Silvia Maria Suter Correia Cadena

Involvement of catalase in the apoptotic mechanism induced by apigenin in HepG2 human hepatoma cells

Apigenin has been reported to inhibit proliferation of cancer cells; however, the mechanism underlying its action is not completely understood. Here, we evaluated the effects of apigenin on the levels of expression and activity of antioxidant enzymes, and the involvement of ROS in the mechanism of cell death induced by apigenin in HepG2 human hepatoma cells. Upon treatment with apigenin, HepG2 cells displayed a reduction in cell viability in a dose- and time-dependent manner, and some morphological changes. In addition, apigenin treatment induced ROS generation and significantly decreased the mRNA levels and activity of catalase and levels of intracellular GSH. On the other hand, apigenin treatment did not alter the expression or activity levels of other antioxidant enzymes. Addition of exogenous catalase significantly reduced the effects of apigenin on HepG2 cell death. We also demonstrated that HepG2 cells are more sensitive to apigenin-mediated cell death than are primary cultures of mouse hepatocytes, suggesting a differential toxic effect of this agent in tumor cells. Our results suggest that apigenin-induced apoptosis in HepG2 cells may be mediated by a H(2)O(2)-dependent pathway via reduction of the antioxidant defenses.

Hispidulin: Antioxidant properties and effect on mitochondrial energy metabolism†

Hispidulin (6-methoxy-5,7,4′-trihydroxyflavone) and eupafolin (6-methoxy-5,7,3′,4′-tetrahydroxyflavone), are flavonoids found in the leaves of Eupatorium litoralle. They have recognized antioxidant and antineoplastic properties, although their action mechanisms have not been previously described. We now report the effects of hispidulin on the oxidative metabolism of isolated rat liver mitochondria (Mit) and have also investigated the prooxidant and antioxidant capacity of both flavonoids. Hispidulin (0.05–0.2 mM) decreased the respiratory rate in state III and stimulated it in state IV, when glutamate or succinate was used as oxidizable substrate. Hispidulin inhibited enzymatic activities between complexes I and III of the respiratory chain. In broken Mit hispidulin (0.2 mM) slightly inhibited ATPase activity (25%). However, when intact Mit were used, the flavonoid stimulated this activity by 100%. Substrate energized mitochondrial swelling was markedly inhibited by hispidulin. Both hispidulin and eupafolin were able to promote iron release from ferritin, this effect being more accentuated with eupafolin with the suggestion of a possible involvement of H2O2 in the process. Hispidulin was incapable of donating electrons to the stable free radical DPPH, while eupafolin reacted with it in a similar way to ascorbic acid. The results indicate that hispidulin as an uncoupler of oxidative phosphorylation, is able to release iron from ferritin, but has distinct prooxidant and antioxidant properties when compared to eupafolin.

Effect of MI-D, a new mesoionic compound, on energy-linked functions of rat liver mitochondria.

MI‐D (4‐phenyl‐5‐(4‐nitro‐cinnamoyl)‐1,3,4‐thiadiazolium‐2‐phenylamine chloride), a new mesoionic compound, depressed the phosphorylation efficiency of liver mitochondria as deduced from an accentuated decrease of the respiratory control coefficient and ADP/O ratio. Analysis of segments of the respiratory chain suggested that the MI‐D inhibition site is further on than complex I and between complexes II and III. The transmembrane electrical potential (Δψ) was collapsed dependent on MI‐D concentration. ATPase activity was dramatically increased by MI‐D in intact mitochondria, but inhibited in carbonylcyanide p‐trifluoromethoxyphenylhydrazone (FCCP)‐uncoupled mitochondria. These results suggest that MI‐D acts as an uncoupler agent, a property closely related to its structural characteristics.

Converting potent indeno[1,2-b]indole inhibitors of protein kinase CK2 into selective inhibitors of the breast cancer resistance protein ABCG2.

A series of indeno[1,2-b]indole-9,10-dione derivatives were synthesized as human casein kinase II (CK2) inhibitors. The most potent inhibitors contained a N(5)-isopropyl substituent on the C-ring. The same series of compounds was found to also inhibit the breast cancer resistance protein ABCG2 but with totally different structure-activity relationships: a N(5)-phenethyl substituent was critical, and additional hydrophobic substituents at position 7 or 8 of the D-ring or a methoxy at phenethyl position ortho or meta also contributed to inhibition. The best ABCG2 inhibitors, such as 4c, 4h, 4i, 4j, and 4k, behaved as very weak inhibitors of CK2, whereas the most potent CK2 inhibitors, such as 4a, 4p, and 4e, displayed limited interaction with ABCG2. It was therefore possible to convert, through suitable substitutions of the indeno[1,2-b]indole-9,10-dione scaffold, potent CK2 inhibitors into selective ABCG2 inhibitors and vice versa. In addition, some of the best ABCG2 inhibitors, which displayed a very low cytotoxicity, thus giving a high therapeutic ratio, and appeared not to be transported, constitute promising candidates for further investigations.

Effect of temperature acclimation on the liver antioxidant defence system of the Antarctic nototheniids Notothenia coriiceps and Notothenia rossii.

