Evidence of Paraoxonases 1, 2, and 3 Expression in Human Ovarian Granulosa Cells

Increasing evidence suggests that the antioxidant paraoxonase proteins, PON1, PON2, and PON3, have a role in reproduction and may be synthesized by ovarian cells. The aim of this work was to investigate whether human ovarian granulosa cells (GC) express paraoxonases 1, 2, and 3 (PON1, PON2, and PON3) at both the transcriptional and protein levels. Cells were purified from follicle samples of women undergoing ovarian stimulation at oocyte retrieval. We analyzed mRNA by polymerase chain reaction using specific primers for the different variants and quantified the proteins by Western blot using commercially available human recombinant PON proteins as standards. The protein subcellular distribution was determined by immunofluorescence and confocal microscopy and the cell cycles by flow cytometry. Thymidine was used for cellular synchronization at G1/S. Human hepatoma HepG2 and immortalized granulosa COV434 cell lines were used to optimize methodologies. mRNAs from PON1, the two variants of PON2, and PON3 were detected in GC. The cells actively secreted PON1 and PON3, as evidenced by the protein detection in the incubation medium. PON1 and PON3 were mainly distributed in the cytoplasm and notably in the nucleus, while PON2 colocalized with mitochondria. Subcellular nucleo-cytoplasmic distribution of PON1 was associated with the cell cycle. This is the first evidence describing the presence of mRNAs and proteins of the three members of the PON family in human ovarian GC. This study provides the basis of further research to understand the role of these proteins in GC, which will contribute to a better understanding of the reproduction process.This work was supported by research grants from the University of the Basque Country UPV/EHU (General Support to Research Groups, refs. GIU16/62 and GIU20/021), and Basque Government (pre-doctoral grant to I.P.-R.)

Metabolic adaptations in spontaneously immortalized PGC-1a knock-out mouse embryonic fibroblasts increase their oncogenic potential

Trabajo presentado en la SEBBN 19 Madrid, celebrada en Madrid del 16 al 19 de julio de 2019.PGC-1a controls, to a large extent, the capacity of cells to respond to changing nutritional requirements and energetic demands. The key role of metabolic reprogramming in tumor development has highlighted the potential role of PGC-1a in cancer. To investigate how loss of PGC-1a activity in primary cells impacts the oncogenic characteristics of spontaneously immortalized cells, and the mechanisms involved, we used the classic 3T3 protocol to generate spontaneously immortalized mouse embryonic fibroblasts (iMEFs) from wild-type (WT) and PGC-1a knockout (KO) mice and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1a KO iMEFs formed larger and more proliferative primary tumors than WT counterparts, and fostered the formation of lung metastasis by B16 melanoma cells. These characteristics were associated with the reduced capacity of KO iMEFs to respond to cell contact inhibition, in addition to an increased ability to form colonies in soft agar, an enhanced migratory capacity, and a reduced growth factor dependence. The mechanistic basis of this phenotype is likely associated with the observed higher levels of nuclear b-catenin and c-myc in KO iMEFs. Evaluation of the metabolic adaptations of the immortalized cell lines identified a decrease in oxidative metabolism and an increase in glycolytic flux in KO iMEFs, which were also more dependent on glutamine for their survival. Furthermore, glucose oxidation and tricarboxylic acid cycle forward flux were reduced in KO iMEF, resulting in the induction of compensatory anaplerotic pathways. Indeed, analysisi of aminoacid and lipid patterns supported the efficient use of tricarboxylic acid cycle intermediates to synthesize lipids and proteins to support elevated cell growth rates. All these characteristics have been observed in aggressive tumors and support a tumor suppressor role for PGC-1a, restraining metabolic adaptations in cancer

Influence Of Oxygen Partial Pressure On The Characteristics Of Human Hepatocarcinoma Cells

Most of the in vitro studies using liver cell lines have been performed under atmospheric oxygen partial pressure (21% O-2). However, the oxygen concentrations in the liver and cancer cells are far from this value. In the present study, we have evaluated the influence of oxygen on 1) the tumor cell lines features (growth, steadystate ROS levels, GSH content, activities of antioxidant enzymes, p66 Shc and SOD expressions, metalloproteinases secretion, migration, invasion, and adhesion) of human hepatocellular carcinoma cell lines, and b) the response of the cells to an oxidant stimulus (aqueous leaf extract of the V. baccifera plant species). For this purpose, three hepatocarcinoma cell lines with different p53 status, HepG2 (wild-type), Huh7 (mutated), and Hep3B (deleted), were cultured (6-30 days) under atmospheric (21%) and more physiological (8%) pO(2). Results showed that after long-term culturing at 8% versus 21% O-2, the cellular proliferation rate and the steady-state levels of mitochondrial O-2-were unaffected. However, the intracellular basal ROS levels were higher independently of the characteristics of the cell line. Moreover, the lower pO(2) was associated with lower glutathione content, the induction of p66 Shc and Mn-SOD proteins, and increased SOD activity only in HepG2. This cell line also showed a higher migration rate, secretion of active metalloproteinases, and a faster invasion. HepG2 cells were more resistant to the oxidative stress induced by V. baccifera. Results suggest that the longterm culturing of human hepatoma cells at a low, more physiological pO(2) induces antioxidant adaptations that could be mediated by p53, and may alter the cellular response to a subsequent oxidant challenge. Data support the necessity of validating outcomes from studies performed with hepatoma cell cultures under ambient O-2.This work was supported by research grants from the Basque Government (Department of Education, Universities and Research, ref. IT687-13), and University of the Basque Country, UPV/EHU (CLUMBER UFI11/20 and pre-doctoral and post-doctoral grants to J.T.). We thank Jose Antonio Lopez for his valuable technical assistance with cell cultures. Technical and human support provided by SGIKer (UPV/EHU, MICINN, GV/EJ, ESF) is gratefully acknowledged

