Differential response of hepatocellular carcinoma glycolytic metabolism and oxidative stress markers after exposure to human amniotic membrane proteins.

BACKGROUND: The human Amniotic Membrane (hAM) has been studied as a potential therapeutic option in cancer, namely in hepatocellular carcinoma. Previously, our research group evaluated the effect of human Amniotic Membrane Protein Extracts (hAMPE) in cancer therapy, demonstrating that hAMPE inhibit the metabolic activity of human hepatocellular carcinoma cell lines: Hep3B2.1-7, HepG2 and Huh7. Therefore, and considering the close relationship between metabolic activity and oxidative stress, the aim of this study was to evaluate the effect of hAMPE treatment in glucose metabolism and its role in oxidative stress of hepatocellular carcinoma. METHODS AND RESULTS: Glucose uptake and lactate production was assessed by 1 H-NMR, and the expression of several mediators of the glycolytic pathway was evaluated by Western blot or fluorescence. Total antioxidant capacity (TAC) and biomarkers of oxidative stress effects in proteins were detected. Our results showed that hAMPE treatment increased glucose consumption on Hep3B2.1-7, HepG2, and Huh7 through the increase of GLUT1 in Hep3B2.1-7 and Huh7, and GLUT3 in HepG2 cells. It was observed an increased expression of 6-phosphofrutokinase (PFK-1L) in all cell lines though glucose was not converted to lactate on HepG2 and Huh7 cells, suggesting that hAMPE treatment may counteract the Warburg effect observed in carcinogenesis. In Hep3B2.1-7, hAMPE treatment induced an increase in expression of lactate dehydrogenase (LDH) and monocarboxylate transporter isoform 4 (MCT4). We further detected that hAMPE enhances the TAC of culture media after 2 and 8 h. This was followed by a degree of protection against proteins nitration and carbonylation. CONCLUSIONS: Overall, this work highlights the potential usefulness of hAMPE as anticancer therapy through the modulation of the glycolytic and oxidative profile in human hepatocellular carcinoma.

Amniotic membrane extract differentially regulates human peripheral blood T cell subsets, monocyte subpopulations and myeloid dendritic cells.

The discovery of the immunoregulatory potential of human amniotic membrane (hAM) propelled several studies focusing on its application for the treatment of immunological disorders. However, there is little information regarding the effects of hAM on distinct activation and differentiation stages of immune cells. Here, we aim to investigate the effect of human amniotic membrane extract (hAME) on the pattern of cytokine production by T cells, monocytes and myeloid dendritic cells (mDCs). For this purpose, peripheral blood mononuclear cells (PBMCs) from eight healthy individuals were stimulated in vitro in the presence or absence of hAME. Mitogen-induced proliferation of PBMCs and cytokine production among the distinct T cell functional compartments, monocyte subpopulations and mDCs were evaluated. hAME displayed an anti-proliferative effect and decreased the frequency of T cells producing tumor necrosis factor (TNF)α, interferon (IFN)γ and interleukin (IL)-2, for all T cell functional compartments. The frequency of IL-17 and IL-9-producing T cells was also reduced. The inhibition of mRNA expression of granzyme B, perforin and NKG2D by CD8+ T cells and γδ T cells and the augment of FoxP3 and IL-10 in CD4+ T cells and IL-10 in regulatory T cells were also observed. Furthermore, hAME inhibited IFNγ-induced protein (IP)-10 expression by classical and non-classical monocytes, without hampering the production of TNFα and IL-6 by monocytes and mDCs. These results suggest that hAME exerts an anti-inflammatory effect on T cells, still at a different extent for distinct T cell functional compartments.

The protective effect of regucalcin against radiation-induced damage in testicular cells.

AIMS: Regucalcin (RGN), a protein broadly expressed in the male reproductive tract, has shown to have beneficial effects on spermatogenesis suppressing chemical-induced apoptosis. This study aimed to evaluate whether RGN overexpression ameliorates the spermatogenic phenotype after radiation treatment. MAIN METHODS: Transgenic rats overexpressing RGN (Tg-RGN) and their wild-type (Wt) counterparts were exposed to a single dose of X-rays (6Gy), and at ten weeks after irradiation, the testicular status and the epididymal sperm parameters were evaluated. The expression of RGN and several cell cycle and apoptosis regulators, the enzymatic activity of caspase-3, and RGN immunostaining were also assessed. KEY FINDINGS: Tg-RGN animals displayed higher gonadosomatic index, and augmented sperm viability and motility relatively to their Wt counterparts after irradiation, as well as higher frequency of normal sperm morphology and a diminished incidence of head-defects. The differences in reproductive parameters were underpinned by a lower rate of apoptosis, as evidenced by the reduced activity of caspase-3, lower levels of caspase-8, and increased Bcl-2/Bax ratio in the testis of Tg-RGN animals. Supporting the involvement of RGN in the anti-apoptotic response, an enhanced expression of RGN was observed in irradiated rats. SIGNIFICANCE: Transgenic-overexpression of RGN protected against radiation-induced testicular damage, which strengthens the role of this protein protecting cells from the damage of external agents. These findings also indicated that the modulation of RGN testicular levels would be a mechanism for fertility preservation in men undergoing oncological treatment.