Cytotoxic and antioxidant activity of hypericum perforatum L. extracts against human melanoma cells from different stages of cancer progression, cultured under normoxia and hypoxia

Oxidative stress and the hypoxic microenvironment play a key role in the progression of human melanoma, one of the most aggressive skin cancers. The aim of our study was to evaluate the effect of Hypericum perforatum extracts of different origins (both commercially available (HpEx2) and laboratory-prepared from wild grown (HpEx12) and in vitro cultured (HpEx13) plants) and hyperforin salt on WM115 primary and WM266-4 lymph node metastatic human melanoma cells cultured under normoxic and hypoxic conditions. The polyphenol content, radical scavenging activity, and hyperforin concentration were determined in the extracts, while cell viability, apoptosis, ROS production, and expression of NRF2 and HO-1, important oxidative stress-related factors, were analyzed after 24 h of cell stimulation with HpExs and hyperforin salt. We found that cytotoxic, pro-apoptotic and antioxidant effects depend on the extract composition, the stage of melanoma progression, and the oxygen level. Hyperforin salt showed lower activity than H. perforatum extracts. Our study for the first time showed that the anticancer activity of H. perforatum extracts differs in normoxia and hypoxia. Importantly, the composition of extracts of various origins, including in vitro cultured, resulting in their unique properties, may be important in the selection of plants for therapeutic application

IgG fucosylation in Graves' disease

Przeciwciała klasy G (IgG) wiążące swoiście antygeny, biorą udział w rozpoznawaniu, neutralizacji i eliminacji patogenów. IgG są modyfikowane potranslacyjnie w procesie N-glikozylacji, polegającym na przyłączeniu reszt cukrowych do białka przy udziale enzymów zwanych glikozylotransferazami. IgG posiada konserwatywne miejsce N-glikozylacji zlokalizowane na Asn297 w domenie CH2 fragmentu Fc. N-glikozylacja IgG zachodzi także w domenie zmiennej fragmentu Fab. N-oligosacharydy fragmentu Fc IgG w ok. 92% podlegają fukozylacji. Fukoza (Fuc) jest deoksyheksozą o konfiguracji L przyłączaną wiązaniem α-glikozydowym przez fukozylotransferazy do części rdzeniowej glikanów lub anten struktur kompleksowych. Ekspresja fukozylowanych glikanów zmienia się w procesach patologicznych. Celem badań była analiza fukozylacji IgG w chorobie Gravesa-Basedowa (GD) o podłożu autoimmunologicznym. IgG izolowano metodą chromatografii powinowactwa z surowicy krwi zdrowych ochotników (CTR, n=9), pacjentów z GD przed włączeniem leczenia (GD, n=7) i po ustabilizowaniu TSH w efekcie leczenia (GD/L, n=2). Fukozylację IgG analizowano metodą lektynową na błonie z fluorku poliwinylidenu (PVDF) po rozdziale elektroforetycznym w warunkach redukujących w żelach wykonanych w technologii stain-free. Do oceny fukozylacji rdzeniowej wykorzystano lektynę LCA a do antenowej UEA-I. AAL, trzecia z użytych lektyn, wykazuje powinowactwo do Fuc obydwu typów, przy czym mocniej wiąże fukozę proksymalną. Intensywność reakcji N-glikanów łańcucha lekkiego i ciężkiego IgG z daną lektyną mierzono densytometrycznie i normalizowano względem ilości białka wizualizowanego w świetle UV. W celu oceny istotności statystycznej uzyskanych wyników wykonano test Kruskala-Wallisa, przyjmując poziom istotności dla p<0,05. Lektyna LCA nie wiązała się z łańcuchem lekkim IgG, co wskazuje, że N-glikany fragmentu Fab nie są fukozylowane w części rdzeniowej. Intensywność wiązania lektyn AAL i UEA-I była znacząco mocniejsza dla łańcucha ciężkiego niż lekkiego, co wynika głównie z tego, że tylko ok. 20% glikanów Fab jest glikozylowana. Uzyskane wyniki wskazują, że fukozylacja N-glikanów IgG wykazuje tendencję do zmian w chorobie Gravesa-Basedowa, chociaż w żadnym z eksperymentów nie stwierdzono różnic istotnych statystycznie. Najbardziej spójne wyniki dla trzech powtórzeń eksperymentu uzyskano w przypadku reakcji z lektyną AAL, które wskazują na tendencję do wzrostu fukozylacji obydwu łańcuchów IgG u chorych a GD oraz tendencję spadkową u osób, które poddały się leczeniu w porównaniu z GD. Brak istotności statystycznej wynika prawdopodobnie ze zbyt małej liczby próbek użytych do analizy. Ocena zmian fukozylacji IgG w GD jest istotna do określania udziału zmienionej zawartości Fuc w autoagresji z udziałem przeciwciał w tej chorobie autoimmunizacyjnej.Immunoglobulins class G (IgGs) that specifically bind antigens, are involved in the recognition, neutralization and elimination of pathogens. IgGs are post-translationally modified in the N-glycosylation process, which involves the attachment of sugar residues to a protein by enzymes called glycosyltransferases. IgG has a conserved glycosylation site located at Asn297 in the CH2 domain of the Fc fragment. N-glycosylation of IgG also occurs in the variable domain of a Fab fragment. Approximately 92% of Fc N-oligosaccharides are fucosylated. Fucose (Fuc) is a L-deoxyhexose attached via α-glycosidic linkages by fucosyltransferases to the core part of glycans or antennas of complex structures. The expression of fucosylated glycans changes in pathological processes. The aim of the study was to analyze IgG fucosylation in Graves' disease (GD) with autoimmune background. IgG was isolated by G protein affinity chromatography from the serum of healthy volunteers (CTR, n=9), patients with GD before treatment initiation (GD, n=7) and after stabilization of TSH level as a result of treatment (GD/L, n=2). IgG fucosylation was analyzed by the lectin method on polyvinylidene fluoride (PVDF) membrane after electrophoretic separation under reducing conditions in stain-free gels. LCA lectin was used to assess core fucosylation and UEA-I for Fuc attached to antenna. AAL, the third of the used lectins, has affinity for both types of Fuc, with the higher affinity to proximal fucose. The intensity of the reactions between the light and heavy chain N-glycans with the given lectin was measured densitometrically and normalized to the amount of protein visualized under UV light. The Kruskal-Wallis test was performed to assess the statistical significance of the obtained results, for p<0,05. LCA lectin did not bind to the IgG light chain, indicating that the Fab fragment N-glycans are not core fucosylated. The binding intensity of AAL and UEA-I lectins was significantly stronger for the heavy chain than the light chain, which is mainly due to the fact that only about 20% of Fab N-glycans are glycosylated. The results suggest that fucosylation of IgG N-glycans tends to change in Graves' disease, although no experiments showed statistically significant differences. The most consistent results for the three replicates of the experiment were obtained in the case of reactions with the AAL lectin, which indicates a tendency to the fucosylation increase of both IgG chains in patients with GD and a downward trend in donors who underwent treatment compared to GD. The results are not statistically significant, probably due to not enough samples used for analysis. The assessment of changes in IgG fucosylation in GD is important to determine the contribution of altered fucosylation to autoimmunity process in this disease

