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1.
Sci Rep ; 10(1): 19914, 2020 11 16.
Article in English | MEDLINE | ID: mdl-33199752

ABSTRACT

Desalted Salicornia europaea L. (SE) inhibits acetylcholine esterase, attenuates oxidative stress and inflammatory cytokines, and activates neurotrophic pathway. We performed 12-week, randomized, double-blind, placebo-controlled study to evaluate the efficacy of PhytoMeal(a desalted SE)-ethanol extract (PM-EE), in improving the cognitive performance in patients with subjective memory impairment. 63 participants complaining memory dysfunction without dementia (Korean Mini-Mental State Examination [K-MMSE] score ≥ 23) were assigned to PM-EE 600 mg/day or placebo. The cognitive domain of the Alzheimer's disease assessment scale-Korean version (ADAS-K) was set as the primary outcome. After 12 weeks, there was no differences in the changes in the primary outcome or the frequency of adverse events between the groups. In the subgroup analysis for the 30 subjects with mild cognitive impairment (MCI, baseline K-MMSE scores ≤ 28), PM-EE significantly improved the color-reading score of the Korean color-word stroop test (8.2 ± 25.0 vs. - 4.7 ± 13.2, P = 0.018). Our findings suggest that PM-EE is safe but might not be effective in this setting of this study. However, PM-EE may improve the frontal executive function in the patients with MCI. Further large-sized studies with longer follow-up period is warranted (trial registration number KCT0003418).


Subject(s)
Alzheimer Disease/physiopathology , Chenopodiaceae/chemistry , Ethanol/chemistry , Memory Disorders/drug therapy , Plant Extracts/pharmacology , Double-Blind Method , Female , Humans , Male , Memory Disorders/etiology , Memory Disorders/pathology , Middle Aged , Prognosis
2.
Oxid Med Cell Longev ; 2019: 6764756, 2019.
Article in English | MEDLINE | ID: mdl-31379989

ABSTRACT

Hyperactivation of microglia, the resident innate immune cells of the central nervous system, exacerbates various neurodegenerative disorders, including Parkinson's disease (PD). Parkinson's disease is generally characterized by a severe loss of dopaminergic neurons in the nigrostriatal pathway, with substantial neuroinflammation and motor deficits. This was experimentally replicated in animal models, using neurotoxins, i.e., LPS (lipopolysaccharides) and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Salicornia europaea L. (SE) has been used as a dietary supplement in Korea and Europe for several years, due to its nutritional and therapeutic value. In this study, we intend to investigate the antineuroinflammatory and anti-PD-like effects of the bioactive fraction/candidate of the SE extract. Initially, we screened various fractions of SE extract using an in vitro antioxidant assay. The optimal fraction was investigated for its in vitro antineuroinflammatory potential in LPS-stimulated BV-2 microglial cells and in vivo anti-PD-like potential in MPTP-intoxicated mice. Subsequently, to identify the potential candidate responsible for the elite therapeutic potential of the optimal fraction, we conducted antioxidant activity-guided isolation and purification; the bioactive candidate was structurally characterized using nuclear magnetic resonance spectroscopy and chromatographic techniques and further investigated for its in vitro antioxidative and antineuroinflammatory potential. The results of our study indicate that SE-EA and its bioactive candidate, Irilin B, effectively alleviate the deleterious effect of microglia-mediated neuroinflammation and promote antioxidative effects. Thus, they exhibit potential as therapeutic candidates against neuroinflammatory and oxidative stress-mediated PD-like neurodegenerative complications.


Subject(s)
Chenopodiaceae/chemistry , Isoflavones/pharmacology , MPTP Poisoning/prevention & control , Neuroprotective Agents/pharmacology , Plant Extracts/chemistry , Acetates/chemistry , Animals , Antioxidants/metabolism , Chenopodiaceae/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Disease Models, Animal , Isoflavones/therapeutic use , Lipopolysaccharides/pharmacology , MPTP Poisoning/metabolism , MPTP Poisoning/pathology , Male , Mice , Mice, Inbred C57BL , Microglia/cytology , Microglia/drug effects , Microglia/metabolism , Neuroprotective Agents/chemistry , Neuroprotective Agents/therapeutic use , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Reactive Oxygen Species/metabolism
3.
Food Chem Toxicol ; 129: 444-457, 2019 Jul.
Article in English | MEDLINE | ID: mdl-31077737

ABSTRACT

Recently, our research team reported the anti-amnesic potential of desalted-hydroethanolic extracts of Salicornia europaea L. (SE-EE). In this study, we performed bioactivity-guided isolation and identification of Acanthoside B (Aca.B), from SE-EE, as the potential bioactive candidate and examined anti-amnesic activity with its potential mechanism of action using an in vivo model. S7-L3-3 purified from SE-EE showed enhanced in vitro acetylcholinesterase (AChE) inhibitory activity. The isolated S7-L3-3 was identified and characterized as Aca.B using varied spectral analyses, i.e., Nuclear magnetic resonance (NMR), Ultraviolet-visible (UV-Vis), and Electrospray ionization-mass spectrometry (ESI-MS). In the in vitro studies, Aca.B exhibited negligible toxicity and showed a dose-dependent nitric oxide inhibitory potential in Lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. In the in vivo studies, the oral administration of Aca.B to mice showed enhanced bioavailability and dose-dependent repression of the behavioral/cognitive impairment by regulating the cholinergic function, restoring the antioxidant status, attenuating the inflammatory cytokines/mediators and actively enriching neurotropic proteins in the hippocampal regions of the scopolamine-administered mice.


