NT5E and FcGBP as key regulators of TGF-1-induced epithelial–mesenchymal transition (EMT) are associated with tumor progression and survival of patients with gallbladder cancer

Epithelial–mesenchymal transitions (EMTs) are essential manifestations of epithelial cell plasticity during tumor progression. Transforming growth factor-β(TGF-β) modulates epithelial plasticity in tumor physiological contexts by inducing EMT, which is associated with the altered expression of genes. In the present study, we used DNA micro-array analysis to search for differentially expressed genes in the TGF-β1 induced gallbladder carcinoma cell line (GBC-SD cells), as compared with normal GBC-SD cells. We identified 225 differentially expressed genes, including 144 that were over-expressed and 81 that were under-expressed in the TGF-β1 induced GBC-SD cells. NT5E (CD73) is the most increased gene, while the Fc fragment of the IgG binding protein (FcGBP) is the most decreased gene. The expression patterns of these two genes in gallbladder adenocarcinoma and chronic cholecystitis tissue were consistent with the micro-array data. Immunochemistry and clinicopathological results showed that the expression of NT5E and FcGBP in gallbladder adenocarcinoma is an independent marker for evaluation of the disease progression, clinical biological behaviors and prognosis. The data from the current study indicate that differential NT5E and FcGBP expressions could be further evaluated as biomarkers for predicting survival of patients with gallbladder cancer and that NT5E and FcGBP could be promising targets in the control of gallbladder cancer progression.     

Retigeric acid B exhibits antitumor activity through suppression of nuclear factor-κB signaling in prostate cancer cells in vitro and in vivo

Previously, we reported that retigeric acid B (RB), a natural pentacyclic triterpenic acid isolated from lichen, inhibited cell growth and induced apoptosis in androgen-independent prostate cancer (PCa) cells. However, the mechanism of action of RB remains unclear. In this study, we found that using PC3 and DU145 cells as models, RB inhibited phosphorylation levels of IκBα and p65 subunit of NF-κB in a time- and dosage-dependent manner. Detailed study revealed that RB blocked the nuclear translocation of p65 and its DNA binding activity, which correlated with suppression of NF-κB-regulated proteins including Bcl-2, Bcl-x(L), cyclin D1 and survivin. NF-κB reporter assay suggested that RB was able to inhibit both constitutive activated-NF-κB and LPS (lipopolysaccharide)-induced activation of NF-κB. Overexpression of RelA/p65 rescued RB-induced cell death, while knockdown of RelA/p65 significantly promoted RB-mediated inhibitory effect on cell proliferation, suggesting the crucial involvement of NF-κB pathway in this event. We further analyzed antitumor activity of RB in in vivo study. In C57BL/6 mice carrying RM-1 homografts, RB inhibited tumor growth and triggered apoptosis mainly through suppressing NF-κB activity in tumor tissues. Additionally, DNA microarray data revealed global changes in the gene expression associated with cell proliferation, apoptosis, invasion and metastasis in response to RB treatment. Therefore, our findings suggested that RB exerted its anti-tumor effect by targeting the NF-κB pathway in PCa cells, and this could be a general mechanism for the anti-tumor effect of RB in other types of cancers as well.     

Transforming growth factor beta (TGF-β) and inflammation in cancer

The transforming growth factor beta (TGF-β) has been studied with regard to the regulation of cell behavior for over three decades. A large body of research has been devoted to the regulation of epithelial cell and derivative carcinoma cell populations in vitro and in vivo. TGF-β has been shown to inhibit epithelial cell cycle progression and promote apoptosis that together significantly contribute to the tumor suppressive role for TGF-β during carcinoma initiation and progression. TGF-β is also able to promote an epithelial to mesenchymal transition that has been associated with increased tumor cell motility, invasion and metastasis. However, it has now been shown that loss of carcinoma cell responsiveness to TGF-β stimulation can also promote metastasis. Interestingly, enhanced metastasis in the absence of a carcinoma cell response to TGF-β stimulation has been shown to involve increased chemokine production resulting in recruitment of pro-metastatic myeloid derived suppressor cell (MDSC) populations to the tumor microenvironment at the leading invasive edge. When present, MDSCs enhance angiogenesis, promote immune tolerance and provide matrix degrading enzymes that promote tumor progression and metastasis. Further, the recruitment of MDSC populations in this context likely enhances the classic role for TGF-β in immune suppression since the MDSCs are an abundant source of TGF-β production. Importantly, it is now clear that carcinoma-immune cell cross-talk initiated by TGF-β signaling within the carcinoma cell is a significant determinant worth consideration when designing therapeutic strategies to manage tumor progression and metastasis.     

