Activation of Heme Oxygenase-1 by Mangiferin in Human Retinal Pigment Epithelial Cells Contributes to Blocking Oxidative Damage.

Mangiferin is a kind of natural xanthone glycosides and is known to have various pharmacological activities. However, since the beneficial efficacy of this compound has not been reported in retinal pigment epithelial (RPE) cells, this study aimed to evaluate whether mangiferin could protect human RPE ARPE-19 cells from oxidative injury mimicked by hydrogen peroxide (H2O2). The results showed that mangiferin attenuated H2O2-induced cell viability reduction and DNA damage, while inhibiting reactive oxygen species (ROS) production and preserving diminished glutathione (GSH). Mangiferin also antagonized H2O2-induced inhibition of the expression and activity of antioxidant enzymes such as manganese superoxide dismutase and GSH peroxidase, which was associated with inhibition of mitochondrial ROS production. In addition, mangiferin protected ARPE-19 cells from H2O2-induced apoptosis by increasing the Bcl-2/Bax ratio, decreasing caspase-3 activation, and blocking poly(ADP-ribose) polymerase cleavage. Moreover, mangiferin suppressed the release of cytochrome c into the cytosol, which was achieved by interfering with mitochondrial membrane disruption. Furthermore, mangiferin increased the expression and activity of heme oxygenase-1 (HO-1) and nuclear factor-erythroid-2 related factor 2 (Nrf2). However, the inhibition of ROS production, cytoprotective and anti-apoptotic effects of mangiferin were significantly attenuated by the HO-1 inhibitor, indicating that mangiferin promoted Nrf2-mediated HO-1 activity to prevent ARPE-19 cells from oxidative injury. The results of this study suggest that mangiferin, as an Nrf2 activator, has potent ROS scavenging activity and may have the potential to protect oxidative stress-mediated ocular diseases.

Correlation analysis of cancer stem cell marker CD133 and human endogenous retrovirus (HERV)-K env in SKOV3 ovarian cancer cells.

BACKGROUND: Human endogenous retrovirus (HERV)-K is a type of retrovirus that is present in the human genome, and its expression is usually silenced in healthy tissues. The precise mechanism by which HERV-K env influences cancer stemness is not fully understood, but it has been suggested that HERV-K env may activate various signaling pathways that promote stemness traits in cancer cells. OBJECTIVE: To establish the connection between HERV-K env expression and cancer stemness in ovarian cancer cells, we carried out correlation analyses between HERV-K env and the cancer stem cell (CSC) marker known as the cluster of differentiation 133 (CD133) gene in SKOV3 ovarian cancer cells. METHOD: To perform correlation analysis between HERV-K env and CSCs, ovarian cancer cells were cultured in a medium designed for cancer stem cell induction. The expression of HERV-K env and CD133 genes was verified using quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Additionally, the expression of stemness-related markers, such as OCT-4 and Nanog, was also confirmed using RT-qPCR. RESULTS: In the stem cell induction medium, the number of tumorsphere-type SKOV3 cells increased, and the expression of CD133 and HERV-K env genes was up-regulated. Additionally, other stemness-related markers like OCT-4 and Nanog also exhibited increased expression when cultured in the cancer stem cell induction medium. However, when HERV-K env knockout (KO) SKOV3 cells were cultured in the same cancer stem cell induction medium, there was a significant decrease in the number of tumorsphere-type cells compared to mock SKOV3 cells subjected to the same conditions. Furthermore, the expression of CD133, Nanog, and OCT-4 did not show a significant increase in HERV-K env KO SKOV3 cells compared to mock SKOV3 cells cultured in the same cancer stem cell induction medium. CONCLUSION: These findings indicate that the expression of HERV-K env increased in SKOV3 cells when cultured in cancer stem cell induction media, and cancer stem cell induction was inhibited by KO of HERV-K env in SKOV3 cells. These results suggest a strong association between HERV-K env and stemness in SKOV3 ovarian cancer cells.

Functional Analysis of Membrane-Associated Scaffolding Tight Junction (TJ) Proteins in Tumorigenic Characteristics of B16-F10 Mouse Melanoma Cells.

