Synthetic Spirocyclohexadienones as New Anti-Migratory Compounds in Triple- Negative Breast Cancer Cell Migration.

BACKGROUND: Triple-negative BC is the most aggressive type of breast cancer and its lack of responsiveness to conventional therapies requires screening of new chemical entities. Anti-migratory compounds are promising to treat metastatic cancer since they inhibit one of the main steps of the metastatic cascade. Spirocyclic compounds are non-conventional structures used as building blocks for the synthesis of biologically active molecules and considered interesting structures in the search for new targets in cancer research. OBJECTIVE: Here, we evaluated the potential of eight synthetic spirocyclohexadienones as cell migration inhibitors. METHODS: The anti-migratory ability of compounds was tested by wound healing and Boyden chamber approaches. Experiments in tubulin were performed by fluorescence and tubulin polymerization techniques. Finally, compounds were submitted to cell proliferation inhibition and flow cytometry assays to explore the mechanism by which they inhibit cell migration. RESULTS: Four compounds inhibited cell migration significantly. Analogs containing the 3,4,5-trimethoxyphenil ring at R1 position were the most potent and, thus, selected for additional experiments. Tubulin polymerization and fluorescence assays highlighted a possible binding of spirocyclohexadienones in the colchicine binding site; however, these compounds did not affect the cell cycle to the same extent as colchicine. Cell proliferation was affected and, notably, the most potent analogs induced apoptosis of tumor cells, suggesting a different mechanism by which they inhibit cell migration. CONCLUSION: We presented, for the first time, a series of eight synthetic spirocyclohexadienones with the ability to inhibit TNBC cell migration. These compounds represent a new category to be explored as anticancer agents.

Design, Synthesis and Evaluation of 2,4-Diaminoquinazoline Derivatives as Potential Tubulin Polymerization Inhibitors.

Microtubules are highly dynamic polymers composed of α- and ß-tubulin proteins that have been shown to be potential therapeutic targets for the development of anticancer drugs. Currently, a wide variety of chemically diverse agents that bind to ß-tubulin have been reported. Nocodazole (NZ) and colchicine (COL) are well-known tubulin-depolymerizing agents that have close binding sites in the ß-tubulin. In this study, we designed and synthesized a set of nine 2,4-diaminoquinazoline derivatives that could occupy both NZ and COL binding sites. The synthesized compounds were evaluated for their antiproliferative activities against five cancer cell lines (PC-3, HCT-15, MCF-7, MDA-MB-231, and SK-LU-1), a noncancerous one (COS-7), and peripheral blood mononuclear cells (PBMC). The effect of compounds 4 e and 4 i on tubulin organization and polymerization was analyzed on the SK-LU-1 cell line by indirect immunofluorescence, western blotting, and tubulin polymerization assays. Our results demonstrated that both compounds exert their antiproliferative activity by inhibiting tubulin polymerization. Finally, a possible binding pose of 4 i in the NZ/COL binding site was determined by using molecular docking and molecular dynamics (MD) approaches. To our knowledge, this is the first report of non-N-substituted 2,4-diaminoquinazoline derivatives with the ability to inhibit tubulin polymerization.

S-(4-Methoxyphenyl)-4-methoxybenzenesulfonothioate as a Promising Lead Compound for the Development of a Renal Carcinoma Agent.

Organosulfur compounds show cytotoxic potential towards many tumor cell lines. Disulfides and thiosulfonates act through apoptotic processes, inducing proteins associated with apoptosis, endoplasmic reticulum stress, and the unfolded protein response. Three p-substituted symmetric diaryl disulfides and three diaryl thiosulfonates were synthesized and analyzed for inhibition of tubulin polymerization and for human cancer cell cytotoxic activity against seven tumor cell lines and a non-tumor cell line. S-(4-methoxyphenyl)-4-methoxybenzenesulfonothioate (6) exhibited inhibition of tubulin polymerization and showed the best antiproliferative potential, especially against the 786-0 cell line, being six times more selective as compared with the non-tumor cell line. In addition, compound 6 was able to activate caspase-3 after 24 and 48 h treatments of the 786-0 cell line and induced cell-cycle arrest in the G2/M stage at the highest concentration evaluated at 24 and 48 h. Compound 6 was able to cause complete inhibition of proliferation, inducing the death of 786-0 cells, by increasing the number of cells at G2/M and greater activation of caspase-3.

