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1.
Cancer Lett ; 524: 131-143, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34678434

RESUMO

Aberrant angiogenesis is a hallmark of cancer and is critically associated with tumor progression. Perivascular cells are essential components of blood vessels, and the role of tumor perivascular cell-derived extracellular vesicles (TPC-EVs) in angiogenesis remains elusive. In the present study, using genetic mouse models and pharmacological inhibitors, we found that ablation of perivascular cells inhibited angiogenesis in allografted colorectal cancer tumors. Further studies demonstrated that TPC-EVs promoted the proliferation, migration, invasion, viability, and tube formation of HUVECs. They also facilitated vessel spouting in rat aortic rings and induced neovascularization in chick chorioallantoic membranes (CAMs). Silencing of Gas6 or blockade of the Axl pathway suppressed TPC-EV-induced angiogenesis in vitro and ex vivo. Moreover, inhibition of the Gas6/Axl signaling pathway impaired TPC-EV-mediated angiogenesis in vivo. Our findings present a deeper insight into the biological functions of TPCs and TPC-EVs in tumor angiogenesis and demonstrate that TPC-EV-derived Gas6 could be an attractive and innovative regulator of tumor angiogenesis.


Assuntos
Neoplasias Colorretais/genética , Vesículas Extracelulares/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Animais , Aorta/crescimento & desenvolvimento , Aorta/patologia , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Galinhas , Membrana Corioalantoide/enzimologia , Membrana Corioalantoide/patologia , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neovascularização Patológica/patologia , Neoplasias de Células Epitelioides Perivasculares/genética , Neoplasias de Células Epitelioides Perivasculares/patologia , Ratos
2.
PLoS One ; 16(12): e0261122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34914770

RESUMO

Fowlpox (FP) is an economically important viral disease of commercial poultry. The fowlpox virus (FPV) is primarily characterised by immunoblotting, restriction enzyme analysis in combination with PCR, and/or nucleotide sequencing of amplicons. Whole-genome sequencing (WGS) of FPV directly from clinical specimens prevents the risk of potential genome modifications associated with in vitro culturing of the virus. Only one study has sequenced FPV genomes directly from clinical samples using Nanopore sequencing, however, the study didn't compare the sequences against Illumina sequencing or laboratory propagated sequences. Here, the suitability of WGS for strain identification of FPV directly from cutaneous tissue was evaluated, using a combination of Illumina and Nanopore sequencing technologies. Sequencing results were compared with the sequence obtained from FPV grown in chorioallantoic membranes (CAMs) of chicken embryos. Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of single nucleotide polymorphisms (SNPs) in the two sequences when compared to the reference genome, providing essentially the same strain identification information. Comparative genome analysis of the map to reference consensus sequences from the two genomes revealed that this field isolate had the highest nucleotide identity of 99.5% with an FPV strain from the USA (Fowlpox virus isolate, FWPV-MN00.2, MH709124) and 98.8% identity with the Australian FPV vaccine strain (FWPV-S, MW142017). Sequencing results showed that WGS directly from cutaneous tissues is not only rapid and cost-effective but also provides essentially the same strain identification information as in-vitro grown virus, thus circumventing in vitro culturing.


Assuntos
Membrana Corioalantoide/virologia , Vírus da Varíola das Aves Domésticas/isolamento & purificação , Varíola Aviária/diagnóstico , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Pele/virologia , Sequenciamento Completo do Genoma/métodos , Animais , Austrália , Embrião de Galinha , Galinhas , Varíola Aviária/virologia , Vírus da Varíola das Aves Domésticas/classificação , Vírus da Varíola das Aves Domésticas/genética , Vírus da Varíola das Aves Domésticas/crescimento & desenvolvimento , Polimorfismo Genético
3.
Biomater Sci ; 9(21): 7297-7310, 2021 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-34617526

