Quercetin Shows the Pharmacological Activity to Simultaneously Downregulate the Inflammatory and Fibrotic Responses to Tissue Injury in Association with its Ability to Target Multi-Kinases.

AIM: Previous studies have suggested that quercetin is effective for treating diverse chronic disorders including organ fibrosis and airway and cardiovascular disorders. To access the pharmacological background for its broad efficacy, we examined the ability of quercetin to modulate the inflammatory and fibrotic responses associated with organ injury that commonly underlie the pathogenesis of those disorders. METHODS: A cutaneous wound model on rabbit ear was used for in vivo study. Quercetin was topically applied to the wounds, and the number of macrophages and myofibroblasts and the size of the hypertrophic scar formed were estimated. An in vitro study examined the ability of quercetin to inhibit cell-signaling pathways that activate RAW264.7 macrophages and primary dermal fibroblasts and the tyrosine kinase activity of discoidin domain receptor 2. RESULTS: Quercetin reduced the population of macrophages and myofibroblasts and the scar formation in cutaneous wound healing. Quercetin suppressed the signaling pathways activating RAW264.7 macrophages and dermal fibroblasts, which is associated with its inhibition of multiple tyrosine kinases to regulate the pathways. This pharmacological activity of quercetin to simultaneously inhibit the inflammatory and fibrotic responses upon tissue damage by targeting multi-kinases could be the action mechanism to support its broad efficacy for various chronic disorders.

Anti-angiogenic activity of thienopyridine derivative LCB03-0110 by targeting VEGFR-2 and JAK/STAT3 Signalling.

Vascular endothelial growth factor receptor-2 (VEGFR-2) and Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signalling are important for tumor angiogenesis and metastasis. In this study, we identified (3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2-b]pyridin-7-ylamino)phenol (LCB03-0110) as a potent angiogenesis inhibitor. LCB03-0110 inhibited VEGFR-2 and JAK/STAT3 signalling in primary cultured human endothelial cells and cancer cells. An in vitro kinase assay and molecular modelling revealed that LCB03-0110 inhibited VEGFR-2, c-SRC and TIE-2 kinase activity via preferential binding at the ATP-binding site of their kinases. LCB03-0110 successfully occupied the hydrophobic pocket of VEGFR-2, c-SRC and TIE-2. LCB03-0110 also inhibited hypoxia-induced HIF/STAT3 and EGF- or angiopoietin-induced signalling cascades. In addition, LCB03-0110 inhibited VEGF-induced proliferation, viability, migration and capillary-like tube formation. LCB03-0110 also suppressed the sprouting of endothelial cells in the rat aorta and the formation of new blood vessels in the mouse Matrigel plug assay, but also suppressed pulmonary metastasis and tumor xenograft in mice. Our results suggest that LCB03-0110 is a potential candidate small molecule for blocking angiogenesis mediated by aberrant activation of VEGFR-2 and JAK/STAT3 signalling.

Defective Ca(2+) binding in a conserved binding site causes incomplete N-glycan processing and endoplasmic reticulum trapping of discoidin domain receptors.

An X-ray crystallographic study has suggested that vertebrate discoidin domain receptors (DDRs) have a conserved Ca(2+) binding site. DDR1 and DDR2 transfected in HEK293 cells were expressed mainly as 120 and 130 kDa forms, respectively, as they are sufficiently N-glycosylated. However, both of them showed the molecular weight of 110 kDa predominantly in the cells cultured with Ca(2+)-depleted media. DDR2-carrying D234A mutation at the conserved Ca(2+)-binding site expressed the 110 kDa form dominantly even in normal culture condition. DDR2 becomes 100 kDa form in glucose-depleted culture condition and its molecular weight increases up to 130 kDa with re-feeding glucose. However, in the mutant DDR2, the increase came to a halt at 110 kDa. The 110 kDa form had premature N-glycosyl carbohydrates and located predominantly within the endoplasmic reticulum. These results suggest that DDRs require Ca(2+)-binding to complete their N-glycan processing and generate the form targeted to cell membrane.