Effect of radiant exposure and UV accelerated aging on physico-chemical and mechanical properties of composite resins

Abstract Currently, there is no consensus in terms of defining the minimum radiant exposure values necessary for achieving adequate properties of composite resin. In addition, the long-term influence that radiant exposure has on the properties of composite resins is still questionable. Objective: The objective of this study was to evaluate the effect of radiant exposure and UV accelerated aging on the physico-chemical and mechanical properties of micro-hybrid and nanofilled composite resins. Material and Methods: A nanofilled (Filtek Supreme; 3M ESPE) and a micro-hybrid composite resin (Filtek Z250; 3M ESPE) were investigated under different radiant exposures (3.75, 9, and 24 J/cm2) and UV accelerated aging protocols (0, 500, 1000, and 1500 aging hours). The degree of conversion (DC), flexural strength (FS), modulus (M), water sorption (WS), and solubility (WL) were evaluated. The results obtained were analyzed using two-way ANOVA and Tukey's test. Comparisons were performed using a significance level of α=0.05. Results: The DC, FS, and M were found to be significantly influenced by both radiant exposure and accelerated aging time. The DC and EM increased with radiant exposure in the no-aging group (0-hour aging) for both micro-hybrid and nanofilled composites, whereas no correlation was found after accelerated aging protocols. WS and WL of micro-hybrid and nanofilled composite resins were scarcely affected by radiant exposure (p>0.05), whereas they were significantly reduced by accelerated aging (p<0.001). Conclusions: Although increasing radiant exposure affected the degree of conversion and mechanical properties of micro-hybrid and nanofilled composites, no influence on the hydrolytic degradation of the material was observed. In contrast, UV accelerated aging affected both the physico-chemical and mechanical properties of the composites.

Inhibición de la formación de ß-hematina de especies colombianas de Piper spp. y Calophyllum spp. como potenciales agentes antimaláricos / Inhibition of ß-hematin formation of Colombian species of Piper spp. and Calophyllum spp. as potential antimalarial agents

Resumen Nuevos agentes antimaláricos a partir de plantas son estudiados como alternativas en el tratamiento de la malaria. Los principales antimaláricos como la cloroquina tienen varios mecanismos de acción contra parásitos, uno de ellos es la inhibición de polimerización del grupo hemo, modelo que ha permitido el diseño de nuevos candidatos antimaláricos. En este sentido, el objetivo de este trabajo fue evaluar extractos de plantas de género Piper y Calophyllum sobre la capacidad de inhibición de la β-hematina. Se informa las concentraciones inhibitorias de la formación de β-hematina por parte de 40 extractos de diferente polaridad obtenidos a partir de las especies P. piedecuestanum, C. brasiliense, C. longinforium, y Calophyllum. sp. 19 extractos mostraron un mayor potencial para inhibir la formación de β−hematina con CI50 < 3mg / ml. Estas actividades respaldan principalmente, futuros estudios con el género Calophyllum, en el desarrollo y descubrimiento de nuevas sustancias antiplasmodiales con modos de acción conocido.(AU)

Abstract New antimalarial agents from plants are studied as alternatives in the treatment of malaria. The main antimalarials such as chloroquine have several mechanisms of action against parasites, one of which is the inhibition of polymerization of the heme group, a model that has allowed the design of new antimalarial candidates. In this sense the objective of this work was to evaluate extracts of genus Piper and Calophyllum plants on the inhibition capacity of β-hematin. Inhibitory concentrations of β-hematin are reported from 40 extracts of different polarity obtained from the species P. piedecuestanum, C. brasiliense, C. longinforium, and Calophyllum. sp. 19 extracts showed a greater potential to inhibit β-hematin with IC50 < 3 mg/ml. These activities mainly support future studies with the genus Calophyllum in the development and discovery of new antiplasmodial substances with known modes of action.(AU)

Effect of different bonding protocols on degree of monomer conversion and bond strength between orthodontic brackets and enamel.

