Plant-Derived Anti-Human Epidermal Growth Factor Receptor 2 Antibody Suppresses Trastuzumab-Resistant Breast Cancer with Enhanced Nanoscale Binding.

Traditional monoclonal antibodies such as Trastuzumab encounter limitations when treating Human Epidermal Growth Factor Receptor 2 (HER2)-positive breast cancer, particularly in cases that develop resistance. This study introduces plant-derived anti-HER2 variable fragments of camelid heavy chain domain (VHH) fragment crystallizable region (Fc) KEDL(K) antibody as a potent alternative for overcoming these limitations. A variety of biophysical techniques, in vitro assays, and in vivo experiments uncover the antibody's nanoscale binding dynamics with transmembrane HER2 on living cells. Single-molecule force spectroscopy reveals the rapid formation of two robust bonds, exhibiting approximately 50 pN force resistance and bond lifetimes in the second range. The antibody demonstrates a specific affinity for HER2-positive breast cancer cells, including those that are Trastuzumab-resistant. Moreover, in immune-deficient mice, the plant-derived anti-HER2 VHH-FcK antibody exhibits superior antitumor activity, especially against tumors that are resistant to Trastuzumab. These findings underscore the plant-derived antibody's potential as an impactful immunotherapeutic strategy for treating Trastuzumab-resistant HER2-positive breast cancer.

Anti-obesity effects of a standardized ethanol extract of Eisenia bicyclis by regulating the AMPK signaling pathway in 3T3-L1 cells and HFD-induced mice.

Obesity requires treatment to mitigate the potential development of further metabolic disorders, including diabetes, hyperlipidemia, tumor growth, and non-alcoholic fatty liver disease. We investigated the anti-obesity effect of a 30% ethanol extract of Eisenia bicyclis (Kjellman) Setchell (EEB) on 3T3-L1 preadipocytes and high-fat diet (HFD)-induced obese C57BL/6 mice. Adipogenesis transcription factors including peroxisome proliferator-activated receptor (PPAR)γ, CCAAT/enhancer-binding protein-alpha (C/EBPα), and sterol regulatory element-binding protein-1 (SREBP-1) were ameliorated through the AMP-activated protein kinase (AMPK) pathway by EEB treatment in differentiated 3T3-L1 cells. EEB attenuated mitotic clonal expansion by upregulating cyclin-dependent kinase inhibitors (CDKIs) while downregulating cyclins and CDKs. In HFD-fed mice, EEB significantly decreased the total body weight, fat tissue weight, and fat in the tissue. The protein expression of PPARγ, C/EBPα, and SREBP-1 was increased in the subcutaneous fat and liver tissues, while EEB decreased the expression levels of these transcription factors. EEB also inhibited lipogenesis by downregulating acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) expression in the subcutaneous fat and liver tissues. Moreover, the phosphorylation of AMPK and ACC was downregulated in the HFD-induced mouse group, whereas the administration of EEB improved AMPK and ACC phosphorylation; thus, EEB treatment may be related to the AMPK pathway. Histological analysis showed that EEB reduced the adipocyte size and fat accumulation in subcutaneous fat and liver tissues, respectively. EEB promotes thermogenesis in brown adipose tissue and improves insulin and leptin levels and blood lipid profiles. Our results suggest that EEB could be used as a potential agent to prevent obesity.

Plant-Produced Therapeutic Crizanlizumab Monoclonal Antibody Binds P-Selectin to Alleviate Vaso-occlusive Pain Crises in Sickle Cell Disease.

