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Mitochondria-related oxidative stress contributes to ovarian cancer-promoting activity of mesothelial cells subjected to malignant ascites
International Journal of Biochemistry and Cell Biology 98 (2018) 82–88
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International Journal of Biochemistry and Cell Biology journal homepage: www.elsevier.com/locate/biocel
Mitochondria-related oxidative stress contributes to ovarian cancerpromoting activity of mesothelial cells subjected to malignant ascites
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Martyna Pakułaa, Justyna Mikuła-Pietrasika, Łukasz Stryczyńskia, Paweł Uruskia, Sebastian Szubertb, Rafał Moszyńskib, Dariusz Szpurekb, Stefan Sajdakb, Andrzej Tykarskia, ⁎ Krzysztof Książeka, a b
Department of Hypertensiology, Angiology and Internal Medicine, Poznań University of Medical Sciences, Długa 1/2 Str., 61-848 Poznań, Poland Division of Gynecological Surgery, Poznań University of Medical Sciences, Polna 33 Str., 60-535 Poznań, Poland
A R T I C LE I N FO
A B S T R A C T
Keywords: Malignant ascites Mesothelial cells Ovarian cancer Oxidative stress Senescence
Very little is known about the mechanisms by which malignant ascites modulates the cancer-promoting activity of human peritoneal mesothelial cells (HPMCs). Because malignant ascites induces pro-tumoral senescence in HPMCs, here we examined if this effect could be driven by oxidative stress. The study showed that malignant ascites generated by serous ovarian tumors induced oxidative damage to the DNA (γH2A.X, 53BP1, 8-hydroxy2′-deoxyguanosine) and lipids (8-isoprostane) in HPMCs as well as increased the production of mitochondrial superoxides and cellular peroxides in these cells. This activity coincided with increased activity of two enzymes involved in the mitochondrial production of oxidants, i.e. cytochrome c oxidase and NADH dehydrogenase, decreased mitochondrial inner membrane potential, increased mitochondrial mass, and increased the activity of peroxisome proliferator-activated receptor gamma coactivator-1 alpha. Increased production of superoxides and peroxides in cells subjected to the malignant ascites was effectively reduced when the fluid was pre-incubated with neutralizing antibodies against hepatocyte growth factor. Moreover, when HPMCs subjected to the malignant ascites were protected against oxidative stress with a spin-trap scavenger of reactive oxygen species, they displayed decreased expression of senescence-associated β-galactosidase and their potential to stimulate cancer cell adhesion, proliferation, and migration was significantly diminished. Collectively, our findings indicate that improved ovarian cancer cell progression in response to HPMCs exposed to malignant ascites may be associated with the development of profound oxidative stress in these cells.
1. Introduction Although a plethora of evidence has emerged that malignant ascites contributes to progression of ovarian cancer, more knowledge is being gained about the mechanistic aspects of its activity. In general, it is believed that malignant ascites creates a hospitable peritoneal milieu that helps ovarian cancer cells survive, invade, and disseminate (Ahmed and Stenvers, 2013). This is linked, in turn, with the ascites’ ability to suppress peritoneal inflammation (Simpson-Abelson et al., 2013) and with the presence of large amounts of agents that support angiogenesis and cancer cell motility in this fluid (Kipps et al., 2013). Last but not least, malignant ascites modulates the behavior of normal peritoneal cells, e.g. by inhibiting the cancer cells’ apoptosis driven by the peritoneal mesothelium (Matte et al., 2014). It has been shown that human peritoneal mesothelial cells (HPMCs), which constitute the largest fraction of cells within the peritoneal
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cavity, support the progression of ovarian cancer in vitro and in vivo when they become senescent (Mikula-Pietrasik et al., 2016b). Interestingly, recent findings indicate that malignant ascites accelerates the senescence of HPMCs via the activity of growth-related oncogene 1 (GRO-1) and hepatocyte growth factor (HGF) (Mikula-Pietrasik et al., 2016a). The intracellular mechanism underlying this activity of the ascites remains, however, unknown. Taking into account that the senescence of normal somatic cells is often causatively linked with deleterious activity of reactive oxygen species (ROS) (Davalli et al., 2016), the goal of this study was to examine if the pro-senescence activity of malignant ascites and concomitant pro-tumoral effects of HPMCs may be caused by oxidative stress. To this end, several parameters associated with oxidative cell status were examined, including oxidative DNA damage, lipid peroxidation, production of ROS, activity of enzymes involved in ROS release, mitochondrial membrane potential, and mitochondrial
Corresponding author. E-mail address: [email protected] (K. Książek).
https://doi.org/10.1016/j.biocel.2018.03.011 Received 7 December 2017; Received in revised form 18 February 2018; Accepted 13 March 2018 Available online 14 March 2018 1357-2725/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Effect of growth medium (GM), benign ascites (BA) and malignant ascites (MA) on activation of DNA damage response (DDR) and magnitude of lipid peroxidation in cultured HPMCs. Quantification of fluorescence of γH2A.X (A) and 53BP1 foci (B) was used as a measure of DDR. Quantification of 8-OH-dG was treated as a measure of nonspecific DNA injury (C). Quantification of 8isoprostane was used to estimate the magnitude of lipid peroxidation (D). Representative pictures showing γH2A.X and 53BP1 foci (green dots) in the nuclei (blue DAPI staining) of cells exposed to growth medium, benign and malignant fluid (E). The results derive from experiments performed with HPMCs obtained from 8 different donors, and with benign and malignant fluids collected from 8 different patients. (+) in the boxes indicate means, while the horizontal lines indicate medians. RFU – relative fluorescence units. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
undergoing abdominal surgery (institutional consent number 187/14), as described in detail elsewhere (Ksiazek, 2013). Cells were identified as pure mesothelium by their epithelial-like morphology at confluency and uniform positive staining for cytokeratins and the HBME-1 antigen. During the experiments only HPMCs obtained from the first two passages, which corresponds to about 25% of their replicative lifespan, were used. The viability of these cells exceeded 98%. Ovarian cancer cells, SKOV-3, were obtained from the ECCC (Porton Down, UK) and maintained in RPMI 1640 medium with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 g/ml), and 10% FBS. Primary epithelial ovarian cancer cells (EOCs) were isolated from tumors excised during cytoreductive surgery from patients with serous ovarian cancer (stage IV). Briefly, the tumors were cut into pieces of equal weight and then placed in a solution of 0.05% trypsin and 0.02% EDTA for 20 min at 37 °C with gentle shaking. After resuspension in RPMI1640 containing 20% FBS, the cells were probed with an antibody directed against the epithelial-related antigen (MOC-31; Abcam, Cambridge, UK) to confirm their cancerous nature. Finally, the ovarian cancer cells were cultured in RPMI 1640 supplemented with L-glutamine (2 mM) and 20% FBS.