Topical application of ALK5 inhibitor A-83-01 reduces burn wound contraction in rats by suppressing myofibroblast population.

Burn scar contracture that follows the healing of deep dermal burns causes severe deformation and functional impairment. However, its current therapeutic interventions are limited with unsatisfactory outcomes. When we treated deep second-degree burns in rat skin with activin-like kinase 5 (ALK5) inhibitor A-83-01, it reduced wound contraction and enhanced the area of re-epithelialization so that the overall time for wound closing was not altered. In addition, it reduced myofibroblast population in the dermis of burn scar with a diminished deposition of its biomarker proteins such as α-SMA and collagen. Treatment of rat dermal fibroblast with A-83-01 inhibited transforming growth factor-ß1 (TGF-ß1)-dependent induction of α-SMA and collagen type I. Taken together, these results suggest that topical application of ALK5 inhibitor A-83-01 could be effective in preventing the contraction of burn wound without delaying the wound closure by virtue of its inhibitory activity against the TGF-ß-induced increase of myofibroblast population.

Single nucleotide polymorphisms in fibroblast growth factor 23 gene, FGF23, are associated with prostate cancer risk.

OBJECTIVE: To determine whether sequence variants within the FGF23 gene are associated with the risk of developing prostate cancer in a Korean population. PATIENTS AND METHODS: Five common single nucleotide polymorphisms (SNPs) in the FGF23 gene were assessed in 272 patients with prostate cancer and 173 control subjects with benign prostatic hyperplasia. Single-locus analyses were conducted using conditional logistic regression. In addition, we performed a haplotype analysis for the five FGF23 SNPs tested. RESULTS: Three SNPs in the FGF23 gene (rs11063118, rs13312789 and rs7955866) were associated with an increased risk of prostate cancer in our study population. Odds ratios for homozygous variants vs wild-type variants ranged from 1.68 (95% confidence interval [CI]: 1.15-2.46) to 1.79 (95% CI: 1.16-2.75). CONCLUSION: This is the first study showing that genetic variations in FGF23 increase prostate cancer susceptibility.

Low stability and a conserved N-glycosylation site are associated with regulation of the discoidin domain receptor family by glucose via post-translational N-glycosylation.

Cell-surface expression of the discoidin domain receptor (DDR) tyrosine kinase family in high molecular mass form was controlled sensitively by the glucose concentration through a post-translational N-glycosylation process. Cycloheximide time-course experiments revealed that the high-molecular-mass forms of DDR1 and DDR2 were significantly less stable than control receptor tyrosine kinases. Site-directed mutational analysis of the consensus N-glycosylation sites of the DDRs revealed that mutations of asparagine 213 of DDR2 and asparagine 211 of DDR1, a conserved N-glycosylation site among vertebrate DDRs, inhibited the generation of the high-molecular-mass isoform. Taken together, these results suggest a mechanism to control the activity of the DDR family by regulating its cell-surface expression. Due to low stability, the steady-state population of functional DDR proteins in the cell surface depends sensitively on its maturation process via post-translational N-glycosylation, which is controlled by the glucose supply and the presence of a conserved N-glycosylation site.

Topical Administration of Manuka Oil Prevents UV-B Irradiation-Induced Cutaneous Photoaging in Mice.

Manuka tree is indigenous to New Zealand, and its essential oil has been used as a traditional medicine to treat wounds, fever, and pain. Although there is a growing interest in the use of manuka oil for antiaging skin care products, little is known about its bioactivity. Solar ultraviolet (UV) radiation is the primary environmental factor causing skin damage and consequently premature aging. Therefore, we evaluated manuka oil for its effects against photoaging in UV-B-irradiated hairless mice. Topical application of manuka oil suppressed the UV-B-induced increase in skin thickness and wrinkle grading in a dose-dependent manner. Application of 10% manuka oil reduced the average length, depth, and % area of wrinkles significantly, and this was correlated with inhibition of loss of collagen fiber content and epidermal hyperplasia. Furthermore, we observed that manuka oil could suppress UV-B-induced skin inflammation by inhibiting the production of inflammatory cytokines. Taken together, this study provides evidence that manuka oil indeed possesses antiphotoaging activity, and this is associated with its inhibitory activity against skin inflammation induced by UV irradiation.

