH (3.5~5) and exerts its activity inside lysosomal structures.58 Therefore, the change of lysosomal pH or release of CSTD into the cytosol is associated with lysosomal dysfunction.59 Western blot analysis showed an increase of cytosolic CSTD after P-KLAK treatment (Figure 5B). Simultaneously, BODIPY FL-pepstatin A, which binds to the lysosomal protease CSTD in a pH-dependent way,60 decreased in the P-KLAK treated cell, while the control P and KLAK treatments exhibited a normal staining under the same experimental conditions (Figure 5C). BODIPY FL-pepstatin A selectively binds to the active site of CSTD under acidic pH around 3.5~5, and this binding is blocked when the pH is increased to neutral.60 Hence, the decreased fluorescence of BODIPY FL-pepstatin A after P-KLAK treatment indicates unsuccessful binding. This indicated increased pH in the lysosomes, and an 18


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increase in pH would be expected to decrease the lysosomal degradation capability, since acidic pH is optimal for lysosome enzyme activity.52 Furthermore, variations in pH can decrease the fusion capability of lysosomes.61 All the results suggested lysosome impairment was induced by P-KLAK nanoparticles, affecting the formation of autolysosomes and resulting in autophagosome accumulation. Many inorganic nanoparticles have been reported to be able to change the basal level of autophagy.62 Silver and SiO2 nanoparticles19,63 as well as COOH-carbon nanotubes64 can induce autophagy through the PI3K/Akt/mTOR signaling pathway, gold nanoparticles can block autophagy flux through lysosome impairment,20 while Fe3O4 nanoparticles induce autophagosome accumulation through multiple mechanisms including inhibiting the activity of mTOR and lysosome impairment.65 However, for soft nanoparticles, especially for polymer-peptide conjugate (PPC) nanoparticles, the related reported are few. Although many PPCs have been reported for cancer treatments, the mechanism of the bioeffects of nanoscaled PPCs except the conjugated peptide itself is less clear. Here we found that P-KLAK could induce lysosome impairment and disturb autophagic flux, and this bioeffect has not been reported in the KLAK mechanism as free peptide. This could possibly be due to the “proton sponge” effect66 of nanoscaled P-KLAK, as the amine group in the conjugated peptide allows it to be protonated in the acidic condition, and the polymer part of P-KLAK allows it to have the flexibility to swell when protonated. The protonation of P-KLAK in lysosomes will induce the flow of ions (protons and Cl-) and water (osmotic swelling) into the lysosome,66 which subsequently causes a lysosomal pH change, and a rupture of the membrane results in lysosome impairment and autophagic flux blockage. Meanwhile, autophagy is an evolutionarily conserved process in which cellular proteins and organelles, such as redox-damaged proteins, damaged mitochondria, and peroxisomes are engulfed in autophagosomes, which then fuse 19



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with lysosomes to form autolysosome for degradation.67 Previous studies have also shown that autophagy may play a cytoprotective role in tumor cells under metabolic stress and the cytotoxicity of chemotherapy.63 The disturbance of autophagy is recognized as a potential mechanism of cell death, resulting in either apoptosis or autophagic cell death.68 Hence our result of P-KLAK opened a new avenue for the biological mechanism of PPC nanoparticles, revealing that well designed PPC nanoparticles especially when the conjugated peptide is cationic can induce cell death not only by the functional peptide itself but also through autophagy blockage due to the lysosome impairment by nanoparticles.

CONCLUSION While PPCs exhibit many advanced features compared to free peptides, the mechanisms of the enhanced bioeffects of nanoscaled PPCs can be complicated. From this work using a quantitative proteomics method, we found that P-KLAK can induce mitochondrial disorder as free KLAK. In addition, the proteomic data suggested that the downstream Nrf2/ARE pathway was activated under oxidative stress and ROS. Meanwhile, lysosome impairment mediated autophagosome accumulation was observed; this phenomenon has not been reported for the KLAK mechanism and is specific to nanoscaled P-KLAK. Therefore, we propose that the over-produced ROS due to mitochondrial disruption related apoptosis and lysosome mediated autophagic interruption act synergistically, resulting in the high antitumor effect of P-KLAK. Our findings also suggest that new strategies using PPCs for cancer treatment may consider the Nrf2/ARE pathway as a synergetic target by designing PPCs with the Nrf2 inhibitor brusatol.

