• Journal of Animal Reproduction and Biotechnology
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• v.37 no.4
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• pp.231-238
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• 2022

Reactive oxygen species (ROS) production and F-actin cytoskeleton dynamics play important roles in the survival rate of blastocysts after the vitrified-warming process. However, the protective effects of Mito-TEMPO against cryo-injury and viability through F-actin aggregation and mitochondrial-specific ROS production in vitrificated-warmed bovine embryos have not been investigated. The aims of the present study were to: (1) determine the effects of Mito-TEMPO on embryonic developmental competence and quality by F-actin stabilization during in vitro culturing (IVC), and (2) confirm the effects of Mito-TEMPO through F-actin structure on the cryotolerance of vitrification-warming in Mito-TEMPO exposed in vitro production (IVP) of bovine blastocysts. Bovine zygotes were cultured with 0.1 μM Mito-TEMPO treatment for 2 days of IVC. Mito-TEMPO (0.1 μM) exposed bovine embryos slightly improved in blastocyst developmental rates compared to the non-treated group. Moreover, the viability of vitrified-warmed blastocysts from Mito-TEMPO treated embryos significantly increased (p < 0.05, non-treated group: 66.7 ± 3.2% vs Mito-TEMPO treated group: 79.2 ± 5.9%; re-expanded at 24 hours). Mito-TEMPO exposed embryos strengthened the F-actin structure and arrangement in the blastocyst after vitrification-warming. Furthermore, the addition of Mito-TEMPO into the IVC medium enhanced embryonic survival and quality through F-actin stabilization after the vitrification-warming procedure. Overall, our results suggest that supplementing the culture with 0.1 μM Mito-TEMPO improves the embryonic quality and cryo-survival of IVP bovine blastocysts.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.4
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• pp.299-306
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• 2021

Vitrification methods are commonly used for mammalian reproduction through the long-term storage of blastocyst produced in vitro. However, the survival and quality of embryos following vitrification are significantly low compared with blastocyst from in vitro production (IVP). This study evaluates that the survival of frozen-thawed bovine embryos was relevant to mitochondrial superoxide derived mitochondrial activity. Here we present supplementation of the cryopreservation medium with Mito-TEMPO (0.1 µM) induced a significant (p < 0.001; non-treated group: 56.8 ± 8.7%, reexpanded at 24 h vs Mito-TEMPO treated group: 77.5 ± 8.9%, re-expanded at 24 h) improvement in survival rate of cryopreserved-thawed bovine blastocyst. To confirm the quality of vitrified blastocyst after thawing, DNA fragmentation of survived embryos was examined by TUNEL assay. As a result, TUNEL positive cells rates of frozen-thawed embryos were lower in the Mito-TEMPO treated group (4.2 ± 1.4%) than the non-treated group (7.1 ± 3.5%). In addition, we investigated the intracellular ROS and mitochondrial specific superoxide production using DCF-DA and Mito-SOX staining in survived bovine embryos following vitrification depending on Mito-TEMPO treatment. As expected, intracellular ROS levels and superoxide production of vitrified blastocysts after cryopreservation were significantly reduced (p < 0.05) according to Mito-TEMPO supplement in freezing medium. Also, mitochondrial activity measured by MitoTracker Orange staining increased in the frozen-thawed embryos with Mito-TEMPO compared with non-treated group. These results indicate that the treatment of Mito-TEMPO during cryopreservation might induce reduction in DNA fragmentation and apoptosis-related ROS production, consequently increasing mitochondrial activation for developmental capacity of frozen-thawed embryos.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.1
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• pp.10-19
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• 2019

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: $79.9{\pm}3.8%$ vs G2: $57.5{\pm}4.6%$) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO ($0.1{\mu}M$, MT) treatment (G2: $68.4{\pm}3.2%$ vs G2 + MT: $73.9{\pm}1.4%$). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.3
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• pp.199-205
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• 2020

Cryopreservation of semen is useful for animal breeding via artificial insemination (AI). However, the use of frozen-thawed boar semen is limited due to cryodamage. The aim of this study was to investigate the effects of different concentrations of MitoTEMPO (a mitochondria-targeted antioxidant) in lactose-egg yolk (LEY) extenders on kinetic characteristics of frozen-thawed boar sperms. Semen samples were collected from mature Duroc boars (2~3 years old) and cryopreserved in LEY extenders containing 0, 0.5, 5, 50, and 500 μM MitoTEMPO. The kinetic characteristics of frozen-thawed sperms were determined 0 and 30 min after thawing using computer-assisted sperm analysis (CASA). Results indicated that sperm motility immediately after thawing was significantly higher with 5 and 50 μM (50.46±2.71% and 46.96±2.66%, respectively) than with 500 μM MitoTEMPO (35.40±2.95%) (P<0.05). However, there were no significant differences in other kinetic characteristics except motility. In conclusion, the addition of MitoTEMPO to the sperm freezing extender may have a beneficial effect on motility of post-thawed boar semen.

