Synthesis and anti-prion aggregation activity of acylthiosemicarbazide analogues.

Prions are infectious protein particles known to cause prion diseases. The biochemical entity of the pathogen is the misfolded prion protein (PrPSc) that forms insoluble amyloids to impair brain function. PrPSc interacts with the non-pathogenic, cellular prion protein (PrPC) and facilitates conversion into a nascent misfolded isoform. Several small molecules have been reported to inhibit the aggregation of PrPSc but no pharmacological intervention was well established thus far. We, here, report that acylthiosemicarbazides inhibit the prion aggregation. Compounds 7x and 7y showed almost perfect inhibition (EC50 = 5 µM) in prion aggregation formation assay. The activity was further confirmed by atomic force microscopy, semi-denaturing detergent agarose gel electrophoresis and real-time quaking induced conversion assay (EC50 = 0.9 and 2.8 µM, respectively). These compounds also disaggregated pre-existing aggregates in vitro and one of them decreased the level of PrPSc in cultured cells with permanent prion infection, suggesting their potential as a treatment platform. In conclusion, hydroxy-2-naphthoylthiosemicarbazides can be an excellent scaffold for the discovery of anti-prion therapeutics.

Ursonic acid inhibits migration and invasion of human osteosarcoma cells through the suppression of mitogen-activated protein kinases and matrix metalloproteinases.

INTRODUCTION: Osteosarcoma (OS) is the most common form of bone malignancy. Although contemporary chemotherapy and surgery have improved the prognosis of those with OS, developing new OS therapies has proven difficult for some time. The activation of the matrix metalloproteinase (MMP) and mitogen-activated protein kinase (MAPK) signaling pathways can induce metastasis, which is an obstacle to OS treatment. Ursonic acid (UNA) is a phytochemical with the potential to cure a variety of human ailments, including cancer. METHODS AND RESULTS: In this study, we investigated the anti-tumor properties of UNA in MG63 cells. We conducted colony formation assay, wound healing assay, and Boyden chamber assays to investigate the anti-OS effects of UNA. UNA was found to significantly inhibit the proliferative, migratory, and invasive abilities of MG63 cells. This bioactivity of UNA was mediated by the inhibition of extracellular signal-regulated kinase (ERK) and p38 and reduction of MMP-2 transcriptional expression as observed in western blot analysis, gelatin zymography and RT-PCR. Anti-OS activities of UNA were also observed in Saos2 and U2OS cells, indicating that its anti-cancer properties are not specific to cell types. CONCLUSION: Our findings suggest that UNA has the potential for use in anti-metastatic drugs in the treatment of OS.

A three-dimensional spheroid co-culture system of neurons and astrocytes derived from Alzheimer's disease patients for drug efficacy testing.

Cell culture systems derived from the progenitor cells of human patients have many advantages over animal models for therapeutic drug testing and studies of disease pathogenesis. Here we describe a three-dimensional (3D) spheroid co-culture system of neurons and astrocytes derived from induced pluripotent stem cells-neural precursor cells (iPSCs-NPCs) of Alzheimer's disease (AD) patients or healthy individuals that can provide information on drug efficacy unobtainable by 2D co-culture or monoculture approaches. iPSCs-NPCs of healthy controls or AD patients were seeded onto 96-well U-bottom plates and incubated with neuronal differentiation medium for one week and with astrocytic medium for two weeks to replicate the temporal order of cell maturation during brain development. These 3D spheroid models expressed marker proteins for mature neurons and astrocytes. In particular, patient-derived spheroids showed beta-amyloid (Aß) accumulation as revealed by thioflavin T (ThT) staining and ELISA. Aggregation of Aß induced caspase activation and cell death, while the neuroprotectants nordihydroguaiaretic acid (NDGA) and curcumin (CU) reduced the levels of both ThT and caspase staining. Taken together, these results demonstrate the feasibility of our 3D spheroids combined with ThT and caspase staining as a patient-based anti-AD drug screening platform.

Phthalate plasticizer decreases the prion-like protein doppel essential for structural integrity and function of spermatozoa.

