Upregulation of CYP1B1 by hypoxia is mediated by ERα activation in breast cancer cells.

Endocrine therapy for breast cancer often leads to drug resistance and tumor recurrence; tumor hypoxia is also associated with mortality and tumor relapse. Cytochrome P450 1B1 (CYP1B1) regulates estrogen metabolism in breast cells and is known to be overexpressed in breast cancer tissue. Although the individual association of hypoxia-induced hypoxia-inducible factor-1-alpha (HIF-1α) and CYP1B1 with tumorigenesis is well known, the association between HIF-1α and CYP1B1 leading to tumorigenesis has not been investigated. Here, we investigated the correlation between hypoxia and CYP1B1 expression in breast cancer cells for tumorigenesis-related mechanisms. Hypoxia was induced in the human breast cancer cell lines MCF-7 (Er-positive) and MDA-MB-231 (triple-negative) and the normal breast epithelial cell line MCF10A; these cell lines were then subjected to immunoblotting, transient transfection, luciferase assays, gene silencing using small interfering RNA, polymerase chain reaction analysis, chromatin immunoprecipitation, co-immunoprecipitation, and mammalian two-hybrid assays. Furthermore, immunofluorescence analysis of the tumor microarrays was performed, and the pub2015 and the Cancer Genome Atlas patient datasets were analyzed. HIF-1α expression in response to hypoxia occurred in both normal and breast cancer cells, whereas CYP1B1 was induced only in estrogen receptor α (ERα)-positive breast cancer cells under hypoxia. HIF-1α activated ERα through direct binding and in a ligand-independent manner to promote CYP1B1 expression. Therefore, we suggested the mechanism by which hypoxia and ER-positivity orchestrate breast cancer relapse.

Pillar-Based Mechanical Induction of an Aggressive Tumorigenic Lung Cancer Cell Model.

Tissue microarchitecture imposes physical constraints to the migration of individual cells. Especially in cancer metastasis, three-dimensional structural barriers within the extracellular matrix are known to affect the migratory behavior of cells, regulating the pathological state of the cells. Here, we employed a culture platform with micropillar arrays of 2 µm diameter and 16 µm pitch (2.16 micropillar) as a mechanical stimulant. Using this platform, we investigated how a long-term culture of A549 human lung carcinoma cells on the (2.16) micropillar-embossed dishes would influence the pathological state of the cell. A549 cells grown on the (2.16) micropillar array with 10 µm height exhibited a significantly elongated morphology and enhanced migration even after the detachment and reattachment, as evidenced in the conventional wound-healing assay, single-cell tracking analysis, and in vivo tumor colonization assays. Moreover, the pillar-induced morphological deformation in nuclei was accompanied by cell-cycle arrest in the S phase, leading to suppressed proliferation. While these marked traits of morphology-migration-proliferation support more aggressive characteristics of metastatic cancer cells, typical indices of epithelial-mesenchymal transition were not found, but instead, remarkable traces of amoeboidal transition were confirmed. Our study also emphasizes the importance of mechanical stimuli from the microenvironment during pathogenesis and how gained traits can be passed onto subsequent generations, ultimately affecting their pathophysiological behavior. Furthermore, this study highlights the potential use of pillar-based mechanical stimuli as an in vitro cell culture strategy to induce more aggressive tumorigenic cancer cell models.

Identification of Nucleolin as a Novel AEG-1-Interacting Protein in Breast Cancer via Interactome Profiling.

Breast cancer is one of the most common malignant diseases worldwide. Astrocyte elevated gene-1 (AEG-1) is upregulated in breast cancer and regulates breast cancer cell proliferation and invasion. However, the molecular mechanisms by which AEG-1 promotes breast cancer have yet to be fully elucidated. In order to delineate the function of AEG-1 in breast cancer development, we mapped the AEG-1 interactome via affinity purification followed by LC-MS/MS. We identified nucleolin (NCL) as a novel AEG-1 interacting protein, and co-immunoprecipitation experiments validated the interaction between AEG-1 and NCL in breast cancer cells. The silencing of NCL markedly reduced not only migration/invasion, but also the proliferation induced by the ectopic expression of AEG-1. Further, we found that the ectopic expression of AEG-1 induced the tyrosine phosphorylation of c-Met, and NCL knockdown markedly reduced this AEG-1 mediated phosphorylation. Taken together, our report identifies NCL as a novel mediator of the oncogenic function of AEG-1, and suggests that c-Met could be associated with the oncogenic function of the AEG-1-NCL complex in the context of breast cancer.

