Novel Analytical Platform For Robust Identification of Cell Migration Inhibitors.

Wound healing assay is a simple and cost-effective in vitro assay for assessing therapeutic impacts on cell migration. Its key limitation is the possible confoundment by other cellular phenotypes, causing misinterpretation of the experimental outcome. In this study, we attempted to address this problem by developing a simple analytical approach for scoring therapeutic influences on both cell migration and cell death, while normalizing the influence of cell growth using Mitomycin C pre-treatment. By carefully mapping the relationship between cell death and wound closure rate, contribution of cell death and cell migration on the observed wound closure delay can be quantitatively separated at all drug dosing. We showed that both intrinsic cell motility difference and extrinsic factors such as cell seeding density can significantly affect final interpretation of therapeutic impacts on cellular phenotypes. Such discrepancy can be rectified by using the actual wound closure time of each treatment condition for the calculation of phenotypic scores. Finally, we demonstrated a screen for strong pharmaceutical inhibitors of cell migration in cholangiocarcinoma cell lines. Our approach enables accurate scoring of both migrastatic and cytotoxic effects, and can be easily implemented for high-throughput drug screening.

Proteomic and bioinformatic analyses of possible target-related proteins of gambogic acid in human breast carcinoma MDA-MB-231 cells.

Gambogic acid (GA) is an anticancer agent in phase ‖b clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early (3 h) and late stage (24 h) of treatment were searched using a proteomic technology, two-dimensional electrophoresis (2-DE). The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related proteins such as cytoskeleton-related proteins.

Critical role for reactive oxygen species in apoptosis induction and cell migration inhibition by diallyl trisulfide, a cancer chemopreventive component of garlic.

Diallyl trisulfide (DATS) is a structurally simple but biologically active constituent of processed garlic with in vivo activity against chemically induced as well as oncogene-driven cancer in experimental rodents. This study offers novel insights into the mechanisms underlying anticancer effects of DATS using human breast cancer cells as a model. Exposure of human breast cancer cells (MCF-7 and MDA-MB-231) and a cell line derived from spontaneously developing mammary tumor of a transgenic mouse (BRI-JM04) to DATS resulted in a dose-dependent inhibition of cell viability that was accompanied by apoptosis induction. A non-tumorigenic normal human mammary cell line (MCF-10A) was resistant to growth inhibition and apoptosis induction by DATS. The DATS-induced apoptosis in MDA-MB-231, MCF-7, and BRI-JM04 cells was associated with reactive oxygen species (ROS) production as evidenced by fluorescence microscopy and flow cytometry using a chemical probe (MitoSOX Red). Overexpression of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) as well as Mn-SOD conferred significant protection against DATS-induced ROS production and apoptotic cell death in MDA-MB-231 and MCF-7 cells. Activation of Bak, but not Bax, resulting from DATS treatment was markedly suppressed by overexpression of Mn-SOD. The DATS treatment caused ROS generation, but not activation of Bax or Bak, in MCF-10A cells. Furthermore, the DATS-mediated inhibition of cell migration was partially but significantly attenuated by Cu,Zn-SOD and Mn-SOD overexpression in association with changes in levels of proteins involved in epithelial-mesenchymal transition. The DATS-mediated induction of heme oxygenase-1 was partially attenuated by overexpression of Mn-SOD. These results provide novel mechanistic insights indicating a critical role for ROS in anticancer effects of DATS.

1,25(OH)2 vitamin D suppresses macrophage migration and reverses atherogenic cholesterol metabolism in type 2 diabetic patients.