Sickle Cell Disease (SCD) is a severe genetic disorder causing vascular occlusion and pain by upregulating the adhesion molecule P-selectin on endothelial cells and platelets. It primarily affects infants and children, causing chronic pain, circulatory problems, organ damage, and complications. Thus, effective treatment and management are crucial to reduce SCD-related risks. Anti-P-selectin antibody Crizanlizumab (Crimab) has been used to treat SCD. In this study, the heavy and light chain (HC and LC) genes of anti-P-Selectin antibody Crimab were cloned into a plant expression binary vector. The HC gene was under control of the duplicated 35S promoter and nopaline synthase (NOS) terminator, whereas the LC gene was under control of the potato proteinase inhibitor II (PIN2) promoter and PIN2 terminator. Agrobacterium tumefaciens LBA4404 was used to transfer the genes into the tobacco (Nicotiana tabacum cv. Xanthi) plant. In plants the genomic PCR and western blot confirmed gene presence and expression of HC and LC Crimab proteins in the plant, respectively. Crimab was successfully purified from transgenic plant leaf using protein A affinity chromatography. In ELISA, plant-derived Crimab (CrimabP) had similar binding activity to P-selectin compared to mammalian-derived Crimab (CrimabM). In surface plasmon resonance, the KD (dissociation binding constant) and response unit values were lower and higher than CrimabP, respectively. Taken together, these results demonstrate that the transgenic plant can be applied to produce biofunctional therapeutic monoclonal antibody.

Translation efficiency driven by CNOT3 subunit of the CCR4-NOT complex promotes leukemogenesis.

Protein synthesis is frequently deregulated during tumorigenesis. However, the precise contexts of selective translational control and the regulators of such mechanisms in cancer is poorly understood. Here, we uncovered CNOT3, a subunit of the CCR4-NOT complex, as an essential modulator of translation in myeloid leukemia. Elevated CNOT3 expression correlates with unfavorable outcomes in patients with acute myeloid leukemia (AML). CNOT3 depletion induces differentiation and apoptosis and delayed leukemogenesis. Transcriptomic and proteomic profiling uncovers c-MYC as a critical downstream target which is translationally regulated by CNOT3. Global analysis of mRNA features demonstrates that CNOT3 selectively influences expression of target genes in a codon usage dependent manner. Furthermore, CNOT3 associates with the protein network largely consisting of ribosomal proteins and translation elongation factors in leukemia cells. Overall, our work elicits the direct requirement for translation efficiency in tumorigenesis and propose targeting the post-transcriptional circuitry via CNOT3 as a therapeutic vulnerability in AML.

Efficacy of IFN-γ-Primed Umbilical Cord-Derived Mesenchymal Stem Cells on Temporomandibular Joint Osteoarthritis.

BACKGROUND: Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease affecting the cartilage and subchondral bone, leading to temporomandibular joint pain and dysfunction. The complex nature of TMJOA warrants effective alternative treatments, and mesenchymal stem cells (MSCs) have shown promise in regenerative therapies. The aim of this study is twofold: firstly, to ascertain the optimal interferon-gamma (IFN-γ)-primed MSC cell line for TMJOA treatment, and secondly, to comprehensively evaluate the therapeutic efficacy of IFN-γ-primed mesenchymal stem cells derived from the human umbilical cord matrix in a rat model of TMJOA. METHODS: We analyzed changes in the expression of several key genes associated with OA protection in MSC-secreted compounds. Following this, we performed co-culture experiments using a transwell system to predict gene expression changes in primed MSCs in the TMJOA environment. Subsequently, we investigated the efficacy of the selected IFN-γ-primed human umbilical cord matrix-derived MSCs (hUCM-MSCs) for TMJOA treatment in a rat model. RESULTS: IFN-γ-primed MSCs exhibited enhanced expression of IDO, TSG-6, and FGF-2. Moreover, co-culturing with rat OA chondrocytes induced a decrease in pro-inflammatory and extracellular matrix degradation factors. In the rat TMJOA model, IFN-γ-primed MSCs with elevated IDO1, TSG-6, and FGF2 expression exhibited robust anti-inflammatory and therapeutic capacities, promoting the improvement of the inflammatory environment and cartilage regeneration. CONCLUSION: These findings underscore the importance of prioritizing the mitigation of the inflammatory milieu in TMJOA treatment and highlight IFN-γ-primed MSCs secreting these three factors as a promising, comprehensive therapeutic strategy.