Tight junction (TJ) proteins (Tjps), Tjp1 and Tjp2, are tight junction-associated scaffold proteins that bind to the transmembrane proteins of tight junctions and the underlying cytoskeleton. In this study, we first analyzed the tumorigenic characteristics of B16-F10 melanoma cells, including cell proliferation, migration, invasion, metastatic potential, and the expression patterns of related proteins, after the CRISPR-Cas9-mediated knockout (KO) of Tjp genes. The proliferation of Tjp1 and Tjp2 KO cells significantly increased in vitro. Other tumorigenic characteristics, including migration and invasion, were significantly enhanced in Tjp1 and Tjp2 KO cells. Zonula occludens (ZO)-associated protein Claudin-1 (CLDN-1), which is a major component of tight junctions and functions in controlling cell-to-cell adhesion, was decreased in Tjp KO cells. Additionally, Tjp KO significantly stimulated tumor growth and metastasis in an in vivo mouse model. We performed a transcriptome analysis using next-generation sequencing (NGS) to elucidate the key genes involved in the mechanisms of action of Tjp1 and Tjp2. Among the various genes affected by Tjp KO-, cell cycle-, cell migration-, angiogenesis-, and cell-cell adhesion-related genes were significantly altered. In particular, we found that the Ninjurin-1 (Ninj1) and Catenin alpha-1 (Ctnna1) genes, which are known to play fundamental roles in Tjps, were significantly downregulated in Tjp KO cells. In summary, tumorigenic characteristics, including cell proliferation, migration, invasion, tumor growth, and metastatic potential, were significantly increased in Tjp1 and Tjp2 KO cells, and the knockout of Tjp genes significantly affected the expression of related proteins.

The Role of Human Endogenous Retrovirus (HERV)-K119 env in THP-1 Monocytic Cell Differentiation.

Human endogenous retrovirus (HERV)-K was reportedly inserted into the human genome millions of years ago and is closely related to various diseases, including cancer and immune regulation. In our previous studies, CRISPR-Cas9-enabled knockout (KO) of the HERV-K env gene was found to potentially reduce cell proliferation, cell migration, and invasion in colorectal and ovarian cancer cell lines. The immune response involves the migration and invasion of cells and is similar to cancer; however, in certain ways, it is completely unlike cancer. Therefore, we induced HERV-K119 env gene KO in THP-1, a monocytic cell that can be differentiated into a macrophage, to investigate the role of HERV-K119 env in immune regulation. Cell migration and invasion were noted to be significantly increased in HERV-K119 env KO THP-1 cells than in MOCK, and these results were contrary to those of cancer cells. To identify the underlying mechanism of HERV-K119 env KO in THP-1 cells, transcriptome analysis and cytokine array analysis were conducted. Semaphorin7A (SEMA7A), which induces the production of cytokines in macrophages and monocytic cells and plays an important role in immune effector cell activation during an inflammatory immune response, was significantly increased in HERV-K119 env KO THP-1 cells. We also found that HERV-K119 env KO THP-1 cells expressed various macrophage-specific surface markers, suggesting that KO of HERV-K119 env triggers the differentiation of THP-1 cells from monocytic cells into macrophages. In addition, analysis of the expression of M1 and M2 macrophage markers showed that M1 macrophage marker cluster of differentiation 32 (CD32) was significantly increased in HERV-K119 env KO cells. These results suggest that HERV-K119 env is implicated in the differentiation of monocytic cells into M1 macrophages and plays important roles in the immune response.

Inhibition of Urban Particulate Matter-Induced Airway Inflammation by RIPK3 through the Regulation of Tight Junction Protein Production.

Urban particulate matter (UPM) is a high-hazard cause of various diseases in humans, including in the respiratory tract, skin, heart, and even brain. Unfortunately, there is no established treatment for the damage caused by UPM in the respiratory epithelium. In addition, although RIPK3 is known to induce necroptosis, its intracellular role as a negative regulator in human lungs and bronchial epithelia remains unclear. Here, the endogenous expression of RIPK3 was significantly decreased 6 h after exposure to UPM. In RIPK3-ovexpressed cells, RIPK3 was not moved to the cytoplasm from the nucleus. Interestingly, the overexpression of RIPK3 dramatically decreased TEER and F-actin formation. Its overexpression also decreased the expression of genes for pro-inflammatory cytokines (IL-6 and IL-8) and tight junctions (ZO-1, -2, -3, E-cadherin, and claudin) during UPM-induced airway inflammation. Importantly, overexpression of RIPK3 inhibited the UPM-induced ROS production by inhibiting the activation of iNOS and eNOS and by regulating mitochondrial fission processing. In addition, UPM-induced activation of the iκB and NF-κB signaling pathways was dramatically decreased by RIPK3, and the expression of pro-inflammatory cytokines was decreased by inhibiting the iκB signaling pathway. Our data indicated that RIPK3 is essential for the UPM-induced inflammatory microenvironment to maintain homeostasis. Therefore, we suggest that RIPK3 is a potential therapeutic candidate for UPM-induced pulmonary inflammation.

PDZ Peptide of the ZO-1 Protein Significantly Increases UTP-Induced MUC8 Anti-Inflammatory Mucin Overproduction in Human Airway Epithelial Cells.