biogenesis. These tests were followed by intervention studies in which HPMCs treated with ascites were protected against oxidative stress by using a spin-trap ROS scavenger, then their senescence and their impact on cancer cell progression were evaluated. 2. Materials and methods 2.1. Chemicals Unless otherwise stated, all chemicals were from Sigma (St. Louis, MO) and culture plastics were from Thermo Fisher Scientific (Waltham, MA, USA). 2.2. Ascitic fluids Malignant ascites were obtained during cytoreductive surgery from patients with high-grade serous ovarian cancer at stage IV (n = 8). The histopathology, grade, and stage of the tumors were assigned in keeping with the criteria of the International Federation of Gynecology and Obstetrics. Benign fluids were obtained from age-matched patients with cystadenoma mucinosum multiloculare (n = 8). After collection in sterile conditions, the fluids were centrifuged at 2500 rpm for 5 min and the acellular supernatants were stored at −20 °C until required. The study was approved by an institutional ethics committee (consent number 543/14).
2.4. Experimental conditions HPMCs were placed onto culture dishes and allowed to grow until reaching sub-confluency. Then the cells were exposed to standard growth medium (GM), 10% malignant ascites (MA), and 10% benign ascites (BA) for 72 h. In some experiments, the HPMCs were pre-incubated for 6 h with a spin-trap ROS scavenger, N-tert-butyl-alphaphenylnitrone (PBN, Sigma; 800 μM), before the addition of malignant and benign ascites. Starting from pre-incubation, PBN was constantly
2.3. Cell cultures Human peritoneal mesothelial cells (HPMCs) were isolated from fragments of the omentum derived from 8 non-oncological patients 83
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Fig. 2. Effect of growth medium (GM), benign ascites (BA) and malignant ascites (MA) on the production of reactive oxygen species in HPMCs. Generation of mitochondrial superoxides was monitored using MitoSOX fluorescence (A), whereas generation of cellular peroxides was monitored with DHR fluorescence (B). Moreover, the activity of two enzymes engaged in ROS production was quantified, i.e. cytochrome c oxidase (C) and NADH dehydrogenase (D). The results derive from experiments performed with HPMCs obtained from 8 different donors, and with benign and malignant fluids collected from 8 different patients. (+) in the boxes indicate means, while the horizontal lines indicate medians. RFU – relative fluorescence units.
Fig. 3. Effect of HGF neutralization on the production of mitochondrial superoxides (A) and cellular peroxides (B) by HPMCs exposed to malignant ascites (MA). The fluid was pre-incubated with specific neutralizing antibodies against HGF for 4 h before its addition to cell culture. The results derive from experiments performed with HPMCs obtained from 6 different donors, and with benign and malignant fluids collected from 8 different patients. (+) in the boxes indicate means, while the horizontal lines indicate medians. RFU – relative fluorescence units.
2.6. Oxidative stress-related parameters
present in the culture environment throughout the whole experiment.
Elements of senescence-associated DNA damage, i.e. the foci of the phosphorylated variant of histone H2A.X (γH2A.X) and p53-binding protein 1 (53BP1), were quantified using immunofluorescence as described in (Mikula-Pietrasik et al., 2017). The concentration of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), one of the major products of DNA oxidation, was quantified using DNA Damage (8OHdG) ELISA Kit
2.5. Cellular senescence Development of the senescence phenotype in the HPMCs was determined according to quantification of the activity of senescence-associated β-galactosidase (SA-β-Gal) using a fluorescence-based method as described by Gary and Kindell (Gary and Kindell, 2005). 84
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Fig. 4. Effect of growth medium (GM), benign ascites (BA) and malignant ascites (MA) on mitochondrial metabolism. The fluorescence of JC-1 was used to determine mitochondrial inner membrane potential (A), whereas the fluorescence of NAO was used to estimate mitochondrial mass (B). Panel C shows the activity of PGC1α, an enzyme involved in mitochondrial biogenesis. The results derive from experiments performed with HPMCs obtained from 8 different donors, and with benign and malignant fluids collected from 8 different patients. (+) in the boxes indicate means, while the horizontal lines indicate medians. RFU – relative fluorescence units.
accumulates in the vital mitochondria, forming red fluorescent J-aggregates (at 590 nm), while mitochondrial de-energization leads to the formation of green fluorescent monomers (at 535 nm). A rise in the green/red fluorescence intensity ratio indicates a drop in mitochondrial membrane potential values. Mitochondrial mass was quantified upon cell treatment with 10 μM of 10-n-nonyl-acridine orange (NAO; Sigma) for 10 min at 37 °C in the dark. The fluorescence was recorded at an excitation wavelength of 435 nm and an emission wavelength of 535 nm. Enzymes engaged in the generation of mitochondrial ROS, i.e. cytochrome c oxidase and NADH dehydrogenase, as well as those contributing to the biogenesis of mitochondria (peroxisome proliferatoractivated receptor gamma coactivator-1 alpha; PGC-1α) were analyzed using commercial ELISA-based assays obtained from Wuhan EIAab Science Co., Ltd. (Wuhan, China), as per manufacturer’s instructions. Fig. 5. Effect of growth medium (GM), benign ascites (BA) and malignant ascites (MA) on the activity of SA-β-Gal in HPMCs, in either the presence or absence of the spin-trap ROS scavenger, PBN. The results derive from experiments performed with HPMCs obtained from 8 different donors, and with benign and malignant fluids collected from 8 different patients. (+) in the boxes indicate means, while the horizontal lines indicate medians. RFU – relative fluorescence units.