HEG1 Protects Against Atherosclerosis by Regulating Stable Flow-Induced KLF2/4 Expression in Endothelial Cells.

BACKGROUND: Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow, and stable flow (s-flow) protects against atherosclerosis by incompletely understood mechanisms. METHODS: Our single-cell RNA-sequencing data using the mouse partial carotid ligation model was reanalyzed, which identified Heart-of-glass 1 (HEG1) as an s-flow-induced gene. HEG1 expression was studied by immunostaining, quantitive polymerase chain reaction, hybridization chain reaction, and Western blot in mouse arteries, human aortic endothelial cells (HAECs), and human coronary arteries. A small interfering RNA-mediated knockdown of HEG1 was used to study its function and signaling mechanisms in HAECs under various flow conditions using a cone-and-plate shear device. We generated endothelial-targeted, tamoxifen-inducible HEG1 knockout (HEG1iECKO) mice. To determine the role of HEG1 in atherosclerosis, HEG1iECKO and littermate-control mice were injected with an adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9] and fed a Western diet to induce hypercholesterolemia either for 2 weeks with partial carotid ligation or 2 months without the surgery. RESULTS: S-flow induced HEG1 expression at the mRNA and protein levels in vivo and in vitro. S-flow stimulated HEG1 protein translocation to the downstream side of HAECs and release into the media, followed by increased messenger RNA and protein expression. HEG1 knockdown prevented s-flow-induced endothelial responses, including monocyte adhesion, permeability, and migration. Mechanistically, HEG1 knockdown prevented s-flow-induced KLF2/4 (Kruppel-like factor 2/4) expression by regulating its intracellular binding partner KRIT1 (Krev interaction trapped protein 1) and the MEKK3-MEK5-ERK5-MEF2 pathway in HAECs. Compared with littermate controls, HEG1iECKO mice exposed to hypercholesterolemia for 2 weeks and partial carotid ligation developed advanced atherosclerotic plaques, featuring increased necrotic core area, thin-capped fibroatheroma, inflammation, and intraplaque hemorrhage. In a conventional Western diet model for 2 months, HEG1iECKO mice also showed an exacerbated atherosclerosis development in the arterial tree in both sexes and the aortic sinus in males but not in females. Moreover, endothelial HEG1 expression was reduced in human coronary arteries with advanced atherosclerotic plaques. CONCLUSIONS: Our findings indicate that HEG1 is a novel mediator of atheroprotective endothelial responses to flow and a potential therapeutic target.

Single-cell transcriptome sequencing of plant leaf expressing anti-HER2 VHH-FcK cancer therapeutic protein.

The transgenic plant is a promising strategy for the production of highly valuable biotherapeutic proteins such as recombinant vaccines and antibodies. To achieve an efficient level of protein production, codon sequences and expression cassette elements need to be optimized. However, the systematical expression of recombinant proteins in plant biomass can generally be controlled for the production of therapeutic proteins after the generation of transgenic plants. Without understanding the transgene expression patterns in plant tissue, it is difficult to enhance further production levels. In this study, single-cell RNA-sequencing (scRNA-seq) analysis of transgenic tobacco (Nicotiana tabacum) leaf, expressing an immunotherapeutic llama antibody against breast cancer, anti-HER2 VHH-Fc, was conducted to obtain data on the expression pattern of tissue-specific cells. These high-quality scRNA-seq data enabled the identification of gene expression patterns by cell types, which can be applied to select the best cell types or tissues for the high production of these recombinant antibodies. These data provide a foundation to elucidate the mechanisms that regulate the biosynthesis of recombinant proteins in N. tabacum.

Blood flow patterns switch VEGFR2 activity through differential S-nitrosylation and S-oxidation.