A Novel Protein-Protein Interaction between RSK3 and IκBα and a New Binding Inhibitor That Suppresses Breast Cancer Tumorigenesis.

Multiple cancer-related biological processes are mediated by protein-protein interactions (PPIs). Through interactions with a variety of factors, members of the ribosomal S6 kinase (RSK) family play roles in cell cycle progression and cell proliferation. In particular, RSK3 contributes to cancer viability, but the underlying mechanisms remain unknown. We performed a kinase library screen to find IκBα PPI binding partners and identified RSK3 as a novel IκBα binding partner using a cell-based distribution assay. In addition, we discovered a new PPI inhibitor using mammalian two-hybrid (MTH) analysis. We assessed the antitumor effects of the new inhibitor using cell proliferation and colony formation assays and monitored the rate of cell death by FACS apoptosis assay. IκBα is phosphorylated by the active form of the RSK3 kinase. A small-molecule inhibitor that targets the RSK3/IκBα complex exhibited antitumor activity in breast cancer cells and increased their rate of apoptosis. RSK3 phosphorylation and RSK3/IκBα complex formation might be functionally important in breast tumorigenesis. The RSK3/IκBα-specific binding inhibitor identified in this study represents a lead compound for the development of new anticancer drugs.

Co-relation with novel phosphorylation sites of IκBα and necroptosis in breast cancer cells.

BACKGROUND: Phosphorylation of NF-kappaB inhibitor alpha (IκBα) is key to regulation of NF-κB transcription factor activity in the cell. Several sites of IκBα phosphorylation by members of the IκB kinase family have been identified, but phosphorylation of the protein by other kinases remains poorly understood. We investigated a new phosphorylation site on IκBα and identified its biological function in breast cancer cells. METHODS: Previously, we observed that aurora kinase (AURK) binds IκBα in the cell. To identify the domains of IκBα essential for phosphorylation by AURK, we performed kinase assays with a series of IκBα truncation mutants. AURK significantly promoted activation of IκBα at serine 32 but not serine 36; by contrast, IκB kinase (IKK) family proteins activated both of these residues. We also confirmed phosphorylation of IκBα by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and nano-liquid chromatography hybrid quadrupole orbitrap mass spectrometer (nanoLC-MS/MS; Q-Exactive). RESULTS: We identified two novel sites of serine phosphorylation, S63 and S262. Alanine substitution of S63 and S262 (S63A and S262A) of IκBα inhibited proliferation and suppressed p65 transcription activity. In addition, S63A and/or S262A of IκBα regulated apoptotic and necroptotic effects in breast cancer cells. CONCLUSIONS: Phosphorylation of IκBα by AURK at novel sites is related to the apoptosis and necroptosis pathways in breast cancer cells.

Activity-Based Protein Profiling Reveals Potential Dasatinib Targets in Gastric Cancer.

Dasatinib is a multi-target kinase inhibitor, whose targets include BCR-ABL, SRC family kinases, and various cancer kinases. The elevated SRC activity in gastric cancer (GC) has prompted the need for the therapeutic application of dasatinib in GC. We observed that the efficacy of dasatinib varied with the GC cell lines. The differential effect of dasatinib was not correlated with the basal SRC activity of each cell line. Moreover, the GC cell lines showing the strong antitumor effects of dasatinib were refractory to other SRC inhibitors, i.e., bosutinib and saracatinib, suggesting that unexpected dasatinib's targets could exist. To profile the targets of dasatinib in GC, we performed activity-based protein profiling (ABPP) via mass spectrometry using a desthiobiotin-ATP probe. We identified 29 and 18 kinases as potential targets in dasatinib-sensitive (SNU-216, MKN-1) and -resistant (SNU-484, SNU-601) cell lines, respectively. The protein-protein interaction mapping of the differential drug targets in dasatinib-sensitive and -resistant GC using the STRING database suggested that dasatinib could target cellular energy homeostasis in the drug-sensitive GC. RNAi screening for identified targets indicated p90RSK could be a novel dasatinib target, which is important for maintaining the viability and motility of GC cells. Further functional validation of dasatinib off-target actions will provide more effective therapeutic options for GC.