2.8. Collection of conditioned media Sub-confluent HPMCs were subjected to standard growth medium, 10% malignant ascites, and 10% benign ascites for 72 h, and then they were washed carefully with PBS and incubated with serum-free medium for 48 h to generate conditioned medium (CM). Samples of the CM were centrifuged, filtered through a 0.2 μm pore size filter, and stored at –80 °C until required.
(Biorbyt Ltd., Cambridge, UK), as per manufacturer’s instructions. The magnitude of oxidative modifications of cellular lipids was evaluated according to quantification of 8-isoprostane (8-iso Prostaglandin F2α) using the 8-Isoprostane ELISA kit from Cayman Chemical (Ann Arbor, MI, USA), as per manufacturer’s instructions. The generation of mitochondrial superoxides was examined using a fluorescent dye, MitoSOX Red (Thermo Fisher Scientific). In brief, the cells were stained with 0.1 μM MitoSOX Red for 15 min and then fluorescence was recorded at an excitation wavelength of 390 nm and an emission wavelength of 580 nm. Parallel measurement at an excitation wavelength of 510 nm was performed to monitor the nonsuperoxide-dependent processes that can oxidize HE probes to ethidium (Robinson et al., 2006). Cellular peroxides were detected in cells incubated with 30 μM dihydrorhodamine 123 (DHR) for 15 min at 37 °C. Fluorescence was recorded at 507 nm and 540 nm, for excitation and emission wavelengths, respectively. In some experiments, the production of superoxides and peroxides was examined upon HPMC exposure to malignant ascites pre-incubated for 4 h with specific neutralizing antibodies against hepatocyte growth factor (HGF; R&D Systems; 1 μg/ ml).
2.9. Cancer cell progression Adhesion of ovarian cancer cells probed with a fluorescent dye, calcein AM, to HPMCs exposed to malignant and benign ascites was evaluated according to a methodology described in (Mikula-Pietrasik et al., 2014). Proliferation of ovarian cancer cells towards the CM harvested from HPMCs treated with malignant and benign ascites was tested using Cell Proliferation Kit I (PromoKine; Heidelberg, Germany), as per manufacturer’s instructions. Migration of ovarian cancer cells towards a chemotactic gradient generated by the CM from HPMCs treated with malignant and benign ascites was quantified with ChemoTx migration chambers (Neuro Probe, Gaithersburg, MD, USA), as per manufacturer’s instructions.
2.10. Statistics Statistical analysis was performed using GraphPad Prism™ 5.00 (GraphPad Software, San Diego, USA). The groups were compared with repeated measures analysis of variance (ANOVA) with the NewmanKeuls test as post-hoc. The results are shown as boxes and whiskers, in which sample distribution (minimum, maximum) as well as means and medians are presented. Differences with a P value < 0.05 were treated as statistically significant.
2.7. Mitochondrial metabolism Mitochondrial membrane potential (ΔΨm) was measured in cells probed with 1 μM of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Cayman Chemical), which is a lipophilic cationic fluorescent dye exhibiting potential-dependent accumulation in the mitochondria (Passos et al., 2007). JC-1 85
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Fig. 6. Effect of HPMCs subjected to benign ascites (BA) and malignant ascites (MA) on adhesion (A, B), proliferation (C, D), and migration (E, F) of commercial ovarian cancer cell line SKOV-3 and primary epithelial ovarian cancer cells (EOCs) in either the presence or absence of PBN. The results derive from experiments performed with HPMCs obtained from 8 different donors, and with benign and malignant fluids collected from 8 different patients. Cancer cells were used for each experiment in quadruplicates. (+) in the boxes indicate means, while the horizontal lines indicate medians. RFU – relative fluorescence units.
3. Results and discussion
2018) and increased progression of gastric cancer (Miao et al., 2016). Notably, peritoneal metastases of ovarian tumors develop in late stages of the disease (III/IV) (Halkia et al., 2012), which means that their development coincides not only with the accumulation of senescent HPMCs (Mikula-Pietrasik et al., 2017) but also with the formation of ascites, which becomes clinically significant in the advanced disease (Kipps et al., 2013). In this report, we extended the panel of senescence markers, which are increased in HPMCs subjected to malignant ascites, thus strengthening the evidence for the fluid’s senescence-promoting activity. More precisely, we found that HPMCs exposed to malignant ascites for 3 days
It has recently been shown that the activity of senescence-associated β-galactosidase (SA-β-Gal), a valuable marker of cellular senescence in vitro and in vivo, is elevated in HPMCs subjected to malignant ascites generated by serous ovarian tumors in culture conditions for 3 days. Moreover, HPMCs maintained under such a regimen appeared to promote adhesion, proliferation, and migration of ovarian cancer cells (Mikula-Pietrasik et al., 2016a), which is in line with well-established knowledge on the causative role of senescent HPMCs in the formation of peritoneal metastases of ovarian cancer (Mikula-Pietrasik et al., 86
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display increased expression of γH2A.X and 53BP1 (by 23% and 24%, respectively, vs. benign ascites), which indicates activation of the DNA damage response (DDR) pathway – a phenomenon typical of senescent cells (Passos et al., 2007) (Fig. 1A, B, E). Malignant ascites also increases (by 87%) cellular concentration of DNA adduct, 8-OH-dG (Valavanidis et al., 2009), which means that oxidative modifications of DNA in response to malignant fluid are not solely associated with the induction of pathways related to senescence, but also with the accumulation of nonspecific DNA injury (Fig. 1C). This effect coincided with an increased concentration of 8-isoprostane (by 522%), a marker of cellular phospholipid peroxidation (Swardfager et al., 2017), which implies that malignant ascites potentiates oxidative damage to various classes of macromolecules (Fig. 1D). Indeed, increased oxidative damage to DNA has also been reported in peripheral blood lymphocytes from patients gathering malignant ascites (Wang et al., 2012). Interestingly, in the case of γH2A.X and 8-isoprostane, but not 53BP1, the level of DNA and lipids injury in cells treated with benign ascites was higher (by 13% and 103%, respectively) than in cells maintained in standard growth medium, which indicates that the environment created in the peritoneal cavity by the benign fluid is far from physiological. An initiating event in the activation of DDR is the formation of γH2A.X foci close to DNA double-strand breaks, elicited primarily by deleterious activity of reactive oxygen species (ROS) (Woodbine et al., 2011). In order to establish if ROS contribute to malignant ascites-dependent accumulation of DNA injury, we