Vascular endothelial growth factor receptor-2 (VEGFR2) plays a key role in maintaining vascular endothelial homeostasis. Here, we show that blood flows determine activation and inactivation of VEGFR2 through selective cysteine modifications. VEGFR2 activation is regulated by reversible oxidation at Cys1206 residue. H2O2-mediated VEGFR2 oxidation is induced by oscillatory flow in vascular endothelial cells through the induction of NADPH oxidase-4 expression. In contrast, laminar flow induces the expression of endothelial nitric oxide synthase and results in the S-nitrosylation of VEGFR2 at Cys1206, which counteracts the oxidative inactivation. The shear stress model study reveals that disturbed blood flow operated by partial ligation in the carotid arteries induces endothelial damage and intimal hyperplasia in control mice but not in knock-in mice harboring the oxidation-resistant mutant (C1206S) of VEGFR2. Thus, our findings reveal that flow-dependent redox regulation of the VEGFR2 kinase is critical for the structural and functional integrity of the arterial endothelium.

Etiology and Pathogenesis of Rheumatoid Arthritis-Interstitial Lung Disease.

Interstitial lung disease (ILD) is one of the most serious extra-articular complications of rheumatoid arthritis (RA), which increases the mortality of RA. Because the pathogenesis of RA-ILD remains poorly understood, appropriate therapeutic strategies and biomarkers have not yet been identified. Thus, the goal of this review was to summarize and analyze the reported data on the etiology and pathogenesis of RA-ILD. The incidence of RA-ILD increases with age, and is also generally higher in men than in women and in patients with specific genetic variations and ethnicity. Lifestyle factors associated with an increased risk of RA-ILD include smoking and exposure to pollutants. The presence of an anti-cyclic citrullinated peptide antibody, high RA disease activity, and rheumatoid factor positivity also increase the risk of RA-ILD. We also explored the roles of biological processes (e.g., fibroblast-myofibroblast transition, epithelial-mesenchymal transition, and immunological processes), signaling pathways (e.g., JAK/STAT and PI3K/Akt), and the histopathology of RA involved in RA-ILD pathogenesis based on published preclinical and clinical models of RA-ILD in animal and human studies.

Organoid Establishment of Long-Term Culture Using Primary Mouse Hepatocytes and Evaluation of Liver Function.

Primary hepatocytes and various animal models have traditionally been used in liver function tests to assess the effects of nutrients. However, these approaches present several limitations such as time consumption, high cost, the need for facilities, and ethical issues in primary mouse hepatocytes and animal models. In this study, we constructed liver organoids from primary mouse hepatocytes (OrgPH) to replace primary hepatocytes and animal models. We isolated primary mouse hepatocytes from 6- to 10-week-old male C57BL/6J mice using the two-step collagenase method, and generated liver organoids by clustering the cells in Matrigel. To assess the hepatic function of OrgPH, we examined specific liver markers and gene expressions related to hepatic glucose, ethanol, and cholesterol metabolism. Over a 28-day culture period, liver-specific markers, including Alb, Arg1, G6pc, and Cyp1a1, increased or remained stable in the OrgPH. However, they eventually decreased in primary hepatocytes. Glucose and ethanol metabolism-related gene expression levels exhibited a similar tendency in AML12 cells and OrgPH. However, the expression levels of cholesterol metabolism-related genes displayed an opposite trend in OrgPH compared with those in AML12 cells. These results agree with those of previous studies involving in vivo models. In conclusion, our study indicates that OrgPH can retain liver function and mimic the hepatocytic physiology of mouse in vivo models. Therefore, organoids originating from primary mouse hepatocytes are potentially useful as an animal-free method for evaluating the safety and toxicity of health functional foods and a replacement for animal models.

Co-transient expression of PSA-Fc and PAP-Fc fusion protein in plant as prostate cancer vaccine candidates and immune responses in mice.