Subject(s)
Amnesia/chemically induced , Furans/pharmacology , Glucosides/pharmacology , Inflammation/metabolism , Lignans/pharmacology , Receptors, Cholinergic/metabolism , Animals , Brain-Derived Neurotrophic Factor/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Disease Models, Animal , Membrane Glycoproteins/metabolism , Mice , Oxidation-Reduction , Protein-Tyrosine Kinases/metabolism , Scopolamine/pharmacology
4.
Sci Rep ; 8(1): 7174, 2018 05 08.
Article in English | MEDLINE | ID: mdl-29740000

ABSTRACT

The Salicornia europaea L. (SE) plant is a halophyte that has been widely consumed as a seasoned vegetable, and it has been recently reported to counteract chronic diseases related to oxidative and inflammatory stress. In this study, we performed an initial phytochemical analysis with in vitro biochemical tests and chromatographic profiling of desalted and enzyme-digested SE ethanol extract (SE-EE). Subsequently, we evaluated the anti-neuroinflammatory and ameliorative potential of SE-EE in LPS-inflicted BV-2 microglial cells and scopolamine-induced amnesic C57/BL6N mice, respectively. SE-EE possess considerable polyphenols and flavonoids that are supposedly responsible to improve its bio-efficacy. SE-EE dose-dependently attenuated LPS-induced inflammation in BV-2 cells, significantly repressed behavioural/cognitive impairment, dose-dependently regulated the cholinergic function, suppressed oxidative stress markers, regulated inflammatory cytokines/associated proteins expression and effectively ameliorated p-CREB/BDNF levels, neurogenesis (DCX stain), neuron proliferation (Ki67 stain) in scopolamine-administered mice. Thus, SE-EE extract shows promising multifactorial disease modifying activities and can be further developed as an effective functional food, drug candidate, or supplemental therapy to treat neuroinflammatory mediated disorders.


Subject(s)
Alzheimer Disease/drug therapy , Amnesia/drug therapy , Antioxidants/administration & dosage , Chenopodiaceae/chemistry , Inflammation/drug therapy , Alzheimer Disease/chemically induced , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amnesia/chemically induced , Amnesia/genetics , Amnesia/pathology , Animals , Antioxidants/chemistry , Brain-Derived Neurotrophic Factor/genetics , Cyclic AMP Response Element-Binding Protein/genetics , Disease Models, Animal , Doublecortin Protein , Gene Expression Regulation/drug effects , Humans , Inflammation/chemically induced , Inflammation/genetics , Inflammation/pathology , Maze Learning/drug effects , Mice , Microglia/drug effects , Microglia/pathology , Oxidative Stress/drug effects , Plant Extracts/administration & dosage , Plant Extracts/chemistry , Scopolamine/toxicity
5.
Pharm Biol ; 56(1): 183-191, 2018 Dec.
Article in English | MEDLINE | ID: mdl-29521146

ABSTRACT

CONTEXT: Salicornia europaea (Amaranthaceae) (SE) has been shown to reduce obesity, but it remains a problem as a food supplement because of its high salt content (25-35% NaCl). OBJECTIVES: This study investigated the anti-obesity effects and mechanism of action of desalted SE powder (DSP). MATERIALS AND METHODS: Sprague-Dawley rats (n = 50) were divided into a normal control group (NC), a high-fat diet (HFD)-induced obesity control group (HFD), and HFD groups co-administered DSP (250 and 500 mg/kg) or Garcinia cambogia (Clusiaceae) extract (GE, 200 mg/kg, standard control) orally each day for 12 weeks. RESULTS: The body weight was significantly reduced by co-administration of DSP (596.51 ± 19.84 kg, 4.60% and 562.08 ± 9.74 kg, 10.10%, respectively) and GE (576.00 ± 11.29 kg, 7.88%) relative to the HFD group (625.25 ± 14.02 kg) and was accompanied by reduced abdominal fat mass, and serum lipid levels, with no effects on feed intake. To find the underlying mechanism of the anti-obesity effects, trans-ferulic acid (TFA) was identified as the main ingredient and investigated with regard to whether it attenuated adipogenesity in 3T3L-1 cells. DSP-derived TFA suppressed adipocyte differentiation and accumulation of intracellular lipids. TFA also down-regulated the adipogenesis-related gene expression of sterol regulatory element-binding protein 1, peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein-α and fatty acid synthase. CONCLUSIONS: These findings suggest that DSP may be considered for use as a food supplement intent of controlling obesity through its antiobesity and antiadipogenic properties.


Subject(s)
Adipogenesis/drug effects , Anti-Obesity Agents/therapeutic use , Chenopodiaceae , Coumaric Acids/therapeutic use , Obesity/drug therapy , Plant Extracts/therapeutic use , 3T3-L1 Cells , Adipogenesis/physiology , Animals , Anti-Obesity Agents/isolation & purification , Anti-Obesity Agents/pharmacology , Body Weight/drug effects , Body Weight/physiology , Coumaric Acids/isolation & purification , Coumaric Acids/pharmacology , Dose-Response Relationship, Drug , Male , Mice , Obesity/metabolism , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Rats , Rats, Sprague-Dawley
6.
Int J Syst Evol Microbiol ; 67(10): 4240-4245, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28920849

ABSTRACT

A Gram-stain-negative, motile-by-gliding, aerobic, non-spore-forming, rod-shaped and yellow-pigmented bacterium was isolated from Salicornia herbacea in the Yellow Sea and designated as strain MBLN091T. It belonged to the family Flavobacteriaceae. The 16S rRNA gene sequence of this isolated strain was similar to that of Flavimarina pacifica IDSW-73T with 94.8 % similarity, and with 92.3-92.8 % similarities to those of other closely related species of the genus Leeuwenhoekiella. The similarities of the RNA polymerase subunit B gene between this strain and F. pacifica KCTC 32466T and Leeuwenhoekiella marinoflava DSM 3653T were 80.5 and 80.2 %, respectively. Growth of strain MBLN091T was observed in the presence of 0.5‒15.0 % (w/v) NaCl at 4‒35 °C and pH 6.0-8.0, with optimal growth in the presence of 2.5‒5.0 % (w/v) NaCl at 20‒25 °C and pH 7.0. This isolate was able to hydrolyse gelatin. The only respiratory quinone was MK-6. The major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and two unidentified lipids. Major fatty acids of the isolate were iso-C15 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), iso-C17 : 0 3-OH and iso-C15 : 1 G. The genomic DNA G+C content was 39.6 mol%. The physiological features were closely related to F. pacifica. Therefore, strain MBLN091T is considered to represent a novel species within the genus Flavimarina, for which the name Flavimarina flava sp. nov. is proposed. The type strain is MBLN091T (=KCTC 52527T=JCM 31731T).


