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1.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281277

RESUMO

The aim of this research was to analyze the heterologous expression, purification, and immunoregulatory activity of recombinant YGP40 (rYGP40), the potential precursor of the yolkin peptide complex. The ygp40 coding sequence was codon optimized, successfully expressed in the E. coli system, and purified from inclusion bodies with a yield of about 1.1 mg/L of culture. This study showed that the protein exhibits immunomodulatory activity, expressed by the stimulation of TNF-α and IL-10 production and nitric oxide induction at a level comparable to that of the natural yolkin peptide complex obtained by other authors from hen egg yolk. At the highest dose of 100 µg/mL, rYGP40 also caused the up-regulation of iNOS expression in murine bone marrow-derived macrophages (BMDM). Moreover, no cytotoxic effects of rYGP40 on the BMDM cell line were observed.


Assuntos
Vitelogeninas/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Clonagem Molecular , Gema de Ovo/química , Feminino , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/genética , Fatores Imunológicos/farmacologia , Técnicas In Vitro , Interleucina-10/biossíntese , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Peso Molecular , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Vitelogeninas/genética , Vitelogeninas/farmacologia
2.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34198981

RESUMO

Recently, adipose-derived stem cells (ADSCs) are considered to be ideal for application in cell therapy or tissue regeneration, mainly due to their wide availability and easy access. In this study, we examined the anti-inflammatory effects of membrane-free stem cell extract (MFSC-Ex) derived from ADSCs against lipopolysaccharide (LPS)/interferon-gamma (IFN-γ) on RAW 264.7 macrophage cells. Exposure of RAW macrophages to LPS and IFN-γ stimuli induced high levels of nitric oxide (NO), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) production. However, pretreatment with MFSC-Ex inhibited LPS/IFN-γ-induced these pro-inflammatory mediators. To clarify the molecular mechanisms underlying the anti-inflammatory property of MFSC-Ex, we analyzed nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) protein expressions by Western blotting. Our study showed that treatment of MFSC-Ex significantly down-regulated inducible nitric oxide synthase (iNOS) and COX-2 protein expressions. Furthermore, phosphorylation of extracellular signal-regulated kinase (ERK) and p38 was also blocked by treatment with MFSC-Ex, indicating that inhibitory effect of MFSC-Ex on MAPK signaling cascade may attribute to inactivation of NF-κB. From these findings, we suggest that MFSC-Ex exert anti-inflammatory activities, which suppressed LPS/IFN-γ-induced production of NO, COX-2 and PGE2 by regulation of NF-κB and MAPK signaling pathway in RAW 264.7 macrophages. In conclusion, MFSC-Ex might provide a new therapeutic opportunity to treatment of inflammatory-related diseases.


Assuntos
Tecido Adiposo/citologia , Anti-Inflamatórios/farmacologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Células-Tronco/metabolismo , Animais , Anti-Inflamatórios/química , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/biossíntese , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Camundongos , Modelos Biológicos , Substâncias Protetoras/metabolismo , Células RAW 264.7
3.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199016

RESUMO

Paeonia suffruticosa is a magnificent and long-lived woody plant that has traditionally been used to treat various diseases including inflammatory, neurological, cancer, and cardiovascular diseases. In the present study, we demonstrated the biological mechanisms of paeonoside (PASI) isolated from the dried roots of P. suffruticosa in pre-osteoblasts. Herein, we found that PASI has no cytotoxic effects on pre-osteoblasts. Migration assay showed that PASI promoted wound healing and transmigration in osteoblast differentiation. PASI increased early osteoblast differentiation and mineralized nodule formation. In addition, PASI enhanced the expression of Wnt3a and bone morphogenetic protein 2 (BMP2) and activated their downstream molecules, Smad1/5/8 and ß-catenin, leading to increases in runt-related transcription factor 2 (RUNX2) expression during osteoblast differentiation. Furthermore, PASI-mediated osteoblast differentiation was attenuated by inhibiting the BMP2 and Wnt3a pathways, which was accompanied by reduction in the expression of RUNX2 in the nucleus. Taken together, our findings provide evidence that PASI enhances osteoblast differentiation and mineralized nodules by regulating RUNX2 expression through the BMP2 and Wnt3a pathways, suggesting a potential role for PASI targeting osteoblasts to treat bone diseases including osteoporosis and periodontitis.


Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Glicosídeos/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Extratos Vegetais/farmacologia , Biomarcadores , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Glicosídeos/química , Humanos , Imuno-Histoquímica , Espectroscopia de Ressonância Magnética/efeitos adversos , Osteogênese/efeitos dos fármacos , Extratos Vegetais/química , Via de Sinalização Wnt
4.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199017

RESUMO

Venoms are a rich source of potential lead compounds for drug discovery, and descriptive studies of venom form the first phase of the biodiscovery process. In this study, we investigated the pharmacological potential of crude Pseudocerastes and Eristicophis snake venoms in haematological disorders and cancer treatment. We assessed their antithrombotic potential using fibrinogen thromboelastography, fibrinogen gels with and without protease inhibitors, and colourimetric fibrinolysis assays. These assays indicated that the anticoagulant properties of the venoms are likely induced by the hydrolysis of phospholipids and by selective fibrinogenolysis. Furthermore, while most fibrinogenolysis occurred by the direct activity of snake venom metalloproteases and serine proteases, modest evidence indicated that fibrinogenolytic activity may also be mediated by selective venom phospholipases and an inhibitory venom-derived serine protease. We also found that the Pseudocerastes venoms significantly reduced the viability of human melanoma (MM96L) cells by more than 80%, while it had almost no effect on the healthy neonatal foreskin fibroblasts (NFF) as determined by viability assays. The bioactive properties of these venoms suggest that they contain a number of toxins suitable for downstream pharmacological development as candidates for antithrombotic or anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Fibrinolíticos/farmacologia , Venenos de Serpentes/farmacologia , Venenos de Víboras/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibrinólise/efeitos dos fármacos , Humanos , Inibidores de Serino Proteinase/farmacologia
5.
Molecules ; 26(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199157

RESUMO

The influence of p-terphenyl polyketides 1-3 from Aspergillus candidus KMM 4676 and cerebroside flavuside B (4) from Penicillium islandicum (=Talaromyces islandicus) against the effect of neurotoxins, rotenone and paraquat, on Neuro-2a cell viability by MTT and LDH release assays and intracellular ROS level, as well as DPPH radical scavenging activity, was investigated. Pre-incubation with compounds significantly diminished the ROS level in rotenone- and paraquat-treated cells. It was shown that the investigated polyketides 1-3 significantly increased the viability of rotenone- and paraquat-treated cells in two of the used assays but they affected only the viability of paraquat-treated cells in the LDH release assay. Flavuside B statistically increased the viability of paraquat-treated cells in both MTT and LDH release assays, however, it increased the viability of rotenone-treated cells in the LDH release assay. Structure-activity relationships for p-terphenyl derivatives, as well as possible mechanisms of cytoprotective action of all studied compounds, were discussed.


Assuntos
Aspergillus/química , Citoproteção/efeitos dos fármacos , Glicoesfingolipídeos/farmacologia , Neuroblastoma/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/toxicidade , Policetídeos/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Herbicidas/toxicidade , Inseticidas/toxicidade , Camundongos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fármacos Neuroprotetores/química , Paraquat/toxicidade , Policetídeos/química , Espécies Reativas de Oxigênio , Rotenona/toxicidade
6.
Molecules ; 26(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199287

RESUMO

High-grade epithelial ovarian cancer is a fatal disease in women frequently associated with drug resistance and poor outcomes. We previously demonstrated that a marine-derived compound MalforminA1 (MA1) was cytotoxic for the breast cancer cell line MCF-7. In this study, we aimed to examine the effect of MA1 on human ovarian cancer cells. The potential cytotoxicity of MA1was tested on cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780CP) ovarian cancer cell lines using AlamarBlue assay, Hoechst dye, flow cytometry, Western blot, and RT-qPCR. MA1 had higher cytotoxic activity on A2780S (IC50 = 0.23 µM) and A2780CP (IC50 = 0.34 µM) cell lines when compared to cisplatin (IC50 = 31.4 µM and 76.9 µM, respectively). Flow cytometry analysis confirmed the cytotoxic effect of MA1. The synergistic effect of the two drugs was obvious, since only 13% of A2780S and 7% of A2780CP cells remained alive after 24 h of treatment with both MA1 and cisplatin. Moreover, we examined the expression of bcl2, p53, caspase3/9 genes at RNA and protein levels using RT-qPCR and Western blot, respectively, to figure out the cell death mechanism induced by MA1. A significant down-regulation in bcl2 and p53 genes was observed in treated cells compared to non-treated cells (p < 0.05), suggesting that MA1 may not follow the canonical pathway to induce apoptosis in ovarian cancer cell lines. MalforminA1 showed promising anticancer activity by inducing cytotoxicity in cisplatin-sensitive and cisplatin-resistant cancer cell lines. Interestingly, a synergistic effect was observed when MA1 was combined with cisplatin, leading to it overcoming its resistance to cisplatin.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cisplatino/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Peptídeos Cíclicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Peptídeos Cíclicos/administração & dosagem
7.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34201962