In vitro and in vivo evaluation of the antifungal activity of fluoxetine combined with antifungals against Candida albicans biofilms and oral candidiasis

Abstract

Candida albicans biofilms are responsible for oral candidiasis. Fluoxetine is a widely used antidepressant, with certain anti-Candida activities. The antifungal activity of fluoxetine combined with various antifungals against C. albicans biofilms and oral candidiasis was evaluated in this study. The morphological change in the inhibition of fluoxetine on C. albicans biofilms was observed using SEM. The interactions between fluoxetine and antifungals against C. albicans biofilms were evaluated using microdilution checkerboard methods, FICI and the ΔE model. The synergistic combination was tested in vivo on the mice model of oral candidiasis. SEM imaging showed fluoxetine inhibited hyphal growth and biofilm formation. Fluoxetine combined with caspofungin exhibited synergistic effects against C. albicans biofilms. Antagonistic effects occurred when fluoxetine was combined with amphotericin B or terbinafine. Further, the fluoxetine combined with caspofungin significantly reduced the lesion score and CFU of C. albicans on the murine tongue (p?<?0.05), and relieved oral candidiasis of the infected mice.     

Migration speed and directionality switch of normal epithelial cells after TGF-β1-induced EMT (tEMT) on micro-structured polydimethylsiloxane (PDMS) substrates with variations in stiffness and topographic patterning

Abstract

The epithelial to mesenchymal transition (EMT) involves several physiological and pathological phenomena and endows cells with invasive and migratory properties. However, the effects of substrate stiffness and topography on the migration of cells before or after transforming growth factor-β1 (TGF-β1)-induced EMT (tEMT) are unknown.

Herein, we seed control or tEMT NMuMG cells on the 2D patterns consisted of 1 μm or 5 μm line-widths and groove or cone patterns on either 2 MPa (1.96 ± 0.48 MPa) or 4 MPa (3.70 ± 0.74 MPa) polydimethylsiloxane (PDMS) substrates.

After tEMT, the increased expression of α-SMA with vinculin in focal adhesion (FA) sites led to an acceleration of tEMT cell motility. On the 2 MPa substrate, the most influenced substrate was the 1 μm, cone-patterned substrate, where the tEMT cells’ motility decelerated by 0.13 μm/min (36% slower than the cells on groove pattern). However, on the 5 μm, groove-patterned substrate, where the tEMT cells demonstrated the most rapid motility relative to the control cells, with an increment of 0.18 μm/min (100%).

Among the different physical cues from substrate, the cone pattern could impede the migration speed of tEMT cells. Furthermore, we recommend the groove-patterned with a 5 μm line-width substrate as a useful tool to differentiate control and tEMT cells by migration speed.     

Evaluation of cytotoxic activity and genotoxicity of structurally well characterized potent cobalt(II) phen–based antitumor drug entities: An in vitro and in vivo approach