Di-n-butyl phthalate (DBP), a well-known endocrine disruptor, causes male reproductive dysfunction. To understand the underlying mechanisms, we performed histological, endocrinological, and biochemical analyses and assessed the expression of genes involved in spermatogenesis and sperm function according to OECD test guideline 407. Following 28 days of administration of the lowest observed adverse effect level dose of DBP to mice, no significant changes in body weight, testis and epididymis weights and histology, serum testosterone level, or testicular daily sperm production were found. Nonetheless, the motility of the epididymal sperm of the DBP group was significantly decreased together with an increase in the incidence of bent tails and abnormal heads. In the testes of the DBP group, lipid peroxidation (LPO) level was significantly increased and testicular Bcl-2 mRNA level was significantly decreased together with an increase in the Bax/Bcl-2 mRNA ratio. In the testes of the DBP group, levels of Prnd mRNA and protein and Pou4f1 mRNA, an activator of the Prnd promotor, were significantly decreased. Of note, prion-like protein doppel (PRND) was significantly decreased together with decreased PRND immunoreactivity in the head, midpiece, and tail of sperm. In the testes of the DBP group, levels of Sox9, Sgp1, and Sgp2 mRNA, which are functional Sertoli cell markers, were significantly decreased. Level of Amh mRNA, a Sertoli cell immaturity marker, was significantly increased together with that of Inha mRNA, suggesting deregulation of the brain-gonadal axis. Together, our findings suggest that DBP at present dosage may potentiate LPO generation and Sertoli cell immaturity via downregulation of Sox9 and disruption of the Pou4f1-Prnd gene network in post-meiotic germ cells without visible changes in spermatogenesis or testosterone level. This may result in structural and functional abnormalities in spermatozoa. Additionally, our findings suggest that assessment of the male reproductive toxicity of phthalate ester plasticizers based on conventional OECD test guidelines should be reconsidered.

Endoproteolysis of cellular prion protein by plasmin hinders propagation of prions.

Many questions surround the underlying mechanism for the differential metabolic processing observed for the prion protein (PrP) in healthy and prion-infected mammals. Foremost, the physiological α-cleavage of PrP interrupts a region critical for both toxicity and conversion of cellular PrP (PrP C ) into its misfolded pathogenic isoform (PrP Sc ) by generating a glycosylphosphatidylinositol (GPI)-anchored C1 fragment. During prion diseases, alternative ß-cleavage of PrP becomes prominent, producing a GPI-anchored C2 fragment with this particular region intact. It remains unexplored whether physical up-regulation of α-cleavage can inhibit disease progression. Furthermore, several pieces of evidence indicate that a disintegrin and metalloproteinase (ADAM) 10 and ADAM17 play a much smaller role in the α-cleavage of PrP C than originally believed, thus presenting the need to identify the primary protease(s) responsible. For this purpose, we characterized the ability of plasmin to perform PrP α-cleavage. Then, we conducted functional assays using protein misfolding cyclic amplification (PMCA) and prion-infected cell lines to clarify the role of plasmin-mediated α-cleavage during prion propagation. Here, we demonstrated an inhibitory role of plasmin for PrP Sc formation through PrP α-cleavage that increased C1 fragments resulting in reduced prion conversion compared with non-treated PMCA and cell cultures. The reduction of prion infectious titer in the bioassay of plasmin-treated PMCA material also supported the inhibitory role of plasmin on PrP Sc replication. Our results suggest that plasmin-mediated endoproteolytic cleavage of PrP may be an important event to prevent prion propagation.

The Effect of Curcuma phaeocaulis Valeton (Zingiberaceae) Extract on Prion Propagation in Cell-Based and Animal Models.

Prion diseases are neurodegenerative disorders in humans and animals for which no therapies are currently available. Here, we report that Curcuma phaeocaulis Valeton (Zingiberaceae) (CpV) extract was partly effective in decreasing prion aggregation and propagation in both in vitro and in vivo models. CpV extract inhibited self-aggregation of recombinant prion protein (PrP) in a test tube assay and decreased the accumulation of scrapie PrP (PrPSc) in ScN2a cells, a cultured neuroblastoma cell line with chronic prion infection, in a concentration-dependent manner. CpV extract also modified the course of the disease in mice inoculated with mouse-adapted scrapie prions, completely preventing the onset of prion disease in three of eight mice. Biochemical and neuropathological analyses revealed a statistically significant reduction in PrPSc accumulation, spongiosis, astrogliosis, and microglia activation in the brains of mice that avoided disease onset. Furthermore, PrPSc accumulation in the spleen of mice was also reduced. CpV extract precluded prion infection in cultured cells as demonstrated by the modified standard scrapie cell assay. This study suggests that CpV extract could contribute to investigating the modulation of prion propagation.