KEY MESSAGE: PAP-FcK and PSA-FcK prostate cancer antigenic proteins transiently co-expressed in plant induce their specific humoral immune responses in mice. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been considered as immunotherapeutic antigens for prostate cancer. The use of a single antigenic agent is unlikely to be effective in eliciting immunotherapeutic responses due to the heterogeneous and multifocal nature of prostate cancer. Thus, multiple antigens have been combined to enhance their anti-cancer effects. In the current study, PSA and PAP were fused to the crystallizable region (Fc region) of immunoglobulin G1 and tagged with KDEL, the endoplasmic reticulum (ER) retention signal motif, to generate PSA-FcK and PAP-FcK, respectively, and were transiently co-expressed in Nicotiana benthamiana. Western blot analysis confirmed the co-expression of PSA-FcK and PAP-FcK (PSA-FcK + PAP-FcK) with a 1:3 ratios in the co-infiltrated plants. PSA-FcK, PAP-FcK, and PSA-FcK + PAP-FcK proteins were successfully purified from N. benthamiana by protein A affinity chromatography. ELISA showed that anti-PAP and anti-PSA antibodies successfully detected PAP-FcK and PSA-FcK, respectively, and both detected PSA-FcK + PAP-FcK. Surface plasmon resonance (SPR) analysis confirmed the binding affinity of the plant-derived Fc fusion proteins to FcγRI/CD64. Furthermore, we also confirmed that mice injected with PSA-FcK + PAP-FcK produced both PSA- and PAP-specific IgGs, demonstrating their immunogenicity. This study suggested that the transient plant expression system can be applied to produce the dual-antigen Fc fusion protein (PSA-FcK + PAP-FcK) for prostate cancer immunotherapy.

FAT10 Induces cancer cell migration by stabilizing phosphorylated ABI3/NESH.

The WAVE regulatory complex (WRC) is involved in various cellular processes by regulating actin polymerization. The dysregulation of WRC components is associated with cancer development. ABI family member 3 (ABI3)/new molecule including SH3 (NESH) is one of the WRC components and it has been reported that ABI3 phosphorylation can affect WRC function. Although several residues of ABI3 have been reported to be possible phosphorylation sites, it is still unclear which residues are important for the function of ABI3. Furthermore, it is unclear how the phosphorylated form of ABI3 is regulated. Here, we demonstrate that ABI3 is stabilized by its interaction with human leukocyte antigen-F adjacent transcript 10 (FAT10). Using phospho-dead or phospho-mimetic mutants of ABI3, we showed that serine 213 and 216 are important phosphorylation sites of ABI3. In particular, FAT10 has a higher affinity for the phosphorylated form of ABI3 than the non-phosphorylated form, and it stabilizes the phosphorylated form more than the non-phosphorylated form through this differential affinity. The interaction between FAT10 and the phosphorylated form of ABI3 promoted cancer cell migration. Therefore, our results suggest that FAT10 stabilizes the phosphorylated form of ABI3, which may lead to WRC activation, thereby promoting cancer cell migration.

ß-Carotene suppresses cancer cachexia by regulating the adipose tissue metabolism and gut microbiota dysregulation.

Cancer cachexia is a metabolic disease affecting multiple organs and characterized by loss adipose and muscle tissues. Metabolic dysregulated of adipose tissue has a crucial role in cancer cachexia. ß-Carotene (BC) is stored in adipose tissues and increases muscle mass and differentiation. However, its regulatory effects on adipose tissues in cancer cachexia have not been investigated yet. In this study, we found that BC supplementations could inhibit several cancer cachexia-related changes, including decreased carcass-tumor (carcass weight after tumor removal), adipose weights, and muscle weights in CT26-induced cancer cachexia mice. Moreover, BC supplementations suppressed cancer cachexia-induced lipolysis, fat browning, hepatic gluconeogenesis, and systemic inflammation. Altered diversity and composition of gut microbiota in cancer cachexia were restored by the BC supplementations. BC treatments could reverse the down-regulated adipogenesis and dysregulated mitochondrial respiration and glycolysis in adipocytes and colon cancer organoid co-culture systems. Taken together, these results suggest that BC can be a potential therapeutic strategy for cancer cachexia.

