RESUMO
Secreted quiescin/sulfhydryl oxidase (QSOX) is overexpressed in many tumor cell lines, including melanoma, and is usually associated with a pro-invasive phenotype. Our previous work described that B16-F10 cells enter in a quiescent state as a protective mechanism against damage generated by reactive oxygen species (ROS) during melanogenesis stimulation. Our present results show that QSOX activity was two-fold higher in cells with stimulated melanogenesis when compared to control cells. Considering that glutathione (GSH) is one of the main factor responsible for controlling redox homeostasis in cells, this work also aimed to investigate the relationship between QSOX activity, GSH levels and melanogenesis stimulation in B16-F10 murine melanoma cell line. The redox homeostasis was impaired by treating cells with GSH in excess or depleting its intracellular levels through BSO treatment. Interestingly, GSH-depleted cells without stimulation of melanogenesis kept high levels of viability, suggesting a possible adaptive mechanism of survival even under low GSH levels. They also showed lower extracellular activity of QSOX, and higher QSOX intracellular immunostaining, suggesting that this enzyme was less excreted from cells and corroborating with a diminished extracellular QSOX activity. On the other hand, cells under melanogenesis stimulation showed a lower GSH/GSSG ratio (8:1) in comparison with control (non-stimulated) cells (20:1), indicating a pro-oxidative state after stimulation. This was accompanied by decreased cell viability after GSH-depletion, no alterations in QSOX extracellular activity, but higher QSOX nucleic immunostaining. We suggest that melanogenesis stimulation and redox impairment caused by GSH-depletion enhanced the oxidative stress in these cells, contributing to additional alterations of its metabolic adaptive response.
Assuntos
Melanoma , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Animais , Camundongos , Glutationa/metabolismo , Melanoma/metabolismo , Oxirredução , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismoRESUMO
Melanogenesis-stimulated B16-F10 cells enter in a quiescent state, present inhibited mitochondrial respiration and increased reactive oxygen species levels. These alterations suggest that these cells may be under redox signaling, allowing tumor survival. The aim of this study was to evaluate redox-modified proteins in B16-F10 cells after melanogenesis stimulation and rose bengal-photodynamic therapy (RB-PDT). A redox proteomics label-free approach based on the biotin switch assay technique with biotin-HPDP and N-ethylmaleimide was used to assess the thiol-oxidized protein profile. Aconitase was oxidized at Cys-448 and Cys-451, citrate synthase was oxidized at Cys-202 and aspartate aminotransferase (Got2) was oxidized at Cys-272 and Cys-274, exclusively after melanogenesis stimulation. After RB-PDT, only guanine nucleotide-binding protein subunit beta-2-like 1 (Gnb2l1) was oxidized (Cys-168). In contrast, melanogenesis stimulation followed by RB-PDT led to the oxidation of different cysteines in Gnb2l1 (Cys-153 and Cys-249). Besides that, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) presented oxidation at Cys-245, peptidyl-prolyl cis-trans isomerase A (Ppia) was oxidized at Cys-161 and 5,6-dihydroxyindole-2-carboxylic acid oxidase (Tyrp1) was oxidized at Cys-65, Cys-30, and Cys-336 after melanogenesis stimulation followed by RB-PDT. The redox alterations observed in murine melanoma cells and identification of possible target proteins are of great importance to further understand tumor resistance mechanisms.
RESUMO
Hemicellulose-type polysaccharides were isolated from Campomanesia xanthocarpa fruits by alkaline extraction and submitted to fractionation processes giving rise to eluted (GE-300) and retained (GR-300) fractions. GE-300 presented a mixture of galactoglucomannans (GGM) and glucuronoxylans (MGX), while the GR-300 fraction is composed only of MGX. In this way, the chemical structure of MGX, investigated by 1D 1H, 13C and 2D 1H-13C HSQC, 1H-1H COSY and 1H-13C HMBC NMR spectroscopy, revealed that the chemical structure of polysaccharide is a (4-O-methyl-α-D-glucurono)-D-xylan. Deep and precise NMR chemical shift determination of clean and specific 1H NMR glycosyl units were developed by 1D TOCSY and 1D NOESY analysis. This approach demonstrated unequivocally that 4-O-methyl-α-D-glucopyranosyl uronic acid group is linked to O-2 of a (1 â 4)-ß-D-xylan in the main chain. Furthermore, MGX scavenged DPPH radical (0.5 to 1.0 mg mL-1) and was not cytotoxic to human dermal fibroblasts at concentrations up to 1.0 mg mL-1, as demonstrated by neutral red and crystal violet assays, evidencing in vitro biocompatibility. The structure elucidation of GR-300 together with its bioactivity assessment contributed to better understand the chemical characteristics of C. xanthocarpa hemicelluloses and may provide structural basis for future structure-property studies.
