Oleuropein Induces Cytotoxicity and Peroxiredoxin Over-expression in MCF-7 Human Breast Cancer Cells.

BACKGROUND/AIM: As a fundamental staple of the Mediterranean diet, olive oil has long been recognized for its health benefits, including its ability to reduce cardiovascular and neurological disease. Oleuropein is the primary phenolic chemical found in all parts of the olive tree, especially in the leaves and fruit. Oleuropein exhibits anti-inflammatory and antioxidant properties, and has been associated with cancer inhibition in various animal and cell models. We investigated the effects of oleuropein on the MCF-7 human breast cancer cell line, and compared it to the non-cancerous MCF-10A breast epithelial line. MATERIALS AND METHODS: Both cell lines were treated with two different concentrations of oleuropein for 48 and 72 hours. Cytotoxicity, apoptosis, and peroxiredoxin expression were measured. RESULTS: Forty-eight hours of oleuropein treatment induced cytotoxicity in MCF-7 cells, whereas it had no effect on MCF-10A cells. Furthermore, oleuropein-induced cytotoxicity in MCF-7 cells involved a measurable increase in apoptosis. Oleuropein treatment of MCF-7 cells significantly and dramatically increased expression of all six peroxiredoxin mRNAs (Prdx1-Prdx6), whereas oleuropein treatment of MCF-10A cells resulted in only a small increase in Prdx1 and Prdx6 expression, with no change in the expression of the other peroxiredoxins. Together, these data demonstrate differential susceptibility to oleuropein-induced cell death between the two lines, and differential regulation of peroxiredoxins. CONCLUSION: Oleuropein-induced over-expression of peroxiredoxins in MCF-7 cells may either facilitate its cancer-specific cytotoxicity or, alternatively, is a consequence of an altered response of cancer cells.

Undergraduate lab series using the K562 human leukemia cell line: Model for cell growth, death, and differentiation in an advanced cell biology course.

This sequence of labs was developed for an upper level undergraduate cell biology course at Fairfield University. The labs are based on the use of the K562 human erythroleukemia cell line, a model system that is exceptionally amenable to an undergraduate cell biology lab course due to its ease of maintenance and propagation and usefulness for studies of growth, death, and differentiation. The sequence of labs is conducted over a 6-week period, following a series of weekly cell biology labs covering basic cell and molecular biology techniques. Together, the lab series has four primary objectives 1) to teach students how to culture and maintain mammalian cells; 2) to build student competency in standard cell biology techniques; 3) to demonstrate the role of growth factors on cell proliferation and viability; and 4) to provide students with an opportunity to use these cells in an independent investigation on cell differentiation. We provide examples of student data and offer a range of experimental measurements depending on institutional capacity and facilities. Our assessment data suggest that students find great value in this lab series, enhancing their comprehension of key concepts, acquisition of important lab skills, and depth of understanding of the research process. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):263-271, 2019.

Overexpression of Prdx6 and resistance to peroxide-induced death in Hepa1-6 cells: Prdx suppression increases apoptosis.

Peroxiredoxins are thiol-specific antioxidants that catalyze the reduction of cellular peroxides and protect cells from ROS-mediated damage and death. Peroxiredoxin gene expression is up-regulated in a number of cancers, suggesting a possible role in cancer cell maintenance. Prdx6, a cytoplasmic protein elevated in certain cancers, is highly expressed in liver and transcriptionally regulated by various oxidative stresses. In the present study, we found that the cancerous Hepa1-6 hepatoma cell line is significantly more resistant to peroxide-induced cytotoxicity than the non-cancerous H2.35 cell line. We also demonstrated that Hepa1-6 cells express approximately 3-fold more Prdx6 mRNA and 2.5-fold more Prdx6 protein than H2.35 cells. Treatment with mithramycin A resulted in a nearly 20% reduction in Prdx6 mRNA in Hepa1-6 cells, suggesting a possible role for Sp1 in Prdx6 up-regulation. We hypothesized that suppression of Prdx6 in Hepa1-6 cells would increase susceptibility to peroxide-induced cell death. Transient transfection of Hepa1-6 cells with Prdx6 siRNA led to a marked reduction in Prdx6 expression, and an increase in peroxide-induced cytotoxicity by apoptosis. Together, these data demonstrate an important anti-apoptotic function for Prdx6 in cancerous liver cells, and suggest that its up-regulation may be a tumor-supportive adaptation in cancerous states.

