Nanostructured doping of WSe2via block copolymer patterns and its self-powered photodetector application.

Transition metal dichalcogenides (TMDs), e.g., MoS2, MoSe2, ReS2, and WSe2, are effective materials for advanced optoelectronics owing to their intriguing optical, structural, and electrical properties. Various approaches for manipulating the surface of the TMDs have been suggested to unleash the optoelectronic potential of the TMDs. Herein, we employed the self-assembly of the poly(styrene-b-methyl methacrylate) (PS-b-PMMA) block copolymer (BCP) to prepare a nanoporous pattern and generate nanostructured charge-transfer p-doping on the WSe2 surface, maximizing the depletion region in the absorber layer. After the spin coating and thermal annealing of PS-b-PMMA, followed by the selective etching of PMMA cylindrical microdomains using oxygen reactive-ion plasma, nanopatterned WOx with high electron affinity was grown on the WSe2 surface, forming a three-dimensional homojunction. The nanopatterned WOx significantly expanded the depletion region in the WSe2 layer, thus enhancing optoelectronic performance and self-powered photodetection. The proposed approach based on the nanostructured doping of the TMDs via BCP nanolithography can help create a promising platform for highly functional optoelectrical devices.

Can We Better Differentiate Type A Dissections: Evaluating the Role of Aortic Ratios.

OBJECTIVES: Type A aortic dissection (ATAAD) is hypothesised as a progression of aneurysmal dilation, but 60% of patients in the International Registry of Acute Aortic Dissection (iRAD) registry had a maximum aortic diameter (MAD)<55 mm. We aim to demonstrate that size ratios and aortic wall stress, assessed using a simplified markers, are unique to aortic patients who have had adverse events (ATAAD) compared to those who have not (thoracic aortic aneurysm [TAA]). METHODS: A retrospective cohort analysis of patients who underwent aortic intervention at Waikato Hospital, New Zealand between 2015-2020, comparing dissection (ATAAD) to TAA patients. MAD; ratio of MAD to standardised-points within the aorta; and MAD-to-height collected from computed tomography (CT)-scans of all patients was undertaken. Receiver operating characteristic (ROC)-analysis to determine cut-off point for each marker was undertaken together with multivariable logistic regression comparing both cohorts, cross-validated by propensity-score matched analysis. RESULTS: Cohort of 215 patients, 78 (36.3%) ATAAD and 137 (63.7%) TAA; median age at intervention 63.3 years, 52 (24.2%) females, both cohorts matched for size. Using the entire cohort, the MAD: sinus of Valsalva (SoV) ratio>1.06 (cut-off value) had 4.5-times greater association with ATAAD (95%CI 1.46-13.8) and a 0.1-unit increased conferred 1.45-times greater association with ATAAD (95%CI 1.00-2.08). MAD>55 mm only seen in 33.3% of ATAAD (n=26/78), and not associated with ATAAD (OR 1.88, 95%CI 0.64-5.51). Compared to MAD, MAD:SoV ratio had greater sensitivity (33% vs 73%), lower number-needed-to-treat (17.9 vs 2.7) and superior discrimination (area under the curve [AUC] 0.54 vs 0.71). Findings were consistent with propensity score matched analysis. CONCLUSIONS: MAD:SoV ratio significantly correlates with ATAAD (4.5 times), with superior sensitivity, discrimination, and attributable-risk-percentage compared to MAD alone.

An in vitro study on anti-carcinogenic effect of remdesivir in human ovarian cancer cells via generation of reactive oxygen species.

BACKGROUND: Remdesivir is an anti-viral drug that inhibits RNA polymerase. In 2020, remdesivir was recognized as the most promising therapeutic agents against coronavirus disease 2019 (COVID-19). However, the effects of remdesivir on cancers have hardly been studied. PURPOSE: Here, we reported that the anti-carcinogenic effect of remdesivir on SKOV3 cells, one of human ovarian cancer cell lines. RESEARCH DESIGN: We anlalyzed the anti-carcarcinogenic effect of remdesivir in SKOV3 cells by performing in vitro cell assay and western blotting. RESULTS: WST-1 showed that remdesivir decreased cell viability in SKOV3 cells. Experiments conducted by Muse Cell Analyzer showed that remdesivir-induced apoptosis in SKOV3 cells. We found that the expression level of FOXO3, Bax, and Bim increased, whereas Bcl-2, caspase-3, and caspase-7 decreased by remdesivir in SKOV3 cells. Furthermore, we observed that intracellular reactive oxygen species (ROS) level increased after treatment of remdesivir in SKOV3 cells. Interestingly, cytotoxicity of remdesivir decreased after treatment of N-Acetylcysteine. CONCLUSION: Taken together, our results demonstrated that remdesivir has an anti-carcinogenic effect on SKOV3 cells vis up-regulation of reactive oxygen species, which suggests that remdesivir could be a promising reagent for treatment of ovarian cancer.