• Biomolecules & Therapeutics
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• v.31 no.6
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• pp.692-699
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• 2023

The lack of molecular targets hampers the treatment of triple-negative breast cancer (TNBC). In this study, we determined the cytotoxicity of domperidone, a dopamine D2 receptor (DRD2) antagonist in human TNBC BT-549 and CAL-51 cells. Domperidone inhibited cell growth in a dose- and time-dependent manner. The annexin V/propidium iodide staining showed that domperidone induced apoptosis. The domperidone-induced apoptosis was accompanied by the generation of mitochondrial superoxide and the down-regulation of cyclins and CDKs. The apoptotic effect of domperidone on TNBC cells was prevented by pre-treatment with Mito-TEMPO, a mitochondria-specific antioxidant. The prevention of apoptosis with Mito-TEMPO even at concentrations as low as 100 nM, implies that the generation of mitochondrial ROS mediated the domperidone-induced apoptosis. Immunoblot analysis showed that domperidone-induced apoptosis occurred through the down-regulation of the phosphorylation of JAK2 and STAT3. Moreover, domperidone downregulated the levels of D2-like dopamine receptors including DRD2, regardless of their mRNA levels. Our results support further development of DRD2 antagonists as potential therapeutic strategy treating TNBC.

• Lettere Italiane
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• no.27
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• pp.155-169
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• 2009

Al centro dell'interesse di Strehler ci sono sempre l'uomo e le sue azioni, e l'uomo e la posizione del teatro. Inoltre, nei suoi teatri vengono alla luce i rapporti che interessano l'uomo e la societa', l'uomo e se stesso, l'uomo e la storia, l'uomo e la politica. Strehler interpreta sempre i testi prendendo in considerazione la contemporaneita' e completa la rappresentazione strutturandola in un sistema implacabilmente coerente di segni scrulolosi. Poiche' le sue opere, anche se le stesse, sono rappresentate ogni volta con significati diversi a secondo del tempo, non si tratta soltanto di rivivere le opere, ma di metterle in scena in nuove versioni che hanno significati che interpretano i cambiamenti avvenuti. Questo e' anche il caso de I giganti della montagna. Nella prima regia di quest'opera, Strehler cerca di mettere in evidenza una concreta e generale storia umana attraverso il mito, e un messaggio che è al tempo stesso di disperazione e di fede nell'uomo. Un messaggio di perdono e di pietà. Pietà per tutti. Nella seconda regia, egli incarna, attraverso il vuoto dell'immobilita' e il silenzio, la vita della poesia. Cerca di rappresentare l'estremo degli effetti scenici attraverso la minima mobilita' in una scena immobile, e attraverso il massimo grido di dolore in una scena di silenzio. E anche i personaggi sono divisi in due diverse categorie. 'Gli scalognati', che creano 'teatro puro' non per un pubblico ma per puro giuoco, e 'la compagnia dei comici', che crea 'teatro rappresentato' che implica la possibilita' dell'interprete condizionato dai rischi, dagli errori di valutazione e d'interpretazione e che resta legato ad una realta' concreta. Attraverso questo contrasto egli vuole esprimere "il timore che nonostante tanta "rivoluzione" giovanile intorno, il cammino della societa' degli uomini verso l'abbandono di alti ideali e valori, fosse accelerato e quasi ineluttabile". Nella terza regia, i due mondi si incontrano in uno spazio dove realta' e sogno si confondono. Non sono piu' contrapposti. E' come "una tragica contestazione che i Giganti hanno vinto e che ci hanno, consapevoli o inconsapevoli, travolti, che non siamo piu gli stessi, che stiamo precipitando". Ma egli non dispera, benche' rappresenti la condizione tragica, dice al pubblico di "comunicare in silenzio la reciprocita' delle sconfitte. al di la' delle parole che non esistono".

• Biomolecules & Therapeutics
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• v.30 no.6
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• pp.585-592
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• 2022

Treatment of triple-negative breast cancer (TNBC) has been limited due to the lack of molecular targets. In this study, we evaluated the cytotoxicity of hydroxyzine, a histamine H1 receptor antagonist in human triple-negative breast cancer BT-20 and HCC-70 cells. Hydroxyzine inhibited the growth of cells in dose- and time-dependent manners. The annexin V/propidium iodide double staining assay showed that hydroxyzine induced apoptosis. The hydroxyzine-induced apoptosis was accompanied down-regulation of cyclins and CDKs, as well as the generation of reactive oxygen species (ROS) without cell cycle arrest. The effect of hydroxyzine on the induction of ROS and apoptosis on TNBC cells was prevented by pre-treatment with ROS scavengers, N-acetyl cysteine or Mito-TEMPO, a mitochondria-targeted antioxidant, indicating that an increase in the generation of ROS mediated the apoptosis induced by hydroxyzine. Western blot analysis showed that hydroxyzine-induced apoptosis was through down-regulation of the phosphorylation of JAK2 and STAT3 by hydroxyzine treatment. In addition, hydroxyzine induced the phosphorylation of JNK and p38 MAPK. Our results indicate that hydroxyzine induced apoptosis via mitochondrial superoxide generation and the suppression of JAK2/STAT3 signaling.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.4
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• pp.1993-1998
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• 2016