The objective of the present study was to evaluate the effect of different surface treatments and polymerization protocols on the bond strength of brackets to enamel, and the degree of conversion of the bonding agents. 120 bovine crowns were embedded in acrylic resin blocks and sanded. Next, the blocks were randomly assigned into 12 groups. Metal brackets were bonded to enamel according to the "surface treatment" factor (A: Phosphoric Acid; ATxt: Phosphoric Acid + Transbond XT Primer®; Tse: Transbond Plus Self Etching Primer®; and SBU: Scotchbond Universal®) and "polymerization" factor (R20: Radii-Cal®/20 seconds; V20: Valo Cordless®/20 seconds; and V3: Valo Cordless®/3 seconds). All samples were stored for 6 months (water, 37ºC) and then subjected to a shear bond strength test (SBS). Bond failures were classified according to the Adhesive Remnant Index (ARI) and analyzed with the Kruskal-Wallis and Mann-Whitney tests (5%). Using the same factors, 120 resin discs were made to assess the degree of conversion (DC) of the monomer. Data from the SBS (MPa) and DC (%) were analyzed by analysis of variance (2 factors) and Tukey's test (5%). For the SBS, the factors "polymerization" (R20 = 8.1B; V20 = 13.2A; V3 = 5.2C, p = 0.0001) and "surface treatment" (A = 3.1C; ATxt = 13.6A; Tse = 12.3A; SBU = 6.3B, p = 0.0001) were statistically significant among groups. The highest adhesion value were found for the ATxt/V20 group (22.2A) and the lowest value for the A/R20 group (1.2E). Regarding ARI, score 2 was the most prevalent in groups A, ATxt, V20 and V3, while score 4 was the most prevalent in the Tse, SBU and R20 groups, with no significant difference between them (p = 1.0). Regarding DC, the factors "polymerization" (R20 = 66.6A; V20 = 58.4B; V3 = 45.1C, p = 0.0001) and "surface treatment" (A = 52B, ATxt = 59.7A, Tse = 51.4B, SBU = 63.8A, p = 0.0001) were statistically significant. Tse was more sensitive to the variations in polymerization protocols than the other surface treatments. Treatment A did not present suitable bond strength or degree of conversion.

Effect on adhesion of a nanocapsules-loaded adhesive system.

This study aimed to evaluate the in situ degree of conversion, contact angle, and immediate and long-term bond strengths of a commercial primer and an experimental adhesive containing indomethacin- and triclosan-loaded nanocapsules (NCs). The indomethacin- and triclosan-loaded NCs, which promote anti-inflammatory and antibacterial effects through controlled release, were incorporated into the primer at a concentration of 2% and in the adhesive at concentrations of 1, 2, 5, and 10%. The in situ degree of conversion (DC, n=3) was evaluated by micro-Raman spectroscopy. The contact angle of the primer and adhesive on the dentin surface (n = 3) was determined by an optical tensiometer. For the microtensile bond strength µTBS test (12 teeth per group), stick-shaped specimens were tested under tensile stress immediately after preparation and after storage in water for 1 year. The data were analyzed using two-way ANOVA, three-way ANOVA and Tukey's post hoc tests with α=0.05. The use of the NC-loaded adhesive resulted in a higher in situ degree of conversion. The DC values varied from 75.07 ± 8.83% to 96.18 ± 0.87%. The use of NCs in only the adhesive up to a concentration of 5% had no influence on the bond strength. The contact angle of the primer remained the same with and without NCs. The use of both the primer and adhesive with NCs (for all concentrations) resulted in a higher contact angle of the adhesive. The longitudinal µTBS was inversely proportional to the concentration of NCs in the adhesive system, exhibiting decreasing values for the groups with primer containing NCs and adhesives with increasing concentrations of NCs. Adhesives containing up to 5% of nanocapsules and primer with no NCs maintained the in situ degree of conversion, contact angle, and immediate and long-term bond strengths. Therefore, the NC-loaded adhesive can be an alternative method for combining the bond performance and therapeutic effects. The use of an adhesive with up to 5% nanocapsules containing indomethacin and triclosan and a primer with no nanocapsules maintained the long-term bond performance.