LCB 03-0110, a novel pan-discoidin domain receptor/c-Src family tyrosine kinase inhibitor, suppresses scar formation by inhibiting fibroblast and macrophage activation.

Wound healing generally induces an inflammatory response associated with tissue fibrosis in which activated macrophage and myofibroblast cells are primarily involved. Although this is known to be the underlying mechanism for scarring and various fibrotic pathologies, no effective intervention is currently available. We identified (3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2-b]pyridin-7-ylamino)phenol (LCB 03-0110), a thienopyridine derivative, as a potent inhibitor of discoidin domain receptor family tyrosine kinases and discovered that this compound strongly inhibits several tyrosine kinases, including the c-Src family, spleen tyrosine kinase, Bruton's tyrosine kinase, and vascular endothelial growth factor receptor 2, which are important for immune cell signaling and inflammatory reactions. LCB 03-0110 suppressed the proliferation and migration of primary dermal fibroblasts induced by transforming growth factor ß1 and type I collagen, and this result correlated with the inhibition ability of the compound against enhanced expression of α-smooth muscle actin and activation of Akt1 and focal adhesion kinase. In J774A.1 macrophage cells activated by lipopolysaccharide LCB 03-0110 inhibited cell migration and nitric oxide, inducible nitric-oxide synthase, cyclooxygenase 2, and tumor necrosis factor-α synthesis. LCB 03-0110 applied topically to full excisional wounds on rabbit ears suppressed the accumulation of myofibroblast and macrophage cells in the healing wound and reduced hypertrophic scar formation after wound closing, without delaying the wound closing process. Taken together, the pharmacological activities of LCB 03-0110 suggest that it could be an effective agent for suppressing fibroinflammation by simultaneously targeting activated fibroblasts and macrophages.

Actinomycin D identified as an inhibitor of discoidin domain receptor 2 interaction with collagen through an insect cell based screening of a drug compound library.

Discoidin domain receptors belong to the cell surface receptor tyrosine kinase family and recognize collagens for their activating ligands. They have been implicated for cell growth and migration and their elevated expressions were observed in various human cancers. When we expressed human Discoidin domain receptor 2 (DDR2) in insect cells, the protein was targeted properly into the cell membrane and this could enforce the cells to adhere on culture plate coated with type I collagen. By taking advantage of this, we established a novel insect cell based screening protocol to identify chemicals which inhibit the interaction between DDR2 and collagen. We screened a drug-compound library to select an anti-cancer drug, actinomycin D, as the inhibitory compound. Actinomycin D prevented the activation of DDR2 by type I collagen in human embryonic kidney 293 cells with an IC(50) value of 9 microM, while it did not interfere with the activation of other receptor tyrosine kinases by their ligands. In conclusion we identified a new biological function of actinomycin D and the insect cell based method provides a useful protocol for screening inhibitors against the association of DDR2 with collagen.

Natural compounds from Danshen suppress the activity of hepatic stellate cells.

Danshen is an herbal medication frequently used in oriental medicine to treat liver or kidney malfunction. In the course of our studies, we observed that compounds purified from Danshen exhibit an inhibitory activity against Discoidin Domain Receptor 2 (DDR2) tyrosine kinase. Through this inhibition, these compounds also inhibited the growth of HSC T6 cells and suppressed the expression of alpha-smooth muscle actin and MMP2, as well as collagen synthesis, all of which are increased in activated liver stellate cells. Given that activation of liver stellate cells is the hallmark of liver fibrosis and that DDR2 plays a critical role in this activation, these results suggest that one of the pharmacological activities of Danshen extract that protects the liver is the inhibition of key cell-signaling kinases, such as DDR2, in liver stellate cells.