RESUMO

Sphingosine-1-phosphate (S1P) is a crucial mediator involved in the progression of different cancers, including glioblastoma multiforme (GBM), the most frequent and deadly human brain tumor, characterized by extensive invasiveness and rapid cell growth. Most of GBMs overexpress the epidermal growth factor receptor (EGFR), and we investigated the possible link between S1P and EGFR signaling pathways, focusing on its role in GBM survival, using the U87MG human cell line overexpressing EGFR (EGFR+). We previously demonstrated that EGFR+ cells have higher levels of extracellular S1P and increased sphingosine kinase-1 (SK1) activity than empty vector expressing cells. Notably, we demonstrated that EGFR+ cells are resistant to temozolomide (TMZ), the standard chemotherapeutic drug in GBM treatment, and the inhibition of SK1 or S1P receptors made EGFR+ cells sensitive to TMZ; moreover, exogenous S1P reverted this effect, thus involving extracellular S1P as a survival signal in TMZ resistance in GBM cells. In addition, both PI3K/AKT and MAPK inhibitors markedly reduced cell survival, suggesting that the enhanced resistance to TMZ of EGFR+ cells is dependent on the increased S1P secretion, downstream of the EGFR-ERK-SK1-S1P pathway. Altogether, our study provides evidence of a functional link between S1P and EGFR signaling pathways enhancing the survival properties of GBM cells.


Assuntos
Regulação Neoplásica da Expressão Gênica , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Humanos , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingosina/metabolismo
8.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34201963

RESUMO

Pancreatic ductal adenocarcinoma is a devastating disease with a 5-year overall survival of 9% for all stages. Gemcitabine-based chemoradiotherapy for locally advanced pancreatic cancer is highly toxic. We conducted an in vitro study to determine whether poly(ADP-ribose) polymerase-1 inhibition radiosensitized gemcitabine-based chemotherapy. Human pancreatic cancer cell lines, MIA PaCa-2, AsPC-1, BxPC-3 and PANC-1 were treated with gemcitabine (10 nM) and/or olaparib (1 µM). Low-LET gamma single dose of 2, 5 and 10 Gy radiations were carried out. Clonogenic assay, PAR immunoblotting, cell cycle distribution, γH2Ax, necrotic and autophagic cell death quantifications were performed. Treatment with olaparib alone was not cytotoxic, but highly radiosensitized cell lines, particularly at high dose per fraction A non-cytotoxic concentration of gemcitabine radiosensitized cells, but less than olaparib. Interestingly, olaparib significantly enhanced gemcitabine-based radiosensitization in PDAC cell lines with synergistic effect in BxPC-3 cell line. All cell lines were radiosensitized by the combination of gemcitabine and olaparib, through an increase of unrepaired double-strand, a G2 phase block and cell death. Radiosensitization was increased with high dose of radiation. The combination of olaparib with gemcitabine-based chemoradiotherapy could lead to an enhancement of local control in vivo and an improvement in disease-free survival.