Development of a Polo-like Kinase-1 Polo-Box Domain Inhibitor as a Tumor Growth Suppressor in Mice Models.

Polo-like kinase-1 (Plk1) plays a key role in mitosis and has been identified as an attractive anticancer drug target. Plk1 consists of two drug-targeting sites, namely, N-terminal kinase domain (KD) and C-terminal polo-box domain (PBD). As KD-targeting inhibitors are associated with severe side effects, here we report on the pyrazole-based Plk1 PBD inhibitor, KBJK557, which showed a remarkable in vitro anticancer effect by inducing Plk1 delocalization, mitotic arrest, and apoptosis in HeLa cells. Further, in vivo optical imaging analysis and antitumorigenic activities in mouse xenograft models demonstrate that KBJK557 preferentially accumulates in cancer cells and selectively inhibits cancer cell proliferation. Pharmacokinetic profiles and partition coefficients suggest that KBJK557 was exposed in the blood and circulated through the organs with an intermediate level of clearance (t1/2, 7.73 h). The present investigation offers a strategy for specifically targeting cancer using a newly identified small-molecule inhibitor that targets the Plk1 PBD.

Mitochondrial carnitine palmitoyltransferase 2 is involved in Nε-(carboxymethyl)-lysine-mediated diabetic nephropathy.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease in the world. Advanced glycation end products (AGEs) are thought to be involved in the pathogenesis of DN via multifactorial mechanisms including the generation of oxidative stress and overproduction of various growth factors and cytokines. AGEs are heterogeneous cross-linked sugar-derived proteins, and Nε-(carboxymethyl)-lysine (CML)-conjugated BSA is a major component of AGEs. However, the proteins involved in DN induction by CML have never been reported. Herein, we investigated specific protein regulators of AGE-mediated DN via proteomic analysis of streptozotocin (STZ)-induced diabetic mice kidneys. We identified 937, 976, and 870 proteins in control, STZ, and STZ + CML-BSA samples, respectively. Bioinformatics analysis identified several CML-mediated proteins potentially involved in kidney damage, activation of fatty acid oxidation (FAO), and mitochondrial dysfunction. Furthermore, we identified the CML-specific differential protein carnitine palmitoyltransferase 2 (CPT2), related to FAO. To confirm the effect of CPT2 and the CML-mediated mechanism, human renal tubular HK-2 cells were treated with CML-BSA and cpt2 siRNA, and examined for FAO-mediated fibrosis and mitochondrial dysfunction. CML-BSA and CPT2 knockdown induced fibrosis-related gene expression and damage to mitochondrial membrane potential. Moreover, CPT2 overexpression recovered CML-induced fibrosis-related gene expression. Based on these results, a decrease in CML-induced CPT2 expression causes mitochondrial FAO damage, leading to renal fibrosis and DN.

Amphiphilic Triazine Polymer Derivatives as Antibacterial And Anti-atopic Agents in Mice Model.

Considering the emergence of bacterial resistance and low proteolytic stability of antimicrobial peptides (AMPs), herein we developed a series of ultra-short triazine based amphipathic polymers (TZP) that are connected with ethylene diamine linkers instead of protease sensitive amide bond. The most potent oligomers, TZP3 and TZP5 not only displayed potent antibacterial action on various drug-resistant pathogens but also exhibited a strong synergic antibacterial activity in combination with chloramphenicol against multidrug-resistant Pseudomonas aeruginosa (MDRPA). Since most of atopic dermatitis (AD) infections are caused by bacterial colonization, we evaluated the potency of TZP3 and TZP5 on AD in vitro and in vivo. In vitro AD analysis of these two polymers showed significant inhibition against the release of ß-hexosaminidase and tumor necrosis factor (TNF-α) from RBL-2H3 cells. In AD-like skin lesions in BALB/c mice model, these two polymers displayed significant potency in suppressing dermal and epidermal thickness, mast cell infiltration and pro-inflammatory cytokines expression. Moreover, these polymers exhibited remarkable efficacy over the allergies caused by the imbalance of Th1/Th2 by regulating total IgE and IgG2a. Finally, the impact of treatment effects of these polymers was examined through analyzing the weights and sizes of spleen and lymph node of AD-induced mice.