Adiponectin induces growth arrest and apoptosis of MDA-MB-231 breast cancer cell.

Recently, it was reported that reduction in serum adiponectin levels is correlated with the incidence of breast cancer. As an effort to explain this, we screened various human breast cancer cell lines to identify those in which proliferation is directly controlled by adiponectin. Among the five tested cell lines, proliferation of MDA-MB-231 cancer cell was significantly suppressed by adiponectin within the range of physiological concentration. Furthermore, prolonged adiponectin treatment caused cell growth arrest and even apoptosis of MDA-MB-231. This result is the first to show that adiponectin can directly control cancer cell growth and provides a rationale for the theory that reduction in plasma adiponectin levels could be a risk factor for breast cancer.

Synthesis and biological evaluation of bis(methoxy methyl)-7,8-dihydro-[1,4]dioxino[2,3-g]quinazolines as EGFR tyrosine kinase inhibitors.

A series of 7,8-bis(methoxymethyl)-7,8-dihydro-[1,4]dioxino[2,3-g]quinazolines were prepared and evaluated for their inhibition of phosphorylation of the isolated epidermal growth factor receptor (EGFR) enzyme and for their growth inhibition of the A431 cell line. Among them, compound 3c having a 3-iodophenyl ring was most potent (IC(50) = 1.66 nM) against the isolated EGFR enzyme and also showed meaningful potency (GI(50) = 1.99 microM) against the A431 cell line, although less than PD153035 (GI(50) = 1.03 microM). However, compound 3e as the exact rigidified analogue of Erlotinib (Tarceva) was inferior to the original compound when compared to its reported data.

Tyrosine 740 phosphorylation of discoidin domain receptor 2 by Src stimulates intramolecular autophosphorylation and Shc signaling complex formation.

DDR2 is a receptor tyrosine kinase whose activating ligands are various collagens. DDR2-mediated cellular signaling has been shown to require Src activity. However, the precise mechanism underlying the Src dependence of DDR2 signaling is unknown. Here, using baculoviral co-expression of the DDR2 cytosolic domain and Src, we show that Src targets three tyrosine residues (Tyr-736, Tyr-740, and Tyr-741) in the activation loop of DDR2 for phosphorylation. This phosphorylation by Src stimulates DDR2 cis-autophosphorylation of additional tyrosine residues. In vitro Shc binding assays demonstrate that phosphotyrosines resulting from DDR2 autophosphorylation are involved in Shc binding to the DDR2 cytosolic domain. Mutating tyrosine 740 of DDR2 to phenylalanine stimulates autophosphorylation of DDR2 to an extent similar to that resulting from Src phosphorylation of DDR2. In addition, the DDR2 Y740F mutant protein displays collagen-independent, constitutively activated signaling. These findings suggest that tyrosine 740 inhibits DDR2 autophosphorylation. Collectively, our findings are consistent with the following mechanism for Src-dependent DDR2 activation and signaling: 1) ligand binding promotes phosphorylation of Tyr-740 in the DDR2 activation loop by Src; 2) Tyr-740 phosphorylation stimulates intramolecular autophosphorylation of DDR2; 3) DDR2 autophosphorylation generates cytosolic domain phosphotyrosines that promote the formation of DDR2 cytosolic domain-Shc signaling complexes.

N'-(phenyl-pyridin-2-yl-methylene)-hydrazine carbodithioic acid methyl ester enhances radiation-induced cell death by targeting Bcl-2 against human lung carcinoma cells.