measured the production of mitochondrial superoxides and cellular peroxides and found that both types of ROS are overproduced (by 75% and 242%, respectively) in HPMCs exposed to the malignant fluid (Fig. 2A, B). In addition, the magnitude of cellular peroxide production by cells treated with benign ascites was by 33% higher compared with cells grown in the presence of standard medium, which may explain increased level of the macromolecules’ injury in this group. Mechanistically, increased generation of mitochondrial ROS was plausibly associated with intensified activity of two enzymes involved in oxidant efflux during respiratory chain reactions, i.e. cytochrome c oxidase (by 18%) and NADH dehydrogenase (by 14%) (Fig. 2C, D). The activity of the latter enzyme in cells subjected to benign ascites was by 24% higher than in cells cultured under physiological conditions. Increased activity of cytochrome c oxidase and NADH dehydrogenase was previously causatively linked with increased generation of ROS in senescent fibroblasts (Allen et al., 1999). Moreover, the contribution of cytochrome c oxidase has also been evidenced in HPMCs, whose premature senescence was triggered by ovarian cancer-derived hepatocyte growth factor (HGF) (Mikula-Pietrasik et al., 2017). Interestingly, HGF present in malignant ascites from patients suffering from serous ovarian carcinoma has recently been identified as a mediator of this fluid’s pro-senescent activity (Mikula-Pietrasik et al., 2016a). For this reason, it cannot be excluded that the mitochondrial machinery responsible for the overproduction of ROS in cells exposed to malignant ascites may be launched by HGF. In order to verify this assumption, the production of ROS was examined in the presence of malignant ascites in which HGF was neutralized using specific antibodies. The experiment showed that, indeed, the production of either mitochondrial superoxides or cellular peroxides that was initially upregulated in response to the malignant fluid was significantly attenuated (by 35% and by 28% for superoxides and peroxides, respectively) when the activity of HGF in the fluid was chemically inhibited (Fig. 3). The relationship between the activity of HGF and increased production of ROS has also been evidenced in experiments on keratinocytes (Nam et al., 2010). A specific feature of mitochondria in senescent cells is the so-called retrograde signaling response, in which progressing cell de-energization evokes a compensatory increase in the biogenesis of mitochondria, leading to further overproduction of ROS and deterioration of mitochondrial metabolism (Passos et al., 2007). The same vicious circle has been observed in HPMCs subjected to malignant ascites in which
decreased ability to generate ATP, reflected by decreased (by 31%) values of the inner mitochondrial membrane potential (ΔΨm) was accompanied by increased (by 35%) mitochondrial mass, likely due to their improved (by 12%) PGC-1α-dependent biogenesis (LeBleu et al., 2014) (Fig. 4). In order to prove that oxidative stress truly participates in malignant ascites-related senescence of HPMCs, the activity of SA-β-Gal, a marker of senescent cells (Dimri et al., 1995), was quantified in cultures exposed to ascites in either the presence or absence of the spin-trap ROS scavenger, PBN. It is worth mentioning that normal cell exposure to PBN efficiently improved their replicative capacity (Chen et al., 1995) and reduced the development of molecular characteristics of senescent cells (Passos et al., 2010). In this project, cells exposed to malignant ascites displayed increased (by 58%) activity of SA-β-Gal, however, their treatment with PBN decreased enzyme activity to values characterizing cells subjected to benign fluids (Fig. 5). Significantly, analogical cell protection against oxidative stress restricted the malignant ascites-dependent stimulatory effect of HPMCs on ovarian cancer cell adhesion (by 23% and 36% for SKOV-3 and primary EOCs, respectively), proliferation (by 29% and 47% for SKOV-3 and primary EOCs, respectively), and migration (Fig. 6). Interestingly, these experiments revealed that the reactions of the established SKOV-3 cell line and primary ovarian cancer cells to malignant ascites may sometimes differ significantly. A particular difference was observed in the case of migration, where SKOV-3 cells did not react to the fluid at all, whereas the primary cells experienced strong stimulation (by 71%). One may speculate that this diversified effect may be related, at least to some extent, to the fact that majority of primary ovarian cancer cells display the spindle-shaped morphology, suggesting that they may undergo epithelial-mesenchymal transition (EMT). Taking into account that EMT promotes cancer cell motility (Son and Moon, 2010), one may envisage that this specific state can make the primary cells more sensitive to the migration-promoting stimuli than the established SKOV-3 cells which maintain typical epithelial-like appearance. In conclusion, our project revealed that the induction of senescence in HPMCs and the concomitant development of these cells’ pro-tumor characteristics in response to malignant ascites are strongly determined by oxidative cells elicited intracellularly by the fluid. Successful inhibition of these processes by administrating the ROS scavenger may suggest that strategies aimed at reducing the intraperitoneal spread of cancer via the addition of antioxidative substances, similarly as was tested on mice using exogenous catalase-loaded hydrogels (Hyoudou et al., 2007), are worth conducting further investigations for both conceptual and clinical purposes. This conclusion has strong support in previous results of Belotte and colleagues, who observed that the survival of patients with ovarian cancer may be determined by a single nucleotide polymorphism in catalase (Belotte et al., 2015). Conflict of interest The authors declare no conflict of interest. Acknowledgment The study was supported by a grant from the National Science Centre, Poland (registration number 2014/15/B/NZ3/00421). References Ahmed, N., Stenvers, K.L., 2013. Getting to know ovarian cancer ascites: opportunities for targeted therapy-based translational research. Front. Oncol. 3, 256. Allen, R.G., Tresini, M., Keogh, B.P., Doggett, D.L., Cristofalo, V.J., 1999. Differences in electron transport potential, antioxidant defenses, and oxidant generation in young and senescent fetal lung fibroblasts (WI-38). J. Cell Physiol. 180, 114–122. Belotte, J., Fletcher, N.M., Saed, M.G., Abusamaan, M.S., Dyson, G., Diamond, M.P., et al., 2015. A single nucleotide polymorphism in catalase is strongly associated with
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International Journal of Biochemistry and Cell Biology journal homepage: www.elsevier.com/locate/biocel
Mitochondria-related oxidative stress contributes to ovarian cancerpromoting activity of mesothelial cells subjected to malignant ascites