Assuntos
Frutas , Myrtaceae , Frutas/química , Humanos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Myrtaceae/química , XilanosRESUMO
Polysaccharides from seaweed have been shown to present a variety of antitumor effects, however the understanding of which structural patterns are responsible for these biological effects are still unclear. This review aimed to gather and critically evaluate published data of seaweed polysaccharide's chemical structure elucidation and their relation with antimelanoma effects. Data were collected at the electronic article databases Science Direct, NCBI/Pubmed and Google Scholar, selecting papers with polysaccharide structural information and biological effects on melanoma models. Most of the papers referred to sulfated polysaccharides as fucans and fucoidans, and to a lesser extent galactans, rhamnans, alginates, and neutral one's glucans. Fine chemical features as presence and position of sulfate groups, monosaccharide composition, linear or branched backbones, and glycosidic linkage type are crucial to antimelanoma effects, as well as molecular weight and macromolecular conformation.
Assuntos
Alga Marinha , Sulfatos , Galactanos/química , Polissacarídeos/química , Polissacarídeos/farmacologia , Óxidos de Enxofre , VerdurasRESUMO
Melanoma is a type of tumor that originates from melanocytes. Irradiation of melanin with UVA and visible light can produce reactive oxygen species (ROS) such as singlet molecular oxygen (1 O2 ). The objective of this study was to examine DNA damage in melanoma cells (B16-F10) with different melanin contents, subjected to 1 O2 generation. To this end, we used the photosensitizer Rose Bengal acetate (RBAc) and irradiation with visible light (526 nm) (RBAc-PDT). We used the modified comet assay with the repair enzymes hOGG1 and T4 endonuclease V to detect the DNA damage associated with 8-oxo-7,8-dihydro-2'-deoxyguanosine and cyclobutane pyrimidine dimers lesions, respectively. We observed increased formation of hOGG1- and T4endoV-sensitive DNA lesions after light exposure (with or without RBAc). Furthermore, 18 h after irradiation, hOGG1-sensitive DNA lesions increased compared to that at the initial time point (0 h), which shows that a high melanin content contributes to post-irradiation formation of them, mainly via sustained oxidative stress, as confirmed by the measurement of ROS levels and activity of antioxidant enzymes. Contrastingly, the number of T4endoV-sensitive DNA lesions decreased over time (18 h). Our data indicate that in melanoma cells, a higher amount of melanin may affect DNA damage levels when subjected to RBAc-PDT.