EXPERIMENTAL PROCEDURES Preparation of P-KLAK. The syntheses of P-KLAK and self-assembly of P-KLAK in phosphate-buffered saline (PBS) were operated following the protocol described in the literature 7. 20


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The chemical structure of P-KLAK is shown in Scheme S1, in which the peptides CCGGG(KLAKLAK)2 and CCGGGRGD (molar ratio = 8:2, named KLAK) are conjugated onto the polymer backbone. The peptide CCGGG conjugated polymer is used as a control polymer, called P.

Cell viability and apoptosis assay. U-87 MG (U87) cells were seeded in confocal dishes with high-glucose Dulbecco’s modified Eagle’s medium (DMEM), 10% FBS (Gibco), and 1% penicillin/streptomycin (Gibco). When the cells reached 80% confluence, 10 µM of KLAK, P, and P-KLAK were added and incubated for 24 h separately. Cell viability was analyzed by CCK-8 analysis. Briefly, CCK-8 was added to each well and incubated for 2 h, and then absorbance was measured at 450 nm. The cell viability (%) was calculated as (Asample/Acontrol) × 100, where Asample and Acontrol denote the absorbance of the sample well and the control well, respectively. The experiments were performed in triplicate. The apoptosis of U87 cells was further determined by flow cytometry (FCM) using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide double staining after treatment with 12 µM KLAK, P, and P-KLAK for 24 h. Briefly, the treated cells were trypsinized and collected by centrifugation for 5 min. Then the cells were washed and re-suspended with 100 µL of PBS. Finally, the cells were stained with annexin V/FITC kits (Beyotime, Jiangsu, China) before FCM analysis.

Cell SILAC labeling. A SILAC™ Protein ID & Quantitation Media Kit was used to label U87. The cells were cultured in DMEM/F12 medium with a light (L-arginine and L-lysine) or heavy (L-13C15N-arginine and L-13C14N-lysine) medium and supplemented with 10% (v/v) dialyzed fetal bovine serum, 50 IU/ml penicillin, and 50 mg/ml streptomycin (Gibco), according to the instructions provided. After six passages, KLAK, P, and P-KLAK (12 µM for peptides) were added to the medium of heavy labeled cells respectively and incubated for 24 h for the following labeling analysis. An equal volume of hanks balanced salt solution (HBSS) was added to the medium with 21



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light labeled cells to serve as the control for each of the above treatments. A reverse labeling was also designed to eliminate systematic errors caused by labeling efficiency. This was performed with the control cells grown in the heavy medium, while cells treated with KLAK, P, and P-KLAK were grown in the light medium.

LC-MS/MS. After the protein extraction, in-solution digestion, and fraction, approximately 2 µg of peptides were loaded for LC-MS/MS analysis. The peptides were separated on an in-house made 30 cm reversed phase capillary emitter column (inner diameter 75 µm, 2.5 µm Venusil MP C18 resin [Agela Technologies, China]) using 240 min gradients and analyzed on the Q Exactive instrument (Thermo Fisher Scientific). MS data were acquired by Thermo Xcalibur (2.0) with data-dependent MS/MS scans (TopN = 15); the target value for the full MS scan was 3 × 106 in the 300−1,700 m/z range, with a maximum injection time of 20 ms and a resolution of 70,000 at m/z 200. The isolation window was 1.6 m/z, and the normalized collision energy was 28. The MS/MS scans’ resolution was 17,500 at m/z 200, with an ion target value of 1 × 105 and a maximum injection time of 60 ms. To avoid peptides being sequenced repeatedly, the exclusion time was set to 60 s.