Mucus hyperproduction and hypersecretion are observed often in respiratory diseases. MUC8 is a glycoprotein synthesized by epithelial cells and generally expressed in the respiratory track. However, the physiological mechanism by which extracellular nucleotides induce MUC8 gene expression in human airway epithelial cells is unclear. Here, we show that UTP could induce MUC8 gene expression through P2Y2-PLCß3-Ca2+ activation. Because the full-length cDNA sequence of MUC8 has not been identified, a specific siRNA-MUC8 was designed based on the partial cDNA sequence of MUC8. siRNA-MUC8 significantly increased TNF-α production and decreased IL-1Ra production, suggesting that MUC8 may downregulate UTP/P2Y2-induced airway inflammation. Interestingly, the PDZ peptide of ZO-1 protein strongly abolished UTP-induced TNF-α production and increased IL-1Ra production and MUC8 gene expression. In addition, the PDZ peptide dramatically increased the levels of UTP-induced ZO proteins and TEER (trans-epithelial electrical resistance). These results show that the anti-inflammatory mucin MUC8 may contribute to homeostasis, and the PDZ peptide can be a novel therapeutic candidate for UTP-induced airway inflammation.

Human Endogenous Retrovirus-H-Derived miR-4454 Inhibits the Expression of DNAJB4 and SASH1 in Non-Muscle-Invasive Bladder Cancer.

Although most human endogenous retroviruses (HERVs) have been silenced and lost their ability to translocate because of accumulated mutations during evolution, they still play important roles in human biology. Several studies have demonstrated that HERVs play pathological roles in numerous human diseases, especially cancer. A few studies have revealed that long non-coding RNAs that are transcribed from HERV sequences affect cancer progression. However, there is no study on microRNAs derived from HERVs related to cancer. In this study, we identified 29 microRNAs (miRNAs) derived from HERV sequences in the human genome. In particular, we discovered that miR-4454, which is HERV-H-derived miRNA, was upregulated in non-muscle-invasive bladder cancer (NMIBC) cells. To figure out the effects of upregulated miR-4454 in NMIBC, genes whose expression was downregulated in NMIBC, as well as tumor suppressor genes, were selected as putative target genes of miR-4454. The dual-luciferase assay was used to determine the negative relationship between miR-4454 and its target genes, DNAJB4 and SASH1, and they were confirmed to be promising target genes of miR-4454. Taken together, this study suggests that the upregulation of miR-4454 derived from HERV-H in NMIBC reduces the expression of the tumor suppressor genes, DNAJB4 and SASH1, to promote NMIBC progression.

ß-Asarone Alleviates High-Glucose-Induced Oxidative Damage via Inhibition of ROS Generation and Inactivation of the NF-κB/NLRP3 Inflammasome Pathway in Human Retinal Pigment Epithelial Cells.

Diabetic retinopathy (DR) is the leading cause of vision loss and a major complication of diabetes. Hyperglycemia-induced accumulation of reactive oxygen species (ROS) is an important risk factor for DR. ß-asarone, a major component of volatile oil extracted from Acori graminei Rhizoma, exerts antioxidant effects; however, its efficacy in DR remains unknown. In this study, we investigated whether ß-asarone inhibits high-glucose (HG)-induced oxidative damage in human retinal pigment epithelial (RPE) ARPE-19 cells. We found that ß-asarone significantly alleviated cytotoxicity, apoptosis, and DNA damage in HG-treated ARPE-19 cells via scavenging of ROS generation. ß-Asarone also significantly attenuated the excessive accumulation of lactate dehydrogenase and mitochondrial ROS by increasing the manganese superoxide dismutase and glutathione activities. HG conditions markedly increased the release of interleukin (IL)-1ß and IL-18 and upregulated their protein expression and activation of the nuclear factor-kappa B (NF-κB) signaling pathway, whereas ß-asarone reversed these effects. Moreover, expression levels of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome multiprotein complex molecules, including thioredoxin-interacting protein, NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain, and cysteinyl aspartate-specific proteinase-1, were increased in ARPE-19 cells under HG conditions. However, their expression levels remained similar to those in the control group in the presence of ß-asarone. Therefore, ß-asarone protects RPE cells from HG-induced injury by blocking ROS generation and NF-κB/NLRP3 inflammasome activation, indicating its potential as a therapeutic agent for DR treatment.

Molecular mechanisms underlying the vulnerability of Pacific abalone (Haliotis discus hannai) to Vibrio harveyi infection at higher water temperature.