Reduced monocyte infiltration into the vessel wall and increased macrophage cholesterol efflux are critical components in atherosclerotic plaque regression. During inflammation, monocyte chemotactic protein 1 (MCP-1) signaling activation and cholesterol deposition in macrophages induce endoplasmic reticulum (ER) stress, which promotes an increased inflammatory response. Increased macrophage ER stress shifts macrophages into an M2 macrophage phenotype with increased cholesterol uptake and deposition. In type 2 diabetes, a population with elevated baseline risk of cardiovascular disease (CVD), vitamin D deficiency doubles that risk. We have found that 1,25-dihydroxy vitamin D [1,25(OH)2D] prevents foam cell formation during macrophage differentiation by suppressing ER stress. However, it is unknown whether suppression of ER stress by 1,25(OH)2D decreases monocyte infiltration and reverses atherogenic cholesterol metabolism in previously differentiated, vitamin D-deplete macrophages. We collected peripheral monocytes from type 2 diabetic patients and differentiated them into macrophages under vitamin D-deplete or 1,25(OH)2D-supplemented conditions. 1,25(OH)2D supplementation suppressed macrophage migration in response to MCP-1 and mRNA expression of chemokine (C-C motif) receptor 2 (CCR2), the MCP-1 receptor, compared to vitamin D-deplete cells. Furthermore, inhibition of ER stress with phenyl butyric acid resulted in similar effects even in vitamin D-deplete cells, while induction of ER stress with Thapsigargin under 1,25(OH)2D-supplemented conditions increased macrophage migration and CCR2 expression, suggesting that the effects of vitamin D on migration are mediated through ER stress suppression. To determine whether the detrimental pattern of macrophage cholesterol metabolism in vitamin D depletion is reversible, we assessed cholesterol uptake in macrophages differentiated under vitamin D-deplete conditions as described above, then supplemented with 1,25(OH)2D or maintained in vitamin D-deplete conditions. Cholesterol uptake was decreased in 1,25(OH)2D-supplemented compared to vitamin D-deplete cells, suggesting slowed cholesterol deposition with active vitamin D. 1,25(OH)2D supplementation also suppressed cholesteryl ester formation and enhanced cholesterol efflux in M2 macrophages compared to vitamin D-deplete cells, suggesting facilitation of cholesterol egress in the presence of 1,25(OH)2D. We thus provide further evidence that active vitamin D is an ER stress reliever that may have a role in atherosclerotic plaque regression. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Stereochemical assignment and anti-inflammatory properties of the omega-3 lipid mediator resolvin E3.

Uncontrolled inflammation is now considered to be a link between many widely occurring diseases. Thus, controlling the innate inflammatory response and its local chemical mediators has been receiving increasing attention. We recently identified a novel family of eicosapentaenoic acid (EPA)-derived mediators produced by eosinophils, denoted as resolvin E3 (RvE3), that possess potent anti-inflammatory actions both in vitro and in vivo. Carbons at 17 and 18 positions are asymmetric and thus the molecule has a total of four potential stereoisomers. Here, we assigned the stereochemistry of the conjugated double bonds and chirality of alcohols present in two natural isomers of RvE3 with four different stereoisomers prepared by total organic synthesis. The complete structures of two natural isomers of RvE3 were determined to be 17R,18S- and 17R,18R-dihydroxy-5Z,8Z,11Z,13E,15E-EPA, respectively. These natural isomers prepared by total organic synthesis displayed a potent anti-inflammatory action by limiting neutrophil infiltrations both in vitro and in vivo. The unnatural stereoisomers were much less active compared with the natural isomers, demonstrating the stereoselective action of RvE3.

Mechanisms of osteoclastogenesis inhibition by a novel class of biphenyl-type cannabinoid CB(2) receptor inverse agonists.

The cannabinoid CB(2) receptor is known to modulate osteoclast function by poorly understood mechanisms. Here, we report that the natural biphenyl neolignan 4'-O-methylhonokiol (MH) is a CB(2) receptor-selective antiosteoclastogenic lead structure (K(i) < 50 nM). Intriguingly, MH triggers a simultaneous G(i) inverse agonist response and a strong CB(2) receptor-dependent increase in intracellular calcium. The most active inverse agonists from a library of MH derivatives inhibited osteoclastogenesis in RANK ligand-stimulated RAW264.7 cells and primary human macrophages. Moreover, these ligands potently inhibited the osteoclastogenic action of endocannabinoids. Our data show that CB(2) receptor-mediated cAMP formation, but not intracellular calcium, is crucially involved in the regulation of osteoclastogenesis, primarily by inhibiting macrophage chemotaxis and TNF-α expression. MH is an easily accessible CB(2) receptor-selective scaffold that exhibits a novel type of functional heterogeneity.

Appraisal of the antichemotactic activity of flavonoids on polymorphonuclear neutrophils.