Design, Synthesis, and Cytotoxicity and Topoisomerase I/IIα Inhibition Activity of Pyrazolo[4,3-f]quinoline Derivatives.

With the several targets of cancer treatment, inhibition of DNA topoisomerase activity is one of the well-known focuses in cancer chemotherapy. Here, we describe the design and synthesis of a novel series of pyrazolo[4,3-f]quinolines with potential anticancer/topoisomerase inhibition activity. Forty newly designed pyrazolo[4,3-f]quinoline derivatives were synthesized via inverse imino Diels-Alder reaction. The antiproliferative activity of the synthesized derivatives was initially measured in the human NUGC-3 cancer cell line. Then, the selected compounds 1B, 1C, 1M, 2A, 2D, 2E, 2F, and 2R with higher activity among tested compounds were screened against six cancer cell lines, including ACHN, HCT-15, MM231, NCI-H23, NUGC-3, and PC-3. The results demonstrated that the compounds 1M, 2E, and 2P were most effective in all cancer cell lines exhibiting GI50 below 8 µM. Among them, 2E showed an equivalent inhibition pattern of topoisomerase IIα activity to that of etoposide, positive control at a 100 µM dose.

ß-carotene regulates cancer stemness in colon cancer in vivo and in vitro.

BACKGROUND/OBJECTIVES: Colorectal cancer (CRC) is the third most common cancer worldwide and has a high recurrence rate, which is associated with cancer stem cells (CSCs). ß-carotene (BC) possesses antioxidant activity and several anticancer mechanisms. However, no investigation has examined its effect on colon cancer stemness. MATERIALS/METHODS: CD133+CD44+ HCT116 and CD133+CD44+ HT-29 cells were isolated and analyzed their self-renewal capacity by clonogenic and sphere formation assays. Expressions of several CSCs markers and Wnt/ß-catenin signaling were examined. In addition, CD133+CD44+ HCT116 cells were subcutaneously injected in xenograft mice and analyzed the effect of BC on tumor formation, tumor volume, and CSCs markers in tumors. RESULTS: BC inhibited self-renewal capacity and CSC markers, including CD44, CD133, ALDH1A1, NOTCH1, Sox2, and ß-catenin in vitro. The effects of BC on CSC markers were confirmed in primary cells isolated from human CRC tumors. BC supplementation decreased the number and size of tumors and delayed the tumor-onset time in xenograft mice injected with CD133+CD44+ HCT116 cells. The inhibitory effect of BC on CSC markers and the Wnt/ß-catenin signaling pathway in tumors was confirmed in vivo as well. CONCLUSIONS: These results suggest that BC may be a potential therapeutic agent for colon cancer by targeting colon CSCs.

Neohesperidin Dihydrochalcone and Neohesperidin Dihydrochalcone-O-Glycoside Attenuate Subcutaneous Fat and Lipid Accumulation by Regulating PI3K/AKT/mTOR Pathway In Vivo and In Vitro.