Subject(s)
Chenopodiaceae/microbiology , Flavobacteriaceae/classification , Phylogeny , Seawater/microbiology , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Fatty Acids/chemistry , Flavobacteriaceae/genetics , Flavobacteriaceae/isolation & purification , Phosphatidylethanolamines/chemistry , Pigmentation , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Vitamin K 2/analogs & derivatives , Vitamin K 2/chemistry
7.
Biomed Pharmacother ; 94: 430-438, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28778046

ABSTRACT

Salicornia europaea L. (SE) has been used as folk medicine for the treatment of various diseases such as obesity, diabetes, and cancer. However, its effects on atherosclerotic events in vascular smooth muscle cells (VSMCs) remain unknown. The present study explored the effects of the ethyl acetate fraction of desalted SE hot water extract (SEWEAF) on atherosclerotic responses (especially migration and proliferation) in VSMCs and vascular neointima formation. Treatment with the SEWEAF significantly suppressed the platelet-derived growth factor (PDGF)-BB-induced VSMC migration and proliferation as well the phosphorylation of mitogen-activated protein kinases (MAPKs) such as the p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2. Moreover, oral administration of the SEWEAF resulted in the attenuation of neointima formation in balloon-injured rat carotid arteries. Additionally, HPLC analysis showed that the major components in the two subfractions of the SEWEAF were five phenolic acids and four flavonols. In the SEWEAF components, for which atherosclerosis-linked responses in VSMCs have not been known, p-coumaric acid, quercetin-3-ß-d-glucoside, and isorhamnetin-3-ß-d-glucoside inhibited both PDGF-BB-induced migration and proliferation and isorhamnetin attenuated only PDGF-BB-stimulated VSMC proliferation. These results suggest that the SEWEAF may suppress PDGF-BB-induced VSMC migration by downregulating the phosphorylation of p38 MAPK and ERK1/2, thus leading to the reduction of neointimal hyperplasia during vascular remodeling. Therefore, the desalted SE extract, SEWEAF may be a potential ingredient for dietary supplements or nutraceuticals to ameliorate and/or prevent vascular remodeling-related disorders.


Subject(s)
Cell Movement/drug effects , Chenopodiaceae/chemistry , MAP Kinase Signaling System/drug effects , Myocytes, Smooth Muscle/pathology , Neointima/enzymology , Neointima/pathology , Plant Extracts/pharmacology , Acetates/chemistry , Animals , Becaplermin , Cell Proliferation/drug effects , Chromatography, High Pressure Liquid , Flavonols/pharmacology , Hydroxybenzoates/pharmacology , Male , Mitogen-Activated Protein Kinases/metabolism , Myocytes, Smooth Muscle/drug effects , Phosphorylation/drug effects , Proto-Oncogene Proteins c-sis/pharmacology , Rats, Sprague-Dawley , Water
8.
J Food Sci ; 82(7): 1765-1774, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28608557

ABSTRACT

High-fat and high-salt intakes are among the major risks of chronic diseases including obesity, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). Salicornia is a halophytic plant known to exert antioxidant, antidiabetic, and hypolipidemic effects, and Salicornia-extracted salt (SS) has been used as a salt substitute. In this study, the effects of SS and purified salt (PS) on the aggravation of NAFLD/NASH were compared. C57BL/6J male mice (8-wk-old) were fed a high-fat diet (HFD) for 6 mo and divided into 3 dietary groups, which were additionally fed HFD, HFD + SS, and HFD + PS for 13 wk. PS induced aggravation of NAFLD/NASH in HFD-fed mice. Although the actual salt intake was same between the PS and SS groups as 1% of the diet (extrapolated from the World Health Organization [WHO] guideline), SS induced less liver injury and hepatic steatosis compared to PS. The hepatic mRNA expressions of inflammatory cytokines and fibrosis marker were significantly lower in the SS group than the PS group. Oxidative stress is one of the major causes of inflammation in NAFLD/NASH. Results of the component analysis showed that the major polyphenols that exhibited antioxidant activity in the Salicornia water extract were ferulic acid, caffeic acid, and isorhamnetin. These results suggest that even the level of salt intake recommended by WHO can accelerate the progression of liver disease in obese individuals consuming HFD. It is proposed that SS can be a salt substitute for obese individuals who consume HFD.