T
Martyna Pakułaa, Justyna Mikuła-Pietrasika, Łukasz Stryczyńskia, Paweł Uruskia, Sebastian Szubertb, Rafał Moszyńskib, Dariusz Szpurekb, Stefan Sajdakb, Andrzej Tykarskia, ⁎ Krzysztof Książeka, a b
Department of Hypertensiology, Angiology and Internal Medicine, Poznań University of Medical Sciences, Długa 1/2 Str., 61-848 Poznań, Poland Division of Gynecological Surgery, Poznań University of Medical Sciences, Polna 33 Str., 60-535 Poznań, Poland
A R T I C LE I N FO
A B S T R A C T
Keywords: Malignant ascites Mesothelial cells Ovarian cancer Oxidative stress Senescence
Very little is known about the mechanisms by which malignant ascites modulates the cancer-promoting activity of human peritoneal mesothelial cells (HPMCs). Because malignant ascites induces pro-tumoral senescence in HPMCs, here we examined if this effect could be driven by oxidative stress. The study showed that malignant ascites generated by serous ovarian tumors induced oxidative damage to the DNA (γH2A.X, 53BP1, 8-hydroxy2′-deoxyguanosine) and lipids (8-isoprostane) in HPMCs as well as increased the production of mitochondrial superoxides and cellular peroxides in these cells. This activity coincided with increased activity of two enzymes involved in the mitochondrial production of oxidants, i.e. cytochrome c oxidase and NADH dehydrogenase, decreased mitochondrial inner membrane potential, increased mitochondrial mass, and increased the activity of peroxisome proliferator-activated receptor gamma coactivator-1 alpha. Increased production of superoxides and peroxides in cells subjected to the malignant ascites was effectively reduced when the fluid was pre-incubated with neutralizing antibodies against hepatocyte growth factor. Moreover, when HPMCs subjected to the malignant ascites were protected against oxidative stress with a spin-trap scavenger of reactive oxygen species, they displayed decreased expression of senescence-associated β-galactosidase and their potential to stimulate cancer cell adhesion, proliferation, and migration was significantly diminished. Collectively, our findings indicate that improved ovarian cancer cell progression in response to HPMCs exposed to malignant ascites may be associated with the development of profound oxidative stress in these cells.
1. Introduction Although a plethora of evidence has emerged that malignant ascites contributes to progression of ovarian cancer, more knowledge is being gained about the mechanistic aspects of its activity. In general, it is believed that malignant ascites creates a hospitable peritoneal milieu that helps ovarian cancer cells survive, invade, and disseminate (Ahmed and Stenvers, 2013). This is linked, in turn, with the ascites’ ability to suppress peritoneal inflammation (Simpson-Abelson et al., 2013) and with the presence of large amounts of agents that support angiogenesis and cancer cell motility in this fluid (Kipps et al., 2013). Last but not least, malignant ascites modulates the behavior of normal peritoneal cells, e.g. by inhibiting the cancer cells’ apoptosis driven by the peritoneal mesothelium (Matte et al., 2014). It has been shown that human peritoneal mesothelial cells (HPMCs), which constitute the largest fraction of cells within the peritoneal
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cavity, support the progression of ovarian cancer in vitro and in vivo when they become senescent (Mikula-Pietrasik et al., 2016b). Interestingly, recent findings indicate that malignant ascites accelerates the senescence of HPMCs via the activity of growth-related oncogene 1 (GRO-1) and hepatocyte growth factor (HGF) (Mikula-Pietrasik et al., 2016a). The intracellular mechanism underlying this activity of the ascites remains, however, unknown. Taking into account that the senescence of normal somatic cells is often causatively linked with deleterious activity of reactive oxygen species (ROS) (Davalli et al., 2016), the goal of this study was to examine if the pro-senescence activity of malignant ascites and concomitant pro-tumoral effects of HPMCs may be caused by oxidative stress. To this end, several parameters associated with oxidative cell status were examined, including oxidative DNA damage, lipid peroxidation, production of ROS, activity of enzymes involved in ROS release, mitochondrial membrane potential, and mitochondrial
Corresponding author. E-mail address: [email protected] (K. Książek).
https://doi.org/10.1016/j.biocel.2018.03.011 Received 7 December 2017; Received in revised form 18 February 2018; Accepted 13 March 2018 Available online 14 March 2018 1357-2725/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Effect of growth medium (GM), benign ascites (BA) and malignant ascites (MA) on activation of DNA damage response (DDR) and magnitude of lipid peroxidation in cultured HPMCs. Quantification of fluorescence of γH2A.X (A) and 53BP1 foci (B) was used as a measure of DDR. Quantification of 8-OH-dG was treated as a measure of nonspecific DNA injury (C). Quantification of 8isoprostane was used to estimate the magnitude of lipid peroxidation (D). Representative pictures showing γH2A.X and 53BP1 foci (green dots) in the nuclei (blue DAPI staining) of cells exposed to growth medium, benign and malignant fluid (E). The results derive from experiments performed with HPMCs obtained from 8 different donors, and with benign and malignant fluids collected from 8 different patients. (+) in the boxes indicate means, while the horizontal lines indicate medians. RFU – relative fluorescence units. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
undergoing abdominal surgery (institutional consent number 187/14), as described in detail elsewhere (Ksiazek, 2013). Cells were identified as pure mesothelium by their epithelial-like morphology at confluency and uniform positive staining for cytokeratins and the HBME-1 antigen. During the experiments only HPMCs obtained from the first two passages, which corresponds to about 25% of their replicative lifespan, were used. The viability of these cells exceeded 98%. Ovarian cancer cells, SKOV-3, were obtained from the ECCC (Porton Down, UK) and maintained in RPMI 1640 medium with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 g/ml), and 10% FBS. Primary epithelial ovarian cancer cells (EOCs) were isolated from tumors excised during cytoreductive surgery from patients with serous ovarian cancer (stage IV). Briefly, the tumors were cut into pieces of equal weight and then placed in a solution of 0.05% trypsin and 0.02% EDTA for 20 min at 37 °C with gentle shaking. After resuspension in RPMI1640 containing 20% FBS, the cells were probed with an antibody directed against the epithelial-related antigen (MOC-31; Abcam, Cambridge, UK) to confirm their cancerous nature. Finally, the ovarian cancer cells were cultured in RPMI 1640 supplemented with L-glutamine (2 mM) and 20% FBS.