Neohesperidin dihydrochalcone (NHDC), a semi-natural compound from bitter orange, is an intense sweetener. The anti-obesity effects of NHDC and its glycosidic compound, NHDC-O-glycoside (GNHDC), were investigated. C57BLKS/J db/db mice were supplemented with NHDC or GNHDC (100 mg/kg b.w.) for 4 weeks. Body weight gain, subcutaneous tissues, and total adipose tissues (sum of perirenal, visceral, epididymal, and subcutaneous adipose tissue) were decreased in the NHDC and GNHDC groups. Fatty acid uptake, lipogenesis, and adipogenesis-related genes were decreased, whereas ß-oxidation and fat browning-related genes were up-regulated in the sweetener groups. Furthermore, both sweeteners suppressed the level of triacylglycerol accumulation, lipogenesis, adipogenesis, and proinflammatory cytokines in the 3T3-L1 cells. The PI3K/AKT/mTOR pathway was also down-regulated, and AMP-acttvated protein kinase (AMPK) was phosphorylated in the treatment groups. These results suggest that NHDC and GNHDC inhibited subcutaneous fat and lipid accumulation by regulating the PI3K/AKT/mTOR pathway and AMPK-related lipogenesis and fat browning.

The transcription factor ORA59 exhibits dual DNA binding specificity that differentially regulates ethylene- and jasmonic acid-induced genes in plant immunity.

Jasmonic acid (JA) and ethylene (ET) signaling modulate plant defense against necrotrophic pathogens in a synergistic and interdependent manner, while JA and ET also have independent roles in certain processes, e.g. in responses to wounding and flooding, respectively. These hormone pathways lead to transcriptional reprogramming, which is a major part of plant immunity and requires the roles of transcription factors. ET response factors are responsible for the transcriptional regulation of JA/ET-responsive defense genes, of which ORA59 functions as a key regulator of this process and has been implicated in the JA-ET crosstalk. We previously demonstrated that Arabidopsis (Arabidopsis thaliana) GDSL LIPASE 1 (GLIP1) depends on ET for gene expression and pathogen resistance. Here, promoter analysis of GLIP1 revealed ERELEE4 as the critical cis-element for ET-responsive GLIP1 expression. In a yeast one-hybrid screening, ORA59 was isolated as a specific transcription factor that binds to the ERELEE4 element, in addition to the well-characterized GCC box. We found that ORA59 regulates JA/ET-responsive genes through direct binding to these elements in gene promoters. Notably, ORA59 exhibited a differential preference for GCC box and ERELEE4, depending on whether ORA59 activation is achieved by JA and ET, respectively. JA and ET induced ORA59 phosphorylation, which was required for both activity and specificity of ORA59. Furthermore, RNA-seq and virus-induced gene silencing analyses led to the identification of ORA59 target genes of distinct functional categories in JA and ET pathways. Our results provide insights into how ORA59 can generate specific patterns of gene expression dynamics through JA and ET hormone pathways.

Contribution of dipeptidyl peptidase 4 to non-typeable Haemophilus influenzae-induced lung inflammation in COPD.

Dipeptidyl peptidase 4 (DPP4) expression is increased in the lungs of chronic obstructive pulmonary disease (COPD). DPP4 is known to be associated with inflammation in various organs, including LPS-induced acute lung inflammation. Since non-typeable Haemophilus influenzae (NTHi) causes acute exacerbations in COPD patients, we examined the contribution of DPP4 in NTHi-induced lung inflammation in COPD. Pulmonary macrophages isolated from COPD patients showed higher expression of DPP4 than the macrophages isolated from normal subjects. In response to NTHi infection, COPD, but not normal macrophages show a further increase in the expression of DPP4. COPD macrophages also showed higher expression of IL-1ß, and CCL3 responses to NTHi than normal, and treatment with DPP4 inhibitor, diprotin A attenuated this response. To examine the contribution of DPP4 in NTHi-induced lung inflammation, COPD mice were infected with NTHi, treated with diprotin A or PBS intraperitoneally, and examined for DPP4 expression, lung inflammation, and cytokine expression. Mice with COPD phenotype showed increased expression of DPP4, which increased further following NTHi infection. DPP4 expression was primarily observed in the infiltrated inflammatory cells. NTHi-infected COPD mice also showed sustained neutrophilic lung inflammation and expression of CCL3, and this was inhibited by DPP4 inhibitor. These observations indicate that enhanced expression of DPP4 in pulmonary macrophages may contribute to sustained lung inflammation in COPD following NTHi infection. Therefore, inhibition of DPP4 may reduce the severity of NTHi-induced lung inflammation in COPD.