Data analysis. MS raw files were processed with MaxQuant software (version 1.4.1.2), and peak lists were searched against the human Uniprot FASTA version released on April 20, 2014 (88,725 entries) with its reversed protein sequences and a common contaminants database (247 entries) by the Andromeda search engine. The search parameters included cysteine carbamidomethylation as a fixed modification, variable modifications of methionine oxidation, and protein N-terminal acetylation. Enzyme specificity was set to cleave the C-terminal of arginine and lysine, and a maximum of two missed cleavages was allowed in the database search. Peptides with at least six amino acids were considered for identification. The false discovery rate for both peptides 22


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and proteins was ≤ 0.01. The quantification of SILAC pairs was carried out by MaxQuant with the requantification option. For the remaining parameters in the software, default values were used.

Bioinformatics analysis. Data analysis was performed with Perseus software in the MaxQuant computational platform and R statistical computing environment. Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, WikiPathways, and REACTOME enrichment analyses of differentially expressed proteins and their interaction network were performed by Cytoscape69 plugged ClueGO+CluePedia70 with a p-value (Benjamini-Hochberg correction) cutoff of < 0.05.

Lysosome co-localization.U87 cells were seeded in confocal dishes with the DMEM medium containing 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). When the cells reached 60% confluency, 12 µM of KLAK, FITC-P, and FITC-P-KLAK were added and incubated for 12 h or 24 h separately. Lysosome was stained by LysoTracker Red (Beyotime), as described in the protocol. Co-localization analysis was performed by confocal laser scanning microscopy (CLSM, Zeiss LSM710, Germany).

Autophagy observation. CLSM was employed to observe GFP-LC3 dots induced by PPCs. A density of 5 × 105 U87/GFP-LC3 cells were seeded in the 15φ culture dishes in the RPMI1640 medium containing 10% FBS and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO2. The cells were first cultured with KLAK, P, and P-KLAK for 24 h and then washed with PBS three times. The cells were imaged using a Zeiss LSM710 CLSM with a 63× objective lens. Exposure settings were unchanged throughout acquisition. The quantification of GFP-LC3 puncta was analyzed using ImageJ software. Approximately 100 GFP-LC3-positive cells were counted per group after the selection of cells with > 10 LC3-labeled vesicles. The experiments were performed in triplicate. 23



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Isolation of cell nucleus. Cell nuclei were isolated using sucrose gradients. Briefly, cells were washed three times with cold PBS before being scraped and pelleted, then re-suspended in a homogenization buffer (0.25 M sucrose, 0.5 mM EGTA, 20 mM Tris-HCl pH = 7.4) with a protease inhibitor cocktail (Roche), and homogenized on ice in a Dounce homogenizer. The completement of lysis was confirmed by trypan blue stain. The lysate was centrifuged at 4 °C 500 g for 5 min. The pellet was re-suspended in the homogenization buffer and repeated twice for homogenate and centrifugation to collect the nuclei.

Western blot. U87 cells were re-suspended in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (vol/vol) Triton-X 100, and a protease inhibitor. The protein content was estimated using a BCA kit (Applygen). Each sample (20 µg of protein) was subjected to SDS-PAGE and then transferred to a nitrocellulose membrane. Blots were blocked in a blocking buffer containing 5% (wt/vol) non-fat milk and 0.1% (vol/vol) Tween 20 in 0.01 M Tris-buffered saline (TBS) and were incubated with primary antibodies overnight at 4 °C and then an appropriate secondary antibody (ZSGB-BIO) for 1 h at room temperature, subsequently scanned on a Typhoon Trio Variable Mode Imager. Band intensity was calculated using NIH ImageJ software.

ROS analysis. To quantify the release of ROS in the U87 cells, free KLAK, P, and P-KLAK dispersed in DMEM media were added into cells grown in the confocal microscope dish or 12-well plate, and the cells were further incubated at 37 °C for 12 h and 24 h. After the cells were washed with PBS three times, the U87 cells were stained with a 10 µM reactive oxygen species (ROS) assay kit (Beyotime Institute of Biothechnology, China) for another 20 min. Finally, the cells were washed and re-suspended in 1 mL of PBS for flow cytometry analysis.