Spatiotemporal expression of aquaporin 9 is critical for the antral growth of mouse ovarian follicles†.

Although a few aquaporins (AQPs) expressed in granulosa cells have been postulated to mediate fluid passage into the antrum, the specific expression of AQPs in different follicle cell types and stages and their roles have not been evaluated extensively. The spatiotemporal expression of aquaporin (Aqp) 7, 8, and 9 and the functional roles of Aqp9 in antral growth and ovulation were examined using a superovulation model and 3-dimensional follicle culture. Aqp9 was expressed at a high level in the rapid growth phase (24-48 h post equine chorionic gonadotropin (eCG) for superovulation induction) compared to Aqp7 (after human chorionic gonadotropin (hCG)) and Aqp8 (8-24 h post eCG and 24 h post hCG). A dramatic increase in the expression and localization of Aqp9 mRNA in theca cells was observed, as evaluated using quantitative reverse transcription-polymerase (RT-PCR) coupled with laser capture microdissection and immunohistochemistry. AQP9 was located primarily on the theca cells of the tertiary and preovulatory follicles but not on the ovulated follicles. In phloretin-treated mice, the diameter of the preovulatory follicles and the number of ovulated oocytes decreased. Consistent with these findings, knocking down Aqp9 expression with an Aqp9 siRNA inhibited follicle growth (0.28:1 = siRNA:control) and decreased the number of ovulated follicles (0.36:1 = siRNA:control) during in vitro growth and ovulation induction. Based on these results, the expression of AQPs is under the control of the physiological status, and AQP9 expression in theca during folliculogenesis is required for antral growth and ovulation in a tissue-specific and stage-dependent manner.

Self-Assembling Peptides and Their Application in the Treatment of Diseases.

Self-assembling peptides are biomedical materials with unique structures that are formed in response to various environmental conditions. Governed by their physicochemical characteristics, the peptides can form a variety of structures with greater reactivity than conventional non-biological materials. The structural divergence of self-assembling peptides allows for various functional possibilities; when assembled, they can be used as scaffolds for cell and tissue regeneration, and vehicles for drug delivery, conferring controlled release, stability, and targeting, and avoiding side effects of drugs. These peptides can also be used as drugs themselves. In this review, we describe the basic structure and characteristics of self-assembling peptides and the various factors that affect the formation of peptide-based structures. We also summarize the applications of self-assembling peptides in the treatment of various diseases, including cancer. Furthermore, the in-cell self-assembly of peptides, termed reverse self-assembly, is discussed as a novel paradigm for self-assembling peptide-based nanovehicles and nanomedicines.

Direct interaction of DNMT inhibitors to PrPC suppresses pathogenic process of prion.

The conversion of the normal cellular prion protein (PrPC) to the misfolded pathogenic scrapie prion protein (PrPSc) is the biochemical hallmark of prion replication. So far, various chemical compounds that inhibit this conformational conversion have been identified. Here, we report the novel anti-prion activity of SGI-1027 and its meta/meta analogue (M/M), previously known only as potent inhibitors of DNA methyltransferases (DNMTs). These compounds effectively decreased the level of PrPSc in cultured cells with permanent prion infection, without affecting PrPC at the transcriptional or translational levels. Furthermore, SGI-1027 prevented effective prion infection of the cells. In a PrP aggregation assay, both SGI-1027 and M/M blocked the formation of misfolded PrP aggregates, implying that binding of these compounds hinders the PrP conversion process. A series of binding and docking analyses demonstrated that both SGI-1027 and M/M directly interacted with the C-terminal globular domain of PrPC, but only SGI-1027 bound to a specific region of PrPC with high affinity, which correlates with its potent anti-prion efficacy. Therefore, we report SGI-1027 and related compounds as a novel class of potential anti-prion agents that preferentially function through direct interaction with PrPC.

Non-ionic detergents Nonidet P-40 and Triton X-100 increase enzymatic activity of plasmin.