RESUMO

Wound healing involves a complex series of events where cell-cell and cell-extracellular matrix (ECM) interactions play a key role. Wounding can be simple, such as the loss of the epithelial integrity, or deeper and more complex, reaching to subcutaneous tissues, including blood vessels, muscles and nerves. Rapid neovascularisation of the wounded area is crucial for wound healing as it has a key role in supplying oxygen and nutrients during the highly demanding proliferative phase and transmigration of inflammatory cells to the wound area. One approach to circumvent delayed neovascularisation is the exogenous use of pro-angiogenic factors, which is expensive, highly dose-dependent, and the delivery of them requires a very well-controlled system to avoid leaky, highly permeable and haemorrhagic blood vessel formation. In this study, we decorated polycaprolactone (PCL)-based polymerised high internal phase emulsion (PolyHIPE) scaffolds with fibroblast-derived ECM to assess fibroblast, endothelial cell and keratinocyte activity in vitro and angiogenesis in ex ovo chick chorioallantoic membrane (CAM) assays. Our results showed that the inclusion of ECM in the scaffolds increased the metabolic activity of three types of cells that play a key role in wound healing and stimulated angiogenesis in ex ovo CAM assays over 7 days. Herein, we demonstrated that fibroblast-ECM functionalised PCL PolyHIPE scaffolds appear to have great potential to be used as an active wound dressing to promote angiogenesis and wound healing.


Assuntos
Matriz Extracelular , Neovascularização Fisiológica , Animais , Membrana Corioalantoide , Polímeros , Estirenos
4.
Molecules ; 26(20)2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34684822

RESUMO

Cyclopeptidic photosensitizer prodrugs (cPPPs) are compounds designed to specifically target overexpressed hydrolases such as serine proteases, resulting in their specific activation in close proximity to tumor cells. In this study, we explored a series of conjugates that can be selectively activated by the urokinase plasminogen activator (uPA). They differ from each other by their pheophorbide a (Pha) loading, their number of PEG chains and the eventual presence of black hole quenchers (BHQ3). The involvement of a peptidic linker between the drugs and the cyclopeptidic carrier allows specific cleavage by uPA. Restoration of the photophysical activity was observed in vitro on A549 lung and MCF7 breast cancer cells that exhibited an increase in red fluorescence emission up to 5.1-fold and 7.8-fold, respectively for uPA-cPPQ2+2/5. While these cPPP conjugates do not show dark toxicity, they revealed their phototoxic potential in both cell lines at 5 µM of Phaeq and a blue light fluence of 12.7 J/cm2 that resulted in complete cell death with almost all conjugates. This suggests, in addition to the promising use for cancer diagnosis, a use as a PDT agent. Intravenous injection of tetrasubstituted conjugates in fertilized hen eggs bearing a lung cancer nodule (A549) showed that a double PEGylation was favorable for the selective accumulation of the unquenched Pha moieties in the tumor nodules. Indeed, the diPEGylated uPA-cPPP4/52 induced a 5.2-fold increase in fluorescence, while the monoPEGylated uPA-cPPP4/5 or uPA-cPPQ2+2/5 led to a 0.4-fold increase only.


Assuntos
Membrana Corioalantoide/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Pró-Fármacos/metabolismo , Células A549 , Animais , Transporte Biológico Ativo , Embrião de Galinha , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Técnicas In Vitro , Células MCF-7 , Modelos Biológicos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacocinética , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacocinética , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacocinética , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638895