IL4Rα and IL13Rα1 Are Involved in the Development of Human Gallbladder Cancer.

BACKGROUND: Gallbladder cancer is commonly associated with inflammation, which indicates that inflammation-related cytokines and cytokine receptors are related to the progression of gallbladder cancers. Interleukin 4 (IL4) is a well-known cytokine that promotes the differentiation of naive helper T cells (Th0) to T helper type 2 cells (Th2). IL13 is a cytokine that is secreted by Th2 cells. IL4 and IL13 are closely related in immune responses. However, the role of IL4Rα and IL13Rα1 signaling pathway has not been fully understood in the development of gallbladder cancer. METHODS: In human gallbladder carcinomas, the expression of IL4Rα and IL13Rα1 were evaluated with immunohistochemical staining in tissue microarray tissue sections. After knockdown of IL4Rα or IL13Rα1, cell assays to measure the proliferation and apoptosis and Western blotting analysis were conducted in SNU308 human gallbladder cancer cells. Since Janus kinases2 (JAK2) was considered as one of the down-stream kinases under IL4Rα and IL13Rα1 complex, the same kinds of experiments were performed in SNU308 cells treated with AZD1480, Janus-associated kinases2 (JAK2) inhibitor, to demonstrate the cytotoxic effect of AZD1480 in SNU308 cells. RESULTS: Immunohistochemical expression of IL4Rα was significantly associated with the expression of IL13Rα1 in human carcinoma tissue. In univariate analysis, nuclear expression of IL4Rα, cytoplasmic expression of IL4Rα, nuclear expression of IL13Rα1, and cytoplasmic expression of IL13Rα1 were significantly associated with shorter overall survival and shorter relapse-free survival. Multivariate analysis revealed nuclear expression of IL4Rα as an independent poor prognostic indicator of overall survival and relapse-free survival. Then, we found that knockdown of IL4Rα or IL13Rα1 decreased viability and induced apoptosis in SNU308 cells via activation of FOXO3 and similarly, AZD1480 decreased viability and induced apoptosis in SNU308 cells with dose dependent manner. CONCLUSIONS: Taken together, our results suggest that IL4Rα and IL13Rα1 might be involved in the development of human gallbladder cancer cells and IL4Rα and IL13Rα1 complex/JAK2 signaling pathway could be efficient therapeutic targets for gallbladder cancer treatment.

IL13Rα2 Is Involved in the Progress of Renal Cell Carcinoma through the JAK2/FOXO3 Pathway.

Previously, we reported a close relationship between type II IL4Rα and IL13Rα1 complex and poor outcomes in renal cell carcinoma (RCC). In this study, we investigated the clinicopathologically significant oncogenic role of IL13Rα2, a kind of the independent receptor for IL13, in 229 RCC patients. The high expression of IL13Rα2 was closely related to relapse-free survival in specific cancers in univariate and multivariate analysis. Then, the oncogenic role of IL13Rα2 was evaluated by performing in vitro assays for cell proliferation, cell cycle arrest, and apoptosis in A498, ACHN, Caki1, and Caki2, four kinds of RCC cells after transfection of siRNA against IL13Rα2. Cell proliferation was suppressed, and apoptosis was induced in A498, ACHN, Caki1, and Caki2 cells by knockdown of IL13Rα2. Interestingly, the knockdown of IL13Rα2 decreased the phosphorylation of JAK2 and increased the expression of FOXO3. Furthermore, the knockdown of IL13Rα2 reduced the protein interaction among IL13Rα2, phosphorylated JAK2, and FOXO3. Since phosphorylation of JAK2 was regulated by IL13Rα2, we tried to screen a novel JAK2 inhibitor from the FDA-approved drug library and selected telmisartan, a clinically used medicine against hypertension, as one of the strongest candidates. Telmisartan treatment decreased the cell proliferation rate and increased apoptosis in A498, ACHN, Caki1, and Caki2 cells. Mechanistically, telmisartan treatment decreased the phosphorylation of JAK2 and increased the expression of FOXO3. Taken together, these results suggest that IL13Rα2 regulates the progression of RCC via the JAK2/FOXO3-signaling path pathway, which might be targeted as the novel therapeutic option for RCC patients.

