RESUMEN
Chemical compounds from skin secretions from toads of Bufonidae family have been long-studied. In the search for new molecules with pharmacological action, the 3ß-OH groups of bufadienolides are commonly derivatised using acetyl groups. This work described the isolation and/or structural elucidation of isolated and derivatised compounds from the venom of the Brazilian anuran Rhinella marina, and their evaluation in in vitro assays. In the methanolic extract of the R. marina venom, compound cholesterol (1) was isolated from the CRV-52 fraction by classic column chromatography, dehydrobufotenine (2) by Sephadex LH-20 from the CRV-28 fraction, and a mix of suberoyl arginine (3) and compound 2 was obtained from the CRV-6-33 fraction. The compounds marinobufagin (4), telocionbufagin (5) and bufalin (6) were isolated by classic column chromatography, followed by separation via HPLC in the CRV-70 fraction, and the compound marinobufotoxin (9) was isolated by classic column chromatography in the CRV-6 fraction, here being isolated for the first time in R. marina specimens. Compounds 4 and 5 were submitted for acetylation with acetic anhydride, in the presence of pyridine and 4-dimethyilaminopiridine (DMAP), in order to obtain the compounds 3-acetyl-marinobufagin (7) and 3-acetyl-telocinobufogin (8). The isolated and derivatised compounds were identified by 1H and 13C NMR, and their molecular mass confirmed by mass spectrometry. All compounds (except 1 and 3) were tested in cytotoxic assays by the MTT method and presented cytotoxic potential against human cancer cell lines, as well as against non-tumoral human embryonic kidney HEK-293 cells. With the exception of compound 2, all molecules presented IC50 values < 4 µM, and none caused hemolysis of human erythrocytes, demonstrating a promising cytotoxic potential of natural and chemically-modified bufadienolides. This study presents a detailed contribution of bioactive chemicals from Brazilian Amazon Rhinella species, and indicates promising areas for further studies and pharmaceutical investments.
Asunto(s)
Venenos de Anfibios/toxicidad , Bufo marinus , Animales , Brasil , Línea Celular Tumoral , Células HEK293 , Humanos , PonzoñasRESUMEN
Caatinga flora which are found in a poor Brazilian region contain a substantial number of endemic taxa with biomedical and social importance for regional communities. This study examined the antioxidant and cytotoxic potential of 35 samples (extracts/fractions) from 12 Caatinga species and determined the antiproliferative and genotoxic action of dichloromethane fraction from Mimosa caesalpiniifolia stem bark (DC-Mca) on human and vegetal cells. Samples were assessed for chemopreventive ability, toxic effects on Artemia salina shrimp as well as cytotoxicity on tumor cell lines and erythrocytes. DC-Mca was also tested with respect to antiproliferative and genotoxic effects upon normal leukocytes and meristematic cells from A. cepa roots. Some extracts reduced free radical levels >95% and 7 samples exhibited a lethal concentration (LC) 50 < 100 µg/ml upon Artemia salina larvae. Eight samples displayed in vitro antitumor effects and three produced hemolysis. Data also demonstrated the pharmacological significance of bioactive extracts from Brazilian semi-arid region. There was no significant relationship between antioxidant, toxic, and antiproliferative activities, and that these properties were dependent upon the extractant. DC-Mca contained betulinic acid as main compound (approximately 70%), which showed higher (1) cytotoxic activity on cancer cell lines and dividing leukocytes, (2) reduced mitotic index of Allium cepa roots, and (3) induced cell cycle arrest and chromosomal bridges, thereby providing native promising sources for phytotherapy development. ABBREVIATIONS: ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); AcOH: ethyl acetate; ANOVA: analysis of variance; SUS: Brazilian Unified