Assuntos
Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimiorradioterapia , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Histonas/metabolismo , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Radiossensibilizantes/farmacologia
9.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202080

RESUMO

Alveolar macrophages are the first line of defense against intruding pathogens and play a critical role in cancer immunology. The Toll-like receptor (TLR) family mediates an important role in recognizing and mounting an immune response against intruding microbes. TLR-9 is a member of the intracellular TLR family, which recognizes unmethylated CG motifs from the prokaryotic genome. Upon its activation, TLR-9 triggers downstream of the MyD-88-dependent transcriptional activation of NF-κB, and subsequently results in abundant inflammatory cytokines expression that induces a profound inflammatory milieu. The present exploratory investigation aimed at elucidating the potency of schizophyllan for entrapping ODN 1826 (SPG-ODN 1826)-mediated stimulation of TLR-9 in provoking an inflammatory-type response in murine alveolar macrophages. Schizophyllan (SPG), a representative of the ß-glucan family, was used in the present study as a nanovehicle for endosomal trafficking of CpG ODN 1826. TEM analysis of SPG-ODN 1826 nanovehicles revealed that the prepared nanovehicles are spherical and have an average size of about 100 nm. Interestingly, SPG-ODN 1826 nanovehicles were competent in delivering their therapeutic payload within endosomes of murine alveolar macrophage (J774A.1) cells. Exposure of these nanovehicles within LPS stimulated J774A.1, resulted in a significant provocation of reactive oxygen species (ROS) (p < 0.01) in comparison to CpG ODN 1826 alone. Moreover, the formulated nanovehicles succeeded in generating a profound Th1-based cytokine profile constituted by enhanced expression of IFN-γ (p < 0.001) and IL-1ß (p < 0.001) inflammatory cytokines. These findings clearly indicated the immunostimulatory potential of SPG-ODN 1826 nanovehicles for inducing the Th1-type phenotype, which would certainly assist in skewing M2 phenotype into the much-desired M1 type during lung cancer.


Assuntos
Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Nanoestruturas/química , Oligodesoxirribonucleotídeos/química , Sizofirano/química , Receptor Toll-Like 9/agonistas , Animais , Sobrevivência Celular , Citocinas/metabolismo , Endossomos , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Ativação de Macrófagos/imunologia , Camundongos , Nanoestruturas/administração & dosagem , Nanoestruturas/ultraestrutura , Tamanho da Partícula
10.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202736

RESUMO

Regardless of the prophylactic vaccine accessibility, persistent infections of high-risk human papillomaviruses (hr-HPVs), recognized as an etiology of cervical cancers, continues to represent a major health problem for the world population. An overexpression of viral early protein 6 (E6) is linked to carcinogenesis. E6 induces anti-apoptosis by degrading tumor suppressor proteins p53 (p53) via E6-E6-associated protein (E6AP)-mediated polyubiquitination. Thus, the restoration of apoptosis by interfering with the E6 function has been proposed as a selective medicinal strategy. This study aimed to determine the activities of andrographolide (Androg) on the disturbance of E6-mediated p53 degradation in cervical cancer cell lines using a proteomic approach. These results demonstrated that Androg could restore the intracellular p53 level, leading to apoptosis-induced cell death in HPV16-positive cervical cancer cell lines, SiHa and CaSki. Mechanistically, the anti-tumor activity of Androg essentially relied on the reduction in host cell proteins, which are associated with ubiquitin-mediated proteolysis pathways, particularly HERC4 and SMURF2. They are gradually suppressed in Androg-treated HPV16-positive cervical cancer cells. Collectively, the restoration of p53 in HPV16-positive cervical cancer cells might be achieved by disruption of E3 ubiquitin ligase activity by Androg, which could be an alternative treatment for HPV-associated epithelial lesions.


Assuntos
Apoptose/efeitos dos fármacos , Diterpenos/farmacologia , Proteoma , Proteômica , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/metabolismo , Biomarcadores , Sobrevivência Celular/efeitos dos fármacos , Biologia Computacional , Suscetibilidade a Doenças , Diterpenos/química , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Papillomavirus Humano 16 , Humanos , Estrutura Molecular , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Proteólise , Proteômica/métodos , Transcriptoma , Neoplasias do Colo do Útero/etiologia
11.
Molecules ; 26(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199025