Cancer Testis Antigen, NOL4, Is an Immunogenic Antigen Specifically Expressed in Small-Cell Lung Cancer.

To identify cancer/testis (CT) antigens and immunogenic proteins, immunoscreening of testicular and small-cell lung cancer cell line NCI-H889 cDNA libraries was performed using serum obtained from a small-cell lung cancer (SCLC) patient. We obtained 113 positive cDNA clones comprised of 74 different genes, designated KP-SCLC-1 through KP-SCLC-74. Of these genes, 59 genes were found to be related to cancers by EMBASE analysis. Three of these antigens, including KP-SCLC-29 (NOL4), KP-SCLC-59 (CCDC83), and KP-SCLC-69 (KIF20B), were CT antigens. RT-PCR and western blot analysis showed that NOL4 was frequently present in small-cell lung cancer cell lines (8/9, 8/9). In addition, NOL4 mRNA was weakly, or at a low frequency, or not detected in various cancer cell lines. Our results reveal that NOL4 was expressed at protein levels in small-cell lung cancer tissues (10/10) but not detected in lung adenocarcinoma and squamous cell carcinoma by immunohistochemical analysis. Serological response to NOL4 was also evaluated by western blot assay using NOL4 recombinant protein. A humoral response against NOL4 proteins was detected in 75% (33/44) of small-cell lung cancer patients and in 65% (13/20) of healthy donors by a serological western blot assay. These data suggest that NOL4 is a specific target that may be useful for diagnosis and immunotherapy in SCLC.

Curcumin and hesperetin attenuate D-galactose-induced brain senescence in vitro and in vivo.

BACKGROUND/OBJECTIVES: Brain senescence causes cognitive impairment and neurodegeneration. It has also been demonstrated that curcumin (Cur) and hesperetin (Hes), both antioxidant polyphenolic compounds, mediate anti-aging and neuroprotective effects. Therefore, the objective of this study was to investigate whether Cur, Hes, and/or their combination exert anti-aging effects in D-galactose (Dg)-induced aged neuronal cells and rats. MATERIALS/METHODS: SH-SY5Y cells differentiated in response to retinoic acid were treated with Cur (1 µM), Hes (1 µM), or a combination of both, followed by 300 mM Dg. Neuronal loss was subsequently evaluated by measuring average neurite length and analyzing expression of ß-tubulin III, phosphorylated extracellular signal-regulated kinases, and neurofilament heavy polypeptide. Cellular senescence and related proteins, p16 and p21, were also investigated, including their regulation of antioxidant enzymes. In vivo, brain aging was induced by injecting 250 mg/kg body weight (b.w.) Dg. The effects of supplementing this model with 50 mg/kg b.w. Cur, 50 mg/kg b.w. Hes, or a combination of both for 3 months were subsequently evaluated. Brain aging was examined with a step-through passive avoidance test and apoptosis markers were analyzed in brain cortex tissues. RESULTS: Cur, Hes, and their combination improved neuron length and cellular senescence by decreasing the number of ß-gal stained cells, down-regulated expression of p16 and p21, and up-regulated expression of antioxidant enzymes, including superoxide dismutase 1, glutathione peroxidase 1, and catalase. Administration of Cur, Hes, or their combination also tended to ameliorate cognitive impairment and suppress apoptosis in the cerebral cortex by down-regulating Bax and poly (ADP-ribose) polymerase expression and increasing Bcl-2 expression. CONCLUSIONS: Cur and Hes appear to attenuate Dg-induced brain aging via regulation of antioxidant enzymes and apoptosis. These results suggest that Cur and Hes may mediate neuroprotective effects in the aging process, and further study of these antioxidant polyphenolic compounds is warranted.