Cationic Amphipathic Triazines with Potent Anti-bacterial, Anti-inflammatory and Anti-atopic Dermatitis Properties.

The emergence of multi-drug resistant bacteria forces the therapeutic world into a position, where the development of new and alternative kind of antibiotics is highly important. Herein, we report the development of triazine-based amphiphilic small molecular antibacterial agents as mimics of lysine- and arginine-based cationic peptide antibiotics (CPAs). These compounds were screened against a panel of both Gram-positive and Gram-negative bacterial strains. Further, anti-inflammatory evaluation of these compounds led to the identification of four efficient compounds, DG-5, DG-6, DL-5, and DL-6. These compounds displayed significant potency against drug-resistant bacteria, including methicillin-resistant S. aureus (MRSA), multidrug-resistant P. aeruginosa (MDRPA), and vancomycin-resistant E. faecium (VREF). Mechanistic studies, including cytoplasmic membrane depolarization, confocal imaging and flow cytometry suggest that DG-5, DG-6, and DL-5 kill bacteria by targeting bacterial membrane, while DL-6 follows intracellular targeting mechanism. We also demonstrate that these molecules have therapeutic potential by showing the efficiency of DG-5 in preventing the lung inflammation of lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. More interestingly, DL-6 exhibited impressive potency on atopic dermatitis (AD)-like skin lesions in BALB/c mice model by suppressing pro-inflammatory cytokines. Collectively, these results suggest that they can serve a new class of antimicrobial, anti-inflammatory and anti-atopic agents with promising therapeutic potential.

Prolonged MEK inhibition leads to acquired resistance and increased invasiveness in KRAS mutant gastric cancer.

Gastric cancer (GC) is one of the most common causes of cancer-associated death. However, traditional therapeutic strategies have failed to significantly improve the survival of patient with advanced GC. While KRAS mutations have been found in some patients with gastric cancer, an effective therapy to treat KRAS-driven gastric cancer has not been established yet. To provide a rationale for clinical application of kinase inhibitors targeting RAS pathways, we first determined the sensitivity of GC cell lines harboring KRAS mutations or amplification to RAS pathway inhibitors. We found that MAPK pathway inhibitors (MEKi and ERKi) were more effective than AKT inhibitor, suggesting that KRAS-driven gastric cancer cells are dependent on MAPK pathway for survival. Further, we established a KRAS mutant GC cell line with acquired resistance to MEK inhibitors in order to mimic clinical situation of kinase inhibitor resistance. A comprehensive analysis of tyrosine phosphorylation in receptor tyrosine kinases in combination with small molecule chemical library screening revealed upregulated c-MET phosphorylation in this resistance cell line with elevated sensitivity to c-MET TKI (crizotinib) and PI3K/mTOR dual inhibitor (BEZ235). We also showed that migration and invasion of resistant cells were promoted, and crizotinib and BEZ235 could inhibit this malignant phenotype. Overall, our results indicate that prolonged MAPK pathway inhibition could result in acquired resistance which is associated with increased malignant phenotype in KRAS mutant GC and pharmacological targeting c-MET and PI3K/mTOR could overcome this problem.

A small-molecule inhibitor targeting the AURKC-IκBα interaction decreases transformed growth of MDA-MB-231 breast cancer cells.