[Antioxidative role of peroxiredoxin 6 in acute lung injury].

OBJECTIVE: To confirm the antioxidant protective effect of peroxiredoxin 6 (Prdx6) in acute lung injury in mice. METHODS: Lung injury or lung alveolar type II epithelial cell (AEC II) injury models were induced in mice by 100% O2 exposure or H2O2 treatments. Mice and AEC II cell survival rate or BALF analysis were applied for evaluating the degree of acute lung injury. Western Blot assay was used to determine Prdx6 or Gpx1 protein expression in lung. Annexin V staining method was applied to detect cell apoptosis on cultured AEC II cell, and thiobarbituric acid reactive substance (TBARS) measurement and diphenyl-1-pyrenyl phospholine (DPPP) assays were separately used to measure the level of lipid peroxidation in mice lung and AEC II cell membrane. RESULTS: Under 100% O2 exposure, Prdx6-/- mice presented 24 h shorter survival time compared to wild type (WT) mice, on the contrary, Prdx6 gene over-expressed (Tg Prdx6) mice showed enhanced mice survival; meanwhile, the degree of AEC II cell injury had H2O2-dose dependent pattern with interactive relationship of Prdx6 protection. Under 100% O2 exposure for 72 h, it caused 7-fold decreased Gpx1 expression in Prdx6-/- mouse lung with no remarkable decrease of Prdx6 expression in Gpx1-/- mice. The percentage of apoptotic cells was significantly increased in AEC II cells from Prdx6-/- mice, and the percentage of AEC II apoptotic cells from Tg Prdx6 kept consistently around 10% under H2O treatments; also, the lipid peroxidation level of AEC II cell membrane was the highest in the group of Prdx6-/- mice, which was about 2 or 4-fold increased compared to the groups of WT or Tg Prdx6, separately; meanwhile, the lipid peroxidation level in Prdx6-/- mice, was also the highest compared to the other groups. CONCLUSIONS: Prdx6 plays a critical role in defending acute oxidative lung injury and its function of defending cell apoptosis and cell membrane lipid peroxidation suggests its unique cell-based protective effect.

Investigating transcriptional regulation of Prdx6 in mouse liver cells.

Prdx6, a unique member of the peroxiredoxin family of antioxidants, is highly expressed in liver and protects cells from oxidative damage by reducing H2O2 and various lipid peroxides. We investigated the transcriptional regulation of Prdx6 in the H2.35 mouse hepatocyte cell line and sought to determine the mechanism of basal and induced expression. We found that Prdx6 expression is down-regulated upon serum deprivation and subsequently induced in a time-dependent manner in response to KGF, TNF-alpha, dexamethasone, and H2O2. Inhibitors of both PKC and MEK largely prevented Prdx6 induction by KGF and, to a lesser extent, TNF-alpha. Interestingly, inhibition of NF-kappaB led to a marked increase in Prdx6 regulation in the absence or presence of inducers, suggesting a normal role for NF-kappaB in Prdx6 suppression. Using reporter constructs from the mouse gene, we found that the first 160 bp of the proximal promoter was sufficient for low levels of expression, and expression increased sixfold with 1200 bp of the proximal promoter. These regions were not, however, sufficient to mediate up-regulation by the known Prdx6 inducers in our system. Together, these data support multiple pathways of Prdx6 regulation and reveal important promoter regions that mediate its transcriptional regulation.

Transgenic mice overexpressing peroxiredoxin 6 show increased resistance to lung injury in hyperoxia.