Subject(s)
Chenopodiaceae/chemistry , Fatty Liver/drug therapy , Non-alcoholic Fatty Liver Disease/drug therapy , Obesity/drug therapy , Plant Extracts/administration & dosage , Sodium Chloride/adverse effects , Animals , Antioxidants/administration & dosage , Cytokines/metabolism , Diet, High-Fat/adverse effects , Fatty Liver/etiology , Fatty Liver/metabolism , Humans , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Obese , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/metabolism , Obesity/etiology , Obesity/metabolism , Oxidative Stress/drug effects , Sodium Chloride/metabolism
9.
Cancer Res ; 67(7): 3475-82, 2007 Apr 01.
Article in English | MEDLINE | ID: mdl-17389758

ABSTRACT

To develop novel mechanism-based preventive approaches for lung cancer, we examined the effect of oral consumption of a human achievable dose of pomegranate fruit extract (PFE) on growth, progression, angiogenesis, and signaling pathways in two mouse lung tumor protocols. Benzo(a)pyrene [B(a)P] and N-nitroso-tris-chloroethylurea (NTCU) were used to induce lung tumors, and PFE was given in drinking water to A/J mice. Lung tumor yield was examined on the 84th day and 140 days after B(a)P dosing and 240 days after NTCU treatment. Mice treated with PFE and exposed to B(a)P and NTCU had statistically significant lower lung tumor multiplicities than mice treated with carcinogens only. Tumor reduction was 53.9% and 61.6% in the B(a)P + PFE group at 84 and 140 days, respectively, compared with the B(a)P group. The NTCU + PFE group had 65.9% tumor reduction compared with the NTCU group at 240 days. Immunoblot analysis and immunohistochemistry were used to determine effect on cell survival pathways and markers of cellular proliferation and angiogenesis. PFE treatment caused inhibition of (a) activation of nuclear factor-kappaB and IkappaBalpha kinase, (b) degradation and phosphorylation of IkappaBalpha, (c) phosphorylation of mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase 1/2, and p38), (d) phosphatidylinositol 3-kinase (p85 and p110), (e) phosphorylation of Akt at Thr(308), (f) activation of mammalian target of rapamycin signaling, (g) phosphorylation of c-met, and (h) markers of cell proliferation (Ki-67 and proliferating cell nuclear antigen) and angiogenesis (inducible nitric oxide synthase, CD31, and vascular endothelial growth factor) in lungs of B(a)P- and NTCU-treated mice. Thus, our data show that PFE significantly inhibits lung tumorigenesis in A/J mice and merits investigation as a chemopreventive agent for human lung cancer.


Subject(s)
Anticarcinogenic Agents/pharmacology , Fruit , Lung Neoplasms/drug therapy , Lythraceae , Phytotherapy/methods , Plant Extracts/pharmacology , Administration, Oral , Animals , Benzo(a)pyrene , Cell Growth Processes/drug effects , Female , I-kappa B Kinase/metabolism , I-kappa B Proteins/metabolism , Lung Neoplasms/blood supply , Lung Neoplasms/metabolism , Mice , Mice, Inbred A , Mitogen-Activated Protein Kinases/metabolism , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , Neovascularization, Pathologic/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Protein Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-met/metabolism , TOR Serine-Threonine Kinases , Urea/analogs & derivatives
10.
J Invest Dermatol ; 127(1): 222-32, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16902416

ABSTRACT

Solar UV radiation, in particular its UVB component, is the primary cause of many adverse biological effects, the most damaging of which is skin cancer. Here, we assessed the photochemopreventive effect of delphinidin, a major anthocyanidin present in many pigmented fruits and vegetables, on UVB-mediated responses in human immortalized HaCaT keratinocytes and SKH-1 hairless mouse skin. We found that pretreatment of cells with delphinidin (1-20 microM for 24 hours) protected against UVB (15-30 mJ/cm2, 24 hours)-mediated (i) decrease in cell viability and (ii) induction of apoptosis. Furthermore, we found that pretreatment of HaCaT cells with delphinidin inhibited UVB-mediated (i) increase in lipid peroxidation; (ii) formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG); (iii) decrease in proliferating cell nuclear antigen expression; (iv) increase in poly(ADP-ribose) polymerase cleavage; (v) activation of caspases; (vi) increase in Bax; (vii) decrease in Bcl-2; (viii) upregulation of Bid and Bak; and (ix) downregulation of Bcl-xL. Topical application of delphinidin (1 mg/0.1 ml DMSO/mouse) to SKH-1 hairless mouse skin inhibited UVB-mediated apoptosis and markers of DNA damage such as cyclobutane pyrimidine dimers and 8-OHdG. Taken together our results suggest that treatment of HaCaT cells and mouse skin with delphinidin inhibited UVB-mediated oxidative stress and reduced DNA damage, thereby protecting the cells from UVB-induced apoptosis.


Subject(s)
Anthocyanins/pharmacology , Apoptosis/drug effects , Keratinocytes/radiation effects , Oxidative Stress/drug effects , Radiation-Protective Agents/pharmacology , Skin/radiation effects , 8-Hydroxy-2'-Deoxyguanosine , Animals , Antioxidants/pharmacology , Apoptosis/radiation effects , Caspases/metabolism , Cell Survival/drug effects , Cell Survival/radiation effects , Cells, Cultured , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Humans , In Situ Nick-End Labeling , Lipid Peroxidation/drug effects , Mice , Mice, Hairless , Oxidative Stress/radiation effects , Proliferating Cell Nuclear Antigen/analysis , Proto-Oncogene Proteins c-bcl-2/analysis , Pyrimidine Dimers/metabolism , Skin/drug effects , bcl-2-Associated X Protein/analysis
11.
Carcinogenesis ; 28(1): 163-73, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16920736