Effect on adhesion of a nanocapsules-loaded adhesive system

Abstract This study aimed to evaluate the in situ degree of conversion, contact angle, and immediate and long-term bond strengths of a commercial primer and an experimental adhesive containing indomethacin- and triclosan-loaded nanocapsules (NCs). The indomethacin- and triclosan-loaded NCs, which promote anti-inflammatory and antibacterial effects through controlled release, were incorporated into the primer at a concentration of 2% and in the adhesive at concentrations of 1, 2, 5, and 10%. The in situ degree of conversion (DC, n=3) was evaluated by micro-Raman spectroscopy. The contact angle of the primer and adhesive on the dentin surface (n = 3) was determined by an optical tensiometer. For the microtensile bond strength µTBS test (12 teeth per group), stick-shaped specimens were tested under tensile stress immediately after preparation and after storage in water for 1 year. The data were analyzed using two-way ANOVA, three-way ANOVA and Tukey's post hoc tests with α=0.05. The use of the NC-loaded adhesive resulted in a higher in situ degree of conversion. The DC values varied from 75.07 ± 8.83% to 96.18 ± 0.87%. The use of NCs in only the adhesive up to a concentration of 5% had no influence on the bond strength. The contact angle of the primer remained the same with and without NCs. The use of both the primer and adhesive with NCs (for all concentrations) resulted in a higher contact angle of the adhesive. The longitudinal μTBS was inversely proportional to the concentration of NCs in the adhesive system, exhibiting decreasing values for the groups with primer containing NCs and adhesives with increasing concentrations of NCs. Adhesives containing up to 5% of nanocapsules and primer with no NCs maintained the in situ degree of conversion, contact angle, and immediate and long-term bond strengths. Therefore, the NC-loaded adhesive can be an alternative method for combining the bond performance and therapeutic effects. The use of an adhesive with up to 5% nanocapsules containing indomethacin and triclosan and a primer with no nanocapsules maintained the long-term bond performance.

Effect of different bonding protocols on degree of monomer conversion and bond strength between orthodontic brackets and enamel

Abstract The objective of the present study was to evaluate the effect of different surface treatments and polymerization protocols on the bond strength of brackets to enamel, and the degree of conversion of the bonding agents. 120 bovine crowns were embedded in acrylic resin blocks and sanded. Next, the blocks were randomly assigned into 12 groups. Metal brackets were bonded to enamel according to the "surface treatment" factor (A: Phosphoric Acid; ATxt: Phosphoric Acid + Transbond XT Primer®; Tse: Transbond Plus Self Etching Primer®; and SBU: Scotchbond Universal®) and "polymerization" factor (R20: Radii-Cal®/20 seconds; V20: Valo Cordless®/20 seconds; and V3: Valo Cordless®/3 seconds). All samples were stored for 6 months (water, 37ºC) and then subjected to a shear bond strength test (SBS). Bond failures were classified according to the Adhesive Remnant Index (ARI) and analyzed with the Kruskal-Wallis and Mann-Whitney tests (5%). Using the same factors, 120 resin discs were made to assess the degree of conversion (DC) of the monomer. Data from the SBS (MPa) and DC (%) were analyzed by analysis of variance (2 factors) and Tukey's test (5%). For the SBS, the factors "polymerization" (R20 = 8.1B; V20 = 13.2A; V3 = 5.2C, p = 0.0001) and "surface treatment" (A = 3.1C; ATxt = 13.6A; Tse = 12.3A; SBU = 6.3B, p = 0.0001) were statistically significant among groups. The highest adhesion value were found for the ATxt/V20 group (22.2A) and the lowest value for the A/R20 group (1.2E). Regarding ARI, score 2 was the most prevalent in groups A, ATxt, V20 and V3, while score 4 was the most prevalent in the Tse, SBU and R20 groups, with no significant difference between them (p = 1.0). Regarding DC, the factors "polymerization" (R20 = 66.6A; V20 = 58.4B; V3 = 45.1C, p = 0.0001) and "surface treatment" (A = 52B, ATxt = 59.7A, Tse = 51.4B, SBU = 63.8A, p = 0.0001) were statistically significant. Tse was more sensitive to the variations in polymerization protocols than the other surface treatments. Treatment A did not present suitable bond strength or degree of conversion.

Influence of polymeric matrix on the physical and chemical properties of experimental composites.