The Aurora kinases, Aurora A (AURKA), Aurora B (AURKB), and Aurora C (AURKC), are serine/threonine kinases required for the control of mitosis (AURKA and AURKB) or meiosis (AURKC). Several Aurora kinase inhibitors are being investigated as novel anticancer therapeutics. Recent studies demonstrated that AURKC activation contributes to breast cancer cell transformation. Therefore, AURKC is both a promising marker and therapeutic target for breast cancer; however, its signaling network has not been fully characterized. Using translocation-based cellular assays, we identified IκBα as a binding partner of AURKC, and found that AURKC phosphorylates IκBα at Ser32, thereby activating it. In silico modeling and computational analyses revealed a small-molecule inhibitor (AKCI) that blocked the AURKC-IκBα interaction and exerted antitumor activity in MDA-MB-231 breast cancer cells. Specifically, AKCI induced G2/M cell-cycle arrest through modulation of the p53/p21/CDC2/cyclin B1 pathways. In addition, the drug significantly inhibited MDA-MB-231 cell migration and invasion, as well as decreasing colony formation and tumor growth. Via its interaction with IκBα, AURKC indirectly induced NF-κB activation; accordingly, AKCI decreased PMA-induced activation of NF-κB. Thus, the small-molecule inhibitor AKCI represents a first step towards developing targeted inhibitors of AURKC protein binding, which may lead to further advances in the treatment of breast cancer.

Evidence for the presence of cell-surface-bound and intracellular bactericidal toxins in the dinoflagellate Heterocapsa circularisquama.

Heterocapsa circularisquama, a harmful dinoflagellate, has multiple haemolytic toxins that are considered to be involved in the toxic mechanism against shellfish and certain species of zooplankton. To evaluate the further nature of the toxins of H. circularisquama, we investigated its effects on several species of bacteria. By colony formation assay, we found that H. circularisquama had antibacterial activity toward the marine bacterium Vibrio alginolyticus in a cell density-dependent manner. When the inoculated bacterial cells were co-cultured with H. circularisquama under dinoflagellate cell culture conditions, the bacterial growth was significantly suppressed, whereas the number of live bacterial cells increased when cultured in the medium alone. Since the cell-free culture supernatant and the ruptured dinoflagellate cell suspension showed no toxic effects on V. alginolyticus, it is speculated that direct cell-to-cell contact mediated by the live dinoflagellate cells may be the major toxic mechanism. The decrease in bactericidal activity of theca-removed dinoflagellate cells may further support this speculation. H. circularisquama also showed bactericidal activities towards Escherichia coli and Staphylococcus aureus. In the dinoflagellate/bacteria co-culture system, the number of live bacterial cells declined with increasing incubation time. Light-dependent antibacterial activity of the ruptured dinoflagellate cells against S. aureus was observed, whereas no such activity was detected against E. coli. These results suggest that intracellular photosensitising bactericidal toxins, which were previously found to be porphyrin derivatives, may have specificity towards gram-positive bacteria. Based on these results together with previous studies, it is obvious that H. circularisquama possesses antibacterial activity, which may be mediated through toxins located on its cell surface. It is likely that such toxins play a role in the defence mechanism against predators and infectious bacteria. Although the exact biological significance of intracellular photosensitising toxins is still unclear, such toxins may have potential to be developed as novel photo-controllable antibiotics.

One-Dimensional Supramolecular Nanoplatforms for Theranostics Based on Co-Assembly of Peptide Amphiphiles.

We report a simple and facile strategy for the preparation of multifunctional nanoparticles with programmable properties using self-assembly of precisely designed block amphiphiles in an aqueous solution-state. Versatile, supramolecular nanoplatform for personalized needs, particularly-theranostics, was fabricated by coassembly of peptide amphiphiles (PAs) in aqueous solution, replacing time-consuming and inaccessible chemical synthesis. Fibrils, driven by the assembly of hydrophobic ß-sheet-forming peptide block, were utilized as a nanotemplate for drug loading within their robust core. PAs were tagged with octreotide [somatostatin (SST) analogue] for tumor-targeting or were conjugated with paramagnetic metal ion (Gd3+)-chelating 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for magnetic resonance (MR) imaging. The two PA types were coassembled to integrate each PA function into original fibrillar nanotemplates. The adoption of a bulky target-specific cyclic octreotide and ß-sheet-forming peptide with enhanced hydrophobicity led to a morphological transition from conventional fibrils to helical fibrils. The resulting one-dimensional nanoaggregates allowed the successful intracellular delivery of doxorubicin (DOX) to MCF-7 cancer cells overexpressing SST receptor (SSTR) and MR imaging by enabling high longitudinal (T1) relaxivity of water protons. Correlation between the structural nature of fibrils formed by PA coassembly and contrast efficacy was elucidated. The coassembly of PAs with desirable functions may thus be a useful strategy for the generation of tailor-made biocompatible nanomaterials.