ABSTRACT

Developing novel mechanism-based chemopreventive approaches for lung cancer through the use of dietary substances which humans can accept has become an important goal. In the present study, employing normal human bronchial epithelial cells (NHBE) and human lung carcinoma A549 cells, we first compared the growth inhibitory effects of pomegranate fruit extract (PFE). Treatment of PFE (50-150 microg/ml) for 72 h was found to result in a decrease in the viability of A549 cells but had only minimal effects on NHBE cells as assessed by the MTT and Trypan blue assays. PFE treatment of A549 cells also resulted in dose-dependent arrest of cells in G0-G1 phase of the cell cycle (as assessed by DNA cell cycle analysis). We further found that PFE treatment also resulted in (i) induction of WAF1/p21 and KIP1/p27, (ii) decrease in the protein expressions of cyclins D1, D2 and E, and (iii) decrease in cyclin-dependent kinase (cdk) 2, cdk4 and cdk6 expression. The treatment of cells with PFE inhibited (i) phosphorylation of MAPK proteins, (ii) inhibition of PI3K, (iii) phosphorylation of Akt at Thr308, (iv) NF-kappaB and IKKalpha, (v) degradation and phosphorylation of IkappaBalpha, and (vi) Ki-67 and PCNA. We also found that PFE treatment to A549 cells resulted in inhibition of NF-kappaB DNA-binding activity. Oral administration of PFE (0.1 and 0.2%, wt/vol) to athymic nude mice implanted with A549 cells resulted in a significant inhibition in tumor growth. Our results provide a suggestion that PFE can be a useful chemopreventive/chemotherapeutic agent against human lung cancer.


Subject(s)
Cell Survival/drug effects , Fruit/chemistry , Lung Neoplasms/drug therapy , Lythraceae/chemistry , Phytotherapy , Plant Extracts/therapeutic use , Signal Transduction , Animals , Bronchi/cytology , Bronchi/drug effects , Bronchi/metabolism , Cell Cycle/drug effects , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Lung Neoplasms/prevention & control , Male , Mice , Mice, Nude , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Phosphorylation/drug effects
12.
J Biol Chem ; 281(44): 33761-72, 2006 Nov 03.
Article in English | MEDLINE | ID: mdl-16950787

ABSTRACT

Epigallocatechin 3-gallate (EGCG), the major polyphenol found in green tea, exerts antiproliferative and proapoptotic effects in many cancer cells. However, we found that among many cancer cells human lung adenocarcinoma A549 cells are markedly resistant to apoptosis induction by EGCG (even at 100 microm for 72 h). Heme oxygenase-1 (HO-1) induced by stress stimuli represents a prime cellular defense mechanism, but it may be associated with enhanced cell proliferation and chemoresistance in some cancer cells. Because we found that A549 cells constitutively overexpress HO-1 and its associated transcription factor Nrf2, we tested an hypothesis that EGCG resistance in these cells may be linked with Nrf2-mediated HO-1 overexpression. HO-1 inhibition with tin-protoporphyrin IX and silencing with RNA interference rendered cells more sensitive to apoptosis induction by EGCG and classical prooxidants. Interestingly, EGCG at high concentration (>200 microm) induced apoptosis by suppressing expression of HO-1 protein and mRNA, and this effect correlated with a decrease in both Nrf2-ARE binding and HO-1-ARE-luciferase activity, suggesting Nrf2-driven transcriptional activation of ho-1. Because we observed notably high levels of phosphorylated protein kinase C alpha and its suppression by EGCG and deferoxamine (an iron chelator), a possible mechanism involving phosphorylated protein kinase C alpha and iron in Nrf2-HO-1 activation was further investigated. Collectively, our findings suggest that Nrf2-mediated HO-1 overexpression confers resistance to apoptosis induction by EGCG; therefore, its inactivation may be a target for overcoming the resistance to chemoprevention and chemotherapy.


Subject(s)
Apoptosis/drug effects , Catechin/analogs & derivatives , Heme Oxygenase-1/metabolism , NF-E2-Related Factor 2/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Antioxidants/metabolism , Catechin/pharmacology , Catechin/toxicity , Cell Line, Tumor , Cell Survival/drug effects , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation , Gene Expression Regulation, Neoplastic , Heme Oxygenase-1/genetics , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , NF-E2-Related Factor 2/genetics , Oxidation-Reduction , Protein Kinase C-alpha/metabolism , RNA, Small Interfering/genetics , Response Elements , Sensitivity and Specificity , Transcriptional Activation/genetics
13.
Proc Natl Acad Sci U S A ; 103(40): 14825-30, 2006 Oct 03.
Article in English | MEDLINE | ID: mdl-16990429

ABSTRACT

We previously showed that the calcium-binding protein S100A4 is overexpressed during the progression of prostate cancer (CaP) in humans and in the TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse model. We tested a hypothesis that the S100A4 gene plays a role in the invasiveness of human CaP and may be associated with its metastatic spread. We observed that siRNA-mediated suppression of the S100A4 gene significantly reduced the proliferative and invasive capability of the highly invasive CaP cells PC-3. We evaluated the mechanism through which the S100A4 gene controls invasiveness of cells by using a macroarray containing 96 well characterized metastatic genes. We found that matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor (TIMP-1) were highly responsive to S100A4 gene suppression. Furthermore, S100A4 suppression significantly reduced the expression and proteolytic activity of MMP-9. By employing an MMP-9-promoter reporter, we observed a significant reduction in the transcriptional activation of the MMP-9 gene in S100A4-siRNA-transfected cells. Cells overexpressing the S100A4 gene (when transfected with pcDNA3.1-S100A4 plasmid) also significantly expressed MMP-9 and TIMP-1 genes with increased proteolytic activity of MMP-9 concomitant to increased transcriptional activation of the MMP-9 gene. S100A4-siRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. Our data demonstrate that the S100A4 gene controls the invasive potential of human CaP cells through regulation of MMP-9 and that this association may contribute to metastasis of CaP cells. We suggest that S100A4 could be used as a biomarker for CaP progression and a novel therapeutic or chemopreventive target for human CaP treatment.