Plasmin is a potent serin protease involved in a variety of biological functions, such as fibrinolysis and tissue remodeling. On performing an in vitro control assay to measure the activity of endogenous plasmin in cell lysates, a stimulatory effect of non-ionic detergent NP-40 on plasmin activity was discovered. Another non-ionic detergent, TX-100, also enhanced plasmin activity, while ionic detergents sodium deoxycholate and sodiem dodecyl sulfate abolished plasmin enzyme activity. Kinetic analysis of plasmin activity in the presence of NP-40 and TX-100 demonstrated an increase in Vmax; however, there was no change in Km values, suggesting that these detergents stimulate plasmin activity in a non-competitive manner. Fibrin plate assay indicates that NP-40 and TX-100 functionally stimulate plasmin activity by showing a dose-dependent increase in fibrinolysis.

Decrease of Protease-Resistant PrPSc Level in ScN2a Cells by Polyornithine and Polyhistidine.

Based on previous studies reporting the anti-prion activity of poly-L-lysine and poly-L-arginine, we investigated cationic poly-L-ornithine (PLO), poly-L-histidine (PLH), anionic poly-L-glutamic acid (PLE) and uncharged poly-L-threonine (PLT) in cultured cells chronically infected by prions to determine their anti-prion efficacy. While PLE and PLT did not alter the level of PrPSc, PLO and PLH exhibited potent PrPSc inhibition in ScN2a cells. These results suggest that the anti-prion activity of poly-basic amino acids is correlated with the cationicity of their functional groups. Comparison of anti-prion activity of PLO and PLH proposes that the anti-prion activity of poly-basic amino acids is associated with their acidic cellular compartments.

Maclurin exerts anti-cancer effects on PC3 human prostate cancer cells via activation of p38 and inhibitions of JNK, FAK, AKT, and c-Myc signaling pathways.

Maclurin is a phenolic compound extracted from purple mangosteen and mulberry twigs. Earlier reports indicated that it exerts antioxidant activity. We hypothesized that maclurin exerts antioxidant activity and anti-cancer effects in small cell neuroendocrine carcinomas (SCNCs), a very aggressive type of human prostate cancer. To verify our hypothesis, we selected PC3 cells as a model system and investigated the antioxidant activity and anti-cancer effects of maclurin. In the reactive oxygen species (ROS) detection assay for the verification of antioxidant activity, we observed the unexpected prooxidant activity of maclurin in PC3 cells. For the anti-cancer activities, we investigated the effects of maclurin on induction of apoptosis and inhibition of metastatic characteristics of PC3 cells. In the apoptosis assay, maclurin significantly induced apoptosis of PC3 cells. Maclurin also showed significant anti-metastatic effects. Maclurin inhibited cell migration in a dosage-dependent manner. In addition, the gelatin zymography assay indicated that maclurin inhibited activities of matrix metalloproteinase-2 and 9 (MMP-2 and MMP-9) that affect cell migration and extracellular matrix (ECM) degradation. Then, we investigated the effects of maclurin on the cancer-related signaling molecules. Maclurin activated p38 signaling and inhibited c-Jun N-terminal kinase (JNK), focal-adhesion kinase (FAK), AKT, and c-Myc signalings in PC3 cells. Finally, we observed prooxidant activity and anti-SCNC effects of maclurin in DU145 cells. This suggests that the effects of maclurin may not be specifically limited to PC3 cells. Our findings suggest that maclurin exerts anti-cancer effects on SCNC cells via activation of p38 and inhibitions of JNK, FAK, AKT and c-Myc signalings.

High-Level Production of High-Purity Human and Murine Recombinant Prion Proteins Functionally Compatible to In Vitro Seeding Assay.

Recombinant (rec) prion protein (PrP) is an extremely useful resource for studying protein misfolding and subsequent protein aggregation events. Here, we report mass production of high-purity rec-polypeptide encoding the C-terminal globular domain of PrP; (90-230) for human and (89-231) for murine PrP. These proteins were expressed as His-tagged fusion proteins in E. coli cultured by a high cell-density aerobic fermentation method. RecPrPs recovered from inclusion bodies were slowly refolded under reducing conditions. Purification was performed by a sequence of metal-affinity, cation-exchange, and reverse-phase chromatography. The current procedure yielded several dozens of milligrams of recPrP per liter with ＞95% purity. The purified recPrPs predominantly adopted an α-helix-rich conformation and were functionally sufficient as substrates to measure the seeding activity of human and animal prions. Establishment of a procedure for high-level production of high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.

Chronic Low-Dose Nonylphenol or Di-(2-ethylhexyl) Phthalate has a Different Estrogen-like Response in Mouse Uterus.