Downregulation of APRIN expression increases cancer cell proliferation via an interleukin-6/STAT3/cyclin D axis.

APRIN is a putative tumor suppressor whose expression is low in a variety of cancer cells. While decreased expression of APRIN leads to increased cell proliferation, unfavorable diagnosis or metastases in various cancer types, there is limited knowledge on the cellular mechanism of APRIN in cellular responses. The effect of APRIN depletion on cancer cell proliferation was examined in the present study, and the IL-6/STAT3/cyclin D axis was identified as a novel regulatory mechanism. Stable depletion of APRIN in cancer cells resulted in increased cell proliferation. Cytokine array analysis of the cells revealed that downregulation of APRIN induced secretion of interleukin-6 (IL-6) with corresponding activation of STAT3, a downstream intracellular mediator. Levels of cyclin D1 were increased in cells with APRIN depletion and cyclin D1 expression was associated with increased STAT3 binding on cyclin D1 promoter sequence; assessed by chromatin immunoprecipitation assay. The addition of an IL-6 neutralizing antibody P620 to the cell culture attenuated STAT3 activation and cyclin D1 expression in APRIN-depleted cells with corresponding decrease in cell proliferation. These experiments suggest that APRIN regulates cancer cell proliferation via an IL-6/STAT3/cyclin D axis and that targeting this axis in APRIN-associated cancer might provide a novel therapeutic approach.

Cannabidiol induces osteoblast differentiation via angiopoietin1 and p38 MAPK.

In this study, we report the potential of cannabidiol, one of the major cannabis constituents, for enhancing osteoblastic differentiation in U2OS and MG-63 cells. Cannabidiol increased the expression of Angiopoietin1 and the enzyme activity of alkaline phosphatase in U2OS and MG-63. Invasion and migration assay results indicated that the cell mobility was activated by cannabidiol in U2OS and MG-63. Western blotting analysis showed that the expression of tight junction related proteins such as Claudin1, Claudin4, Occuludin1, and ZO1 was increased by cannabidiol in U2OS and MG-63. Alizarin Red S staining analysis showed that calcium deposition and mineralization was enhanced by cannabidiol in U2OS and MG-63. Western blotting analysis indicated that the expression of osteoblast differentiation related proteins such as distal-less homeobox 5, bone sialoprotein, osteocalcin, type I collagen, Runt-related transcription factor 2 (RUNX2), osterix (OSX), and alkaline phosphatase was time dependently upregulated by cannabidiol in U2OS and MG-63. Mechanistically, cannabidiol-regulated osteoblastic differentiation in U2OS and MG-63 by strengthen the protein-protein interaction among RUNX2, OSX, or the phosphorylated p38 mitogen-activated protein kinase (MAPK). In conclusion, cannabidiol increased Angiopoietin1 expression and p38 MAPK activation for osteoblastic differentiation in U2OS and MG-63 suggesting that cannabidiol might provide a novel therapeutic option for the bone regeneration.

Metformin coordinates osteoblast/osteoclast differentiation associated with ischemic osteonecrosis.

In this study, we aimed to identify a candidate drug that can activate endogenous Angiopoietin 1 (Ang1) expression via drug repositioning as a pharmacological treatment for avascular osteonecrosis. After incubation with 821 drugs from the Food and Drug Administration (FDA)-approved drug library, Ang1 expression in U2OS cell culture media was examined by ELISA. Metformin, the first-line medication for treatment of type 2 diabetes, was selected as a candidate for in vitro and in vivo experimental evaluation. Ang1 was induced, and alkaline phosphatase activity was increased by metformin treatment in U2OS and MG63 cells. Wound healing and migration assay showed increased osteoblastic cell mobility by metformin treatment in U2OS and MG63 cells. Metformin upregulated expression of protein markers for osteoblastic differentiation in U2OS and MG63 cells but inhibited osteoclastic differentiation in Raw264.7 cells. Metformin (25 mg/kg) protected against ischemic necrosis in the epiphysis of the rat femoral head by maintaining osteoblast/osteocyte function and vascular density but inhibiting osteoclast activity in the necrotic femoral head. These findings provide novel insight into the specific biomarkers that are targeted and regulated by metformin in osteoblast differentiation and contribute to understanding the effects of these FDA-approved small-molecule drugs as novel therapeutics for ischemic osteonecrosis.