RESUMO

Beta-Caryophyllene (BCP), a naturally occurring sesquiterpene abundantly found in cloves, hops, and cannabis, is the active candidate of a relatively new group of vascular-inhibiting compounds that aim to block existing tumor blood vessels. Previously, we have reported the anti-cancer properties of BCP by utilizing a series of in-vitro anti-tumor-related assays using human colorectal carcinoma cells. The present study aimed to investigate the effects of BCP on in-vitro, ex-vivo, and in-vivo models of anti-angiogenic assays and evaluate its anti-cancer activity in xenograft tumor (both ectopic and orthotopic) mice models of human colorectal cancer. Computational structural analysis and an apoptosis antibody array were also performed to understand the molecular players underlying this effect. BCP exhibited strong anti-angiogenic activity by blocking the migration of endothelial cells, tube-like network formation, suppression of vascular endothelial growth factor (VEGF) secretion from human umbilical vein endothelial cells and sprouting of rat aorta microvessels. BCP has a probable binding at Site#0 on the surface of VEGFR2. Moreover, BCP significantly deformed the vascularization architecture compared to the negative control in a chick embryo chorioallantoic membrane assay. BCP showed a remarkable reduction in tumor size and fluorescence molecular tomography signal intensity in all the mice treated with BCP, in a dose-dependent relationship, in ectopic and orthotopic tumor xenograft models, respectively. The histological analysis of the tumor from BCP-treated mice revealed a clear reduction of the density of vascularization. In addition, BCP induced apoptosis through downregulation of HSP60, HTRA, survivin, and XIAP, along with the upregulation of p21 expressions. These results suggest that BCP acts at multiple stages of angiogenesis and could be used as a promising therapeutic candidate to halt the growth of colorectal tumor cells.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Colorretais/prevenção & controle , Neovascularização Patológica/prevenção & controle , Sesquiterpenos Policíclicos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Neoplasias Colorretais/irrigação sanguínea , Células HCT116 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos Nus , Microvasos/efeitos dos fármacos , Ratos Sprague-Dawley
6.
Clin Hemorheol Microcirc ; 79(1): 157-166, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34487030

RESUMO

BACKGROUND: Breast cancer is the most common malignant tumor in women and highly heterogeneous with a variety of different molecular subtypes. The analysis of the individual tumor biology is necessary to develop a specific and individualized treatment plan for every patient. The chick chorioallantoic membrane (CAM) model, a 3D-in-vivo-tumor-model, could potentially provide a methodology that facilitates the gain of additional information regarding the tumor biology as well as the testing of the tumor's individual sensitivity to different therapies. OBJECTIVE: The objective was to establish the grafting of different breast cancer primaries onto the CAM for tumor profiling and the investigation of different parameters. METHODS: Breast cancer primary tissue of different patients was grafted onto the CAM. Subsequently, 3D volume and perfusion measurements were performed during the engraftment period. Histological analyses of the tumors were carried out after the engraftment period. RESULTS: The grafting of the breast cancer primaries onto the CAM was successful. The tumors remained partially vital and displayed angiogenic development on the CAM. CONCLUSIONS: Breast cancer primary material can be grafted onto the CAM and we observed visible and measurable changes of perfusion over time.


Assuntos
Neoplasias da Mama , Animais , Galinhas , Membrana Corioalantoide , Feminino , Humanos
7.
In Vivo ; 35(5): 2711-2718, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34410960

RESUMO

BACKGROUND/AIM: Colon cancer liver metastases with desmoplastic growth pattern (dGP) have a highly heterogeneous therapy response. The aim of the study was to evaluate the dGP liver metastasis molecular profile from a chemo-naive patient by mimicking metastatic process on an experimental chick embryo chorioallantoic membrane (CAM) model. MATERIALS AND METHODS: Three successive CAM passages of dGP human colorectal liver metastases were immunophenotyped for keratin (K) 8, and 20, CLIC1, VEGF, EGFR, CD34, podoplanin, Ki67, E-cadherin and vimentin. RESULTS: Metastatic cells gradually lost K20 while K8, E-cadherin and vimentin heterogeneously increased during passages. VEGF, CLIC 1, EGFR expression increased in metastatic cells especially at the tumor graft periphery. Scattered proliferating and non-proliferating podoplanin-positive tumor cells, lymphatic and blood vessels were heterogeneously detected in tumor xenografts depending on passage stage. CONCLUSION: By mimicking repetitive metastatic processes we proved that metastatic cells change their phenotype. This may explain why not all metastases have a similar response to therapy.


Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Animais , Embrião de Galinha , Canais de Cloreto , Membrana Corioalantoide , Neoplasias Colorretais/genética , Humanos , Neoplasias Hepáticas/genética , Fenótipo
8.
Comput Biol Med ; 136: 104647, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34274599