The effects of carbon-ion irradiation on cancer cell telomere function have not been comprehensively studied. In our previous report cancer cells with telomere dysfunction were more sensitive to carbon-ion irradiation, but the underlying mechanisms remained unclear. Here we found that telomerase activity was suppressed by carbon-ion irradiation via hTERT down-regulation. Inhibition of telomere activity by MST-312 further increased cancer cell radiosensitivity to carbon-ion radiation. hTERT suppression caused by either carbon-ion irradiation or MST-312 impaired mitochondrial function, as indicated by decreased membrane potential, mtDNA copy number, mitochondrial mass, total ATP levels and elevated reactive oxygen species (ROS). PGC-$1{\alpha}$ expression was repressed after carbion-ion irradiation, and hTERT inhibition by MST-312 could further exacerbate this effect. Lowering the mitochondrial ROS level by MitoTEMPO could partially counteract the induction of cellular senescence induced by carbon-ion radiation and MST-312 incubation. Taken together, the current data suggest that telomere-mitochondrion links play a role in the induction of senescence in MCF-7 cells after carbon-ion irradiation.

• Journal of Periodontal and Implant Science
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• v.53 no.1
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• pp.54-68
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• 2023

Purpose: The purpose of this study was to determine whether metformin (MF) could alleviate the expresssion of reactive oxygen species (ROS) and improve the osteogenic ability of bone marrow mesenchymal stem cells derived from diabetic rats (drBMSCs) in vitro, and to evaluate the effect of MF on the ectopic osteogenesis of drBMSCs in a nude mouse model in vivo. Methods: BMSCs were extracted from normal and diabetic rats. In vitro, a cell viability assay (Cell Counting Kit-8), tests of alkaline phosphatase (ALP) activity, and western blot analysis were first used to determine the cell proliferation and osteogenic differentiation of drBMSCs that were subjected to treatment with different concentrations of MF (0, 50, 100, 200, 500 µM). The cells were then divided into 5 groups: (1) normal rat BMSCs (the BMSCs derived from normal rats group), (2) the drBMSCs group, (3) the drBMSCs + Mito-TEMPO (10 µM, ROS scavenger) group, (4) the drBMSCs + MF (200 µM) group, and (5) the drBMSCs + MF (200 µM) + H₂O₂ (50 µM, ROS activator) group. Intracellular ROS detection, a senescence-associated β-galactosidase assay, ALP staining, alizarin red staining, western blotting, and immunofluorescence assays were performed to determine the effects of MF on oxidative stress and osteogenic differentiation in drBMSCs. In vivo, the effect of MF on the ectopic osteogenesis of drBMSCs was evaluated in a nude mouse model. Results: MF effectively reduced ROS levels in drBMSCs. The cell proliferation, ALP activity, mineral deposition, and osteogenic-related protein expression of drBMSCs were demonstrably higher in the MF-treated group than in the non-MF-treated group. H₂O₂ inhibited the effects of MF. In addition, ectopic osteogenesis was significantly increased in drBMSCs treated with MF. Conclusions: MF promoted the proliferation and osteogenic differentiation of drBMSCs by inhibiting the oxidative stress induced by diabetes and enhenced the ectopic bone formation of drBMSCs in nude mice.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.4
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• pp.377-384
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• 2017

Activation of protein kinase C (PKC) is closely linked with endothelial dysfunction. However, the effect of $PKC{\beta}II$ on endothelial dysfunction has not been characterized in cultured endothelial cells. Here, using adenoviral $PKC{\beta}II$ gene transfer and pharmacological inhibitors, the role of $PKC{\beta}II$ on endothelial dysfucntion was investigated in cultured endothelial cells. Phorbol 12-myristate 13-acetate (PMA) increased reactive oxygen species (ROS), p66shc phosphorylation, intracellular adhesion molecule-1, and monocyte adhesion, which were inhibited by $PKC{\beta}i$ (10 nM), a selective inhibitor of $PKC{\beta}II$. PMA increased the phosphorylation of CREB and manganese superoxide dismutase (MnSOD), which were also inhibited by $PKC{\beta}i$. Gene silencing of CREB inhibited PMA-induced MnSOD expression, suggesting that CREB plays a key role in MnSOD expression. Gene silencing of $PKC{\beta}II$ inhibited PMA-induced mitochondrial ROS, MnSOD, and ICAM-1 expression. In contrast, overexpression of $PKC{\beta}II$ using adenoviral $PKC{\beta}II$ increased mitochondrial ROS, MnSOD, ICAM-1, and p66shc phosphorylation in cultured endothelial cells. Finally, $PKC{\beta}II$-induced ICAM-1 expression was inhibited by Mito-TEMPO, a mitochondrial ROS scavenger, suggesting the involvement of mitochondrial ROS in PKC-induced vascular inflammation. Taken together, the results suggest that $PKC{\beta}II$ plays an important role in PMA-induced endothelial dysfunction, and that the inhibition of $PKC{\beta}II$-dependent p66shc signaling acts as a therapeutic target for vascular inflammatory diseases.