Loss of the dermis zinc transporter ZIP13 promotes the mildness of fibrosarcoma by inhibiting autophagy.

Fibrosarcoma is a skin tumor that is frequently observed in humans, dogs, and cats. Despite unsightly appearance, studies on fibrosarcoma have not significantly progressed, due to a relatively mild tumor severity and a lower incidence than that of other epithelial tumors. Here, we focused on the role of a recently-found dermis zinc transporter, ZIP13, in fibrosarcoma progression. We generated two transformed cell lines from wild-type and ZIP13-KO mice-derived dermal fibroblasts by stably expressing the Simian Virus (SV) 40-T antigen. The ZIP13-/- cell line exhibited an impairment in autophagy, followed by hypersensitivity to nutrient deficiency. The autophagy impairment in the ZIP13-/- cell line was due to the low expression of LC3 gene and protein, and was restored by the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza) treatment. Moreover, the DNA methyltransferase activity was significantly increased in the ZIP13-/- cell line, indicating the disturbance of epigenetic regulations. Autophagy inhibitors effectively inhibited the growth of fibrosarcoma with relatively minor damages to normal cells in xenograft assay. Our data show that proper control over autophagy and zinc homeostasis could allow for the development of a new therapeutic strategy to treat fibrosarcoma.

Interleukin4Rα (IL4Rα) and IL13Rα1 Are Associated with the Progress of Renal Cell Carcinoma through Janus Kinase 2 (JAK2)/Forkhead Box O3 (FOXO3) Pathways.

Specific kinds of interleukin (IL) receptors are known to mediate lymphocyte proliferation and survival. However, recent reports have suggested that the high expression of IL4Rα and IL13Rα1 in tumor tissue might be associated with tumorigenesis in several kinds of tumor. We found that a significant association between mRNA level of IL4Rα or IL13Rα1 and the poor prognosis of renal cell carcinoma (RCC) from the public database (http://www.oncolnc.org/). Then, we evaluated the clinicopathological significance of the immunohistochemical expression of IL4Rα and IL13Rα1 in 199 clear cell RCC (CCRCC) patients. The individual and co-expression patterns of IL4Rα and IL13Rα1 were significantly associated with cancer-specific survival (CSS) and relapse-free survival (RFS) in univariate analysis. Multivariate analysis indicated IL4Rα-positivity and co-expression of IL4Rα and IL13Rα1 as the independent indicators of shorter CSS and RFS of CCRCC patients. For the in vitro evaluation of the oncogenic role of IL4Rα and IL13Rα1 in RCC, we knock-downed IL4Rα or IL13Rα1 and observed that the cell proliferation rate was decreased, and the apoptosis rate was increased in A498 and ACHN cells. Furthermore, we examined the possible role of Janus kinase 2 (JAK2), well-known down-stream tyrosine kinase under the heterodimeric receptor complex of IL4Rα and IL13Rα1. Interestingly, JAK2 interacted with Forkhead box O3 (FOXO3) to cause tyrosine-phosphorylation of FOXO3. Silencing IL4Rα or JAK2 in A498 and ACHN cells reduced the interaction between JAK2 and FOXO3. Moreover, pharmacological inhibition of JAK2 induced the nuclear localization of FOXO3, leading to increase apoptosis and decrease cell proliferation rate in A498 and ACHN cells. Taken together, these results suggest that IL4Rα and IL13Rα1 might be involved in the progression of RCC through JAK2/FOXO3 pathway, and their expression might be used as the novel prognostic factor and therapeutic target for RCC patients.

Mitoxantrone induces apoptosis in osteosarcoma cells through regulation of the Akt/FOXO3 pathway.