RESUMO

Angiogenesis, the formation of new blood vessels from pre-existing ones, begins during embryonic development and continues throughout life. Sprouting angiogenesis is a well-defined process, being mainly influenced by vascular endothelial growth factor (VEGF). In this study, we propose a meshless-based model capable of mimicking the angiogenic response to several VEGF concentrations. In this model, endothelial cells migrate according to a diffusion-reaction equation, following the VEGF gradient concentration. The chick chorioallantoic membrane (CAM) assay was used to model the branching process and to validate the obtained numerical results. To analyse the angiogenic response, the total vessel number and the total vessel length presented in the CAM images and in the simulations for all the VEGF concentrations tested were quantified. In both the CAM assay and simulation, the treatments with VEGF increased the total vessel number and the total vessel length. The obtained quantitative results were very similar between the two methodologies used. The proposed model accurately simulates the capillary network pattern concerning its structure and morphology, for the lowest VEGF concentration tested. For the highest VEGF concentration, the capillary network predicted by the model was less accurate when compared to the one presented in the CAM assay but this may be explained by changes in blood vessel width at higher VEGF concentrations. This remains to be tested.


Assuntos
Membrana Corioalantoide , Neovascularização Fisiológica , Animais , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Células Endoteliais , Fator A de Crescimento do Endotélio Vascular
9.
Biomolecules ; 11(6)2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199986

RESUMO

The natural product elaiophylin is a macrodiolide with a broad range of biological activities. However, no direct target of elaiophylin in eukaryotes has been described so far, which hinders a systematic explanation of its astonishing activity range. We recently showed that the related conglobatin A, a protein-protein interface inhibitor of the interaction between the N-terminus of Hsp90 and its cochaperone Cdc37, blocks cancer stem cell properties by selectively inhibiting K-Ras4B but not H-Ras. Here, we elaborated that elaiophylin likewise disrupts the Hsp90/ Cdc37 interaction, without affecting the ATP-pocket of Hsp90. Similarly to conglobatin A, elaiophylin decreased expression levels of the Hsp90 client HIF1α, a transcription factor with various downstream targets, including galectin-3. Galectin-3 is a nanocluster scaffold of K-Ras, which explains the K-Ras selectivity of Hsp90 inhibitors. In agreement with this K-Ras targeting and the potent effect on other Hsp90 clients, we observed with elaiophylin treatment a submicromolar IC50 for MDA-MB-231 and MIA-PaCa-2 3D spheroid formation. Finally, a strong inhibition of MDA-MB-231 cells grown in the chorioallantoic membrane (CAM) microtumor model was determined. These results suggest that several other macrodiolides may have the Hsp90/ Cdc37 interface as a target site.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Chaperoninas/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Macrolídeos/farmacologia , Nanoconjugados , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Galinhas , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Macrolídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
10.
Molecules ; 26(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072397

RESUMO

The formation of new scaffolds to enhance healing magnitude is necessarily required in biomedical applications. Granulation tissue formation is a crucial stage of wound healing in which granulation tissue grows on the surface of a wound by the formation of connective tissue and blood vessels. In the present study, porous hydrogels were synthesized using chitosan incorporating latex of the Calotropis procera plant by using a freeze-thaw cycle to stimulate the formation of granulation tissue and angiogenesis in wound healing applications. Structural analysis through Fourier transform infrared (FTIR) spectroscopy confirmed the interaction between chitosan and Calotropis procera. Latex extract containing hydrogel showed slightly higher absorption than the control during water absorption analysis. Thermogravimetric analysis showed high thermal stability of the 60:40 combination of chitosan (CS) and Calotropis procera as compared to all other treatments and controls. A fabricated scaffold application on a chick chorioallantoic membrane (CAM) showed that all hydrogels containing latex extract resulted in a significant formation of blood vessels and regeneration of cells. Overall, the formation of connective tissues and blood capillaries and healing magnitude decreased in ascending order of concentration of extract.


Assuntos
Calotropis/metabolismo , Quitosana/química , Hidrogéis/química , Neovascularização Fisiológica , Cicatrização , Animais , Materiais Biocompatíveis , Embrião de Galinha , Membrana Corioalantoide/metabolismo , Congelamento , Látex/química , Teste de Materiais , Microscopia Eletrônica de Varredura , Extratos Vegetais/química , Polímeros/química , Regeneração , Espectroscopia de Infravermelho com Transformada de Fourier , Termogravimetria
11.
PLoS One ; 16(6): e0247471, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086696

RESUMO

Although different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. Compared to the other routes of viral vector administration, the embryo's bloodstream at Hamburger-Hamilton (HH) stages 14-15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14-15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.