Subject(s)
Gene Expression Regulation, Neoplastic , Matrix Metalloproteinase 9/genetics , Prostatic Neoplasms/pathology , S100 Proteins/metabolism , Transcription, Genetic , Animals , Genes, Neoplasm/genetics , Humans , Male , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Nude , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplastic Stem Cells , Prostatic Neoplasms/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , S100 Calcium-Binding Protein A4 , S100 Proteins/genetics , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Inhibitor of Metalloproteinase-1/metabolism , Transcriptional Activation/genetics , Transfection , Transplantation, Heterologous , Tumor Cells, Cultured
14.
Free Radic Biol Med ; 40(8): 1349-61, 2006 Apr 15.
Article in English | MEDLINE | ID: mdl-16631525

ABSTRACT

Induction of detoxifying phase II genes by chemopreventive agents represents a coordinated protective response against oxidative stress and neoplastic effects of carcinogens. We have earlier shown that a novel antioxidant from the bamboo leaves constituent 3-O-caffeoyl-1-methylquinic acid (MCGA3) induces heme oxygenase-1 (HO-1) and protects endothelial cells from ROS-induced endothelial injury. The purpose of this study was to elucidate the induction mechanism of HO-1 and other phase II genes by MCGA3 in human umbilical vascular endothelial cells (HUVECs). Using Northern blotting and RT-PCR, we found that treatment of HUVECs with MCGA3 increased, in a dose and time-dependent manner, steady-state mRNA levels of the selected phase II genes including HO-1, ferritin, gamma-glutamylcysteine lygase, glutathione reductase, and glutathione transferase, which were dependent on Nrf2 nuclear translocation. The observed phase II gene induction by MCGA3 was found to be associated with MCGA3-mediated cytoprotective activity, ROS-scavenging potency, and the increase in the cellular levels of both reduced (GSH) and oxidized glutathione (GSSG). Interestingly, exposure to MCGA3 resulted in a decreased ratio of GSH/GSSG, which was negatively related with mRNA level of phase II genes. By employing N-acetylcysteine and GSH biosynthetic enzyme inhibitors as well as prooxidants, hemin and H(2)O(2), we show that a decreased intracellular GSH/GSSG homeostasis, at least in part, may be involved in the MCGA3-mediated phase II gene induction and Nrf2 translocation, although the attenuation of HO-1 expression with SP 600125 supports a partial involvement of JNK signaling.


Subject(s)
Antioxidants/pharmacology , Glutathione/metabolism , Metabolic Detoxication, Phase II/genetics , NF-E2-Related Factor 2/metabolism , Quinic Acid/analogs & derivatives , Acetylcysteine/pharmacology , Active Transport, Cell Nucleus , Carmustine/pharmacology , Cells, Cultured , Dehydroepiandrosterone/pharmacology , Ferritins/metabolism , Gene Expression Regulation , Heme Oxygenase-1/metabolism , Humans , MAP Kinase Signaling System , Oxidation-Reduction/drug effects , Phosphorylation , Quinic Acid/chemistry , Quinic Acid/pharmacology , RNA, Messenger/genetics , Time Factors , Transcriptional Activation , Umbilical Cord/drug effects , Umbilical Cord/metabolism
15.
Carcinogenesis ; 27(7): 1454-64, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16497706

ABSTRACT

Because of unsatisfactory treatment options for prostate cancer (CaP) there is a need to develop novel preventive approaches for this malignancy. One such strategy is through chemoprevention by the use of non-toxic dietary substances and botanical products. We have shown previously that panduratin A isolated from the extract of Kaempferia pandurata (Zingiberaceae) is a strong inhibitor of cyclooxygenase-2 in RAW264.7 cells and induces apoptosis in HT-29 cells. In the present study, we provide evidence that panduratin A treatment to androgen-independent human CaP cells PC3 and DU145 result in a time and dose-dependent inhibition of cell growth with an IC50 of 13.5-14 microM and no to little effect on normal human prostate epithelial cells. To define the mechanism of these anti-proliferative effects of panduratin A, we determined its effect on critical molecular events known to regulate the cell cycle and the apoptotic machinery. Annexin V/propidium iodide staining provided the evidence for the induction of apoptosis which was further confirmed by the observation of cleavage of poly (ADP-ribose) polymerase and degradation of acinus. Panduratin A treatment to cells was found to result in inhibition of procaspases 9, 8, 6 and 3 with significant increase in the ratio of Bax:Bcl-2, suggesting the involvement of a mitochondrial-dependent apoptotic pathway. Panduratin A-mediated apoptosis was accompanied with upregulation of Fas death receptor and TNF-related apoptosis-inducing ligand (TRAIL). Furthermore, cell cycle analysis using flow cytometry showed that panduratin A treatment of cells resulted in a G2/M arrest in a dose-dependent manner. The immunoblot analysis data revealed that in both cell lines panduratin A treatment resulted in a dose-dependent (i) induction of p21WAF1/Cip1 and p27Kip1, (ii) downregulation of cdks 2, 4 and 6 and (iii) decrease in cyclins D1 and E. These findings suggest that panduratin A may be an effective chemopreventive or therapeutic agent against CaP.


Subject(s)
Adenocarcinoma/pathology , Apoptosis/drug effects , Cell Cycle/drug effects , Chalcones/pharmacology , Phytotherapy , Prostatic Neoplasms/pathology , Zingiberaceae/chemistry , Androgens/metabolism , Apoptosis/physiology , Apoptosis Regulatory Proteins/drug effects , Apoptosis Regulatory Proteins/metabolism , Blotting, Western , Caspases/drug effects , Caspases/metabolism , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p27/drug effects , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Cyclin-Dependent Kinases/drug effects , Cyclin-Dependent Kinases/metabolism , Cyclins/drug effects , Cyclins/metabolism , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Inhibitory Concentration 50 , Male , Membrane Glycoproteins/drug effects , Membrane Glycoproteins/metabolism , Plant Extracts/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , TNF-Related Apoptosis-Inducing Ligand , Tumor Necrosis Factor-alpha/drug effects , Tumor Necrosis Factor-alpha/metabolism , bcl-2-Associated X Protein/drug effects , bcl-2-Associated X Protein/metabolism , bcl-Associated Death Protein/drug effects , bcl-Associated Death Protein/metabolism
16.
Cancer Res ; 65(23): 11203-13, 2005 Dec 01.
Article in English | MEDLINE | ID: mdl-16322271