The outcome of chemotherapy for osteosarcoma have improved during the past decade and more patients have access to combination chemotherapy, but there has been no significant clinical progress in the patient survival rate. Recently, forkhead-box O3 (FOXO3) was identified as a pivotal transcription factor responsible for the transcriptional regulation of genes associated with suppression of cancer. The purpose of the present study was to screen small chemicals activating FOXO3 and elucidate their underlying mechanism. Using a drug discovery platform based on the phosphorylation status of FOXO3 in osteosarcoma cells, mitoxantrone (MTZ), a type of DNA-damaging agent, was selected as a possible FOXO3 activator from the food and drug administration-approved drug library. MTZ treatments significantly inhibited the phosphorylation level of Akt-pS473 and caused nuclear localization of FOXO3 in osteosarcoma cells. MTZ treatment inhibited proliferation in osteosarcoma cells in vitro, whereas silencing FOXO3 potently attenuates MTZ-mediated apoptosis in osteosarcoma cells. Taken together, the results indicated that MTZ induces apoptosis in osteosarcoma cells through an Akt/FOXO3-dependent mechanism.

Potential of l-thyroxine to differentiate osteoblast-like cells via Angiopoietin1.

Angiogenesis is closely associated with osteoblast differentiation. Previously, we demonstrated that bone formation can be accelerated by treatment with COMP-Angiopoietin1, a known angiogenic factor. Angiopoietin1 (Ang1) is a specific growth factor that generates stable and mature vasculature through the Tie2 receptor. In this study, we aimed to identify a novel drug that can activate endogenous Ang1 expression as a pharmacological treatment for bone formation. Therefore, Ang1 expression was examined in U2OS osteoblast-like cells treated with 770 drugs from a library of Food and Drug Administration (FDA)-approved drugs by using ELISA for Ang1. l-thyroxine was selected as a novel drug candidate. l-Thyroxine is a synthetic form of the hormone thyroxine, which is used to treat patients with hypothyroidism. Enzyme-linked immunosorbent assays (ELISAs) were performed to test whether Ang1 is induced in a dose-dependent manner in human osteoblast-like cell lines, U2OS and MG63. The effects of l-thyroxine on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and Alizarin red s staining. To determine the molecular mechanism, the expression of proteins related to bone formation and differentiation, such as type I collagen (COL1A1), osteocalcin (OC), bone sialoprotein (BSP), distal-less homeobox 5 (Dlx5), Runt-related transcription factor 2 (Runx2), osterix (OSX), and ALP, was tested by Western blotting analysis. Consequently, l-thyroxine induced Ang1 expression in a dose-dependent manner in both U2OS and M63 cells, which was confirmed by ELISA and Western blotting. Also, l-thyroxine activated ALP activity in U2OS and MG63 cells as well as ALP expression. Furthermore, l-thyroxine enhanced the expression of COL1A1, Runx2, OC, BSP, Dlx5, and OSX mRNA and proteins. Taken together, we demonstrated that l-thyroxine increased Ang1 expression and induces bone formation, differentiation, and mineralization in U2OS and MG63 cell lines, which suggests that l-thyroxine could be a potential bone production agent.

Dynamic nucleotide-dependent interactions of cysteine- and histidine-rich domain (CHORD)-containing Hsp90 cochaperones Chp-1 and melusin with cochaperones PP5 and Sgt1.

Mammals have two cysteine- and histidine-rich domain (CHORD)-containing Hsp90 cochaperones, Chp-1 and melusin, which are homologs of plant Rar1. It has been shown previously that Rar1 CHORD directly interacts with ADP bound to the nucleotide pocket of Hsp90. Here, we report that ADP and ATP can bind to Hsp90 cochaperones Chp-1 and PP5, inducing their conformational changes. Furthermore, we demonstrate that Chp-1 and melusin can interact with cochaperones PP5 and Sgt1 and with each other in an ATP-dependent manner. Based on the known structure of the Rar1-Hsp90 complex, His-186 has been identified as an important residue of Chp-1 for ADP/ATP binding. His-186 is necessary for the nucleotide-dependent interaction of Chp-1 not only with Hsp90 but also with Sgt1. In addition, Ca(2+), which is known to bind to melusin, enhances the interactions of melusin with Hsp90 and Sgt1. Furthermore, melusin acquires the ADP preference for Hsp90 binding in the presence of Ca(2+). Our newly discovered nucleotide-dependent interactions between cochaperones might provide additional complexity to the dynamics of the Hsp90 chaperone system, also suggesting potential Hsp90-independent roles for these cochaperones.