Assuntos
Galinhas/genética , Quimera/genética , Células Germinativas/fisiologia , Animais , Animais Geneticamente Modificados , Membrana Corioalantoide/fisiologia , Técnicas de Transferência de Genes , Técnicas Genéticas , Vetores Genéticos/genética , Lentivirus/genética , Transgenes/genética
12.
PLoS One ; 16(6): e0252233, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34077449

RESUMO

Tumor angiogenesis is critical for the growth and progression of cancer. As such, angiostasis is a treatment modality for cancer with potential utility for multiple types of cancer and fewer side effects. However, clinical success of angiostatic monotherapies has been moderate, at best, causing angiostatic treatments to lose their early luster. Previous studies demonstrated compensatory mechanisms that drive tumor vascularization despite the use of angiostatic monotherapies, as well as the potential for combination angiostatic therapies to overcome these compensatory mechanisms. We screened clinically approved angiostatics to identify specific combinations that confer potent inhibition of tumor-induced angiogenesis. We used a novel modification of the ex ovo chick chorioallantoic membrane (CAM) model that combined confocal and automated analyses to quantify tumor angiogenesis induced by glioblastoma tumor onplants. This model is advantageous due to its low cost and moderate throughput capabilities, while maintaining complex in vivo cellular interactions that are difficult to replicate in vitro. After screening multiple combinations, we determined that glioblastoma-induced angiogenesis was significantly reduced using a combination of bevacizumab (Avastin®) and temsirolimus (Torisel®) at doses below those where neither monotherapy demonstrated activity. These preliminary results were verified extensively, with this combination therapy effective even at concentrations further reduced 10-fold with a CI value of 2.42E-5, demonstrating high levels of synergy. Thus, combining bevacizumab and temsirolimus has great potential to increase the efficacy of angiostatic therapy and lower required dosing for improved clinical success and reduced side effects in glioblastoma patients.


Assuntos
Inibidores da Angiogênese/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Membrana Corioalantoide/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Glioblastoma/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Animais , Bevacizumab/administração & dosagem , Galinhas , Membrana Corioalantoide/patologia , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Humanos , Neovascularização Patológica/patologia , Ratos , Sirolimo/administração & dosagem , Sirolimo/análogos & derivados , Células Tumorais Cultivadas
13.
J Cancer Res Ther ; 17(2): 484-490, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34121696

RESUMO

Introduction: Andrographis echioides is a prevalently used medicinal herb in South Asian countries. Scientific researches with the extracts of A. echioides revealed its antipyretic, anti-inflammatory, antimicrobial, ulceroprotective, and hepatoprotective properties. This study was done to elucidate antiproliferative and antiangiogenic potential of ethanolic extracts of A. echioides (EEAE) by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and chorioallantoic membrane (CAM) assay. Materials and Methods: EEAE was prepared using Soxhlet apparatus with ethanol after being sun-dried and powdered. MCF 7 (human invasive breast ductal carcinoma) cell lines retaining attributes of differentiated mammary epithelium with both estrogen and progesterone receptors were treated with EEAE, and antiproliferative effect was seen using Mosmann method of MTT assay using 5-fluorouracil (5-FU) as a comparator. The evaluation of antiangiogenic potential of EEAE was done by comparing mean vessel density (MVD) in chick CAM after treatment with EEAE, thalidomide, and vascular endothelial growth factor (VEGF) using CAM assay, an in ovo assay. Results: EEAE displayed antiproliferative activity from low to high concentrations with MTT assay. The IC50 of EEAE and 5-FU was 62.5 and 15.6 µg/ml, respectively (P < 0.05). The exhibition of its antiangiogenic activity increased proportionately with increasing concentration. VEGF increased MVD by 45.94%; thalidomide decreased it by 53.76%. There was a decrease of MVD by 5.91%, 20.46%, and 35.95% at concentrations of 25, 50, and 100 µg of EEAE, respectively. Conclusion: EEAE possessed significant antiangiogenic and antiproliferative activity, making them a promising substrate in the development of a novel anticancer drug and can be successfully used in the therapy of various cancers after establishment of the anticancer effects in animal models and subsequently in clinical trials.