biogenesis. These tests were followed by intervention studies in which HPMCs treated with ascites were protected against oxidative stress by using a spin-trap ROS scavenger, then their senescence and their impact on cancer cell progression were evaluated. 2. Materials and methods 2.1. Chemicals Unless otherwise stated, all chemicals were from Sigma (St. Louis, MO) and culture plastics were from Thermo Fisher Scientific (Waltham, MA, USA). 2.2. Ascitic fluids Malignant ascites were obtained during cytoreductive surgery from patients with high-grade serous ovarian cancer at stage IV (n = 8). The histopathology, grade, and stage of the tumors were assigned in keeping with the criteria of the International Federation of Gynecology and Obstetrics. Benign fluids were obtained from age-matched patients with cystadenoma mucinosum multiloculare (n = 8). After collection in sterile conditions, the fluids were centrifuged at 2500 rpm for 5 min and the acellular supernatants were stored at −20 °C until required. The study was approved by an institutional ethics committee (consent number 543/14).
2.4. Experimental conditions HPMCs were placed onto culture dishes and allowed to grow until reaching sub-confluency. Then the cells were exposed to standard growth medium (GM), 10% malignant ascites (MA), and 10% benign ascites (BA) for 72 h. In some experiments, the HPMCs were pre-incubated for 6 h with a spin-trap ROS scavenger, N-tert-butyl-alphaphenylnitrone (PBN, Sigma; 800 μM), before the addition of malignant and benign ascites. Starting from pre-incubation, PBN was constantly
2.3. Cell cultures Human peritoneal mesothelial cells (HPMCs) were isolated from fragments of the omentum derived from 8 non-oncological patients 83
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Fig. 2. Effect of growth medium (GM), benign ascites (BA) and malignant ascites (MA) on the production of reactive oxygen species in HPMCs. Generation of mitochondrial superoxides was monitored using MitoSOX fluorescence (A), whereas generation of cellular peroxides was monitored with DHR fluorescence (B). Moreover, the activity of two enzymes engaged in ROS production was quantified, i.e. cytochrome c oxidase (C) and NADH dehydrogenase (D). The results derive from experiments performed with HPMCs obtained from 8 different donors, and with benign and malignant fluids collected from 8 different patients. (+) in the boxes indicate means, while the horizontal lines indicate medians. RFU – relative fluorescence units.
Fig. 3. Effect of HGF neutralization on the production of mitochondrial superoxides (A) and cellular peroxides (B) by HPMCs exposed to malignant ascites (MA). The fluid was pre-incubated with specific neutralizing antibodies against HGF for 4 h before its addition to cell culture. The results derive from experiments performed with HPMCs obtained from 6 different donors, and with benign and malignant fluids collected from 8 different patients. (+) in the boxes indicate means, while the horizontal lines indicate medians. RFU – relative fluorescence units.
2.6. Oxidative stress-related parameters
present in the culture environment throughout the whole experiment.
Elements of senescence-associated DNA damage, i.e. the foci of the phosphorylated variant of histone H2A.X (γH2A.X) and p53-binding protein 1 (53BP1), were quantified using immunofluorescence as described in (Mikula-Pietrasik et al., 2017). The concentration of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), one of the major products of DNA oxidation, was quantified using DNA Damage (8OHdG) ELISA Kit
2.5. Cellular senescence Development of the senescence phenotype in the HPMCs was determined according to quantification of the activity of senescence-associated β-galactosidase (SA-β-Gal) using a fluorescence-based method as described by Gary and Kindell (Gary and Kindell, 2005). 84
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Fig. 4. Effect of growth medium (GM), benign ascites (BA) and malignant ascites (MA) on mitochondrial metabolism. The fluorescence of JC-1 was used to determine mitochondrial inner membrane potential (A), whereas the fluorescence of NAO was used to estimate mitochondrial mass (B). Panel C shows the activity of PGC1α, an enzyme involved in mitochondrial biogenesis. The results derive from experiments performed with HPMCs obtained from 8 different donors, and with benign and malignant fluids collected from 8 different patients. (+) in the boxes indicate means, while the horizontal lines indicate medians. RFU – relative fluorescence units.
accumulates in the vital mitochondria, forming red fluorescent J-aggregates (at 590 nm), while mitochondrial de-energization leads to the formation of green fluorescent monomers (at 535 nm). A rise in the green/red fluorescence intensity ratio indicates a drop in mitochondrial membrane potential values. Mitochondrial mass was quantified upon cell treatment with 10 μM of 10-n-nonyl-acridine orange (NAO; Sigma) for 10 min at 37 °C in the dark. The fluorescence was recorded at an excitation wavelength of 435 nm and an emission wavelength of 535 nm. Enzymes engaged in the generation of mitochondrial ROS, i.e. cytochrome c oxidase and NADH dehydrogenase, as well as those contributing to the biogenesis of mitochondria (peroxisome proliferatoractivated receptor gamma coactivator-1 alpha; PGC-1α) were analyzed using commercial ELISA-based assays obtained from Wuhan EIAab Science Co., Ltd. (Wuhan, China), as per manufacturer’s instructions. Fig. 5. Effect of growth medium (GM), benign ascites (BA) and malignant ascites (MA) on the activity of SA-β-Gal in HPMCs, in either the presence or absence of the spin-trap ROS scavenger, PBN. The results derive from experiments performed with HPMCs obtained from 8 different donors, and with benign and malignant fluids collected from 8 different patients. (+) in the boxes indicate means, while the horizontal lines indicate medians. RFU – relative fluorescence units.