Cobalt (II) phen–based drug candidates of the formulation Co(phen)₂Cl_2, 1, Co(phen)₂L, 2 where L = 1H–pyrazole–3,5–dicarboxylic acid were synthesized and thoroughly characterized by spectroscopic methods and single X–ray crystallography. DNA binding interaction of 1 and 2 was carried out employing biophysical techniques {UV–visible, fluorescence, thermal denaturation and cyclic voltammetry} to validate their potential to act as antitumor agents. The interpretations of these biophysical studies of 1 and 2 supported the non–covalent intercalative binding mode; furthermore, a higher binding trend of 2 was observed as compared to 1, phen and 1H–pyrazole–3,5–dicarboxylic acid alone. Cleavage studies of 1 and 2 with pBR322 were assessed by gel electrophoresis and it was observed that both drug candidates cleave DNA by hydrolytic pathway involving hydroxyl radical (OH). Cytotoxic activity of 1 and 2 against human cancer cell lines [MCF–7 (breast), HeLa (cervical), MIA–PA–CA 2 (pancreatic), A–498 (kidney), Hep–G2 (hepatoma)] was evaluated by SRB assay. The obtained results showed that drug candidate 1 showed significantly low GI₅₀ value (<10 µg/ml) against MCF–7 and HeLa cell lines. However, candidate 2 revealed excellent cytotoxicity (<10 µg/ml) against all the tested cancer cell lines. The in vivo genotoxicity of 2 was evaluated by micronucleus (MN) test and chromosomal aberration (CA) in bone marrow cells of the Wistar rats to check cobalt(II)–induced systemic toxicity. The results showed that no significant chromosomal aberrations and micronucleus formation was observed at 5 mg/kg and 10 mg/kg in presence of drug candidate 2 implicating that it could be administered safely at a low dosage. However, an elevated percentage of chromosomal aberration and micronucleated polychromatic erythrocytes (MNPCE) was observed only at higher doses (20 mg/kg and 40 mg/kg) of drug candidate 2.     

Poly(β-amino ester)-nanoparticle mediated transfection of retinal pigment epithelial cells in vitro and in vivo

A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer:DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (p<0.001 for both). Ten formulations exceeded 30% transfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×10(3)-fold and 1.5±0.7×10(3)-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p?=?0.003 for RPE/choroid, p<0.001 for neural retina). The successful transfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases.     

In vitro response of leaf tissues from Lolium multiflorum — a comparison with leaf segment position,leaf age and in vivo mitotic activity

Summary Immature gramineous leaves provide a convenient system for comparing the response of cells in culture with their state of differentiation. Callusing frequency is compared with leaf segment position, leaf age and in vivo mitotic activity in Lolium multiflorum. (1) In a succession of one millimeter sections from the immature leaf base, callus was formed from the first and second sections but not the third or subsequent sections. The frequency of those explants callusing decreased with distance from the base of the leaf and with leaf age (or leaf extension growth). (2) In vivo, the proportion of cells in mitosis declined from around 10–14% at the base of young leaves to zero at 5 mm from the base and beyond. Mitotic activity also declined in leaves as they aged, and dividing cells were not observed in leaves 30 days from initiation or older. (3) A high frequency of callus formation was associated with a high mitotic index in the explant. But for corresponding mitotic indices, cells further away from the leaf base were less responsive in culture. (4) It is proposed that cells are becoming differentiated even in highly meristematically active regions of the leaf and concomitantly losing their ability to respond in culture.     

Oral cancer,cigarette smoke and mitochondrial 18 kDa translocator protein (TSPO) — In vitro,in vivo,salivary analysis

Oral cancer features high rates of mortality and morbidity, and is in dire need for new approaches. In the present study we analyzed 18 kDa translocator protein (TSPO) expression in oral (tongue) cancer tumors by immunohistochemistry. We also assayed TSPO binding in human tongue cancer cell lines and in the cellular fraction of saliva from tongue cancer patients, heavy cigarette smokers, and non-smoking healthy people as controls. Concurrently, TSPO protein levels, cell viability, mitochondrial membrane potential (Δψ_m), and general protein levels were analyzed. TSPO expression could be significantly enhanced in oral cancer tumors, compared to unaffected adjacent tissue. We also found that five-year survival probability dropped from 65% in patients with TSPO negative tumors to 7% in patients with highly expressed TSPO (p < 0.001). TSPO binding capacity was also pronounced in the human oral cancer cell lines SCC-25 and SCC-15 (3133 ± 643 fmol/mg protein and 6956 ± 549 fmol/mg protein, respectively). Binding decreased by 56% and 72%, in the SCC-25 and SCC-15 cell lines, respectively (p < 0.05) following CS exposure in cell culture. In the cellular fraction of saliva of heavy smokers TSPO binding was lower than in non-smokers (by 53%, p < 0.05). Also the cellular fraction of saliva exposed to CS in vitro showed decreased TSPO binding compared to unexposed saliva (by 30%, p < 0.001). Interestingly, oral cancer patients also displayed significantly lower TSPO binding in the cellular fraction of saliva compared to healthy controls (by 40%, p < 0.01). Our results suggest that low TSPO binding found in the cellular fraction of saliva may depend on genetic background as well as result from exposure to CS. We suggest that this may be related to a predisposition for occurrence of oral cancer.     