Assuntos
Andrographis/química , Inibidores da Angiogênese/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Extratos Vegetais/farmacologia , Inibidores da Angiogênese/isolamento & purificação , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide , Ensaios de Seleção de Medicamentos Antitumorais , Etanol/química , Humanos , Células MCF-7 , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Extratos Vegetais/isolamento & purificação
14.
Exp Cell Res ; 405(2): 112716, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34186097

RESUMO

The CAM is a widely used experimental assay to study angiogenesis, wound healing, tumor growth and metastatic process. In this study, we have analyzed and compared the existent literature data concerning the growth of the CAM. Moreover, we have analyzed the data concerning the development of the vascular system and the expression of the most important pro-angiogenic and anti-angiogenic factors. The availability of these data and their comparative evaluation allow to better analyze the experimental data concerning the testing of different pro-angiogenic and anti-angiogenic molecules, as well as biomaterials in the CAM assay. Moreover, the dynamic of the angiogenic response to different tumor cell lines and or tumor bioptic specimens, may be also better evaluated and estimated.


Assuntos
Inibidores da Angiogênese/metabolismo , Bioensaio , Membrana Corioalantoide/metabolismo , Neovascularização Patológica/metabolismo , Animais , Bioensaio/métodos , Linhagem Celular Tumoral , Humanos , Neovascularização Fisiológica/fisiologia
15.
J Ethnopharmacol ; 277: 114215, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34033902

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Hypoxia will cause an increase in the rate of fatigue and aging. Chinese cordyceps, a parasitic Thitarodes insect-Ophiocordyceps sinensis fungus complex in the Qinghai-Tibet Plateau, has long been used to ameliorate human conditions associated with aging and senescence, it is principally applied to treat fatigue, night sweating and other symptoms related to aging, and it may play the anti-aging and anti-fatigue effect by improving the body's hypoxia tolerance. AIMS OF THE STUDY: The present study investigated the anti-hypoxia activity of Chinese cordyceps and explore the main corresponding signal pathways and bioactive compounds. MATERIALS AND METHODS: In this study, network pharmacology analysis, molecular docking, cell and whole pharmacodynamic experiments were hired to study the major signal pathways and the bioactive compounds of Chinese cordyceps for anti-hypoxia activity. RESULTS: 17 pathways which Chinese cordyceps acted on seemed to be related to the anti-hypoxia effect, and "VEGF signal pathway" was one of the most important pathway. Chinese cordyceps improved the survival rate and regulated the targets related VEGF signal pathway of H9C2 cells under hypoxia, and also had significant anti-hypoxia effects to mice. Chorioallantoic membrane model experiment showed that Chinese cordyceps and the main constituents of (9Z,12Z)-octadeca-9,12-dienoic acid and cerevisterol had significant angiogenic activity in hypoxia condition. CONCLUSION: Based on the results of network pharmacology and molecular docking analysis, cell and whole pharmacodynamic experiments, promoting angiogenesis by regulating VEGF signal pathway might be one of the mechanisms of anti-hypoxia effect of Chinese cordyceps, (9Z, 12Z)-octadeca-9,12-dienoic acid and cerevisterol were considered as the major anti-hypoxia bioactive compounds in Chinese cordyceps.


Assuntos
Cordyceps/química , Hipóxia/tratamento farmacológico , Fitosteróis/farmacologia , Animais , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Simulação de Acoplamento Molecular , Fitosteróis/isolamento & purificação , Transdução de Sinais/efeitos dos fármacos
16.
Nanotoxicology ; 15(5): 690-705, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33979554