Flavonoids are polyphenols that are ubiquitous in plants and frequently consumed in the diet. They are suggested to have many beneficial actions on human health, including anti-inflammatory activity. Their properties have been studied in a number of cell types, but little is known about their effects on neutrophil biology. Consequently, we selected 25 flavonoids with different structural features to evaluate their in vitro inhibition of rat polymorphonuclear neutrophil (PMN) chemotaxis, employing a modified Boyden chamber. Migratory activity was measured towards a chemotactic stimulant, formyl-Met-Leu-Phe or lipopolysaccharide. Furthermore, the cytotoxic effect of flavonoids on PMNs was determined by the release of cytosolic lactate dehydrogenase (LDH). Ten flavonoids significantly retarded the migration of PMNs with at least one of the concentrations tested in a range between 0.625 and 100 µM; the best antichemotactic agents were flavone, flavonol, quercetin and rutin. None of the flavanones evaluated presented any significant inhibition of migration in this assay. Our findings indicated that non-hydroxylated flavones possess a better antichemotactic activity when compared to flavones with hydroxy groups. The presence of a sugar moiety in rutin did not produce any increase in this effect, when compared to the respective aglycone analogue. Finally, none of the flavonoids exhibited cell toxicity and for many of these flavonoids this is the first report of the inhibition of PMN chemotaxis.

Methyl gallate exhibits potent antitumor activities by inhibiting tumor infiltration of CD4+CD25+ regulatory T cells.

CD4(+)CD25(+) regulatory T (Treg) cells play crucial roles in the host response to tumors. Increasing evidence supports the existence of elevated numbers of Treg cells in solid tumors and hematologic malignancies. In this study, the effects of methyl gallate on Treg cells were examined. Methyl gallate inhibited Treg cell-suppressive effects on effector CD4(+) T cells and Treg migration toward tumor environment. The expression of Treg surface markers including CTLA-4, CCR4, CXCR4, and glucocorticoid-induced TNFR was significantly suppressed upon methyl gallate treatment. Furthermore, forkhead box P3 (Foxp3) expression was also significantly decreased by methyl gallate, suggesting that the suppressive effects of methyl gallate on Treg were medicated by decrease of Treg-specific transcription factor Foxp3. In tumor-bearing hosts, methyl gallate treatment substantially reduced tumor growth and prolonged the survival rate. In contrast, nu/nu mice did not show decreased tumor progression in response to methyl gallate. In addition, in tumor-bearing Treg-depleted mice, tumor growth and the survival rates were not changed by methyl gallate treatment, strongly suggesting that the main therapeutic target of methyl gallate in tumor suppression was related to modulation of the CD4(+)CD25(+) Treg cell functions. In the spleen of tumor-bearing mice, methyl gallate treatment induced a significant decrease in the CD4(+)CD25(+)Foxp3(high) Treg cell population. Especially, the number of tumor-infiltrating CD25(+)Foxp3(high) Treg cells was significantly lower in methyl gallate-treated mice. These results suggest that methyl gallate can be used to reverse immune suppression and as a potentially useful adjunct for enhancing the efficacy of immune-based cancer therapy.

The mechanisms of action of Tianhua(™) on antitumor activity in lung cancer cells.

CONTEXT: Tianhua (TH-R) is extracted from Trichosanthes kirilowii Maxim (Cucurbitaceae) containing trichosanthin, a traditional Chinese medicine, which has been locally reported to have good anticancer effects in vivo in both animal and human models. However, there have been several reports that trichosanthin has an anticancer effect involving apoptosis. OBJECTIVE: To investigate other anticancer effects of TH-R, various tumorigenesis parameters were verified. MATERIALS AND METHODS: Telomerase activity, anti-apoptosis, anti-migration and immunomodulatory activity were estimated by telomeric repeat amplification protocol assay (TRAP), flow cytometry, Boyden chamber assay and ELISA assay, respectively. RESULTS: In our studies, we are the first to find that TH-R had a cytotoxic effect on lung cancer cells in MTS assays; it could change the cell cycle distribution of human lung cancer cells (A549 cell line) and induce apoptosis. Further anti-telomerase effects in human lung adenocarcinoma A549 cells using the TRAP assay were noted. TH-R also had an aggregation effect on peripheral blood lymphocytes, but no effect on stimulating peripheral lymphocytes to produce human interferon-γ(IFN-γ). TH-R could inhibit the migration, or metastatic ability, of A549 cells by Boyden chamber assay. In the oral feeding therapy of an in vivo mouse model, there was an initial inhibition of A549 cancer cell growth, but no statistical difference after one month of therapy. DISCUSSION AND CONCLUSION: It has been proven that medicinal herbs such as Tianhua have positive effects against cancer through preventing or inhibiting the process of lung tumorigenesis.