High-mobility group box 2 (HMGB2) modulates radioresponse and is downregulated by p53 in colorectal cancer cell.

Overexpression of high-mobility group box 2 (HMGB2) is recently reported in several malignant cancers and was correlated with poor response to preoperative chemoradiotherapy of colorectal cancer patients. To enhance the chemoradiotherapy efficacy, the biological function of HMGB2 was investigated with respect to radiation response. HMGB2 gene knockdown cells were constructed by infecting shRNA expressing lentivirus and clonogenic assay was performed to count the radiosensitivity. HMGB2 knockdown sensitized HCT-116 and HT-29 colorectal cancer cells to ionizing radiation. This could be due to an increased DNA damage and an inefficient DNA damage repair in HMGB2 knockdown cells. In addition, an exposure to radiation downregulated HMGB2 expression in colorectal cancer cells with an intact TP53 gene. HMGB2 gene expression of TP53-mutant cell was not affected by irradiation. p53-mediated downregulation of HMGB2 was confirmed by direct activation of p53 using Nutlin-3 or by inducing p53 expression using Tet-On system. Luciferase reporter assay showed that HMGB2 promoter activity was inversely correlated with the amount p53 cotransfected. Our study revealed that HMGB2 is necessary to protect colorectal cancer cells from DNA damage and efficient DNA repair and p53-mediated downregulation is a critical mechanism of modulating HMGB2 expression.

Insulin regulates aging and oxidative stress in Anopheles stephensi.

Observations from nematodes to mammals indicate that insulin/insulin-like growth factor signaling (IIS) regulates lifespan. As in other organisms, IIS is conserved in mosquitoes and signaling occurs in multiple tissues. During bloodfeeding, mosquitoes ingest human insulin. This simple observation suggested that exogenous insulin could mimic the endogenous hormonal control of aging in mosquitoes, providing a new model to examine this phenomenon at the organismal and cellular levels. To this end, female Anopheles stephensi mosquitoes were maintained on diets containing human insulin provided daily in sucrose or three times weekly by artificial bloodmeal. Regardless of delivery route, mosquitoes provided with insulin at 1.7 x 10(-4) and 1.7 x 10(-3) micromol l(-1), doses 0.3-fold and 3.0-fold higher than non-fasting blood levels, died at a faster rate than controls. In mammals, IIS induces the synthesis of reactive oxygen species and downregulates antioxidants, events that increase oxidative stress and that have been associated with reduced lifespan. Insulin treatment of mosquito cells in vitro induced hydrogen peroxide synthesis while dietary supplementation reduced total superoxide dismutase (SOD) activity and manganese SOD activity relative to controls. The effects of insulin on mortality were reversed when diets were supplemented with manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable SOD mimetic agent, suggesting that insulin-induced mortality was due to oxidative stress. In addition, dietary insulin activated Akt/protein kinase B and extracellular signal-regulated kinase (ERK) in the mosquito midgut, suggesting that, as observed in Caenorhabditis elegans, the midgut may act as a 'signaling center' for mosquito aging.

The structure-activity relationship of the series of non-peptide small antagonists for p56lck SH2 domain.

The antagonists for the SH2 domain are regarded as novel therapeutic candidates for cancer, autoimmune disease, and chronic inflammatory disease. Previously, we identified rosmarinic acid (alpha-o-caffeoyl-3,4-dihydroxyphenyl-lactic acid; RosA) from Prunella vulgaris as an antagonist for the p56lck SH2 domain by screening natural products. RosA not containing phosphotyrosine surrogate had a considerable inhibitory activity for T-cell antigen receptor (TCR)-induced interleukin (IL)-2 expression, and subsequent T-cell proliferation in vitro cell assay. To investigate the structure-activity relationship of RosA and to identify a novel p56lck SH2 antagonist with more potent in vitro T-cell inhibitory activity, we synthesized several analogs of RosA by using rational design. All synthesized compounds were tested in vitro binding activity for the SH2 domain and in vitro T-cell inhibitory activity. All four hydroxyl groups of RosA were essential for binding with the p56lck SH2 domain and T-cell inhibitory activity. Unexpectedly, conformationally less constrained analogs 4 and 9 showed a more potent binding affinity for the SH2 domain than that of RosA, and chirality of the analog did not play an important role in protein binding. We successfully identified several RosA analogs with a more potent T-cell inhibitory activity than that of RosA. Overall results revealed important structural requirements of the p56lck SH2 antagonists for in vitro T-cell inhibitory activity and in vitro protein binding activity.