RESUMO

Ginsenoside Rk1 and Rg5 are minor ginseng saponins that have received more attention recently because of their high oral bioavailability. Each of them can effectively inhibit the survival and proliferation of human liver cancer cells, but the underlying mechanism remains largely unknown. Network pharmacology and bioinformatics analysis demonstrated that G-Rk1 and G-Rg5 yielded 142 potential targets, and shared 44 putative targets associated with hepatocellular carcinoma. Enrichment analysis of the overlapped genes showed that G-Rk1 and G-Rg5 may induce apoptosis of liver cancer cells through inhibition of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signal pathways. Methyl thiazolyl tetrazolium (MTT) assay was used to confirm the inhibition of cell viability with G-Rk1 or G-Rg5 in highly metastatic human cancer MHCC-97H cells. We evaluated the apoptosis of MHCC-97H cells by using flow cytometry and 4',6-diamidino-2-phenylindole (DAPI) staining. The translocation of Bax/Bak led to the depolarization of mitochondrial membrane potential and release of cytochrome c and Smac. A sequential activation of caspase-9 and caspase-3 and the cleavage of poly(ADP-ribose) polymerase (PARP) were observed after that. The levels of anti-apoptotic proteins were decreased after treatment of G-Rk1 or G-Rg5 in MHCC-97H cells. Taken together, G-Rk1 and G-Rg5 promoted the endogenous apoptotic pathway in MHCC-97H cells by targeting and regulating some critical liver cancer related genes that are involved in the signal pathways associated with cell survival and proliferation.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Ginsenosídeos/farmacologia , Neoplasias Hepáticas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos Fitogênicos/química , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ginsenosídeos/química , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo
12.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199336

RESUMO

The natural compound ravenelin was isolated from the biomass extracts of Exserohilum rostratum fungus, and its antimicrobial, antiplasmodial, and trypanocidal activities were evaluated. Ravenelin was isolated by column chromatography and HPLC and identified by NMR and MS. The susceptibility of Gram-positive and Gram-negative bacteria strains to ravenelin was determined by microbroth dilution assay. Cytotoxicity was evaluated in hepatocarcinoma cells (HepG2) and BALB/c peritoneal macrophages by using MTT. SYBR Green I-based assay was used in the asexual stages of Plasmodium falciparum. Trypanocidal activity was tested against the epimastigote and intracellular amastigote forms of Trypanosoma cruzi. Ravenelin was active against Gram-positive bacteria strains, with emphasis on Bacillus subtilis (MIC value of 7.5 µM). Ravenelin's antiparasitic activities were assessed against both the epimastigote (IC50 value of 5 ± 1 µM) and the intracellular amastigote forms of T. cruzi (IC50 value of 9 ± 2 µM), as well as against P. falciparum (IC50 value of 3.4 ± 0.4 µM). Ravenelin showed low cytotoxic effects on both HepG2 (CC50 > 50 µM) and peritoneal macrophage (CC50 = 185 ± 1 µM) cells with attractive selectivity for the parasites (SI values > 15). These findings indicate that ravenelin is a natural compound with both antibacterial and antiparasitic activities, and considerable selectivity indexes. Therefore, ravenelin is an attractive candidate for hit-to-lead development.


Assuntos
Antibacterianos/farmacologia , Antiprotozoários/farmacologia , Ascomicetos/química , Macrófagos Peritoneais/citologia , Xantonas/farmacologia , Animais , Antibacterianos/química , Antiprotozoários/química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Biomassa , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Células Hep G2 , Humanos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Estrutura Molecular , Plasmodium falciparum/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Xantonas/química
13.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199541

RESUMO

Quinone methide precursors 1a-e, with different alkyl linkers between the naphthol and the naphthalimide chromophore, were synthesized. Their photophysical properties and photochemical reactivity were investigated and connected with biological activity. Upon excitation of the naphthol, Förster resonance energy transfer (FRET) to the naphthalimide takes place and the quantum yields of fluorescence are low (ΦF ≈ 10-2). Due to FRET, photodehydration of naphthols to QMs takes place inefficiently (ΦR ≈ 10-5). However, the formation of QMs can also be initiated upon excitation of naphthalimide, the lower energy chromophore, in a process that involves photoinduced electron transfer (PET) from the naphthol to the naphthalimide. Fluorescence titrations revealed that 1a and 1e form complexes with ct-DNA with moderate association constants Ka ≈ 105-106 M-1, as well as with bovine serum albumin (BSA) Ka ≈ 105 M-1 (1:1 complex). The irradiation of the complex 1e@BSA resulted in the alkylation of the protein, probably via QM. The antiproliferative activity of 1a-e against two human cancer cell lines (H460 and MCF 7) was investigated with the cells kept in the dark or irradiated at 350 nm, whereupon cytotoxicity increased, particularly for 1e (>100 times). Although the enhancement of this activity upon UV irradiation has no imminent therapeutic application, the results presented have importance in the rational design of new generations of anticancer phototherapeutics that absorb visible light.