Modulation of the TGF-β1-induced epithelial to mesenchymal transition (EMT) mediated by P1 and P2 purine receptors in MDCK cells

Epithelial to mesenchymal transition (EMT) occurs during embryogenesis or under pathological conditions such as hypoxia, injury, chronic inflammation, or tissue fibrosis. In renal tubular epithelial cells (MDCK), TGF-β1 induces EMT by reducing or increasing epithelial or mesenchymal marker expression, respectively. In this study, we confirmed that the cAMP analogues, 8-CPT-cAMP or N6-Ph-cAMP, inhibited the TGF-β1-driven overexpression of the mesenchymal markers ZEB-1, Slug, Fibronectin, and α-SMA. Furthermore, we showed that A1, A2A, P2Y1, P2Y11, and P2X7 purine receptor agonists modulated the TGF-β1-induced EMT through the involvement of PKA and/or MAPK/ERK signaling. The stimulation of A2A receptor reduced the overexpression of the EMT-related markers, mainly through the cAMP-dependent PKA pathway, as confirmed by cell pre-treatment with Myr-PKI. Both A1 and P2Y1 receptor stimulation exacerbated the TGF-β1-driven effects, which were reduced by cell pre-treatment with the MAPK inhibitor PD98059, according to the increased ERK1/2 phosphorylation upon receptor activation. The effects induced by P2Y11 receptor activation were oppositely modulated by PKA or MAPK inhibition, in line with the dual nature of the Gs- and Gq-coupled receptor. Differently, P2X7 receptor induced, per se, similar and not additive effects compared to TGF-β1, after prolonged cell exposure to BzATP. These results suggest a putative role of purine receptors as target for anti-fibrotic agents.     

In vitro and in vivo antitumor activity of a mouse CTL hybridoma expressing chimeric receptors bearing the single chain Fv from HER-2/neu- specific antibody and the γ-chain from Fc(ε) RI

To broaden the applicability of adoptive cellular immunotherapy against HER-2/neu overexpressing human cancers, we constructed a chimeric scFv/gamma gene composed of the variable regions of a HER-2/neu specific monoclonal antibody (mAb) joined to the signaling gamma-chain of the Fc(epsilon)RI receptor. The scFv(anti-HER-2/neu)/gamma chimeric gene was successfully expressed as functional surface receptor in the MD.45 cytolytic T-cell (CTL) hybridoma (MD.45-HER/gamma). Expression of the chimeric protein triggered IL-2 and IFN-gamma secretion in vitro upon encountering cell surface HER-2/neu and mediated non-major-histocompatibility-complex (MHC)-restricted HER-2/neu-specific target cell lysis. We also examined the in vivo activity of the MD.45-HER/gamma transduced cells. Severe combined immunodeficiency disease (SCID) mice that were given HER-2/neu positive (+) human tumor cell lines had significantly increased survival compared to mice treated with saline only, or with MD.45 cells transduced with a control anti-trinitrophenyl (anti-TNP) chimeric receptor gene (MD.45-TNP/gamma). These results demonstrate the feasibility of redirecting MD.45 CTL to react in vitro and in vivo with a variety of HER-2/neu(+) tumor cells by our gene transduction protocol. Moreover, they open the possibility of using the same chimeric gene for transducing primary lymphocytes and thus allowing adoptive immunotherapy against HER-2/neu(+) cancers.     

Transforming growth factor beta 1 (TGF-β1) modulates Epstein-Barr virus reactivation in absence of Helicobacter pylori infection in patients with gastric cancer