Health System; DC-Mca: dichloromethane fraction from Mimosa caesalpiniifolia stem bark; DMSO: dimethylsulfoxide; DPPH: 1,1-diphenyl-2-picrylhydrazyl; EC50: effective concentration 50%; EtOAc: ethyl acetate; FDA: Food and Drug Administration; GC-Qms: gas chromatograph quadrupole mass spectrometer; GI: genotoxic index; HCT-116: colon carcinoma line; HL-60: promyelocytic leukemia line; HPLC: high-performance liquid chromatography; HRAPCIMS: high resolution atmospheric pressure chemical ionization mass spectrum; IC50: inhibitory concentration 50%; LC50: lethal concentration 50%; MeOH = methyl alcohol; MI: mitotic index; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; MutI: mutagenic index; OVCAR-8 = ovarian carcinoma line; PBMC: peripheral blood mononuclear cells; RPMI-1640: Roswell Park Memorial Institute medium; SF-295: glioblastoma line; TEAC: trolox equivalent antioxidant capacity; TLC: thin-layer chromatography; Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Antioxidantes/farmacología , Plantas Medicinales/química , Antineoplásicos Fitogénicos/química , Antioxidantes/química , Brasil , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Citotoxinas/química , Citotoxinas/farmacología , Daño del ADN , Ecosistema , Ecotoxicología , Humanos , Cloruro de Metileno/química , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/química , Extractos Vegetales/farmacología , Plantas Medicinales/clasificación , Plantas Medicinales/toxicidadRESUMEN
Quinoxaline derivatives are reported as antineoplastic agents against a variety of human cancer cell lines, with some compounds being submitted to clinical trials. In this work, we report the synthesis, characterization and cytotoxicity potential of a new series of quinoxalinyl-hydrazones. The most cytotoxic compound was (E)-2-[2-(2-pyridin-2-ylmethylene)hydrazinyl]quinoxaline (PJOV56) that presented a time-dependent effect against HCT-116 cells. After 48 h of incubation, PJOV56 was able to induce autophagy and apoptosis of HCT-116 cells, mediated by upregulation of Beclin 1, upregulation of LC3A/B II and activation of caspase 7. Apoptosis was induced along with G0/G1 cell cycle arrest at the highest concentration of PJOV56 (6.0 µM). Thus, PJOV56 showed a dose-dependent mode of action related to induction of autophagy and apoptosis in HCT-116 cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Hidrazonas/síntesis química , Quinoxalinas/síntesis química , Humanos , Hidrazonas/química , Quinoxalinas/química , Relación Estructura-ActividadRESUMEN
Skin toad secretion present physiologically active molecules to protect them against microorganisms, predators and infections. This work detailed the antiproliferative action of marinobufagin on tumor and normal lines, investigate its mechanism on HL-60 leukemia cells and its toxic effects on Allium cepa meristematic cells. Initially, cytotoxic action was assessed by colorimetric assays. Next, HL-60 cells were analyzed by morphological and flow cytometry techniques and growing A. cepa roots were examined after 72â¯h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC50 values ranging from 0.15 (leukemia) to 7.35 (larynx) µM] and it failed against human erythrocytes and murine lines. Human normal peripheral blood mononuclear cells (PBMC) were up to 72.5-fold less sensitive [IC50: 10.88⯵M] to marinobufagin than HL-60 line, but DNA strand breaks were no detected. Leukemia treaded cells exhibited cell viability reduction, DNA fragmentation, phosphatidylserine externalization, binucleation, nuclear condensation and cytoplasmic vacuoles. Marinobufagin also reduced the growth of A. cepa roots (EC50: 7.5⯵M) and mitotic index, caused cell cycle arrest and chromosomal alterations (micronuclei, delays and C-metaphases) in meristematic cells. So, to find out partially targeted natural molecules on human leukemia cells, like marinobufagin, is an amazing and stimulating way to continue the battle against cancer.