Nowadays, the main reasons for replacement of resin-based composite restorations are fracture or problems with the integrity of their interface, such as marginal staining, microleakage, or secondary caries. The aim of the present study was to evaluate the influence of the organic matrix on polymerization stress (PS), degree of conversion (DC), elastic modulus (E), flexural strength (FS), Knoop hardness (KHN), sorption (SP), and solubility (SL). In order to obtain a material which combines better mechanical properties with lower PS, seven experimental composites were prepared using BisGMA to TEGDMA molar ratios of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3 and 8:2 and 40% of silica. PS was obtained in a universal testing machine, using acrylic as bonding substrate. DC was determined using Fourier Transform Raman spectroscopy. E and FS were obtained by the three-point bending test. KHN was measured by a microindentation test using a load of 25 g for 30 s. SP and SL were assessed according to ISO 4049. The data were submitted to one-way ANOVA. The increase in BisGMA concentration resulted in the decrease of PS, DC, E, FS and KHN. However, it did not change the SP and SL values. FS, E and KHN showed a strong and direct relationship with the DC of the materials. The composite material with a BisGMA to TEGDMA molar ratio of 1:1 was the one with better mechanical properties and lower PS.

Influence of polymeric matrix on the physical and chemical properties of experimental composites

Nowadays, the main reasons for replacement of resin-based composite restorations are fracture or problems with the integrity of their interface, such as marginal staining, microleakage, or secondary caries. The aim of the present study was to evaluate the influence of the organic matrix on polymerization stress (PS), degree of conversion (DC), elastic modulus (E), flexural strength (FS), Knoop hardness (KHN), sorption (SP), and solubility (SL). In order to obtain a material which combines better mechanical properties with lower PS, seven experimental composites were prepared using BisGMA to TEGDMA molar ratios of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3 and 8:2 and 40% of silica. PS was obtained in a universal testing machine, using acrylic as bonding substrate. DC was determined using Fourier Transform Raman spectroscopy. E and FS were obtained by the three-point bending test. KHN was measured by a microindentation test using a load of 25 g for 30 s. SP and SL were assessed according to ISO 4049. The data were submitted to one-way ANOVA. The increase in BisGMA concentration resulted in the decrease of PS, DC, E, FS and KHN. However, it did not change the SP and SL values. FS, E and KHN showed a strong and direct relationship with the DC of the materials. The composite material with a BisGMA to TEGDMA molar ratio of 1:1 was the one with better mechanical properties and lower PS.

Exploring the possible role of lysine acetylation on Entamoeba histolytica virulence: a focus on the dynamics of the actin cytoskeleton.

Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

Effect of silica coating and silane surface treatment on the bond strength of soft denture liner to denture base material.

OBJECTIVE: This study investigated the effects of different surface treatments on the tensile bond strength of an autopolymerizing silicone denture liner to a denture base material after thermocycling. MATERIAL AND METHODS: Fifty rectangular heat-polymerized acrylic resin (QC-20) specimens consisting of a set of 2 acrylic blocks were used in the tensile test. Specimens were divided into 5 test groups (n=10) according to the bonding surface treatment as follows: Group A, adhesive treatment (Ufi Gel P adhesive) (control); Group S, sandblasting using 50-µm Al2O3; Group SCSIL, silica coating using 30-µm Al2O3 modified by silica and silanized with silane agent (CoJet System); Group SCA, silica coating and adhesive application; Group SCSILA, silica coating, silane and adhesive treatment. The 2 PMMA blocks were placed into molds and the soft lining materials (Ufi Gel P) were packed into the space and polymerized. All specimens were thermocycled (5,000 cycles) before the tensile test. Bond strength data were analyzed using 1-way ANOVA and Duncan tests. Fracture surfaces were observed by scanning electron microscopy. X-ray photoelectron spectrometer (XPS) and Fourier Transform Infrared spectrometer (FTIR) analysis were used for the chemical analysis and a profilometer was used for the roughness of the sample surfaces. RESULTS: The highest bond strength test value was observed for Group A (1.35±0.13); the lowest value was for Group S (0.28±0.07) and Group SCSIL (0.34±0.03). Mixed and cohesive type failures were seen in Group A, SCA and SCSILA. Group S and SCSIL showed the least silicone integrations and the roughest surfaces. CONCLUSION: Sandblasting, silica coating and silane surface treatments of the denture base resin did not increase the bond strength of the silicone based soft liner. However, in this study, the chemical analysis and surface profilometer provided interesting insights about the bonding mechanism between the denture base resin and silicone soft liner.