BackgroundTransforming growth factor-beta 1 (TGF-β1), a multifunctional cytokine, acts as a key factor for Epstein-Barr virus (EBV) reactivation. We investigated the role of TGF-β1 in latent and lytic stages of EBV in relation to Helicobacter pylori infection among patients with gastric cancer (GC) and peptic ulcer disease (PUD).MethodGastric mucosal TGF-β1 expression was determined in 95 EBV positive patients with gastroduodenal pathology [GC 40, PUD 19 and non-ulcer dyspepsia (NUD) 36] by quantitative real time PCR. Presence of H. pylori infection was diagnosed when either culture or any two of three tests (RUT, histopathology and specific ureA PCR) were positive. Serum level of TGF-β1 was detected among 60 patients using ELISA.ResultsMucosal TGF-β1 mRNA expression was detected in 85 of 95 EBV positive patients and it was significantly higher in patients with GC (p = 0.042). TGF-β1 expression tended to be higher among H. pylori non-infected than infected patients (3.80 ± 6.24 vs. 2.07 ± 2.50, p = 0.085). Both mRNA and serum level had significant association with lytic stage of EBV in absence of H. pylori infection when compared with its presence (5.21 ± 4.00 vs. 2.29 ± 2.89, p = 0.040 and 842.00 [669.55] vs. 662.63 [628.76], p = 0.049; respectively).ConclusionTGF-β1 expression was significantly associated with GC. TGF-β1 was higher both at expression and translational levels in lytic EBV infection without H. pylori suggests that H. pylori infection might play important role in preventing EBV reactivation through attenuated TGF-β1 expression. This might be a “wise host defense against EBV reactivation”.     

Estrogen receptor alpha (ERα) promoter methylation status in tumor and serum DNA in Egyptian breast cancer patients

Background

Status of DNA methylation is one of the most common molecular alterations in human neoplasia. Because it is possible to detect these epigenetic alterations in the bloodstream of patients, we investigated the aberrant DNA methylation status of estrogen receptor alpha (ERα) in patient pretherapeutic sera and tissue.

Materials and methods

In this case control study the patient series consisted of 120 sporadic primary breast cancer cases and 100 patients with benign breast lesion. ER3, ER4, and ER5 primers were used for methylation-specific polymerase chain reaction (MSP) to analyze the CpG methylation of promoter region of ERα gene. Correlation between ER3, ER4, and ER5 methylation and clinicopathological characteristics of the patients was investigated.

Result

The methylation status of ER3, ER4 and ER5 was 65%, 26.7% and 61.7% in tissue respectively and 57.5%, 21.7% and 55.8% in serum respectively. The concordance between tumor and serum DNA methylation was 80%, 72% and 92% for ER3, ER4 and ER5 respectively.

Conclusions

This study demonstrated the potential utility of serum DNA methylation of ERα gene promoter as a non-invasive diagnostic and/or prognostic marker in patients with breast cancer.     

Inhibitory effect of curcumin on the Al(III)-induced Aβ₄₂ aggregation and neurotoxicity in vitro

The pathogenesis of Alzheimer's disease (AD) involves a key event which changes the morphology of amyloid-β 42 (Aβ)?? peptide from its soluble monomeric form into the fibrillated aggregates in the brain. Aluminum ion, Al(III), is known to act as a pathological chaperone of the Aβ?? in this process; curcumin, a natural phenolic compound, is considered capable of binding Al(III) and Aβ??; nevertheless, little is known about the combined action of curcumin and Al(III) on the Aβ?? fibrillation and neurotoxicity. Here, combinations of circular dichroism spectroscopy, thioflavin T fluorescence, atomic force microscopy, Bradford and MTT assays, it is demonstrated that although Al(III) can promote the Aβ?? fibrillation dose-dependently, leading to the high neurotoxicity to PC12 cells, curcumin can inhibit the events. Besides, we found that curcumin is able not only to inhibit the formation of Al(III)-induced Aβ?? fibrillation, but also to form the Al(III)-curcumin complexes which in turn can remold the preformed, mature, ordered Aβ?? fibrils into the low toxic amorphous aggregates. These findings suggest that curcumin could block the binding of Al(III) with Aβ?? and form the Al(III)-curcumin complexes, so as to inhibit the Al(III)-induced Aβ?? fibrillation and neurotoxicity. The Al(III)-curcumin complexes are worth potentially developing as a therapy agent against the neurodegenerative disorders in the future.     