Asunto(s)
Antineoplásicos/farmacología , Bufanólidos/farmacología , Ciclo Celular/efectos de los fármacos , Roturas del ADN , Cebollas/efectos de los fármacos , Adolescente , Adulto , Animales , Antineoplásicos/aislamiento & purificación , Antineoplásicos/toxicidad , Bufanólidos/aislamiento & purificación , Bufanólidos/toxicidad , Bufonidae/metabolismo , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Relación Dosis-Respuesta a Droga , Eritrocitos/efectos de los fármacos , Células HL-60 , Voluntarios Sanos , Hemólisis/efectos de los fármacos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Meristema/citología , Meristema/efectos de los fármacos , Meristema/genética , Micronúcleos con Defecto Cromosómico/inducido químicamente , Cebollas/citología , Cebollas/genética , Piel/metabolismo , Adulto JovenRESUMEN
O câncer é uma complexa doença de origem genética, considerada uma das principais causas de morte por doença no mundo. A utilização de substâncias derivadas de produtos naturais tem crescido com o passar dos anos, constituindo uma importante fonte no arsenal terapêutico.Os bufadienolídeos são esteróides cardioativos de24 carbonos, isolados de extratosde glândulasde sapos da família Bufonidae. Essas moléculaspossuem grande variedade de atividades biológicas,incluindo atividadeanticâncer. Os bufadienolídeos tem demonstradocomportamento antiproliferativoemvárias linhagens de células cancerígenas humanas por induzirmortee parada do ciclo celular.A marinobufagenina, umbufadienolídeoextraído do anfíbio Rhinella marina,foi escolhida para determinamos o seu padrão citotóxico e mecanismo de ação. Apesar de demonstrar alta citotoxicidade em células tumorais humanas, a amostranão apresentou citotoxicidade para linhagens murinas. Experimentos in vitro foram realizados utilizando-se a linhagem de adenocarcinoma de próstata PC3. As célulasforam tratadas comdiferentes concentrações da amostramarinobufagenina (0,37, 0,75e 1,5μM)por 24 horas...
Cancer is a complex genetic disease, considered one of the leading causes of death in the world. The use of substances derived from natural products has grown over the years and is an important source forthe therapeutic arsenal. Bufadienolides, a group ofcardioactive steroids with a24 carbonstructures are commonly found inglands of toadsfromBufonidae family.These moleculesahave wide range of biological activities, including anti-cancer effects. Bufadienolides haveshown anti-proliferative effect on various human cancer cell lines by inducing death and cell cycle arrest.Marinobufagenin, abufodienolideextracted fromRhinella marinatoad especies was chosen to determine itsstandard cytotoxic and mechanism of action.Despite showinghigh cytotoxicity in human tumor cells, the sample showed no cytotoxicity to murine strains. In vitro experiments were performed using the PC3 prostateadenocarcinoma lineage. Cells were incubated with different concentrations of marinobufagenin (0.37, 0.75 and 1.5 mM) for 24 hours...
Asunto(s)
Humanos , Anticarcinógenos , Ciclo Celular , Muerte Celular , AdenocarcinomaRESUMEN
Mimosa caesalpiniifolia is a native plant of the Brazilian northeast, and few studies have investigated its chemical composition and biological significance. This work describes the identification of the first chemical constituents in the ethanolic extract and fractions of M. caesalpiniifolia stem bark based on NMR, GC-qMS and HRMS analyses, as well as an assessment of their cytotoxic activity. GC-qMS analysis showed fatty acid derivatives, triterpenes and steroid substances and confirmed the identity of the chemical compounds isolated from the hexane fraction. Metabolite biodiversity in M. caesalpiniifolia stem bark revealed the differentiated accumulation of pentacyclic triterpenic acids, with a high content of betulinic acid and minor amounts of 3-oxo and 3ß-acetoxy derivatives. Bioactive analysis based on total phenolic and flavonoid content showed a high amount of these compounds in the ethanolic extract, and ESI-(-)-LTQ-Orbitrap-MS identified caffeoyl hexose at high intensity, as well as the presence of phenolic acids and flavonoids. Furthermore, the evaluation of the ethanolic extract and fractions, including betulinic acid, against colon (HCT-116), ovarian (OVCAR-8) and glioblastoma (SF-295) tumour cell lines showed that the crude extract, hexane and dichloromethane fractions possessed moderate to high inhibitory activity, which may be related to the abundance of betulinic acid. The phytochemical and biological study of M. caesalpiniifolia stem bark thus revealed a new alternative source of antitumour compounds, possibly made effective by the presence of betulinic acid and by chemical co-synergism with other compounds.