Peroxiredoxin 6 (Prd x 6) is a novel peroxidase enzyme that is expressed at a high level in the lung. We tested the hypothesis that transgenic (Tg) mice overexpressing Prd x 6 would exhibit increased resistance to hyperoxia-induced lung injury. Wild-type and Tg mice were exposed to 100% O(2) and evaluated for survival, lung histopathology, total protein, and nucleated cells in bronchoalveolar lavage fluid (BALF), and oxidation of lung protein and lipids. Prd x 6 protein expression and enzyme activity were approximately 3-fold higher in Tg lungs compared with wild-type. Tg mice survived longer during exposure to 100% O(2) (LT(50) 104+/-2.8 h in Tg versus 88.9+/-1.1 h for wild-type). Lung wet/dry weight ratio and total protein and nucleated cell count in lung lavage fluid were significantly greater in wild-type mice at 72 and 96 h of hyperoxia compared with Tg mice. At 96 h of hyperoxia, Tg mice had less epithelial cell necrosis, perivascular edema, and inflammatory cell recruitment by light microscopy, and lower TBARS and protein carbonyls in lung homogenate (P<0.05). These results show that Tg mice have increased defense against lung injury in hyperoxia, providing evidence that Prd x 6 functions as a lung antioxidant enzyme.

Transcripts associated with Prdx6 (peroxiredoxin 6) and related genes in mouse.

PRDX6 is a cytosolic member of the peroxiredoxin family of antioxidant proteins, which protect cells from oxidative damage by reducing cellular peroxides. Knockout studies and transgenic overexpression of Prdx6 in mice have demonstrated an important role for this protein in the defense against oxidative stress. Using Northern blotting with various Prdx6 probes, we have revealed the existence of multiple transcripts with distinct tissue distributions and regulation, including the major 1.4-kb transcript highly expressed in liver and lung, and two additional transcripts expressed primarily in liver. We hypothesized that these additional transcripts correspond either to alternative Prdx6 mRNAs or to highly related genes such as the intronless genes Aop2-rs1 and Aop2-rs2. A combination of Northern blotting, RACE, and EST and genomic sequence analysis has determined that all three liver transcripts are derived from the Prdx6 gene, as they are absent in Prdx6-null mice and differ in their 3' UTRs, suggesting the utilization of different transcription termination signal sequences which we have identified by sequence analysis. We found the Aop2-rs1 gene to be exclusively expressed in testis as a 1.2-kb transcript and have identified putative regulatory elements in its promoter. In contrast, Aop2-rs2 appears not to be expressed in any tissues, although we have evidence for the existence of other related genes that are expressed in a tissue-specific manner. Since the Prdx6 transcripts exhibit differential regulation in response to growth and oxidative stress, further investigation may reveal their distinct roles in the cell and mechanism of regulation.

Peroxiredoxin 6 deficiency and atherosclerosis susceptibility in mice: significance of genetic background for assessing atherosclerosis.

Peroxiredoxin 6 (Prdx6; also called antioxidant protein 2, or Aop2) is a candidate gene for Ath1, a locus responsible for the respective susceptibility and resistance of mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) to diet-induced atherosclerosis. To evaluate if Prdx6 underlies Ath1, we compared the diet-induced atherosclerotic lesions in Prdx6 targeted mutant (Prdx6-/-) mice of different genetic backgrounds: B6, 129, and B6;129. PRDX6 protein and mRNA were expressed in normal and atherosclerotic aortas. B6;129 Prdx6-/- macrophages oxidized LDL significantly more than did controls. Plasma lipid hydroperoxide levels were higher in atherogenic diet-fed Prdx6-/- mice with B6;129 and B6 backgrounds than in controls. Prdx6-/- and controls in a 129 genetic background were equally lesion-resistant, and Prdx6-/- and controls in a B6 background were equally lesion-susceptible. In contrast, Prdx6-/- mice in a B6;129 background had significantly larger aortic root lesions than did littermate wild type controls. Therefore, although PRDX6 protein did not affect atherosclerosis susceptibility in either the resistant 129 background or the susceptible B6 background, it may inhibit atherosclerosis in backgrounds with mixed pro- and anti-atherogenic genes. Thus, genetic background plays an important role in modulating atherogenesis in targeted mutant mice. However, we think it is unlikely that Prdx6 underlies Ath1.