Correction: Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-degrading Bacterium Burkholderia sp. K24.

[This corrects the article DOI: 10.1371/journal.pone.0154233.].

Bakuchiol sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins.

We investigated whether bakuchiol, an analog of resveratrol enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. Bakuchiol enhanced expression of cell death receptor (DR) in TRAIL-sensitive and -resistant colon cancer cells in a dose-dependent manner. A combination of bakuchiol with TRAIL significantly inhibited cell growth of TRAIL sensitive HCT116 and TRAIL resistant HT-29 cells. The expression of TRAIL receptors; DR4 and DR5 was significantly increased by treatment of bakuchiol, however, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed. Moreover, the expression of apoptosis related proteins such as cleaved caspase-3, -8, -9 and PARP was increased by combination treatment of bakuchiol and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory effects of bakuchiol in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the bakuchiol induced cell growth inhibitory effects. The collective results suggest that bakuchiol facilitates TRAIL-induced apoptosis in colon cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals.

Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24.

Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the ß-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX.

Bioflocculation of the oceanic microalga Dunaliella salina by the bloom-forming dinoflagellate Heterocapsa circularisquama, and its effect on biodiesel properties of the biomass.

The aim of this work was to evaluate the flocculation by the dinoflagellate Heterocapsa circularisquama as a means for harvesting three Chlorophyta species, Chlorella vulgaris, Nannochloropsis granulata, and Dunaliella salina. Relative fluorescence of D. salina culture significantly decreased along with 9.3-fold increased flocculation activity within 24 h when mixed with H. circularisquama. Lipid content of bioflocculated D. salina increased about 40%, while fatty acid methyl ester (FAME) profiles exhibited higher levels of C16:0, C18:0, and C18:1, compared to harvest by centrifugation, suggesting higher energy content. Furthermore, bioflocculated D. salina biomass had more suitable biodiesel properties relative to both EN14214 and ASTMD6751, with a cetane number of 49.0 and an iodine value of 95.9. These results suggest that H. circularisquama-induced bioflocculation is applicable for the sustainable and qualitative production of algal biodiesel.

Phenyl 2-pyridyl ketoxime induces cellular senescence-like alterations via nitric oxide production in human diploid fibroblasts.

Phenyl-2-pyridyl ketoxime (PPKO) was found to be one of the small molecules enriched in the extracellular matrix of near-senescent human diploid fibroblasts (HDFs). Treatment of young HDFs with PPKO reduced the viability of young HDFs in a dose- and time-dependent manner and resulted in senescence-associated ß-galactosidase (SA-ß-gal) staining and G2/M cell cycle arrest. In addition, the levels of some senescence-associated proteins, such as phosphorylated ERK1/2, caveolin-1, p53, p16(ink4a), and p21(waf1), were elevated in PPKO-treated cells. To monitor the effect of PPKO on cell stress responses, reactive oxygen species (ROS) production was examined by flow cytometry. After PPKO treatment, ROS levels transiently increased at 30 min but then returned to baseline at 60 min. The levels of some antioxidant enzymes, such as catalase, peroxiredoxin II and glutathione peroxidase I, were transiently induced by PPKO treatment. SOD II levels increased gradually, whereas the SOD I and III levels were biphasic during the experimental periods after PPKO treatment. Cellular senescence induced by PPKO was suppressed by chemical antioxidants, such as N-acetylcysteine, 2,2,6,6-tetramethylpiperidinyloxy, and L-buthionine-(S,R)-sulfoximine. Furthermore, PPKO increased nitric oxide (NO) production via inducible NO synthase (iNOS) in HDFs. In the presence of NOS inhibitors, such as L-NG-nitroarginine methyl ester and L-NG-monomethylarginine, PPKO-induced transient NO production and SA-ß-gal staining were abrogated. Taken together, these results suggest that PPKO induces cellular senescence in association with transient ROS and NO production and the subsequent induction of senescence-associated proteins.