Through the development of organic synthetic skill, chemicals that mimic signaling mediators such as steroid hormones have been exposed to the environment. Recently, it has become apparent that this circumstance should be further studied in the field of physiology. Estrogenic action of chronic low-dose nonylphenol (NP) and di-(2-ethylhexyl) phthalate (DEHP) in mouse uterus was assessed in this study. Ten to twelve-week-old female mice (CD-1) were fed drinking water containing NP (50 or 500 µg/L) or DEHP (133 or 1,330 µg/L) for 10 weeks. Uterine diameter, the thickness of myometrium and endometrium, and the height of luminal epithelial cells were measured and the number of glands were counted. The expression levels of the known 17ß-estradiol (E2)-regulated genes were evaluated with real-time RT-PCR methodology. The ration of uterine weight to body weight increased in 133 µg/L DEHP. Endometrial and myometrial thickness increased in 133 and 1,330 µg/L DEHP treated groups, and in 50, 500 µg/L NP and 133 µg/L DEHP, respectively. The height of luminal epithelial cell decreased in NP groups. The numbers of luminal epithelial gland were decreased in NP groups but increased in 50 µg/L DEHP group. The histological characters of glands were not different between groups. The mRNA expression profiles of the known 17ß-estradiol (E2) downstream genes, Esr1, Esr2, Pgr, Lox, and Muc1, were also different between NP and DEHP groups. The expression levels dramatically increased in some genes by the NP or DEHP. Based on these results, it is suggested that the chronic low-dose NP or DEHP works as estrogen-like messengers in uterus with their own specific gene expression-regulation patterns.

Prion Diagnosis: Application of Real-Time Quaking-Induced Conversion.

Prions composed of pathogenic scrapie prion protein (PrPSc) are infectious pathogens that cause progressive neurological conditions known as prion diseases or transmissible spongiform encephalopathies. Although these diseases pose considerable risk to public health, procedures for early diagnosis have not been established. One of the most recent attempts at sensitive and specific detection of prions is the real-time quaking-induced conversion (RT-QuIC) method, which measures the activity of PrPSc aggregates or amyloid formation triggered by PrPSc seeds in the presence of recombinant PrP. In this review, we summarize prions, prion diseases, and current approaches to diagnosis, including the principle, conditions for assay performance, and current diagnostic applications of RT-QuIC.

Sustained release docetaxel-incorporated lipid nanoparticles with improved pharmacokinetics for oral and parenteral administration.

The aim of this study was to develop docetaxel-incorporated lipid nanoparticles (DTX-NPs) to improve the pharmacokinetic behaviour of docetaxel (DTX) after oral and parenteral administration via sustained release. DTX-NPs were prepared by nanotemplate engineering technique with palmityl alcohol as a solid lipid and Tween-40/Span-40/Myrj S40 as a surfactants mixture. Spherical DTX-NPs below 100 nm were successfully prepared with a narrow particle size distribution, 96% of incorporation efficiency and 686 times increase in DTX solubility. DTX-NPs showed a sustained release over 24 h in phosphate-buffered saline and simulated gastric and intestinal fluids, while DTX-micelles released DTX completely within 12 h. The half-maximal inhibitory concentration (IC50) of DTX-NPs against human breast cancer MCF-7 cells was 1.9 times lower than that of DTX-micelles and DTX solution. DTX-NPs demonstrated 3.7- and 2.8-fold increase in the area under the plasma concentration-time curve compared with DTX-micelles after oral and parenteral administration, respectively.

Biochemical Analysis of Interaction between Kringle Domains of Plasminogen and Prion Proteins with Q167R Mutation.

The conformational change of cellular prion protein (PrPC) to its misfolded counterpart, termed PrPSc, is mediated by a hypothesized cellular cofactor. This cofactor is believed to interact directly with certain amino acid residues of PrPC. When these are mutated into cationic amino acid residues, PrPSc formation and prion replication halt in a dominant negative (DN) manner, presumably due to strong binding of the cofactor to mutated PrPC, designated as DN PrP mutants. Previous studies demonstrated that plasminogen and its kringle domains bind to PrP and accelerate PrPSc generation. In this study, in vitro binding analysis of kringle domains of plasminogen to Q167R DN mutant PrP (PrPQ167R) was performed in parallel with the wild type (WT) and Q218K DN mutant PrP (PrPQ218K). The binding affinity of PrPQ167R was higher than that of WT PrP, but lower than that of PrPQ218K. Scatchard analysis further indicated that, like PrPQ218K and WT PrP, PrPQ167R interaction with plasminogen occurred at multiple sites, suggesting cooperativity in this interaction. Competitive binding analysis using L-lysine or L-arginine confirmed the increase of the specificity and binding affinity of the interaction as PrP acquired DN mutations. Circular dichroism spectroscopy demonstrated that the recombinant PrPs used in this study retained the α-helix-rich structure. The α-helix unfolding study revealed similar conformational stability for WT and DN-mutated PrPs. This study provides an additional piece of biochemical evidence concerning the interaction of plasminogen with DN mutant PrPs.