Analysis of Protein Oxidative Modifications in Follicular Fluid from Fertile Women: Natural Versus Stimulated Cycles

Oxidative stress is associated with obstetric complications during ovarian hyperstimulation in women undergoing in vitro fertilization. The follicular fluid contains high levels of proteins, which are the main targets of free radicals. The aim of this work was to determine specific biomarkers of non-enzymatic oxidative modifications of proteins from follicular fluid in vivo, and the effect of ovarian stimulation with gonadotropins on these biomarkers. For this purpose, 27 fertile women underwent both a natural and a stimulated cycle. The biomarkers, glutamic semialdehyde (GSA), aminoadipic semialdehyde (AASA), N-epsilon-(carboxymethyl)lysine (CML), and N-epsilon-(carboxyethyl)lysine (CEL), were measured by gas-liquid chromatography coupled to mass spectrometry. Results showed that follicular fluid contained products of protein modifications by direct metal-catalyzed oxidation (GSA and AASA), glycoxidation (CML and CEL), and lipoxidation (CML). GSA was the most abundant biomarker (91.5%). The levels of CML amounted to 6% of the total lesions and were higher than AASA (1.3%) and CEL (1.2%). In the natural cycle, CEL was significantly lower (p < 0.05) than in the stimulated cycle, suggesting that natural cycles are more protected against protein glycoxidation. These findings are the basis for further research to elucidate the possible relevance of this follicular biomarker of advanced glycation end product in fertility programs.This research was funded by the Basque Government, Department of Education, Universities and Research (project ref. IT687-13 and predoctoral grant to I.P.) and Department of Economic Development and Competitiveness, SPRI (refs. IG-20130001214 and IG-2014 0000837), University of the Basque Country UPV/EHU (ref. GIU16/62), and Fundacion Jesus Gangoiti (grant to S.M.)

Metabolic adaptations in spontaneously immortalized PGC-1α knock-out mouse embryonic fibroblasts increase their oncogenic potential

PGC-1α controls, to a large extent, the capacity of cells to respond to changing nutritional requirements and energetic demands. The key role of metabolic reprogramming in tumor development has highlighted the potential role of PGC-1α in cancer. To investigate how loss of PGC-1α activity in primary cells impacts the oncogenic characteristics of spontaneously immortalized cells, and the mechanisms involved, we used the classic 3T3 protocol to generate spontaneously immortalized mouse embryonic fibroblasts (iMEFs) from wild-type (WT) and PGC-1α knockout (KO) mice and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1α KO iMEFs formed larger and more proliferative primary tumors than WT counterparts, and fostered the formation of lung metastasis by B16 melanoma cells. These characteristics were associated with the reduced capacity of KO iMEFs to respond to cell contact inhibition, in addition to an increased ability to form colonies in soft agar, an enhanced migratory capacity, and a reduced growth factor dependence. The mechanistic basis of this phenotype is likely associated with the observed higher levels of nuclear β-catenin and c-myc in KO iMEFs. Evaluation of the metabolic adaptations of the immortalized cell lines identified a decrease in oxidative metabolism and an increase in glycolytic flux in KO iMEFs, which were also more dependent on glutamine for their survival. Furthermore, glucose oxidation and tricarboxylic acid cycle forward flux were reduced in KO iMEF, resulting in the induction of compensatory anaplerotic pathways. Indeed, analysis of amino acid and lipid patterns supported the efficient use of tricarboxylic acid cycle intermediates to synthesize lipids and proteins to support elevated cell growth rates. All these characteristics have been observed in aggressive tumors and support a tumor suppressor role for PGC-1α, restraining metabolic adaptations in cancer.This work was funded by grants from the Spanish “Ministerio de Ciencia, Innovación y Universidades” (MICINN) and ERDF/FEDER funds, SAF2012-37693, SAF2015-63904-R, SAF2015-71521-REDC, RTI2018-093864-B-I00 to M.M., SAF2017-83043-R and B2017/BMD-3724 to S·C., PI15/00107 to A.M.R, the University of the Basque Country UPV/EHU grant GIU16/62) to J.l.R.S. and M.B.R.L., and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement 721236-TREATMENT to M.M