ABSTRACT

In prostate cancer, a fine balance between cell proliferation and apoptotic death is lost, resulting in increased cellular mass and tumor progression. One approach to redress this imbalance and control this malignancy is its preventive intervention through the use of dietary natural agents. Here, we investigated the growth-inhibitory effect and associated mechanisms of Lupeol, a triterpene present in fruits and vegetables, in androgen-sensitive human prostate cancer cells. Lupeol treatment resulted in significant inhibition of cell viability in a dose-dependent manner and caused apoptotic death of prostate cancer cells. Lupeol was found to induce the cleavage of poly(ADP-ribose) polymerase protein and degradation of acinus protein with a significant increase in the expression of FADD protein. Among all death receptor targets examined, Lupeol specifically caused a significant increase in the expression of Fas receptor. The small interfering RNA-mediated silencing of the Fas gene and inhibition of caspase-6, caspase-8, and caspase-9 by their specific inhibitors confirmed that Lupeol specifically activates the Fas receptor-mediated apoptotic pathway in androgen-sensitive prostate cancer cells. The treatment of cells with a combination of anti-Fas monoclonal antibody and Lupeol resulted in higher cell death compared with the additive effect of the two compounds alone, suggesting a synergistic effect. Lupeol treatment resulted in a significant inhibition in growth of tumors with concomitant reduction in prostate-specific antigen secretion in athymic nude mice implanted with CWR22Rnu1 cells. Because early clinical prostate cancer growth is an androgen-dependent response, the results of the present study suggest that Lupeol may have a potential to be an effective agent against prostate cancer.


Subject(s)
Apoptosis/drug effects , Neoplasms, Hormone-Dependent/drug therapy , Prostatic Neoplasms/drug therapy , Triterpenes/pharmacology , fas Receptor/physiology , Androgens/physiology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Apoptosis/physiology , Caspase Inhibitors , Caspases/metabolism , Cell Line, Tumor , Humans , Male , Mice , Mice, Nude , Mitochondria/metabolism , Neoplasms, Hormone-Dependent/pathology , Pentacyclic Triterpenes , Poly(ADP-ribose) Polymerases/metabolism , Prostate-Specific Antigen/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , RNA, Small Interfering/genetics , Receptors, Androgen/metabolism , Xenograft Model Antitumor Assays , fas Receptor/genetics , fas Receptor/immunology
17.
Carcinogenesis ; 26(11): 1956-64, 2005 Nov.
Article in English | MEDLINE | ID: mdl-15958516

ABSTRACT

Pancreatic cancer is an exceptionally aggressive disease, the treatment of which has largely been unsuccessful due to higher resistance offered by pancreatic cancer cells to conventional approaches such as surgery, radiation and/or chemotherapy. The aberration of Ras oncoprotein has been linked to the induction of multiple signaling pathways and to the resistance offered by pancreatic cancer cells to apoptosis. Therefore, there is a need for development of new and effective chemotherapeutic agents which can target multiple pathways to induce responsiveness of pancreatic cancer cells to death signals. In this study, human pancreatic adenocarcinoma cells AsPC-1 were used to investigate the effect of Lupeol on cell growth and its effects on the modulation of multiple Ras-induced signaling pathways. Lupeol caused a dose-dependent inhibition of cell growth as assessed by MTT assay and induction of apoptosis as assessed by flow cytometry, fluorescence microscopy and western blotting. Lupeol treatment to cells was found to significantly reduce the expression of Ras oncoprotein and modulate the protein expression of various signaling molecules involved in PKCalpha/ODC, PI3K/Akt and MAPKs pathways along with a significant reduction in the activation of NFkappaB signaling pathway. Our data suggest that Lupeol can adopt a multi-prong strategy to target multiple signaling pathways leading to induction of apoptosis and inhibition of growth of pancreatic cancer cells. Lupeol could be a potential agent against pancreatic cancer, however, further in-depth in vivo studies are warranted.


Subject(s)
Adenocarcinoma/drug therapy , Anti-Inflammatory Agents/pharmacology , Apoptosis/drug effects , Pancreatic Neoplasms/drug therapy , Signal Transduction/drug effects , Triterpenes/pharmacology , ras Proteins/metabolism , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Blotting, Western , Enzyme Activation/drug effects , Flow Cytometry , Fruit/chemistry , Humans , Microscopy, Fluorescence , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Ornithine Decarboxylase/metabolism , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pentacyclic Triterpenes , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Poly(ADP-ribose) Polymerases/metabolism , Protein Kinase C-alpha/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Tumor Cells, Cultured , Vegetables/chemistry
18.
Free Radic Biol Med ; 36(1): 40-52, 2004 Jan 01.
Article in English | MEDLINE | ID: mdl-14732289