Effect of silica coating and silane surface treatment on the bond strength of soft denture liner to denture base material

OBJECTIVE: This study investigated the effects of different surface treatments on the tensile bond strength of an autopolymerizing silicone denture liner to a denture base material after thermocycling. MATERIAL AND METHODS: Fifty rectangular heat-polymerized acrylic resin (QC-20) specimens consisting of a set of 2 acrylic blocks were used in the tensile test. Specimens were divided into 5 test groups (n=10) according to the bonding surface treatment as follows: Group A, adhesive treatment (Ufi Gel P adhesive) (control); Group S, sandblasting using 50-µm Al2O3; Group SCSIL, silica coating using 30-µm Al2O3 modified by silica and silanized with silane agent (CoJet System); Group SCA, silica coating and adhesive application; Group SCSILA, silica coating, silane and adhesive treatment. The 2 PMMA blocks were placed into molds and the soft lining materials (Ufi Gel P) were packed into the space and polymerized. All specimens were thermocycled (5,000 cycles) before the tensile test. Bond strength data were analyzed using 1-way ANOVA and Duncan tests. Fracture surfaces were observed by scanning electron microscopy. X-ray photoelectron spectrometer (XPS) and Fourier Transform Infrared spectrometer (FTIR) analysis were used for the chemical analysis and a profilometer was used for the roughness of the sample surfaces. RESULTS: The highest bond strength test value was observed for Group A (1.35±0.13); the lowest value was for Group S (0.28±0.07) and Group SCSIL (0.34±0.03). Mixed and cohesive type failures were seen in Group A, SCA and SCSILA. Group ...

A glycolytic metabolon in Saccharomyces cerevisiae is stabilized by F-actin.

In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO2 molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi-enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F-actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F-actin, but not the monomeric globular G-actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F-actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies.

Cytotoxic 3,4,5-trimethoxychalcones as mitotic arresters and cell migration inhibitors.

Based on classical colchicine site ligands and a computational model of the colchicine binding site on beta tubulin, two classes of chalcone derivatives were designed, synthesized and evaluated for inhibition of tubulin assembly and toxicity in human cancer cell lines. Docking studies suggested that the chalcone scaffold could fit the colchicine site on tubulin in an orientation similar to that of the natural product. In particular, a 3,4,5-trimethoxyphenyl ring adjacent to the carbonyl group appeared to benefit the ligand-tubulin interaction, occupying the same subcavity as the corresponding moiety in colchicine. Consistent with modeling predictions, several 3,4,5-trimethoxychalcones showed improved cytotoxicity to murine acute lymphoblastic leukemia cells compared with a previously described parent compound, and inhibited tubulin assembly in vitro as potently as colchicine. The most potent chalcones inhibited the growth of human leukemia cell lines at nanomolar concentrations, caused microtubule destabilization and mitotic arrest in human cervical cancer cells, and inhibited human breast cancer cell migration in scratch wound and Boyden chamber assays.

Synthesis and antimalarial activity of urenyl Bis-chalcone in vitro and in vivo.

Some novel derivatives of Bis-chalcone were synthesized and characterized by their physical and spectral data. All the synthesized compounds were subsequently screened for in vitro globin hydrolysis, ß-hematin formation, and murine Plasmodium berghei, using chloroquine as the reference drug. Most of the synthesized compounds exhibited mild to moderate susceptibilities toward the parasite in comparison with the standard. The most active antimalarial compound was 1,1-Bis-[(3',4'-N-(urenylphenyl)-3-(3″,4″,5″-trimethoxyphenyl)]-2-propen-1-one 5, with a percentage of inhibition of heme polymerization of 87.05 ± 0.77, and this compound increased the survival time after infection, reduce the parasitemia and delay the progression of malaria.