In vivo and in vitro effect of Acacia nilotica seed proteinase inhibitors on Helicoverpa armigera (Hübner) larvae

Acacia nilotica proteinase inhibitor (AnPI) was isolated by ammonium sulphate precipitation followed by chromatography on DEAE-Sephadex A-25 and resulted in a purification of 10.68-fold with a 19.5 percentage yield. Electrophoretic analysis of purified AnPI protein resolved into a single band with molecular weight of approximately 18.6+1.00 kDa. AnPI had high stability at different pH values (2.0 to 10.0) except at pH 5.0 and are thermolabile beyond 80 degree C for 10 min. AnPI exhibited effective against total proteolytic activity and trypsin-like activity, but did not show any inhibitory effect on chymotrypsin activity of midgut of Helicoverpa armigera. The inhibition kinetics studies against H. armigera gut trypsin are of non-competitive type. AnPI had low affinity for H. armigera gut trypsin when compared to SBTI. The partially purified and purified PI proteins-incorporated test diets showed significant reduction in mean larval and pupal weight of H. armigera. The results provide important clues in designing strategies by using the proteinase inhibitors (PIs) from the A. nilotica that can be expressed in genetically engineered plants to confer resistance to H. armigera.     

Retracted: Role of CXCL12–CXCR4 axis in ovarian cancer metastasis and CXCL12–CXCR4 blockade with AMD3100 suppresses tumor cell migration and invasion in vitro

Ovarian cancer (OC) is a lethal gynecologic tumor, which brings its mortality to the head. CXCL12 and its receptor chemokine receptor 4 ( CXCR4) have been found to be highly expressed in OC and contribute to the disease progression by affecting tumor cell proliferation and invasion. Here, in this study, we aim to explore whether the blockade of CXCL12–CXCR4 axis with AMD3100 (a selective CXCR4 antagonist) has effects on the progression of OC. On the basis of the gene expression omnibus database of OC gene expression chips, the OC differentially expressed genes were screened by microarray analysis. OC (nonmetastatic and metastatic) and normal ovarian tissues were collected to determine the expressions of CXCL12 and CXCR4. A series of AMD3100, shRNA against CXCR4, and pCNS-CXCR4 were introduced to treat CAOV3 cells with the highest CXCR4 was assessed. Cell viability, apoptosis, migration, and invasion were all evaluated. The microarray analysis screened out the differential expression of CXCL12–CXCR4 in OC. CXCL12 and CXCR4 expressions were increased in OC tissues, particularly in the metastatic OC tissues. Downregulation of CXCR4 by AMD3100 or shRNA was observed to have a critical role in inhibiting cell proliferation, migration, and invasion of the CAOV3 OC cell line while promoting cell apoptosis. Overexpressed CXCR4 brought significantly promoting effects on the proliferation and invasiveness of OC cells. These results reinforce that the blockade of CXCL12–CXCR4 axis with AMD3100 inhibits the growth of OC cells. The antitumor role of the inhibition of CXCL12–CXCR4 axis offers a preclinical validation of CXCL12–CXCR4 axis as a therapeutic target in OC.     

In vitro and in vivo mechanistic study of a novel proanthocyanidin,GC-(4→8)-GCG from cocoa tea (Camellia ptilophylla) in antiangiogenesis

Angiogenesis, the process of blood vessel formation, is critical to tumor growth. Ant-angiogenic strategies demonstrated importance in cancer therapy. Cocoa tea (Camellia ptilophylla), a naturally decaffeinated tea commonly consumed as a healthy drink in southern China, had recently been found to be a potential candidate for antiangiogenesis. A novel proanthocyanidin, GC-(4→8)-GCG, which consisted of gallocatechin and gallocatechin 3-O gallate moieties, was discovered and thought to be one of the effective candidates for antiangiogenesis. Hence, the present study aimed to evaluate the antiangiogenesis activities of GC-(4→8)-GCG in vitro and in vivo, and SU5416 was applied as a positive control. The inhibitory effects of GC-(4→8)-GCG on three important processes involved in angiogenesis, i.e., proliferation, migration and differentiation, were examined using human microvascular endothelial cell line HMEC-1 by MTT assay, scratch assay and tube formation assay, respectively. Using transgenic zebrafish embryos TG(fli1:EGFP)y1/+(AB) as an animal model of angiogenesis, the antiangiogenic effect of GC-(4→8)-GCG was further verified in vivo. Our results demonstrated that GC-(4→8)-GCG significantly inhibited migration (P<.001) and tubule formation (P<.001–.05) of HMEC-1 in dose-dependent manner. Regarding intracellular signal transduction, GC-(4→8)-GCG attenuated the phosphorylation of ERK, Akt and p38 dose-dependently in HMEC-1. In zebrafish embryo, the formation of new blood vessels was effectively inhibited by GC-(4→8)-GCG in a dose-dependent manner after 3 days of treatment (P<.001–.05). In conclusion, these results revealed that our novel proanthocyanidin, GC-(4→8)-GCG might be a potential and promising agent of natural resource to be further developed as an antiangiogenic agent.     