AO staining. A density of 5 × 105 U87/GFP-LC3 cells were seeded in the 15φ culture dishes in an MEM medium containing 10% FBS and 1% penicillin/streptomycin in a humidified atmosphere 24


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with 5% CO2. The cells were first cultured with KLAK, P, and P-KLAK for 24 h and then stained with AO (1 µM) for 15 min and washed with PBS three times. The cells were imaged using CLSM with a 63× objective lens (Zeiss LSM710).

Analysis of change in lysosomal pH. To detect the loss of lysosomal acidification, cells were stained with the pepstatin A-BODIPY FL conjugates (Invitrogen). Pepstatin A-BODIPY FL binds cathepsin D at acidic pH.60 At designated time-points, cells were washed and incubated with 1 µM Pepstatin A-BODIPY FL at 37 °C for 30 min. After staining, the cells were washed and re-suspended in ice-cold PBS and analyzed by flow cytometry.

Statistical analysis. Student’s t test was employed to test the statistical significance of differences. Results were considered significantly different if p < 0.05.

Acknowledgments This work is financially supported by the “Strategic Priority Research Program” of the Chinese Academy of Sciences (XDA09040300), the 100 Talents Program of the Chinese Academy of Sciences (Y4362911ZX), the National Natural Science Foundation of China (21674027, 21374026 and 51303036), and the National Basic Research Program of China (973 Program, 2013CB932701).

Supporting Information The sample preparations for PPCs and detailed results of LC-MS as well as bioinformatics analysis are available free of charge on the ACS Publications website.

REFERENCES (1)

Antosova, Z., Mackova, M., Kral, V., and Macek, T. (2009) Therapeutic application of peptides and proteins: parenteral forever? Trends Biotechnol. 27, 628-635.

(2)

Gaspar, D., Veiga, A. S., and Castanho, M. A. (2013) From antimicrobial to anticancer peptides. A review. Front.

(3)

Kaspar, A. A., and Reichert, J. M. (2013) Future directions for peptide therapeutics development. Drug Discov.

Microbiol. 4, 294. Today 18, 807-817. (4)

Du, A. W., and Stenzel, M. H. (2014) Drug Carriers for the Delivery of Therapeutic Peptides. 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules 15, 1097-1114. (5)

Ulbrich, K., Holá, K., Šubr, V., Bakandritsos, A., Tuček, J., and Zbořil, R. (2016) Targeted Drug Delivery with Polymers and Magnetic Nanoparticles: Covalent and Noncovalent Approaches, Release Control, and Clinical Studies. Chem. Rev. 116, 5338-5431.

(6)

Hamley, I. W., and Castelletto, V. (2017) Self-Assembly of Peptide Bioconjugates: Selected Recent Research

(7)

Qiao, Z.-Y., Lin, Y.-X., Lai, W.-J., Hou, C.-Y., Wang, Y., Qiao, S.-L., Zhang, D., Fang, Q.-J., and Wang, H. (2016) A

Highlights. Bioconjugate Chem. 28, 731-739. General Strategy for Facile Synthesis and In Situ Screening of Self-Assembled Polymer-Peptide Nanomaterials. Adv. Mater. 28, 1859-1867. (8)

Javadpour, M. M., Juban, M. M., Lo, W. C., Bishop, S. M., Alberty, J. B., Cowell, S. M., Becker, C. L., and McLaughlin, M. L. (1996) De novo antimicrobial peptides with low mammalian cell toxicity. J. Med. Chem. 39, 3107-3113.

(9)

Law, B., Quinti, L., Choi, Y., Weissleder, R., and Tung, C. H. (2006) A mitochondrial targeted fusion peptide exhibits remarkable cytotoxicity. Mol. Cancer Ther. 5, 1944-1949.

(10)

Adar, L., Shamay, Y., Journo, G., and David, A. (2011) Pro-apoptotic peptide-polymer conjugates to induce mitochondrial-dependent cell death. Polym. Adv. Technol. 22, 199-208.