Assuntos
Antineoplásicos/farmacologia , Indolquinonas/síntese química , Naftalimidas/química , Naftóis/química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Transferência Ressonante de Energia de Fluorescência , Humanos , Indolquinonas/química , Indolquinonas/farmacologia , Células MCF-7 , Estrutura Molecular , Processos Fotoquímicos , Teoria Quântica
14.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199597

RESUMO

The disruption of iron homeostasis is an important factor in the loss of mitochondrial function in neural cells, leading to neurodegeneration. Here, we assessed the protective action of gossypitrin (Gos), a naturally occurring flavonoid, on iron-induced neuronal cell damage using mouse hippocampal HT-22 cells and mitochondria isolated from rat brains. Gos was able to rescue HT22 cells from the damage induced by 100 µM Fe(II)-citrate (EC50 8.6 µM). This protection was linked to the prevention of both iron-induced mitochondrial membrane potential dissipation and ATP depletion. In isolated mitochondria, Gos (50 µM) elicited an almost complete protection against iron-induced mitochondrial swelling, the loss of mitochondrial transmembrane potential and ATP depletion. Gos also prevented Fe(II)-citrate-induced mitochondrial lipid peroxidation with an IC50 value (12.45 µM) that was about nine time lower than that for the tert-butylhydroperoxide-induced oxidation. Furthermore, the flavonoid was effective in inhibiting the degradation of both 15 and 1.5 mM 2-deoxyribose. It also decreased Fe(II) concentration with time, while increasing O2 consumption rate, and impairing the reduction of Fe(III) by ascorbate. Gos-Fe(II) complexes were detected by UV-VIS and IR spectroscopies, with an apparent Gos-iron stoichiometry of 2:1. Results suggest that Gos does not generally act as a classical antioxidant, but it directly affects iron, by maintaining it in its ferric form after stimulating Fe(II) oxidation. Metal ions would therefore be unable to participate in a Fenton-type reaction and the lipid peroxidation propagation phase. Hence, Gos could be used to treat neuronal diseases associated with iron-induced oxidative stress and mitochondrial damage.


Assuntos
Flavonoides/farmacologia , Ferro/efeitos adversos , Mitocôndrias/metabolismo , Neurônios/citologia , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Cítrico/efeitos adversos , Compostos Ferrosos/efeitos adversos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos
15.
Int J Mol Sci ; 22(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206740

RESUMO

N-Glycosylations are an important post-translational modification of proteins that can significantly impact cell function. Terminal sialic acid in hybrid or complex N-glycans has been shown to be relevant in various types of cancer, but its role in non-malignant cells remains poorly understood. We have previously shown that the motility of human bone marrow derived mesenchymal stromal cells (MSCs) can be modified by altering N-glycoforms. The goal of this study was to determine the role of sialylated N-glycans in MSCs. Here, we show that IFN-gamma or exposure to culture media low in fetal bovine serum (FBS) increases sialylated N-glycans, while PDGF-BB reduces them. These stimuli alter mRNA levels of sialyltransferases such as ST3Gal1, ST6Gal1, or ST3Gal4, suggesting that sialylation of N-glycans is regulated by transcriptional control of sialyltransferases. We next show that 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-Neu5Ac) effectively inhibits sialylations in MSCs. Supplementation with 3F-Neu5Ac increases adhesion and migration of MSCs, as assessed by both videomicroscopy and wound/scratch assays. Interestingly, pre-treatment with 3F-Neu5Ac also increases the survival of MSCs in an in vitro ischemia model. We also show that pre-treatment or continuous treatment with 3F-Neu5Ac inhibits both osteogenic and adipogenic differentiation of MSCs. Finally, secretion of key trophic factors by MSCs is variably affected upon exposure to 3F-Neu5Ac. Altogether, our experiments suggest that sialylation of N-glycans is tightly regulated in response to environmental cues and that glycoengineering MSCs to reduce sialylated N-glycans could be beneficial to increase both cell migration and survival, which may positively impact the therapeutic potential of the cells.