RESUMO

Toxicity tests in rodents are still considered a controversial topic concerning their ethical justifiability. The chick embryo chorioallantoic membrane (CAM) assay may offer a simple and inexpensive alternative. The CAM assay is easy to perform and has low bureaucratic hurdles. At the same time, the CAM assay allows the application of a broad variety of analytical methods in the field of nanotoxicological research. We evaluated the CAM assay as a methodology for the determination of nanotoxicity. Therefore we calculated the median lethal dose (LD50), performed in vivo microscopy and immunohistochemistry to identify organ-specific accumulation profiles, potential organ damage, and the kinetics of the in vivo circulation of the nanoparticles. Zinc oxide nanoparticles were intravascularly injected on day 10 of the egg development and showed an LD50 of 17.5 µM (1.4 µg/mLeggcontent). In comparison, the LD50 of equivalent amounts of Zn2+ was 4.6 µM (0.6 µg/mLeggcontent). Silica encapsulated ZnO@SiO2 nanoparticles conjugated with fluorescein circulated in the bloodstream for at least 24 h. Particles accumulated mostly in the liver and kidney. In immunohistochemical staining, organ damage was detected only in liver tissue after intravascular injection of zinc oxide nanoparticles in very high concentrations. Zinc oxide nanoparticles showed a different pharmacokinetic profile compared to Zn2+ ions. In conclusion, the CAM assay has proven to be a promising methodology for evaluating nanotoxicity and for the assessment of the in vivo accumulation profiles of nanoparticles. These findings may qualify the methodology for risk assessment of innovative nanotherapeutics in the future.


Assuntos
Nanopartículas , Óxido de Zinco , Animais , Bioensaio , Embrião de Galinha , Membrana Corioalantoide , Nanopartículas/toxicidade , Dióxido de Silício
17.
Biomed Res Int ; 2021: 6654683, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33997037

RESUMO

Chick (CE) or duck embryo eggs are known for nutritional supplement foods in traditional East countries for physical fitness enhancement and postpartum conditioning for many years. In this study, we evaluated the effects of different parts of the 10-day CE (embryo: CEr, yolk: CEw, and chorioallantoic membrane: CEp) on the antifatigue and antiaging activities in a D-galactose- (D-gal) induced aging mice model. The results showed CEp obviously increased the muscle weight and the liver and muscle glycogen content and enhanced exercise performance. In the antiaging assay, CEp significantly increased the activity of superoxide dismutase (SOD) and Glutathione Peroxidase (GPx). Moreover, the immunohistochemistry results of NRF-2 and HO-1 were also detected in the livers of mice in the D-gal/CEp group. The only partially potential such as CEr might improve OFT function with TG level, and CEw had strange grip strength. Therefore, we suggest that CEp has a potent antifatigue ability and could minimize the occurrence of age-associated disorders, more than other parts of the 10 days chicken embryo egg.


Assuntos
Envelhecimento/efeitos dos fármacos , Produtos Biológicos/farmacologia , Embrião de Galinha , Suplementos Nutricionais , Animais , Membrana Corioalantoide/química , Gema de Ovo/química , Galactose/efeitos adversos , Força da Mão , Fígado/química , Fígado/efeitos dos fármacos , Masculino , Medicina Tradicional Chinesa , Camundongos , Camundongos Endogâmicos C57BL , Força Muscular/efeitos dos fármacos , Músculos/efeitos dos fármacos , Músculos/metabolismo , Superóxido Dismutase/metabolismo
18.
PLoS One ; 16(5): e0251765, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33999935

RESUMO

Since growing tumors stimulate angiogenesis, via vascular endothelial growth factor (VEGF), angiogenesis inhibitors (AIs, blockers of the VEGF signaling pathway) have been introduced to cancer therapy. However, AIs often yielded only modest and short-lived gains in cancer patients and more invasive tumor phenotypes in animal models. Combining anti-VEGF strategies with lactate uptake blockers may boost both efficacy and safety of AIs. We assessed this hypothesis by using the ex ovo chorioallantoic membrane (CAM) assay. We show that AI-based monotherapy (Avastin®, AVA) increases tumor hypoxia in human CAM cancer cell xenografts and cell spread in human as well as canine CAM cancer cell xenografts. In contrast, combining AVA treatment with lactate importer MCT1 inhibitors (α-cyano-4-hydroxycinnamic acid (CHC) or AZD3965 (AZD)) reduced both tumor growth and cell dissemination of human and canine explants. Moreover, combining AVA+AZD diminished blood perfusion and tumor hypoxia in human explants. Thus, the ex ovo CAM assay as an easy, fast and cheap experimental setup is useful for pre-clinical cancer research. Moreover, as an animal-free experimental setup the CAM assay can reduce the high number of laboratory animals used in pre-clinical cancer research.