Design and synthesis of cell selective α/β-diastereomeric peptidomimetic with potent in vivo antibacterial activity against methicillin resistant S. Aureus

Design of therapeutically viable antimicrobial peptides with cell selectivity against microorganisms is an important step towards the development of new antimicrobial agents. Here, we report four de novo designed, short amphipathic sequences based on a α-helical template comprising of Lys, Trp and Leu or their corresponding D-and/or β-amino acids. Sequence A-12 was protease susceptible whereas its α/β-diastereomeric analogue UNA-12 was resistant to trypsin and proteinase K up to 24 h. A-12 and UNA-12 exhibited broad-spectrum antibacterial activity (MIC: 2–32 µg/mL) against pathogens including methicillin resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE). Interestingly, A-12 was found to be most toxic (>50% haemolytic at 250 µg/mL) whereas UNA-12 was found to be non cytotoxic among the all analogues against hRBCs and human keratinocytes. Interaction studies with artificial membranes by tryptophan fluorescence and acrylamide quenching assay demonstrated A-12 interacted equally in bacterial as well as mammalian mimic membrane whereas UNA-12 was found to be more selective towards bacterial mimic membrane. Further microscopic tool has revealed membrane damaging ability of A-12 and UNA-12 with bactericidal mode of action against MRSA. Encouragingly, peptidomimetics analogue UNA-12 showed remarkable safety and efficacy against MRSA in in-vivo neutropenic mice thigh infection model. In summary, simultaneous replacement of the natural amino acids with D-/β-congeners is a promising strategy for designing of potent, cell selective and protease stable peptide based antibiotics.     

In vitro and in vivo D2-dopamine receptor binding with [123I]S(−)iodobenzamide ([123I]IBZM) in rat and human brain

As a promising dopamine D2-receptor imaging agent for single photon emission computerized tomography (SPECT), [¹²³I](S)-(−)-2-hydroxy-3-iodo-6-methoxy-N [(1-ethyl-2-pyrrolidinyl)methyl]benzamide ([¹²³I]IBZM) has recently been synthesized in a modified way along with its precursor, S(−)BZM, and the stereoisomer R(+)BZM. The present study applied this new product to investigate in vitro and in vivo D2-receptor binding in rat brain and in postmortem human brain. In vitro saturation binding curves with [¹²³I]IBZM for rat crude striatal membrane preparations yielded an affinity constant (K_d) of 0.28 nM confirming data in the literature. Displacement curves revealed an order of increasing potency as follows: R(+)BZM < S(−)sulpiride = < S(−)BZM < S(−)IBZM. A similar order was obtained when [³H]spiperone was used as ligand. For human putamen and caudate nucleus membranes slightly higher K_d values (0.49 nM) were obtained. Rank order of displacing potency for the various drugs was similar to that found in the rat preparations. In vivo uptake of [¹²³I]IBZM in rat brain following injection of 50 μCi (12–16pmol) in the tail vein revealed an increase in the striatum-to-cerebellum ratio from 1.5 at 5 min to 6.9 at 2 h. The olfactory tubercle-to-cerebellum ratio was also raised from 1.6 to 3.3. Other brain regions tested failed to show statistically significant enhancements. Coinjection of 40 nmol S(-)IBZM, 4μ mol S(−)BZM or 200 nmol haloperidol displaced [¹²³I]IBMZ when tested at 90 min. The use of 4μ mol R(+)BZM resulted in minor displacement only, demonstrating that stereospecificity of the displacement was present in vivo and in vitro. Displacements were also observed in substantia nigra and pons-medulla oblongata, but not in hippocampus or frontal and occipital cortex. The data provide the required background needed in order to initiate in vivo binding studies for D2-receptors in basal ganglia of human patients using [¹²³I]IBZM in SPECT analyses.