Small-molecule inhibitors of vascular adhesion protein-1 reduce the accumulation of myeloid cells into tumors and attenuate tumor growth in mice.

Vascular adhesion protein-1 (VAP-1) is an endothelial, cell surface-expressed oxidase involved in leukocyte traffic. The adhesive function of VAP-1 can be blocked by anti-VAP-1 Abs and small-molecule inhibitors. However, the effects of VAP-1 blockade on antitumor immunity and tumor progression are unknown. In this paper, we used anti-VAP-1 mAbs and small-molecule inhibitors of VAP-1 in B16 melanoma and EL-4 lymphoma tumor models in C57BL/6 mice. Leukocyte accumulation into tumors and neoangiogenesis were evaluated by immunohistochemistry, flow cytometry, and intravital videomicroscopy. We found that both anti-VAP-1 Abs and VAP-1 inhibitors reduced the number of leukocytes in the tumors, but they targeted partially different leukocyte subpopulations. Anti-VAP-1 Abs selectively inhibited infiltration of CD8-positive lymphocytes into tumors and had no effect on accumulation of myeloid cells into tumors. In contrast, the VAP-1 inhibitors significantly reduced only the number of proangiogenic Gr-1(+)CD11b(+) myeloid cells in melanomas and lymphomas. Blocking of VAP-1 by either means left tumor homing of regulatory T cells and type 2 immune-suppressing monocytes/macrophages intact. Notably, VAP-1 inhibitors, but not anti-VAP-1 Abs, retarded the growth of melanomas and lymphomas and reduced tumor neoangiogenesis. The VAP-1 inhibitors also reduced the binding of Gr-1(+) myeloid cells to the tumor vasculature. We conclude that tumors use the catalytic activity of VAP-1 to recruit myeloid cells into tumors and to support tumor progression. Small-molecule VAP-1 inhibitors therefore might be a potential new tool for immunotherapy of tumors.

Chronic morphine administration delays wound healing by inhibiting immune cell recruitment to the wound site.

Patients prescribed morphine for the management of chronic pain, and chronic heroin abusers, often present with complications such as increased susceptibility to opportunistic infections and inadequate healing of wounds. We investigated the effect of morphine on wound-healing events in the presence of an infection in an in vivo murine model that mimics the clinical manifestations seen in opioid user and abuser populations. We show for the first time that in the presence of an inflammatory inducer, lipopolysaccharide, chronic morphine treatment results in a marked decrease in wound closure, compromised wound integrity, and increased bacterial sepsis. Morphine treatment resulted in a significant delay and reduction in both neutrophil and macrophage recruitment to the wound site. The delay and reduction in neutrophil reduction was attributed to altered early expression of keratinocyte derived cytokine and was independent of macrophage inflammatory protein 2 expression, whereas suppression of macrophage infiltration was attributed to suppressed levels of the potent macrophage chemoattractant monocyte chemotactic protein-1. When the effects of chronic morphine on later wound healing events were investigated, a significant suppression in angiogenesis and myofibroblast recruitment were observed in animals that received chronic morphine administration. Taken together, our findings indicate that morphine treatment results in a delay in the recruitment of cellular events following wounding, resulting in a lack of bacterial clearance and delayed wound closure.

Antinociceptive and anti-inflammatory kaempferol glycosides from Sedum dendroideum.

AIM OF THE STUDY: To identify the compounds responsible for the antinociceptive and anti-inflammatory effects previously described for Sedum dendroideum, through bioassay-guided fractionation procedures. MATERIALS AND METHODS: Antinociceptive activity was evaluated through mouse acetic acid-induced writhing model. The anti-inflammatory activity was assessed through croton oil-induced mouse ear oedema and carrageenan-induced peritonitis. RESULTS: The Sedum dendroideum juice afforded seven known flavonoids identified with basis on NMR data. The oral administration of the major kaempferol glycosides kaempferitrin [1] (17.29 micromol/kg), kaempferol 3-O-beta-glucopyranoside-7-O-alpha-rhamnopyranoside [2] (16.82 micromol/kg), kaempferol 3-O-neohesperidoside-7-O-alpha-rhamnopyranoside [3] (13.50 micromol/kg) or alpha-rhamnoisorobin [5] (23.13 micromol/kg) inhibited by 47.3%, 25.7%, 60.2% and 58.0%, respectively, the acetic acid-induced nociception (indomethacin: 27.95 micromol/kg, p.o.; 68.9%). Flavonoids 1, 2, 3 or 5, at the same doses, reduced by 39.5%, 46.5%, 35.6% and 33.3%, respectively, the croton oil-induced oedema (dexamethasone: 5.09 micromol/kg, s.c.; 83.7%) and impaired leukocyte migration by 42.9%, 46.3%, 50.4% and 49.6%, respectively (dexamethasone: 5.09 micromol/kg, s.c.; 66.1%). CONCLUSIONS: Our findings show that the major kaempferol glycosides may account for the renowned medicinal use of Sedum dendroideum against pain and inflammatory troubles.