Changes of IgG N-glycosylation in thyroid autoimmunity : the modulatory effect of methimazole in Graves disease and the association with the severity of inflammation in Hashimotos thyroiditis

The N-glycome of immunoglobulin G (IgG), the most abundant glycoprotein in human blood serum, reflects pathological conditions of autoimmunity and is sensitive to medicines applied in disease therapy. Due to the high sensitivity of N-glycosylation, the IgG N-glycan profile may serve as an indicator of an ongoing inflammatory process. The IgG structure and its effector functions are strongly dependent on the composition of N-glycans attached to the Fc fragment, and the binding of antigens is regulated by Fab sugar moieties. Because of the crucial role of N-glycans in IgG function, remodeling of its N-oligosaccharides can induce pathological changes that ultimately contribute to the development of autoimmunity; restoration of their physiological structure is critical to the reduction of disease symptoms. Our recently published data have shown that the pathology of autoimmune thyroid diseases (AITDs), including Hashimoto’s thyroiditis (HT) and Graves’ disease (GD), is accompanied by alterations of the composition of IgG N-glycans. The present study is a more in-depth investigation of IgG glycosylation in both AITDs, designed to determine the relationship between the severity of thyroid inflammation and IgG N-glycan structures in HT, and to assess the impact of immunosuppressive therapy on the N-glycan profile in GD patients. The study material consisted of human serum samples collected from donors with elevated anti-thyroglobulin (Tg) and/or anti-thyroperoxidase (TPO) IgGs without symptoms of hypothyroidism (n=68), HT patients characterized by high autoantibody titers and advanced destruction of the thyroid gland (n=113), GD patients with up-regulated IgG against thyroid-stimulating hormone receptor (TSHR) before (n=62) and after (n=47) stabilization of TSH level as a result of methimazole therapy (study groups), and healthy donors (control group, n=90). IgG was isolated from blood serum using protein G affinity chromatography. N-glycans were released from IgG by PNGase F digestion and analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) after 2-aminobenzamide (2-AB) labeling. UPLC-MS chromatograms were integrated into 25 peaks (GP) in the Waters UNIFI Scientific Information System, and N-glycans were assigned based on the glucose unit values and mass-to-charge ratios (m/z) of the detected ions. The Kruskal-Wallis non-parametric test was used to determine the statistical significance of the results (p<0.05). The obtained results suggest that modifications of IgG sialylation, galactosylation and core-fucosylation are associated with the severity of HT symptoms. Methimazole therapy implemented in GD patients affected the IgG N-glycan profile; as a result, the content of the sialylated and galactosylated oligosaccharides with core fucose differed after treatment. Our results suggest that N-glycosylation of IgG undergoes dynamic changes during the intensification of thyroiditis in HT, and that in GD autoimmunity it is affected significantly by immunosuppressive therapy