ABSTRACT

The novel antioxidant 3-O-caffeoyl-one-methylquinic acid (MCGA3) is a methyl chlorogenic acid derivative isolated from bamboo leaves. MCGA3 scavenges reactive oxygen species (ROS) and inhibits lipid peroxidation and xanthine oxidase in vitro. In this study, we evaluated the cytoprotective effect of MCGA3, which occurs via heme oxygenase-1 (HO-1) induction in bovine vascular endothelial cells exposed to tert-butylhydroperoxide (tBHP). Cells treated with 1 mM tBHP (6-18 h) generated substantial ROS and concomitantly lost most intracellular lactate dehydrogenase (LDH), which then caused necrotic cell death. Of the several MCGA antioxidants and structurally related phenolic acids examined in this study, MCGA3 (0.01-0.15 mM) was found to completely block this necrosis and generation of ROS by tBHP. Surprisingly, MCGA3 by itself was found to be a potent inducer of HO-1. We observed the time- and dose-dependent induction of HO-1 mRNA and protein, which was closely associated with decreased intracellular ROS and necrosis against tBHP. Deesterified or Al-chelated MCGA3 or co-treatment with MCGA3 and actinomycin D abolished HO-1 induction and the antinecrotic effect of MCGA3. Zinc protoporphyrin IX and cycloheximide attenuated the cytoprotection afforded by MCGA3, but did not reduce HO-1 mRNA. Interestingly, N-acetylcysteine (1 mM) enhanced the HO-1 induction of MCGA3, but N-acetylcysteine itself did not induce HO-1. These results suggested that not only ortho-dihydroxyl groups but also aromatic ester and methoxyl ester moieties are necessary for full HO-1 induction and cytoprotection against toxic tBHP-derived ROS. Ferritin mRNA was also upregulated during all HO-1 induction by MCGA3, which might decrease iron and lower ROS levels. Consequently, the combined action of HO-1 and ferritin may protect cells from toxic tBHP-mediated necrosis.


Subject(s)
Cryoprotective Agents/metabolism , Heme Oxygenase (Decyclizing)/metabolism , Quinic Acid/pharmacology , Acetylcysteine/pharmacology , Animals , Antioxidants/metabolism , Cattle , Cell Line , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Dose-Response Relationship, Drug , Enzyme Induction/drug effects , Ferritins/genetics , Flow Cytometry , Heme Oxygenase (Decyclizing)/genetics , Heme Oxygenase-1 , Hydroxybenzoates/chemistry , Hydroxybenzoates/pharmacology , Molecular Structure , Necrosis , Protoporphyrins/pharmacology , Quinic Acid/analogs & derivatives , Quinic Acid/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Time Factors , Up-Regulation/drug effects , tert-Butylhydroperoxide/toxicity
19.
Immunology ; 110(1): 73-9, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12941143

ABSTRACT

Sh-CRIT-ed1 is a potent anti-complement peptide that inhibits the classical complement-activation pathway by interfering with the formation of the C3-convertase complex, C4b2a. C2 is an essential serum glycoprotein that provides the catalytic subunit of the C3 and C5 convertases of the classical pathways of complement activation. Because only in its C4-bound state is C2a capable of cleaving its physiological protein substrates C3 and C5, the interaction of Sh-CRIT-ed1 with C2 plays a decisive role of inhibition in the classical complement-activation process. However, the role of individual Sh-CRIT-ed1 amino acid residues in C2 binding is not fully understood. We constructed nine recombinant Sh-CRIT-ed1 (rSh1) analogues, substituted at conserved residues, and evaluated their anti-complement and C2-binding activities. Results from glutathione S-transferase (GST) pull-down and haemolytic assays suggested that residues 10K, 17E, 19K and 26Y are critical for the interaction of rSh1 with C2. We then constructed an improved anti-complement peptide by duplicating Sh-CRIT-ed1 C-terminal motifs (17H-26Y). This linear homodimer (rH17d) was more potent than rSh1 with respect to binding to C2 and anti-complement activity (the 50% inhibitory concentration value was approximately equal 1.2 micro m versus approximately equal 6.02 micro m for rSh1). Furthermore, rH17d showed higher anti-complement activity in vivo, providing additional evidence that this duplication is a more effective inhibitor of complement activation than rSh1. Taken together, these results identify four key residues in rSh1 and strongly suggest that rH17d is a potent inhibitor of complement activation that may have therapeutic applications.


Subject(s)
Antigens, Helminth/immunology , Complement Pathway, Classical/immunology , Helminth Proteins/immunology , Receptors, Cell Surface/immunology , Amino Acid Sequence , Animals , Antigens, Helminth/genetics , Complement C2/immunology , Complement C2/metabolism , Forssman Antigen/immunology , Guinea Pigs , Helminth Proteins/genetics , Hemolysis/immunology , Male , Molecular Sequence Data , Receptors, Cell Surface/genetics , Recombinant Proteins/immunology , Sequence Alignment , Shock/immunology , Structure-Activity Relationship
20.
Protein Expr Purif ; 27(2): 202-9, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12597878

ABSTRACT

Many complement inhibitors found in plants and other organisms have been recognized as an antiinflammatory drug. Sh-CRIT-ed1 is a complement inhibitory peptide, present on the Schistosoma parasite surface. In the present study, we expressed chemically synthesized oligonucleotides encoding Sh-CRIT-ed1 with an additional hexahistidine codon at the C-terminus and purified in Escherichia coli BL21. The cloned gene, which was multimerized four times in pBlue-script II KS (+) at the isoschizomer sites (BamHI, BglII), was named Sh4, and expressed in E. coli BL21 harboring pGEX-KG. The fusion protein (GST-Sh4) was purified with high yield successively by affinity chromatographies of glutathione-Sepharose 4B and Ni-NTA-agarose. Recombinant Sh-CRIT-ed1 was obtained readily by thrombin digestion and CNBr cleavage of GST-Sh4, and the yield was 9.03 mg from 1-liter culture of E. coli BL21 harboring pGEX-Sh4. The recombinant Sh-CRIT-ed1 showed strong anticomplementary activity (IC(50) = 6.02 microM) by complement haemolysis assay.


Subject(s)
Peptides/chemistry , Base Sequence , Cloning, Molecular , Codon , DNA/metabolism , Dose-Response Relationship, Drug , Escherichia coli/metabolism , Hemolysis , Humans , Molecular Sequence Data , Oligonucleotides/chemistry , Plasmids/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry
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