pH-Dependent In-Cell Self-Assembly of Peptide Inhibitors Increases the Anti-Prion Activity While Decreasing the Cytotoxicity.

The first step in the conventional approach to self-assembled biomaterials is to develop well-defined nanostructures in vitro, which is followed by disruption of the preformed nanostructures at the inside of the cell to achieve bioactivity. Here, we propose an inverse strategy to develop in-cell gain-of-function self-assembled nanostructures. In this approach, the supramolecular building blocks exist in a unimolecular/unordered state in vitro or at the outside of the cell and assemble into well-defined nanostructures after cell internalization. We used block copolypeptides of an oligoarginine and a self-assembling peptide as building blocks and investigated correlations among the nanostructural state, antiprion bioactivity, and cytotoxicity. The optimal bioactivity (i.e., the highest antiprion activity and lowest cytotoxicity) was obtained when the building blocks existed in a unimolecular/unordered state in vitro and during the cell internalization process, exerting minimal cytotoxic damage to cell membranes, and were subsequently converted into high-charge-density vesicles in the low pH endosome/lysosomes in vivo, thus, resulting in the significantly enhanced antiprion activity. In particular, the in-cell self-assembly concept presents a feasible approach to developing therapeutics against protein misfolding diseases. In general, the in-cell self-assembly provides a novel inverse methodology to supramolecular bionanomaterials.

Effect of poly-L-arginine in inhibiting scrapie prion protein of cultured cells.

Biological effect of poly-L-arginine (PLR), the linear homopolymer comprised of L-arginine, was investigated to determine the activity of suppressing prions. PLR decreased the level of scrapie prion protein (PrPSc) in cultured cells permanently infected with prions in a concentration-dependent manner. The PrPSc inhibition efficacy of PLR was greater than that of another prion-suppressant poly-L-lysine (PLK) in a molecular mass-dependent fashion. The effective concentration of PLR to inhibit prions was achieved safely below the cytotoxic concentrations, and overall cytotoxicity of PLR was similar to that of PLK. PLR did not alter the cellular prion protein (PrPC) level and was unable to change the states of preformed recombinant PrP aggregates and PrPSc from prion-infected cells. These data eliminate the possibility that the action mechanism of PLR is through removal of PrPC and pre-existing PrPSc. However, PLR formed complexes with plasminogen that stimulates prion propagation via conversion of PrPC to the misfolded isoform, PrPSc. The plasminogen-PLR complex demonstrated the greater positive surface charge values than the similar complex with PLK, raising the possibility that PLR interferes with the role of cofactor for PrPSc generation better than PLK.

Timosaponin AIII inhibits migration and invasion of A549 human non-small-cell lung cancer cells via attenuations of MMP-2 and MMP-9 by inhibitions of ERK1/2, Src/FAK and ß-catenin signaling pathways.

Timosaponin AIII (TAIII) is a type of steroidal saponins isolated from Anemarrhena asphodeloides. It was known to improve learning and memory deficits through anti-inflammatory effects. TAIII was also reported to induce autophagy preceding mitochondria-mediated apoptosis in HeLa cancer cells and inhibit the growth of human colorectal cancer cells, thus regarded as a potential candidate for anti-cancer agent. In this study, we verified apoptosis-inducing and cell-cycle-arresting effects of TAIII in A549 human non-small-cell lung cancer (NSCLC) cells. Then, we report that TAIII suppresses migration and invasion of A549 human NSCLC cells. We propose that two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, which are well known to be involved in cancer-metastasis, are attenuated by the treatment of TAIII. TAIII exerts its suppressive effects on MMP-2 and MMP-9 via inhibitions of ERK1/2, Src/FAK and ß-catenin signalings which are closely related with the regulations of MMP-2 and MMP-9.