To develop a new radiosensitizer, we screened a chemical library and selected one chemical reagent, N'-(phenyl-pyridin-2-yl-methylene)-hydrazine carbodithioic acid methyl ester (PHCM), which was already known to have antifungal and antimicrobial properties. PHCM enhanced radiation-induced cell death and its mean calculated dose enhancement ratio was 1.17. PHCM was found to induce the phosphorylation of p38 mitogen-activated protein kinase, and combined treatment with PHCM and radiation down-regulated Bcl-2. In a xenograft assay, the combined PHCM and radiation group showed 39.3 days of growth delay versus the control in terms of tumor growth. The enhancement factor of this combined treatment was determined to be 4.02.

Biochemical characterizations reveal different properties between CDK4/cyclin D1 and CDK2/cyclin A.

CDK2 and CDK4 known promoter of cell cycling catalyze phosphorylation of RB protein. Enzyme specificity between two CDKs that work at a different cell cycle phase is not clearly understood. In order to define kinase properties of CDK2 and CDK4 in complex with cycline A or cycline D1 in relation to their respective role in cell cycling regulation, we examined enzymatic properties of both CDK4/cycline D1 and CDK2/cycline A in vitro. Association constant, Km for ATP in CDK4/cyclin D1 was found as 418 microM, a value unusually high whereas CDK2/cyclin A was 23 microM, a value close to most of other regulatory protein kinases. Turnover value for both CDK4/cyclin D1 and CDK2/cyclin A were estimated as 3.4 and 3.9 min(-1) respectively. Kinetic efficiency estimation indicates far over one order magnitude less efficiency for CDK4/cyclin D1 than the value of CDK2/cycline A (9.3 pM(-1) min(-1) and 170 pM(-1) min(-1) respectively). In addition, inhibition of cellular CDK4 caused increase of cellular levels of ATP, even though inhibition of CDK2 did not change it noticeably. These data suggest cellular CDK4/cyclin D1 activity is tightly associated with cellular ATP concentration. Also, analysis of phosphorylated serine/threonine sites on RB catalyzed by CDK4/cyclin D1 and CDK2/cyclin A showed significant differences in their preference of phosphorylation sites in RB C-terminal domain. Since RB is known to regulate various cellular proteins by binding and this binding is controlled by its phosphorylation, these data shown here clearly indicate significant difference in their biochemical properties between CDK4/cyclin D1 and CDK2/cyclin A affecting regulation of cellular RB function.

Synthesis and biological evaluations of pyrazolo[3,4-d]pyrimidines as cyclin-dependent kinase 2 inhibitors.

A series of 1,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines 15-19, 30-38 capable of selectively inhibiting CDK2 activity were synthesized by derivatization at C-4, C-6 and N-1 with various amines and lower alkyl groups. For above synthetic compounds, biological evaluation was carried out and structure-activity relationship was examined. In our series, 4-anilino compounds exhibited better CDK2 inhibitory activity and antitumor activity compared to 4-benzyl compounds. The compounds 33a,b having a 3-fluoroaniline group at C-4 showed comparable or superior CDK2 inhibitory activity to those of olomoucine and roscovitine as reference compounds. In general, the unsubstituted compounds (30a,b, 33a,b, 36a,b) at N-1 possessed higher potency than the substituted compounds (32a,b, 34a,b) for the CDK2 inhibitory activity. As for EGFR inhibitory activity, most compounds didnot have a significant activity. The compounds 32a,b exhibited potent cell growth inhibitory activity against human cancer cell lines, but their CDK2 inhibitory activities were slightly poorer than olomoucine.

7-Substituted-[1,4]dioxano[2,3-g]quinazolines as inhibitors of epidermal growth factor receptor kinase.

With the aim of developing inhibitors of EGFR tyrosine kinase, the 7-methoxymethyl-[1,4]dioxano[2,3-g]quinazolines (3a-b) and 7-mono- or di-alkylaminomethyl-[1,4]dioxano[2,3-g]quinazolines (4a-i) were prepared and evaluated for the inhibition of EGFR tyrosine kinase and the growth inhibition of human tumor cell lines. Among them, compounds 4d and 4h showed potencies against both EGFR tyrosine and the A431 cell line similar to that of PD153035 with greater aqueous solubilities of their HCl salts.