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Supervivencia Celular/efectos de los fármacos , Flavonoides/farmacología , Mimosa/química , Neoplasias/tratamiento farmacológico , Corteza de la Planta/química , Extractos Vegetales/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Flavonoides/aislamiento & purificación , Humanos , Neoplasias/patología , Triterpenos Pentacíclicos , Fenoles/aislamiento & purificación , Fenoles/farmacología , Triterpenos/farmacología , Células Tumorales Cultivadas , Ácido BetulínicoRESUMEN
Eleven phthalimide derivatives were evaluated with regards to their antiproliferative activity on tumor and normal cells and possible toxic effects. Cytotoxic analyses were performed against murine tumors (Sarcoma 180 and B-16/F-10 cells) and peripheral blood mononuclear cells (PBMC) using MTT and Alamar Blue assays. Following, the investigation of cytotoxicity was executed by flow cytometry analysis and antitumoral and toxicological potential by in vivo techniques. The molecules 3b, 3c, 4 and 5 revealed in vitro cytotoxicity against Sarcoma 180, B-16/F-10 and PBMC. Since compound 4 was the most effective derivative, it was chosen to detail the mechanism of action after 24, 48 and 72 h exposure (22.5 and 45 µM). Sarcoma 180 cells treated with compound 4 showed membrane disruption, DNA fragmentation and mitochondrial depolarization in a time- and dose-dependent way. Compounds 3c, 4 and 5 (50 mg/kg/day) did not inhibit in vivo tumor growth. Compound 4-treated animals exhibited an increase in total leukocytes, lymphocytes and spleen relative weight, a decreasing in neutrophils and hyperplasia of spleen white pulp. Treated animals presented reversible histological changes. Molecule 4 had in vitro antiproliferative action possibly triggered by apoptosis, reversible toxic effects on kidneys, spleen and livers and exhibited immunostimulant properties that can be explored to attack neoplasic cells.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Ftalimidas/farmacología , Animales , Antineoplásicos/toxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Ratones , Ftalimidas/toxicidadRESUMEN
The venom of amphibians is a fascinating source of active substances. In view of their medical importance and aiming to explore the amazing Brazilian biodiversity, we conducted bioprospecting of antiproliferative activity in extracts of Rhinella marina and Rhaebo guttatus toads occurring in the Southern Amazon of Mato Grosso, Brazil. LC-MS and HPLC analysis of the venom extracts of R. marina revealed four bufadienolides (telocinobufagin, marinobufagin, bufalin and resibufogenin. R. guttatus venom extracts contained only marinobufagin. First, R. marina and R. guttatus venom extracts were evaluated for cytotoxicity against tumor cell lines by the MTT assay. All extracts revealed cytotoxicity, where R. marina extracts were comparable to doxorubicin (IC50 values ranging from 0.01 to 0.23 µg/mL). Only extracts of R. guttatus toad venom caused membrane disruption of human erythrocytes. The extracts were investigated for selective activity by determining their effect on stimulated human peripheral blood mononuclear cells (PBMC) with the Alamar Blue™ assay. The extracts were up to 80-fold more selective against leukemia cells when compared to dividing leukocytes. Aiming to confirm these antiproliferative effects, BrdU incorporation into DNA was measured in HL-60 treated cells with R. marina venom extracts. These extracts decreased BrdU incorporation at both concentrations tested. In summary, nine extracts of R. marina and R. guttatus venom showed pronounced lethal and discriminating effects on tumor lines, especially those from R. marina, highlighting toad parotoid gland secretions as a promising source for novel lead anticancer chemicals.