(11)

Shamay, Y., Adar, L., Ashkenasy, G., and David, A. (2011) Light induced drug delivery into cancer cells.

(12)

Ellerby, H. M., Arap, W., Ellerby, L. M., Kain, R., Andrusiak, R., Rio, G. D., Krajewski, S., Lombardo, C. R., Rao, R.,

Biomaterials 32, 1377-1386. Ruoslahti, E., et al. (1999) Anti-cancer activity of targeted pro-apoptotic peptides. Nat. Med. 5, 1032-1038. (13)

Mai, J. C., Mi, Z., Kim, S. H., Ng, B., and Robbins, P. D. (2001) A proapoptotic peptide for the treatment of solid tumors. Cancer Res. 61, 7709-7712.

(14)

Dawson, K. A., Salvati, A., and Lynch, I. (2009) Nanotoxicology: nanoparticles reconstruct lipids. Nat.

(15)

Verma, A., Uzun, O., Hu, Y., Hu, Y., Han, H. S., Watson, N., Chen, S., Irvine, D. J., and Stellacci, F. (2008)

Nanotechnol. 4, 84-85. Surface-structure-regulated cell-membrane penetration by monolayer-protected nanoparticles. Nat. Mater. 7, 588-595. (16)

Xu, F., Piett, C., Farkas, S., Qazzaz, M., and Syed, N. I. (2013) Silver nanoparticles (AgNPs) cause degeneration of cytoskeleton and disrupt synaptic machinery of cultured cortical neurons. Mol. Brain 6, 29.

(17)

Cooper, R. J., and Spitzer, N. (2015) Silver nanoparticles at sublethal concentrations disrupt cytoskeleton and neurite dynamics in cultured adult neural stem cells. Neurotoxicology 48, 231-238.

(18)

Wang, Y., Lin, Y. X., Qiao, Z. Y., An, H. W., Qiao, S. L., Wang, L., Rajapaksha, R. P., and Wang, H. (2015) Self-assembled autophagy-inducing polymeric nanoparticles for breast cancer interference in-vivo. Adv. Mater. 27, 2627-2634.

(19)

Duan, J., Yu, Y., Yu, Y., Li, Y., Wang, J., Geng, W., Jiang, L., Li, Q., Zhou, X., and Sun, Z. (2014) Silica nanoparticles induce autophagy and endothelial dysfunction via the PI3K/Akt/mTOR signaling pathway. Int. J. Nanomed. 9, 5131-5141.

(20)

Ma, X., Wu, Y., Jin, S., Tian, Y., Zhang, X., Zhao, Y., Yu, L., and Liang, X. J. (2011) Gold nanoparticles induce autophagosome accumulation through size-dependent nanoparticle uptake and lysosome impairment. ACS Nano 5, 8629-8639.

(21)

Liu, Y., Liu, X., Jin, X., He, P., Zheng, X., Dai, Z., Ye, F., Zhao, T., Chen, W., and Li, Q. (2015) The dependence of radiation enhancement effect on the concentration of gold nanoparticles exposed to low- and high-LET radiations. Phys. Med. 31, 210-218.

(22)

Qiao, Z.-Y., Hou, C., Zhang, D., Liu, Y., Lin, Y.-X., An, H.-W., Li, X.-J., and Wang, H. (2015) Self-assembly of Cytotoxic Peptide Conjugated Poly([small beta]-amino ester)s for Synergistic Cancer Chemotherapy. J. Mater. 26


Page 26 of 36

Page 27 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Chem. B 3, 2943--2953. (23)

Horton, K. L., and Kelley, S. O. (2009) Engineered apoptosis-inducing peptides with enhanced mitochondrial

(24)

Klionsky, D. J., Abdelmohsen, K., Abe, A., Abedin, M. J., Abeliovich, H., Acevedo Arozena, A., Adachi, H., Adams,

localization and potency. J. Med. Chem. 52, 3293-3299. C. M., Adams, P. D., Adeli, K., et al. (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1-222. (25)

Shahabi, S., Treccani, L., Dringen, R., and Rezwan, K. (2015) Utilizing the protein corona around silica nanoparticles for dual drug loading and release. Nanoscale 7, 16251-16265.