2.8. Collection of conditioned media Sub-confluent HPMCs were subjected to standard growth medium, 10% malignant ascites, and 10% benign ascites for 72 h, and then they were washed carefully with PBS and incubated with serum-free medium for 48 h to generate conditioned medium (CM). Samples of the CM were centrifuged, filtered through a 0.2 μm pore size filter, and stored at –80 °C until required.
(Biorbyt Ltd., Cambridge, UK), as per manufacturer’s instructions. The magnitude of oxidative modifications of cellular lipids was evaluated according to quantification of 8-isoprostane (8-iso Prostaglandin F2α) using the 8-Isoprostane ELISA kit from Cayman Chemical (Ann Arbor, MI, USA), as per manufacturer’s instructions. The generation of mitochondrial superoxides was examined using a fluorescent dye, MitoSOX Red (Thermo Fisher Scientific). In brief, the cells were stained with 0.1 μM MitoSOX Red for 15 min and then fluorescence was recorded at an excitation wavelength of 390 nm and an emission wavelength of 580 nm. Parallel measurement at an excitation wavelength of 510 nm was performed to monitor the nonsuperoxide-dependent processes that can oxidize HE probes to ethidium (Robinson et al., 2006). Cellular peroxides were detected in cells incubated with 30 μM dihydrorhodamine 123 (DHR) for 15 min at 37 °C. Fluorescence was recorded at 507 nm and 540 nm, for excitation and emission wavelengths, respectively. In some experiments, the production of superoxides and peroxides was examined upon HPMC exposure to malignant ascites pre-incubated for 4 h with specific neutralizing antibodies against hepatocyte growth factor (HGF; R&D Systems; 1 μg/ ml).
2.9. Cancer cell progression Adhesion of ovarian cancer cells probed with a fluorescent dye, calcein AM, to HPMCs exposed to malignant and benign ascites was evaluated according to a methodology described in (Mikula-Pietrasik et al., 2014). Proliferation of ovarian cancer cells towards the CM harvested from HPMCs treated with malignant and benign ascites was tested using Cell Proliferation Kit I (PromoKine; Heidelberg, Germany), as per manufacturer’s instructions. Migration of ovarian cancer cells towards a chemotactic gradient generated by the CM from HPMCs treated with malignant and benign ascites was quantified with ChemoTx migration chambers (Neuro Probe, Gaithersburg, MD, USA), as per manufacturer’s instructions.
2.10. Statistics Statistical analysis was performed using GraphPad Prism™ 5.00 (GraphPad Software, San Diego, USA). The groups were compared with repeated measures analysis of variance (ANOVA) with the NewmanKeuls test as post-hoc. The results are shown as boxes and whiskers, in which sample distribution (minimum, maximum) as well as means and medians are presented. Differences with a P value < 0.05 were treated as statistically significant.
2.7. Mitochondrial metabolism Mitochondrial membrane potential (ΔΨm) was measured in cells probed with 1 μM of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Cayman Chemical), which is a lipophilic cationic fluorescent dye exhibiting potential-dependent accumulation in the mitochondria (Passos et al., 2007). JC-1 85
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Fig. 6. Effect of HPMCs subjected to benign ascites (BA) and malignant ascites (MA) on adhesion (A, B), proliferation (C, D), and migration (E, F) of commercial ovarian cancer cell line SKOV-3 and primary epithelial ovarian cancer cells (EOCs) in either the presence or absence of PBN. The results derive from experiments performed with HPMCs obtained from 8 different donors, and with benign and malignant fluids collected from 8 different patients. Cancer cells were used for each experiment in quadruplicates. (+) in the boxes indicate means, while the horizontal lines indicate medians. RFU – relative fluorescence units.
3. Results and discussion
2018) and increased progression of gastric cancer (Miao et al., 2016). Notably, peritoneal metastases of ovarian tumors develop in late stages of the disease (III/IV) (Halkia et al., 2012), which means that their development coincides not only with the accumulation of senescent HPMCs (Mikula-Pietrasik et al., 2017) but also with the formation of ascites, which becomes clinically significant in the advanced disease (Kipps et al., 2013). In this report, we extended the panel of senescence markers, which are increased in HPMCs subjected to malignant ascites, thus strengthening the evidence for the fluid’s senescence-promoting activity. More precisely, we found that HPMCs exposed to malignant ascites for 3 days
It has recently been shown that the activity of senescence-associated β-galactosidase (SA-β-Gal), a valuable marker of cellular senescence in vitro and in vivo, is elevated in HPMCs subjected to malignant ascites generated by serous ovarian tumors in culture conditions for 3 days. Moreover, HPMCs maintained under such a regimen appeared to promote adhesion, proliferation, and migration of ovarian cancer cells (Mikula-Pietrasik et al., 2016a), which is in line with well-established knowledge on the causative role of senescent HPMCs in the formation of peritoneal metastases of ovarian cancer (Mikula-Pietrasik et al., 86
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display increased expression of γH2A.X and 53BP1 (by 23% and 24%, respectively, vs. benign ascites), which indicates activation of the DNA damage response (DDR) pathway – a phenomenon typical of senescent cells (Passos et al., 2007) (Fig. 1A, B, E). Malignant ascites also increases (by 87%) cellular concentration of DNA adduct, 8-OH-dG (Valavanidis et al., 2009), which means that oxidative modifications of DNA in response to malignant fluid are not solely associated with the induction of pathways related to senescence, but also with the accumulation of nonspecific DNA injury (Fig. 1C). This effect coincided with an increased concentration of 8-isoprostane (by 522%), a marker of cellular phospholipid peroxidation (Swardfager et al., 2017), which implies that malignant ascites potentiates oxidative damage to various classes of macromolecules (Fig. 1D). Indeed, increased oxidative damage to DNA has also been reported in peripheral blood lymphocytes from patients gathering malignant ascites (Wang et al., 2012). Interestingly, in the case of γH2A.X and 8-isoprostane, but not 53BP1, the level of DNA and lipids injury in cells treated with benign ascites was higher (by 13% and 103%, respectively) than in cells maintained in standard growth medium, which indicates that the environment created in the peritoneal cavity by the benign fluid is far from physiological. An initiating event in the activation of DDR is the formation of γH2A.X foci close to DNA double-strand breaks, elicited primarily by deleterious activity of reactive oxygen species (ROS) (Woodbine et al., 2011). In