Inhibitory effects of Schizandrae Fructus on eotaxin secretion in A549 human epithelial cells and eosinophil migration.

Eosinophilia have been implicated in a broad range of diseases, most notably allergic conditions (e.g. asthma, rhinitis and atopic dermatitis) and inflammatory diseases. These diseases are characterized by an accumulation of eosinophils in the affected tissue. Defining the mechanisms that control the recruitment of eosinophil is fundamental to understanding how these diseases progress and identifying a novel target for drug therapy. Accordingly, this study was conducted to evaluate the regulatory effect of Schizandrae Fructus (SF) on the expression of eotaxin, an eosinophil-specific chemokine released in respiratory epithelium following allergic stimulation, as well as its effects on eosinophil migration. To accomplish this, human epithelial lung cells (A549 cell) were stimulated with a combination of TNF-alpha (100ng/ml) and IL-4 (100ng/ml) for 24h. The cells were then restimulated with TNF-alpha (100ng/ml) and IL-1beta (10ng/ml) to induce the expression of chemokines and adhesion molecules involved in eosinophil chemotaxis for another 24h. Next, the samples were treated with various concentrations of Schizandrae Fructus (SF) (1, 10, 100, 1000microg/ml) or one of the major constituents of SF, schizandrin (0.1, 1, 10, 100microg/ml), after which following inhibition effect assay was performed triplicates in three independence. The levels of eotaxin in secreted proteins were suppressed significantly by SF (100 and 1000microg/ml, p<0.01) and schizandrin (10 and 100microg/ml, p<0.01). In addition, SF (1, 10, 100 and 1000microg/ml) decreased mRNA expression levels in A549 cells significantly (p<0.01). Eosinophil recruitment to lung epithelial cells was also reduced by SF, which indicates that eotaxin plays a role in eosinophil recruitment. Furthermore, treatment with SF suppressed the expression of another chemokine, IL-8 (0.1 and 1microg/ml SF, p<0.01), as well as intercellular adhesion molecule-1 (10 and 100microg/ml SF, p<0.01) and vascular cell adhesion molecule-1 (0.1 and 1microg/ml SF, p<0.05), which are all related to eosinophil migration. Taken together, these findings indicate that SF may be a desirable medicinal plant for the treatment of allergic diseases.

Deoxypodophyllotoxin, flavolignan, from Anthriscus sylvestris Hoffm. inhibits migration and MMP-9 via MAPK pathways in TNF-alpha-induced HASMC.

The matrix metalloproteinases (MMP-9 and MMP-2) in aortic smooth muscle cells (SMC) play key roles in the pathogenesis atherosclerosis. The SMC migration into the vascular wall via the bloodstream is directly linked with MMP-9 expression. Deoxypodophyllotoxin (DPT), a naturally occurring flavolignan with anti-inflammatory activity, was isolated from Anthriscus sylvestris Hoffm. and has been known inhibit the expression of MMP-9 in tumor necrosis factor-alpha (TNF-alpha) stimulated human aortic smooth muscle cells (HASMC). In this study, DPT was purified and demonstrated to inhibit the MMP-9/2 activities in TNF-alpha-induced HASMC. In addition, MMP-9 expression and migration was strongly inhibited by DPT in TNF-alpha-induced HASMC. To examine whether TNF-alpha-induced MMP-9 expressions are involved with migrations of HASMC, reverse transcription-polymerase chain reaction (RT-PCR) and luciferase-tagged promoter analysis were applied. These experiments revealed that DPT inhibited the mRNA transcription of MMP-9 gene expression. Furthermore, Western blot analysis indicated that the TNF-alpha-induced phosphorylation of extracellular signal regulated kinase 1 and 2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK) were strongly inhibited by DPT. From these results, it is concluded that DPT has an inhibitory activities on migration and MMP-2/9 activities, and MMP-9 transcription in HASMC.