Assuntos
Melanoma , Transtornos de Fotossensibilidade , Humanos , Melaninas , Rosa Bengala/farmacologia , Espécies Reativas de Oxigênio , Raios Ultravioleta , Dano ao DNA , Melanoma/patologia , Oxigênio Singlete , DNA/efeitos da radiaçãoRESUMO
A water-soluble sulfated heterorhamnan (Gb1) was isolated from the green seaweed Gayralia brasiliensis and purified by ultrafiltration, yielding a homogeneous polysaccharide (Gb1r). Both fractions contained rhamnose, xylose, galacturonic and glucuronic acids, galactose, and glucose. Chemical and spectroscopic methods allowed the determination of Gb1 and Gb1r chemical structure. Their backbones were constituted by 3-, 2-, and 2,3-linked rhamnosyl units (1:0.49:0.13 and 1:0.58:0.17, respectively), which are unsulfated (13.5 and 14.6%), disulfated (16.6 and 17.8%) or monosulfated at C-2 (8 and 8.6%) and C-4 (24.5 and 23.4%). Gb1 was oversulfated giving rise to Gb1-OS, which presented ~2.5-fold higher content of disulfated rhamnosyl units than Gb1, as determined by methylation analyses and NMR spectroscopy. Gb1 and Gb1-OS potently reduced the viability of U87MG human glioblastoma cells. Gb1 caused cell cycle arrest in the G1 phase, increased annexin V-stained cells, and no DNA fragmentation, while Gb1-OS increased the percentage of cells in the S and G2 phases and the levels of fragmented DNA and cells double-stained with annexin V/propidium iodide, suggesting an apoptosis mechanism. The results suggest that the different effects of Gb1 and Gb1-OS were related to differences in the sulfate content and position of these groups along the polysaccharide chains.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Mananas/farmacologia , Alga Marinha , Sulfatos/farmacologia , Antineoplásicos/isolamento & purificação , Neoplasias Encefálicas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glioma/patologia , Humanos , Mananas/isolamento & purificação , Estrutura Molecular , Alga Marinha/química , Relação Estrutura-Atividade , Sulfatos/isolamento & purificaçãoRESUMO
Melanogenesis- stimulated B16-F10 cells enter in a quiescent state, present inhib-ited mitochondrial respiration and increased reactive oxygen species levels. Thesealterations suggest that these cells may be under redox signaling, allowing tumorsurvival. The aim of this study was to evaluate redox-modified proteins in B16-F10 cells after melanogenesis stimulation and rose bengal-photodynamic therapy(RB-PDT). A redox proteomics label-free approach based on the biotin switchassay technique with biotin-HPDP and N-ethylmaleimide was used to assess thethiol-oxidized protein profile. Aconitase was oxidized at Cys-448 and Cys-451,citrate synthase was oxidized at Cys-202 and aspartate aminotransferase (Got2)was oxidized at Cys-272 and Cys-274, exclusively after melanogenesis stimula-tion. After RB- PDT, only guanine nucleotide-binding protein subunit beta- 2- like1 (Gnb2l1) was oxidized (Cys-168). In contrast, melanogenesis stimulation fol-lowed by RB- PDT led to the oxidation of different cysteines in Gnb2l1 (Cys-153and Cys- 249). Besides that, glyceraldehyde-3-phosphate dehydrogenase (Gapdh)presented oxidation at Cys-245, peptidyl-prolyl cis-trans isomerase A (Ppia) wasoxidized at Cys-161 and 5,6-dihydroxyindole-2-carboxylic acid oxidase (Tyrp1)was oxidized at Cys-65, Cys-30, and Cys-336 after melanogenesis stimulation fol-lowed by RB-PDT. The redox alterations observed in murine melanoma cells andidentification of possible target proteins are of great importance to further under-stand tumor resistance mechanisms.
RESUMO
Native (F2) and carboxyl-reduced (R) ulvans from Ulva fasciata were sequentially oxidized with periodate-chlorite affording the polycarboxyl ulvans C1, C2 and C3 (1.20, 1.41 and 1.81â¯mmolâ¯g-1 of COOH, respectively; 19.7, 21.3 and 21.0% of NaSO3, respectively) and R-C3 (1.86â¯mmolâ¯g-1 of COOH; NaSO3â¯=â¯22.7%), respectively. APTT assay (polysaccharide fractions at 150⯵gâ¯mL-1) showed clotting time of 45.6â¯s for F2 fraction. For polycarboxyl ulvans C1, C2, C3 and R-C3 the clotting times were 101.0, 122.2, 222.0 and 227.0â¯s, respectively. Comparison of the APTT assay results using ulvans chemically modified by carboxyl-reduction, desulfation, periodate oxidation and/or chlorite oxidation showed the anticoagulant activity of polycarboxyl ulvans is dependent of the sulfate groups present in the native polymer. In addition, the increase of the anticoagulant activity was accompanied by the increasing of the carboxyl groups and the content of this acidic substituent seems to be more important than its positioning.