order to establish if ROS contribute to malignant ascites-dependent accumulation of DNA injury, we measured the production of mitochondrial superoxides and cellular peroxides and found that both types of ROS are overproduced (by 75% and 242%, respectively) in HPMCs exposed to the malignant fluid (Fig. 2A, B). In addition, the magnitude of cellular peroxide production by cells treated with benign ascites was by 33% higher compared with cells grown in the presence of standard medium, which may explain increased level of the macromolecules’ injury in this group. Mechanistically, increased generation of mitochondrial ROS was plausibly associated with intensified activity of two enzymes involved in oxidant efflux during respiratory chain reactions, i.e. cytochrome c oxidase (by 18%) and NADH dehydrogenase (by 14%) (Fig. 2C, D). The activity of the latter enzyme in cells subjected to benign ascites was by 24% higher than in cells cultured under physiological conditions. Increased activity of cytochrome c oxidase and NADH dehydrogenase was previously causatively linked with increased generation of ROS in senescent fibroblasts (Allen et al., 1999). Moreover, the contribution of cytochrome c oxidase has also been evidenced in HPMCs, whose premature senescence was triggered by ovarian cancer-derived hepatocyte growth factor (HGF) (Mikula-Pietrasik et al., 2017). Interestingly, HGF present in malignant ascites from patients suffering from serous ovarian carcinoma has recently been identified as a mediator of this fluid’s pro-senescent activity (Mikula-Pietrasik et al., 2016a). For this reason, it cannot be excluded that the mitochondrial machinery responsible for the overproduction of ROS in cells exposed to malignant ascites may be launched by HGF. In order to verify this assumption, the production of ROS was examined in the presence of malignant ascites in which HGF was neutralized using specific antibodies. The experiment showed that, indeed, the production of either mitochondrial superoxides or cellular peroxides that was initially upregulated in response to the malignant fluid was significantly attenuated (by 35% and by 28% for superoxides and peroxides, respectively) when the activity of HGF in the fluid was chemically inhibited (Fig. 3). The relationship between the activity of HGF and increased production of ROS has also been evidenced in experiments on keratinocytes (Nam et al., 2010). A specific feature of mitochondria in senescent cells is the so-called retrograde signaling response, in which progressing cell de-energization evokes a compensatory increase in the biogenesis of mitochondria, leading to further overproduction of ROS and deterioration of mitochondrial metabolism (Passos et al., 2007). The same vicious circle has been observed in HPMCs subjected to malignant ascites in which
decreased ability to generate ATP, reflected by decreased (by 31%) values of the inner mitochondrial membrane potential (ΔΨm) was accompanied by increased (by 35%) mitochondrial mass, likely due to their improved (by 12%) PGC-1α-dependent biogenesis (LeBleu et al., 2014) (Fig. 4). In order to prove that oxidative stress truly participates in malignant ascites-related senescence of HPMCs, the activity of SA-β-Gal, a marker of senescent cells (Dimri et al., 1995), was quantified in cultures exposed to ascites in either the presence or absence of the spin-trap ROS scavenger, PBN. It is worth mentioning that normal cell exposure to PBN efficiently improved their replicative capacity (Chen et al., 1995) and reduced the development of molecular characteristics of senescent cells (Passos et al., 2010). In this project, cells exposed to malignant ascites displayed increased (by 58%) activity of SA-β-Gal, however, their treatment with PBN decreased enzyme activity to values characterizing cells subjected to benign fluids (Fig. 5). Significantly, analogical cell protection against oxidative stress restricted the malignant ascites-dependent stimulatory effect of HPMCs on ovarian cancer cell adhesion (by 23% and 36% for SKOV-3 and primary EOCs, respectively), proliferation (by 29% and 47% for SKOV-3 and primary EOCs, respectively), and migration (Fig. 6). Interestingly, these experiments revealed that the reactions of the established SKOV-3 cell line and primary ovarian cancer cells to malignant ascites may sometimes differ significantly. A particular difference was observed in the case of migration, where SKOV-3 cells did not react to the fluid at all, whereas the primary cells experienced strong stimulation (by 71%). One may speculate that this diversified effect may be related, at least to some extent, to the fact that majority of primary ovarian cancer cells display the spindle-shaped morphology, suggesting that they may undergo epithelial-mesenchymal transition (EMT). Taking into account that EMT promotes cancer cell motility (Son and Moon, 2010), one may envisage that this specific state can make the primary cells more sensitive to the migration-promoting stimuli than the established SKOV-3 cells which maintain typical epithelial-like appearance. In conclusion, our project revealed that the induction of senescence in HPMCs and the concomitant development of these cells’ pro-tumor characteristics in response to malignant ascites are strongly determined by oxidative cells elicited intracellularly by the fluid. Successful inhibition of these processes by administrating the ROS scavenger may suggest that strategies aimed at reducing the intraperitoneal spread of cancer via the addition of antioxidative substances, similarly as was tested on mice using exogenous catalase-loaded hydrogels (Hyoudou et al., 2007), are worth conducting further investigations for both conceptual and clinical purposes. This conclusion has strong support in previous results of Belotte and colleagues, who observed that the survival of patients with ovarian cancer may be determined by a single nucleotide polymorphism in catalase (Belotte et al., 2015). Conflict of interest The authors declare no conflict of interest. Acknowledgment The study was supported by a grant from the National Science Centre, Poland (registration number 2014/15/B/NZ3/00421). References Ahmed, N., Stenvers, K.L., 2013. Getting to know ovarian cancer ascites: opportunities for targeted therapy-based translational research. Front. Oncol. 3, 256. Allen, R.G., Tresini, M., Keogh, B.P., Doggett, D.L., Cristofalo, V.J., 1999. Differences in electron transport potential, antioxidant defenses, and oxidant generation in young and senescent fetal lung fibroblasts (WI-38). J. Cell Physiol. 180, 114–122. Belotte, J., Fletcher, N.M., Saed, M.G., Abusamaan, M.S., Dyson, G., Diamond, M.P., et al., 2015. A single nucleotide polymorphism in catalase is strongly associated with
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