(26)

Frohlich, E. (2013) Cellular targets and mechanisms in the cytotoxic action of non-biodegradable engineered nanoparticles. Curr. Drug Metab. 14, 976-988.

(27)

Miyayama, T., Arai, Y., Suzuki, N., and Hirano, S. (2014) Cellular distribution and behavior of metallothionein in

(28)

Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., and Mann, M. (2002) Stable

mammalian cells following exposure to silver nanoparticles and silver ions. Yakugaku Zasshi. 134, 723-729. isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376-386. (29)

Ellerby, H. M., Arap, W., Ellerby, L. M., Kain, R., Andrusiak, R., Del Rio, G., Krajewski, S., Lombardo, C. R., Rao, R., Ruoslahti, E., et al. (1999) Anti-cancer activity of targeted pro-apoptotic peptides. Nat. Med. 5, 1032-1038.

(30)

Tong, K. I., Katoh, Y., Kusunoki, H., Itoh, K., Tanaka, T., and Yamamoto, M. (2006) Keap1 recruits Neh2 through binding to ETGE and DLG motifs: characterization of the two-site molecular recognition model. Mol. Cell. Biol. 26, 2887-2900.

(31)

Wallace, D. C. (2005) Mitochondria and cancer: Warburg addressed. Cold Spring Harbor Symp. Quant. Biol. 70, 363-374.

(32)

Takeuchi, A., Miyamoto, T., Yamaji, K., Masuho, Y., Hayashi, M., Hayashi, H., and Onozaki, K. (1995) A human erythrocyte-derived growth-promoting factor with a wide target cell spectrum: identification as catalase. Cancer Res. 55, 1586-1589.

(33)

Manevich, Y., and Fisher, A. B. (2005) Peroxiredoxin 6, a 1-Cys peroxiredoxin, functions in antioxidant defense

(34)

Chang, T. S., Jeong, W., Woo, H. A., Lee, S. M., Park, S., and Rhee, S. G. (2004) Characterization of mammalian

and lung phospholipid metabolism. Free Radical Biol. Med. 38, 1422-1432. sulfiredoxin and its reactivation of hyperoxidized peroxiredoxin through reduction of cysteine sulfinic acid in the active site to cysteine. J. Biol. Chem. 279, 50994-51001. (35)

Woo, H. A., Jeong, W., Chang, T. S., Park, K. J., Park, S. J., Yang, J. S., and Rhee, S. G. (2005) Reduction of cysteine sulfinic acid by sulfiredoxin is specific to 2-cys peroxiredoxins. J. Biol. Chem. 280, 3125-3128.

(36)

Rougee, L. R., Riches, Z., Berman, J. M., and Collier, A. C. (2016) The Ontogeny and Population Variability of Human Hepatic NAD(P)H Dehydrogenase Quinone Oxido-Reductase 1 (NQO1). Drug Metab. Dispos. 44, 967-974.

(37)

Smith, L. J., Browne, S., Mulholland, A. J., and Mantle, T. J. (2008) Computational and experimental studies on the catalytic mechanism of biliverdin-IXbeta reductase. Biochem. J. 411, 475-484.

(38)

Alam, J., Stewart, D., Touchard, C., Boinapally, S., Choi, A. M., and Cook, J. L. (1999) Nrf2, a Cap'n'Collar

(39)

Galloway, D. C., Blake, D. G., and McLellan, L. I. (1999) Regulation of gamma-glutamylcysteine synthetase

transcription factor, regulates induction of the heme oxygenase-1 gene. J. Biol. Chem. 274, 26071-26078. regulatory subunit (GLCLR) gene expression: identification of the major transcriptional start site in HT29 cells. Biochim. Biophys. Acta 1446, 47-56. (40)

Pompella, A., Visvikis, A., Paolicchi, A., De Tata, V., and Casini, A. F. (2003) The changing faces of glutathione, a cellular protagonist. Biochem. Pharmacol. 66, 1499-1503.