Gonadotropin-releasing hormone receptor activates GTPase RhoA and inhibits cell invasion in the breast cancer cell line MDA-MB-231.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) and its receptor (GnRHR) are both expressed by a number of malignant tumors, including those of the breast. In the latter, both behave as potent inhibitors of invasion. Nevertheless, the signaling pathways whereby the activated GnRH/GnRHR system exerts this effect have not been clearly established. In this study, we provide experimental evidence that describes components of the mechanism(s) whereby GnRH inhibits breast cancer cell invasion. METHODS: Actin polymerization and substrate adhesion was measured in the highly invasive cell line, MDA-MB-231 transiently expressing the wild-type or mutant DesK191 GnRHR by fluorometry, flow cytometric analysis, and confocal microscopy, in the absence or presence of GnRH agonist. The effect of RhoA-GTP on stress fiber formation and focal adhesion assembly was measured in MDA-MB-231 cells co-expressing the GnRHRs and the GAP domain of human p190Rho GAP-A or the dominant negative mutant GAP-Y1284D. Cell invasion was determined by the transwell migration assay. RESULTS: Agonist-stimulated activation of the wild-type GnRHR and the highly plasma membrane expressed mutant GnRHR-DesK191 transiently transfected to MDA-MB-231 cells, favored F-actin polymerization and substrate adhesion. Confocal imaging allowed detection of an association between F-actin levels and the increase in stress fibers promoted by exposure to GnRH. Pull-down assays showed that the effects observed on actin cytoskeleton resulted from GnRH-stimulated activation of RhoA GTPase. Activation of this small G protein favored the marked increase in both cell adhesion to Collagen-I and number of focal adhesion complexes leading to inhibition of the invasion capacity of MDA-MB-231 cells as disclosed by assays in Transwell Chambers. CONCLUSIONS: We here show that GnRH inhibits invasion of highly invasive breast cancer-derived MDA-MB-231 cells. This effect is mediated through an increase in substrate adhesion promoted by activation of RhoA GTPase and formation of stress fibers and focal adhesions. These observations offer new insights into the molecular mechanisms whereby activation of overexpressed GnRHRs affects cell invasion potential of this malignant cell line, and provide opportunities for designing mechanism-based adjuvant therapies for breast cancer.

The actin cytoskeleton participates in the early events of autophagosome formation upon starvation induced autophagy.

Autophagy is a process by which cytoplasmic material is sequestered in a double-membrane vesicle destined for degradation. Nutrient deprivation stimulates the pathway and the number of autophagosomes in the cell increases in response to such stimulus. In the current report we have demonstrated that actin is necessary for starvation-mediated autophagy. When the actin cytoskeleton is depolymerized, the increase in autophagic vacuoles in response to the starvation stimulus was abolished without affecting maturation of remaining autophagosomes. In addition, actin filaments colocalized with ATG14, BECN1/Beclin1 and PtdIns3P-rich structures, and some of them have a typical omegasome shape stained with the double FYVE domain or ZFYVE1/DFCP1. In contrast, no major colocalization between actin and ULK1, ULK2, ATG5 or MAP1LC3/LC3 was observed. Taken together, our data indicate that actin has a role at very early stages of autophagosome formation linked to the PtdIns3P generation step. In addition, we have found that two members of the Rho family of proteins, RHOA and RAC1 have a regulatory function on starvation-mediated autophagy, but with opposite roles. Indeed, RHOA has an activatory role whereas Rac has an inhibitory one. We have also found that inhibition of the RHOA effector ROCK impaired the starvation-mediated autophagic response. We propose that actin participates in the initial membrane remodeling stage when cells require an enhanced rate of autophagosome formation, and this actin function would be tightly regulated by different members of the Rho family.

2-Aminoethoxydiphenyl borate (2-APB) reduces alkaline phosphatase release, CD63 expression, F-actin polymerization and chemotaxis without affecting the phagocytosis activity in bovine neutrophils.

2-Aminoethoxydiphenyl borate (2-APB) interferes with the Ca(2+) influx and reduces the ROS production, gelatinase secretion and CD11b expression in bovine neutrophils. Moreover, it has been suggested that inhibition of the Ca(2+) channel involved in the store operated Ca(2+) entry (SOCE) is a potential target for the development of new anti-inflammatory drugs in cattle, however it is unknown whether 2-APB affects neutrophil functions associated with the innate immune response. This study describes the effect of 2-APB, a putative SOCE inhibitor, on alkaline phosphatase activity a marker of secretory vesicles, CD63 a marker for azurophil granules, F-actin polymerization and in vitro chemotaxis in bovine neutrophils stimulated with platelet-activating factor (PAF). Also, we evaluated the effect of 2-APB in the phagocytic activity against Escherichia coli and Staphylococcus aureus bioparticles. We observed that doses of 2-APB ≥10 µM significantly reduced alkaline phosphatase activity and in vitro chemotaxis, whereas concentrations of 2-APB ≥50 µM reduced CD63 expression and F-actin polymerization. Finally, we observed that 2-APB did not affect the phagocytic activity in neutrophils incubated with E. coli and S. aureus bioparticles. We concluded that inhibition of Ca(2+) influx could be a useful strategy to reduce inflammatory process in cattle.