The aim of this study was to determine whether endemic Antarctic nototheniid fish are able to adjust their liver antioxidant defence system in response to the temperature increase. The activity of the superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) enzymes as well as the content of non-enzymatic oxidative stress markers such as reduced glutathione (GSH), lipid peroxidation (LPO) and protein carbonyl (PC) were measured in the liver of two Antarctic fish species, Notothenia rossii and Notothenia coriiceps after 1, 3 and 6days of exposure to temperatures of 0°C and 8°C. The GST activity showed a downregulation in N. rossii after 6days of exposure to the increased temperature. The activity profiles of GST and GR in N. rossii and of GPx in N. coriiceps also changed as a consequence of heating to 8°C. The GSH content increased by heating to 8°C after 3days in N. coriiceps and after 6days in N. rossii. The content of malondialdehyde (MDA), a LPO marker, showed a negative modulation by the heating to 8°C in N. rossii after 3days of exposure to temperatures. Present results show that heating to 8°C influenced the levels and profiles of the antioxidant enzymes and defences over time in the nototheniid fish N. rossii and N. coriiceps.

Production of cachexia mediators by Walker 256 cells from ascitic tumors

In neoplasic cachexia, chemical mediators seem to act as initiators or perpetuators of this process. Walker 256 cells, whose metabolic properties have so far been little studied with respect to cancer cachexia, are used as a model for the study of this syndrome. The main objective of this research was to pinpoint the substances secreted by these cells that may contribute to the progression of the cachectic state. Since inflammatory mediators seem to be involved in the manifestation of this syndrome, the in vitro production of nitric oxide (NO), cytokines (tumor necrosis factor alpha (TNF‐α) and interleukin‐6 (IL‐6)), and prostaglandin E2 (PGE2) was evaluated in Walker 256 cells isolated from ascitic tumors. After 4 or 5 h, a significant increase in NO production was observed (2.55 ± 1.56 and 4.05 ± 1.99 nmol NO per 107 cells, respectively). When isolated from a 6‐day‐old tumor, a significantly lower production of IL‐6 and higher production of TNF‐α than in cells from a 4‐day‐old tumor were observed, indicating a relationship between the production of cytokines and the time of tumor development after implantation. Considerable production of PGE2 by Walker 256 cells isolated from the 6‐day‐old tumor was also observed. Polyamines were also determined in Walker 256 cells. Levels of putrescine, spermidine, and spermine did not show significant differences in tumors developed during 4 or 6 days. Direct evidence of the release of proinflammatory cytokines and PGE2 by Walker 256 cells suggests that these mediators can drive the cachectic syndrome in the host, the effect being dependent on tumor development time. Copyright

Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis.

Phenolic indeno[1,2- b ]indoles as ABCG2-selective potent and non-toxic inhibitors stimulating basal ATPase activity

Ketonic indeno[1,2-b]indole-9,10-dione derivatives, initially designed as human casein kinase II (CK2) inhibitors, were recently shown to be converted into efficient inhibitors of drug efflux by the breast cancer resistance protein ABCG2 upon suited substitutions including a N5-phenethyl on C-ring and hydrophobic groups on D-ring. A series of ten phenolic and seven p-quinonic derivatives were synthesized and screened for inhibition of both CK2 and ABCG2 activities. The best phenolic inhibitors were about threefold more potent against ABCG2 than the corresponding ketonic derivatives, and showed low cytotoxicity. They were selective for ABCG2 over both P-glycoprotein and MRP1 (multidrug resistance protein 1), whereas the ketonic derivatives also interacted with MRP1, and they additionally displayed a lower interaction with CK2. Quite interestingly, they strongly stimulated ABCG2 ATPase activity, in contrast to ketonic derivatives, suggesting distinct binding sites. In contrast, the p-quinonic indenoindoles were cytotoxic and poor ABCG2 inhibitors, whereas a partial inhibition recovery could be reached upon hydrophobic substitutions on D-ring, similarly to the ketonic derivatives.

Effects of natural flavones on membrane properties and citotoxicity of HeLa cells

The aim of this study was to determine whether eupafolin and hispidulin, flavones extracted from Eupatorium littorale Cabrera, Asteraceae, have the ability to change properties of biological membranes and promote cytotoxic effects. Eupafolin (50-200 µM) decreased approximately 30% the rate and total amplitude of valinomycin induced swelling and 60-100% the energy-dependent mitochondrial swelling. Moreover, eupafolin (200 µM) reduced 35% the mitochondrial permeability transition, and hispidulin did not change this parameter in any of the doses tested. The evaluation of phase transition of DMPC liposomes with the probe DPH demonstrated that hispidulin and eupafolin affect gel and fluid phase. With mitochondrial membrane as model, hispidulin increased the polarization of fluorescence when used DPH-PA probe. Eupafolin and hispidulin (100 µM) promoted a reduction of 40% in cellular viability of HeLa cells in 24 h. Our results suggest that eupafolin and hispidulin have cytotoxic effects that can be explained, in part, by alterations promoted on biological membranes properties and mitochondrial bioenergetics.

Metabolic switches during the first steps of adipogenic stem cells differentiation.

The understanding of metabolism during cell proliferation and commitment provides a greater insight into the basic biology of cells, allowing future applications. Here we evaluated the energy and oxidative changes during the early adipogenic differentiation of human adipose tissue-derived stromal cells (hASCs). hASCs were maintained under differentiation conditions during 3 and 7days. Oxygen consumption, mitochondrial mass and membrane potential, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) and catalase activities, non-protein thiols (NPT) concentration and lipid peroxidation were analyzed. We observed that 7days of adipogenic induction are required to stimulate cells to consume more oxygen and increase mitochondrial activity, indicating organelle maturation and a transition from glycolytic to oxidative energy metabolism. ROS production was only increased after 3days and may be involved in the differentiation commitment. ROS source was not only the mitochondria and we suggest that NOX proteins are related to ROS generation and therefore adipogenic commitment. ROS production did not change after 7days, but an increased activity of catalase and NPT concentration as well as a decreased lipid peroxidation were observed. Thus, a short period of differentiation induction is able to change the energetic and oxidative metabolic profile of hASCs and stimulate cytoprotection processes.