(41)

Oppermann, U. (2007) Carbonyl reductases: the complex relationships of mammalian carbonyl- and 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

quinone-reducing enzymes and their role in physiology. Annu. Rev. Pharmacol. Toxicol. 47, 293-322. (42)

Ebert, B., Kisiela, M., Malatkova, P., El-Hawari, Y., and Maser, E. (2010) Regulation of human carbonyl

(43)

Bell, K. F., Fowler, J. H., Al-Mubarak, B., Horsburgh, K., and Hardingham, G. E. (2011) Activation of

reductase 3 (CBR3; SDR21C2) expression by Nrf2 in cultured cancer cells. Biochemistry 49, 8499-8511. Nrf2-regulated glutathione pathway genes by ischemic preconditioning. Oxid. Med. Cell. Longevity 2011, 689524. (44)

Hartikainen, J. M., Tengstrom, M., Kosma, V. M., Kinnula, V. L., Mannermaa, A., and Soini, Y. (2012) Genetic polymorphisms and protein expression of NRF2 and Sulfiredoxin predict survival outcomes in breast cancer. Cancer Res. 72, 5537-5546.

(45)

Turpaev, K. T. (2013) Keap1-Nrf2 signaling pathway: mechanisms of regulation and role in protection of cells against toxicity caused by xenobiotics and electrophiles. Biochemistry. Biokhimiia 78, 111-126.

(46)

Ma, Q., and He, X. (2012) Molecular basis of electrophilic and oxidative defense: promises and perils of Nrf2. Pharmacol. Rev. 64, 1055-1081.

(47)

Bryan, H. K., Olayanju, A., Goldring, C. E., and Park, B. K. (2013) The Nrf2 cell defence pathway:

(48)

Kansanen, E., Kuosmanen, S. M., Leinonen, H., and Levonen, A.-L. (2013) The Keap1-Nrf2 pathway:

Keap1-dependent and -independent mechanisms of regulation. Biochem. Pharmacol. 85, 705-717. Mechanisms of activation and dysregulation in cancer. Redox Biol. 1, 45-49. (49)

Ren, D., Villeneuve, N. F., Jiang, T., Wu, T., Lau, A., Toppin, H. A., and Zhang, D. D. (2011) Brusatol enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism. Proc. Natl. Acad. Sci. U.S.A. 108, 1433-1438.

(50)

Jain, A., Lamark, T., Sjøttem, E., Bowitz Larsen, K., Atesoh Awuh, J., Øvervatn, A., McMahon, M., Hayes, J. D., and Johansen, T. (2010) p62/SQSTM1 Is a Target Gene for Transcription Factor NRF2 and Creates a Positive Feedback Loop by Inducing Antioxidant Response Element-driven Gene Transcription. J. Biol. Chem. 285, 22576-22591.

(51)

Nezis, I. P., and Stenmark, H. (2012) p62 at the interface of autophagy, oxidative stress signaling, and cancer. Antioxid. Redox Signal. 17, 786-793.

(52)

Ashoor, R., Yafawi, R., Jessen, B., and Lu, S. (2013) The contribution of lysosomotropism to autophagy perturbation. PloS one 8, e82481.

(53)

Pankiv, S., Clausen, T. H., Lamark, T., Brech, A., Bruun, J. A., Outzen, H., Overvatn, A., Bjorkoy, G., and Johansen, T. (2007) p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J. Biol. Chem. 282, 24131-24145.

(54)

Wang, Y., Lin, Y.-X., Qiao, Z.-Y., An, H.-W., Qiao, S.-L., Wang, L., Rajapaksha, R. P. Y. J., and Wang, H. (2015) Self-Assembled Autophagy-Inducing Polymeric Nanoparticles for Breast Cancer Interference In-Vivo. Adv. Mater. 27, 2627-2634.

(55)

Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., and Yoshimori, T. (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720-5728.