Assuntos
Movimento Celular , Células-Tronco Mesenquimais/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Sialiltransferases/metabolismo , Adipócitos/metabolismo , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Interferon gama/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Osteoblastos/citologia , Sialiltransferases/antagonistas & inibidores
16.
Stem Cell Res Ther ; 12(1): 373, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210363

RESUMO

BACKGROUND: Even though the manufacturing processes of the stromal vascular fraction for clinical use are performed in compliance with the good manufacturing practices applying to advanced therapy medicinal products, specifications related to stromal vascular fraction quality remain poorly defined. We analyzed stromal vascular fraction clinical batches from two independent good manufacturing practices-compliant manufacturing facilities, the Swiss Stem Cell Foundation (SSCF) and Marseille University Hospitals (AP-HM), with the goal of defining appropriate and harmonized release acceptance criteria. METHODS: This retrospective analysis reviewed the biological characteristics of 364 batches of clinical-grade stromal vascular fraction. Collected data included cell viability, recovery yield, cell subset distribution of stromal vascular fraction, and microbiological quality. RESULTS: Stromal vascular fraction from SSCF cohort demonstrated a higher viability (89.33% ± 4.30%) and recovery yield (2.54 × 105 ± 1.22 × 105 viable nucleated cells (VNCs) per mL of adipose tissue) than stromal vascular fraction from AP-HM (84.20% ± 5.96% and 2.25 × 105 ± 1.11 × 105 VNCs per mL). AP-HM batches were significantly less contaminated (95.71% of sterile batches versus 74.15% for SSCF batches). The cell subset distribution was significantly different (higher proportion of endothelial cells and lower proportion of leukocytes and pericytes in SSCF cohort). CONCLUSIONS: Both centers agreed that a good manufacturing practices-compliant stromal vascular fraction batch should exert a viability equal or superior to 80%, a minimum recovery yield of 1.50 × 105 VNCs per mL of adipose tissue, a proportion of adipose-derived stromal cells at least equal to 20%, and a proportion of leukocytes under 50%. In addition, a multiparameter gating strategy for stromal vascular fraction analysis is proposed.


Assuntos
Tecido Adiposo , Células Endoteliais , Sobrevivência Celular , Estudos Retrospectivos , Células Estromais
17.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198683

RESUMO

We noted recently that the injection of cells with a needle through a cystoscope in the urethral sphincter muscle of pigs failed to deposit them nearby or at the intended target position in about 50% of all animals investigated (n > 100). Increasing the chance for precise cell injection by shotgun approaches employing several circumferential injections into the sphincter muscle bears the risk of tissue injury. In this study, we developed and tested a novel needle-free technique to precisely inject cells in the urethral sphincter tissue, or other tissues, using a water-jet system. This system was designed to fit in the working channels of endoscopes and cystoscopes, allowing a wide range of minimally invasive applications. We analyze key features, including the physical parameters of the injector design, pressure ranges applicable for tissue penetration and cell injections and biochemical parameters, such as different compositions of injection media. Our results present settings that enable the high viability of cells post-injection. Lastly, the method is suitable to inject cells in the superficial tissue layer and in deeper layers, required when the submucosa or the sphincter muscle of the urethra is targeted.


Assuntos
Células/metabolismo , Técnicas Citológicas/métodos , Animais , Sobrevivência Celular , Endoscopia , Células HeLa , Humanos , Suínos , Água
18.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198692

RESUMO

The formation of fibrillar aggregates of the amyloid beta peptide (Aß) in the brain is one of the hallmarks of Alzheimer's disease (AD). A clear understanding of the different aggregation steps leading to fibrils formation is a keystone in therapeutics discovery. In a recent study, we showed that Aß40 and Aß42 form dynamic micellar aggregates above certain critical concentrations, which mediate a fast formation of more stable oligomers, which in the case of Aß40 are able to evolve towards amyloid fibrils. Here, using different biophysical techniques we investigated the role of different fractions of the Aß aggregation mixture in the nucleation and fibrillation steps. We show that both processes occur through bimolecular interplay between low molecular weight species (monomer and/or dimer) and larger oligomers. Moreover, we report here a novel self-catalytic mechanism of fibrillation of Aß40, in which early oligomers generate and deliver low molecular weight amyloid nuclei, which then catalyze the rapid conversion of the oligomers to mature amyloid fibrils. This fibrillation catalytic activity is not present in freshly disaggregated low-molecular weight Aß40 and is, therefore, a property acquired during the aggregation process. In contrast to Aß40, we did not observe the same self-catalytic fibrillation in Aß42 spheroidal oligomers, which could neither be induced to fibrillate by the Aß40 nuclei. Our results reveal clearly that amyloid fibrillation is a multi-component process, in which dynamic collisions between different interacting species favor the kinetics of amyloid nucleation and growth.