Mice with targeted mutation of peroxiredoxin 6 develop normally but are susceptible to oxidative stress.

Reactive oxygen species, especially hydrogen peroxide, are important in cellular signal transduction. However, excessive amounts of these species damage tissues and cells by oxidizing virtually all important biomolecules. Peroxiredoxin 6 (PRDX6) (also called antioxidant protein 2, or AOP2) is a novel peroxiredoxin family member whose function in vivo is unknown. Through immunohistochemistry, we have determined that the PRDX6 protein was widely expressed in every tissue examined, most abundantly in epithelial cells. It was found in cytosol, but not in membranes, organelles, and nuclei fractions. Prdx6 mRNA was also expressed in every tissue examined. The widespread expression of Prdx6 suggested that its functions were quite important. To determine these functions, we generated Prdx6-targeted mutant (Prdx6-/-) mice, confirmed the gene disruption by Southern blots, PCR, RT-PCR, Western blots, and immunohistochemistry, and compared the effects of paraquat, hydrogen peroxide, and t-butyl hydroperoxide on Prdx6-/- and wild-type (Prdx6+/+) macrophages, and of paraquat on Prdx6-/- and Prdx6+/+ mice. Prdx6-/- macrophages had higher hydrogen peroxide levels, and lower survival rates; Prdx6-/- mice had significantly lower survival rates, more severe tissue damage, and higher protein oxidation levels. Additionally, there were no differences in the mRNA expression levels of other peroxiredoxins, glutathione peroxidases, catalase, superoxide dismutases, thioredoxins, and glutaredoxins between normal Prdx6-/- and Prdx6+/+ mice and those injected with paraquat. Our study provides in vivo evidence that PRDX6 is a unique non-redundant antioxidant that functions independently of other peroxiredoxins and antioxidant proteins.

Confirmation and high resolution mapping of an atherosclerosis susceptibility gene in mice on Chromosome 1.

Previously, we demonstrated that Ath1 is a quantitative trait locus for aortic fatty streak formation, located on Chromosome (chr) 1, with susceptibility in C57BL/6J mice and resistance in C3H/HeJ and BALB/cJ mice fed an atherogenic diet. In this study, we find an atherosclerosis susceptibility locus in the same region of Chr 1 by constructing two congenic strains with the resistance phenotype transferred from different resistant strains, PERA/EiJ or SPRETUS/EiJ. By backcrossing one congenic strain to C57BL/6J and testing recombinant animals, we reduced the distance of the atherosclerosis susceptibility region to 2.3 cM between D1Mit14 and D1Mit10. Further testing of nine recombinant animals showed that eight of the nine were consistent with a further narrowing between D1Mit159 and D1Mit398 a distance of 0.66 cM. This region encompasses a number of potential candidate genes including the thiol-specific antioxidant gene Aop2, also known as peroxiredoxin 5 (Prdx5). AOP2 is capable of reducing hydroperoxides and lipid peroxides in the cell. To investigate Aop2 as a potential candidate, we mapped Aop2 in our backcross and localized it to the atherosclerosis susceptibility interval. We determined that Aop2 is highly expressed in atherosclerosis-related tissues including liver and heart. We also found an inverse correlation between Aop2 mRNA in liver and atherosclerosis phenotype for strains C57BL/6 and the resistant congenic derived from SPRETUS/EiJ. Since LDL oxidation has been implicated in the pathogenesis of this disease, and AOP2 possesses antioxidant activity, we suggest the role of Aop2 in atherosclerosis susceptibility needs to be further explored.