(56)

Yang, Z., and Klionsky, D. J. (2010) Eaten alive: a history of macroautophagy. Nat. Cell Biol. 12, 814-822.

(57)

Mizushima, N., Yoshimori, T., and Levine, B. (2010) Methods in mammalian autophagy research. Cell 140, 313-326.

(58)

Roberg, K., and Ollinger, K. (1998) A pre-embedding technique for immunocytochemical visualization of

(59)

Gómez-Sintes, R., Ledesma, M. D., and Boya, P. (2016) Lysosomal cell death mechanisms in aging. Ageing Res.

cathepsin D in cultured cells subjected to oxidative stress. J. Histochem. Cytochem. 46, 411-418. Rev. 32, 150-168. (60)

Chen, C. S., Chen, W. N., Zhou, M., Arttamangkul, S., and Haugland, R. P. (2000) Probing the cathepsin D using 28


Page 28 of 36

Page 29 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a BODIPY FL-pepstatin A: applications in fluorescence polarization and microscopy. J. Biochem. Bioph. Methods 42, 137-151. (61)

Kawai, A., Uchiyama, H., Takano, S., Nakamura, N., and Ohkuma, S. (2007) Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells. Autophagy 3, 154-157.

(62)

Panzarini, E., and Dini, L. (2014) Nanomaterial-induced autophagy: a new reversal MDR tool in cancer therapy?

(63)

Lin, J., Huang, Z., Wu, H., Zhou, W., Jin, P., Wei, P., Zhang, Y., Zheng, F., Zhang, J., Xu, J., et al. (2014) Inhibition

Mol. Pharm. 11, 2527-2538. of autophagy enhances the anticancer activity of silver nanoparticles. Autophagy 10, 2006-2020. (64)

Liu, H. L., Zhang, Y. L., Yang, N., Zhang, Y. X., Liu, X. Q., Li, C. G., Zhao, Y., Wang, Y. G., Zhang, G. G., Yang, P., et al. (2011) A functionalized single-walled carbon nanotube-induced autophagic cell death in human lung cells through Akt–TSC2-mTOR signaling. Cell Death Dis. 2, e159.

(65)

Zhang, X., Zhang, H., Liang, X., Zhang, J., Tao, W., Zhu, X., Chang, D., Zeng, X., Liu, G., and Mei, L. (2016) Iron Oxide Nanoparticles Induce Autophagosome Accumulation through Multiple Mechanisms: Lysosome Impairment, Mitochondrial Damage, and ER Stress. Mol. Pharm. 13, 2578-2587.

(66)

Hussain, S., Garantziotis, S., Rodrigues-Lima, F., Dupret, J.-M., Baeza-Squiban, A., and Boland, S. (2014) Intracellular Signal Modulation by Nanomaterials, in Nanomaterial: Impacts on Cell Biology and Medicine (Capco, D. G., and Chen, Y., Eds.) pp 111-134, Springer Netherlands, Dordrecht.

(67)

Mizushima, N. (2007) Autophagy: process and function. Genes Dev. 21, 2861-2873.

(68)

Stern, S. T., Adiseshaiah, P. P., and Crist, R. M. (2012) Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Part. Fibre Toxicol. 9, 20.

(69)

Shannon, P., Markiel, A., Ozier, O., Baliga, N. S., Wang, J. T., Ramage, D., Amin, N., Schwikowski, B., and Ideker, T. (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498-2504.

(70)

Bindea, G., Mlecnik, B., Hackl, H., Charoentong, P., Tosolini, M., Kirilovsky, A., Fridman, W. H., Pages, F., Trajanoski, Z., and Galon, J. (2009) ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics 25, 1091-1093.

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Figure 1 125x155mm (300 x 300 DPI)


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Figure 2 129x170mm (300 x 300 DPI)



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Figure 3 160x219mm (300 x 300 DPI)


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Figure 4 116x137mm (300 x 300 DPI)



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Figure 5 80x119mm (300 x 300 DPI)


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Figure 6 140x150mm (300 x 300 DPI)



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Table of Contents Graphic 65x50mm (300 x 300 DPI)


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