Assuntos
Peptídeos beta-Amiloides/química , Benzotiazóis/química , Fenômenos Biofísicos , Catálise , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Cinética , Peso Molecular , Conformação Proteica
19.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198700

RESUMO

Vacuum cathodic arc TiN coatings with overlaying TiO2 film were deposited on polished and surface roughened by electron beam modification (EBM) Ti6Al4V alloy. The substrate microtopography consisted of long grooves formed by the liner scan of the electron beam with appropriate frequencies (500 (AR500) and 850 (AR850) Hz). EBM transformed the α + ß Ti6Al4V mixed structure into a single α'-martensite phase. Тhe gradient TiN/TiO2 films deposited on mechanically polished (AR) and EBM (AR500 and AR850) alloys share the same surface chemistry and composition (almost stoichiometric TiN, anatase and rutile in different ratios) but exhibit different topographies (Sa equal to approximately 0.62, 1.73, and 1.08 µm, respectively) over areas of 50 × 50 µm. Although the nanohardness of the coatings on AR500 and AR850 alloy (approximately 10.45 and 9.02 GPa, respectively) was lower than that measured on the film deposited on AR alloy (about 13.05 GPa), the hybrid surface treatment offered improvement in critical adhesive loads, coefficient of friction, and wear-resistance of the surface. In phosphate buffer saline, all coated samples showed low corrosion potentials and passivation current densities, confirming their good corrosion protection. The coated EBM samples cultured with human osteoblast-like MG63 cells demonstrated increased cell attachment, viability, and bone mineralization activity especially for the AR500-coated alloy, compared to uncoated polished alloy. The results underline the synergetic effect between the sub-micron structure and composition of TiN/TiO2 coating and microarchitecture obtained by EBM.


Assuntos
Ligas/química , Materiais Biocompatíveis/química , Eletroquímica , Elétrons , Titânio/química , Calcificação Fisiológica/fisiologia , Adesão Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Módulo de Elasticidade , Fricção , Dureza , Humanos , Microscopia de Força Atômica , Difração de Raios X
20.
Int J Mol Sci ; 22(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198821

RESUMO

Photo-polymerized hydrogels are ideally suited for stem-cell based tissue regeneration and three dimensional (3D) bioprinting because they can be highly biocompatible, injectable, easy to use, and their mechanical and physical properties can be controlled. However, photo-polymerization involves the use of potentially toxic photo-initiators, exposure to ultraviolet light radiation, formation of free radicals that trigger the cross-linking reaction, and other events whose effects on cells are not yet fully understood. The purpose of this study was to examine the effects of hydrogen sulfide (H2S) in mitigating cellular toxicity of photo-polymerization caused to resident cells during the process of hydrogel formation. H2S, which is the latest discovered member of the gasotransmitter family of gaseous signalling molecules, has a number of established beneficial properties, including cell protection from oxidative damage both directly (by acting as a scavenger molecule) and indirectly (by inducing the expression of anti-oxidant proteins in the cell). Cells were exposed to slow release H2S treatment using pre-conditioning with glutathione-conjugated-garlic extract in order to mitigate toxicity during the photo-polymerization process of hydrogel formation. The protective effects of the H2S treatment were evaluated in both an enzymatic model and a 3D cell culture system using cell viability as a quantitative indicator. The protective effect of H2S treatment of cells is a promising approach to enhance cell survival in tissue engineering applications requiring photo-polymerized hydrogel scaffolds.


Assuntos
Técnicas de Cultura de Células/métodos , Hidrogéis/farmacologia , Sulfeto de Hidrogênio/farmacologia , Engenharia Tecidual , Sobrevivência Celular/efeitos dos fármacos , Humanos , Luz , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos da radiação , Polimerização/efeitos dos fármacos , Polimerização/efeitos da radiação , Impressão Tridimensional , Tecidos Suporte , Cicatrização/efeitos dos fármacos , Cicatrização/efeitos da radiação
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