Assuntos
Inibidores da Angiogênese/farmacologia , Membrana Corioalantoide , Neoplasias Experimentais , Neovascularização Patológica , Consumo de Oxigênio/efeitos dos fármacos , Pirimidinonas/farmacologia , Tiofenos/farmacologia , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/metabolismo , Membrana Corioalantoide/patologia , Cães , Humanos , Camundongos , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Int J Biol Macromol ; 183: 695-706, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-33932419

RESUMO

Implantation of biomaterials and hybrid constructs in tissue engineering approaches presents major limitations such as inflammatory reaction and the lack of vasculature integration. Therefore, new strategies are needed to enhance implant function, immune protection, and revascularization. In this work, we developed fibrous meshes composed of fucoidan (Fu), a sulfated polysaccharide extracted from brown algae, and polycaprolactone (PCL), a synthetic biodegradable polymer, using the airbrush technique. The chemical characterization by FTIR, EDS, and XPS confirmed the presence of the two polymers in the structure of airbrushed nanofibrous meshes (ANFM). Moreover, these nanofibrous exhibited good wettability and mechanical properties envisaging their application as templates for biomaterials and cell culture. The developed ANFM were directly cultured with human pulmonary microvascular endothelial (HPMEC-ST1.6R) cells for up to 7 days. Biological results demonstrated that ANFM comprising Fu promoted cellular attachment, spreading, and proliferation of human endothelial cells. The angiogenic potential of ANFM was further evaluated by onplantation of PCL and PCL/Fu ANFM in chick chorioallantoic membrane (CAM). In ovo and ex ovo results showed that the incorporation of Fu increased the pro-angiogenic potential of ANFM. Altogether, the results suggest that airbrush biocomposite meshes could be used as a biomaterial substrate to promote vascularization.


Assuntos
Indutores da Angiogênese/farmacologia , Membrana Corioalantoide/irrigação sanguínea , Pulmão/irrigação sanguínea , Poliésteres/química , Polissacarídeos/farmacologia , Indutores da Angiogênese/química , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Microvasos/citologia , Microvasos/efeitos dos fármacos , Nanofibras , Polissacarídeos/química , Telas Cirúrgicas , Engenharia Tecidual
20.
Molecules ; 26(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805741

RESUMO

A series of novel functionalized methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates 2a-2h were synthesized by C-C Pd-catalyzed Suzuki-Miyaura cross-coupling of methyl 3-bromothieno[3,2-b]pyridine-2-carboxylate with (hetero)aryl pinacol boranes, trifluoro potassium boronate salts or boronic acids. Their antitumoral potential was evaluated in two triple negative breast cancer (TNBC) cell lines-MDA-MB-231 and MDA-MB-468, by sulforhodamine B assay. Their effects on the non-tumorigenic MCF-12A cells were also evaluated. The results demonstrated that three compounds caused growth inhibition in both TNBC cell lines, with little or no effect against the non-tumorigenic cells. The most promising compound was further studied concerning possible effects on cell viability (by trypan blue exclusion assay), cell proliferation (by bromodeoxyuridine assay) and cell cycle profile (by flow cytometry). The results demonstrated that the GI50 concentration of compound 2e (13 µM) caused a decreased in MDA-MB-231 cell number, which was correlated with a decreased in the % of proliferating cells. Moreover, this compound increased G0/G1 phase and decreased S phases, when compared to control cells (although was not statistic significant). Interestingly, compound 2e also reduced tumor size using an in ovo CAM (chick chorioallantoic membrane) model. This work highlights the potential antitumor effect of a novel methyl 3-arylthieno[3,2-b]pyridine-2-carboxylate derivative.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Tienopiridinas/síntese química , Tienopiridinas/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Membrana Corioalantoide/cirurgia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/patologia , Estrutura Molecular , Transplante de Neoplasias , Relação Estrutura-Atividade , Tienopiridinas/química , Neoplasias de Mama Triplo Negativas/patologia
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