Heterogeneity of capillary endothelial cells for basic fibroblast growth factor-induced paracrine signaling.

In this study, the authors isolated morphologically different capillary endothelial cells, designated as BCE-1 and BCE-2 cells, from bovine adrenal cortex. By a series of experiments involving proliferation, migration, and tubular-like structure formation assays, the authors found that the two BCE clones showed a clearly different response to basic fibroblast growth factor (bFGF). Similar to these results, the ERK-1/2 in the BCE-1 cells was phosphorylated by bFGF or vascular endothelial growth factor (VEGF), whereas that of the BCE-2 cells was phosphorylated only by VEGF. However, when the BCE-2 cells were transfected with FGF receptor 1 cDNA, the ERK-1/2 of these cells was phosphorylated by exogenous bFGF. Receptor binding experiments revealed that BCE-2 cells expressed high-affinity tyrosine-kinase FGF receptors approximately twofold less than BCE-1 cells. Transfection and receptor binding studies suggest a possibility that the poor response of the BCE-2 cells to exogenous bFGF is derived from the limitation of functional availability of high affinity FGF receptors. On the other hand, when both BCE clones were treated with anti-bFGF antibodies, basal formation of tubular-like structure in both clones were strongly inhibited, indicating that endogenous bFGF plays a role in in vitro angiogenesis of both BCE clones. Taken together, these data show that the isolated capillary endothelial cells are heterogeneous for paracrine but not autocrine bFGF signaling, and suggest that the diversity of capillary endothelial cells can occur by angiogenic factors, such as bFGF.

Synergistic immunosuppressive effects of rosmarinic acid and rapamycin in vitro and in vivo.

BACKGROUND: We previously demonstrated that rosmarinic acid (RosA) inhibits T-cell antigen receptor-induced T-cell activation and proliferation. In this study, we evaluated the ability of RosA alone and in conjunction with currently used immunosuppressive drugs to inhibit in vitro splenic T-cell proliferation and prolong skin graft survival in vivo. METHODS: Mouse splenic T-cell proliferation assays were performed in the presence of RosA alone or in combination with cyclosporine, rapamycin (Rapa), or prednisone (Pred). The in vivo synergistic efficacy of RosA and Rapa was evaluated in the mouse skin allograft model. RESULTS: RosA combined with Rapa or prednisone synergistically inhibited splenic T-cell proliferation, whereas the combination of RosA and cyclosporine additively inhibited T-cell proliferation. The combination of RosA and Rapa synergistically prolonged allograft survival. CONCLUSIONS: We conclude that the combination of RosA and Rapa promotes immunosuppressive effects.

Rosmarinic acid inhibits TCR-induced T cell activation and proliferation in an Lck-dependent manner.

Lck is a T cell-restricted Src family protein tyrosine kinase that plays pivotal roles in TCR-mediated signaling. We aimed to identify novel agents that could disrupt the molecular interaction of the Src homology 2-domain of Lck (Lck SH2) with its binding partners, with the expectation that this would impair TCR signaling and generate immunosuppression. Large-scale screening of plant extracts indicated that rosmarinic acid (RosA) in extracts of Prunella vulgaris consistently inhibits the interaction between Lck SH2 and a peptide containing its consensus binding sequence (pYEEI). The inhibitory effect of RosA was specific for SH2 domains of Src family protein tyrosine kinase. RosA inhibited TCR-induced-Ca(2+) mobilization and IL-2 promoter activation but not phorbol 12-myristate 13-acetate/ionomycin-induced IL-2 promoter activation, indicating its point of inhibition at the membrane proximal site of TCR signaling. Furthermore, RosA inhibited TCR-induced splenocyte proliferation as well as one-way MLR at an IC(50) of 25-50 microM and inhibited cytokine expression such as IL-2 and IFN-gamma. Here, we first report RosA as an inhibitor of TCR-signaling and subsequent T cell proliferation.