Climate change is one of the most important threats to farmed abalone worldwide. Although abalone is more susceptible to vibriosis at higher water temperatures, the molecular mode of action underlying this has not been fully elucidated. Therefore, this study aimed to address the high susceptibility of Halitotis discus hannai to V. harveyi infection using abalone hemocytes exposed to low and high temperatures. Abalone hemocytes were divided into four groups, 20C, 20 V, 25C, and 25 V, depending on co-culture with (V)/without (C) V. harveyi (MOI = 12.8) and incubation temperature (20 °C or 25 °C). After 3 h of incubation, hemocyte viability and phagocytic activity were measured, and RNA sequencing was performed using Illumina Novaseq. The expression of several virulence-related genes in V. harveyi was analyzed using real-time PCR. The viability of hemocytes was significantly decreased in the 25 V group compared to cells in the other groups, whereas phagocytic activity at 25 °C was significantly higher than at 20 °C. Although a number of immune-associated genes were commonly upregulated in abalone hemocyte exposed to V. harveyi, regardless of temperature, pathways and genes regarding pro-inflammatory responses (interleukin-17 and tumor necrosis factor) and apoptosis were significantly overexpressed in the 25 V group compared to the 25C group. Notably, in the apoptosis pathway, genes encoding executor caspases (casp3 and casp7) and pro-apoptotic factor, bax were significantly up-regulated only in the 25 V group, while the apoptosis inhibitor, bcl2L1 was significantly up-regulated only in the 20 V group compared to the control group at the respective temperatures. The co-culture of V. harveyi with abalone hemocytes at 25 °C up-regulated several virulence-related genes involved in quorum sensing (luxS), antioxidant activity (katA, katB, and sodC), motility (flgI), and adherence/invasion (ompU) compared to those at 20 °C. Therefore, our results showed that H. discus hannai hemocytes exposed to V. harveyi at 25 °C were highly stressed by vigorously activated inflammatory responses and that the bacterial pathogen overexpressed several virulence-related genes at the high temperature tested. The transcriptomic profile of both abalone hemocytes and V. harveyi in the present study provide insight into differential host-pathogen interactions depending on the temperature conditions and the molecular backgrounds related to increased abalone vulnerability upon global warming.

Nrf2-mediated activation of HO-1 is required in the blocking effect of compound K, a ginseng saponin metabolite, against oxidative stress damage in ARPE-19 human retinal pigment epithelial cells.

Background: The beneficial effects of compound K (CK) on different chronic diseases have been shown to be at least related to antioxidant action. Nevertheless, since its antioxidant activity in human retinal pigment epithelial (RPE) cells is still unknown, here we investigated whether CK alleviates oxidative stress-stimulated damage in RPE ARPE-19 cells. Methods: The cytoprotective consequence of CK in hydrogen peroxide (H2O2)-treated cells was evaluated by cell viability, DNA damage, and apoptosis assays. Fluorescence analysis and immunoblotting were performed to investigate the inhibitory action of CK on reactive oxygen species (ROS) production and mitochondrial dysfunction. Results: H2O2-promoted cytotoxicity, oxidative stress, DNA damage, mitochondrial impairment, and apoptosis were significantly attenuated by CK in ARPE-19 cells. Furthermore, nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation level and its shuttling to the nucleus were increased, which was correlated with upregulated activation of heme oxygenase-1 (HO-1). However, zinc protoporphyrin, a blocker of HO-1, significantly abrogated the preventive action of CK in H2O2-treated ARPE-19 cells. Conclusion: This study indicates that activation of Nrf2/HO-1 signaling by CK plays an important role in rescuing ARPE-19 cells from oxidative cellular damage.

Fisetin Protects C2C12 Mouse Myoblasts from Oxidative Stress-Induced Cytotoxicity through Regulation of the Nrf2/HO-1 Signaling.

Fisetin is a bioactive flavonol molecule and has been shown to have antioxidant potential, but its efficacy has not been fully validated. The aim of the present study was to investigate the protective efficacy of fisetin on C2C12 murine myoblastjdusts under hydrogen peroxide (H2O2)-induced oxidative damage. The results revealed that fisetin significantly weakened H2O2-induced cell viability inhibition and DNA damage while blocking reactive oxygen species (ROS) generation. Fisetin also significantly alleviated cell cycle arrest by H2O2 treatment through by reversing the upregulation of p21WAF1/CIP1 expression and the downregulation of cyclin A and B levels. In addition, fisetin significantly blocked apoptosis induced by H2O2 through increasing the Bcl-2/Bax ratio and attenuating mitochondrial damage, which was accompanied by inactivation of caspase-3 and suppression of poly(ADP-ribose) polymerase cleavage. Furthermore, fisetin-induced nuclear translocation and phosphorylation of Nrf2 were related to the increased expression and activation of heme oxygenase-1 (HO-1) in H2O2-stimulated C2C12 myoblasts. However, the protective efficacy of fisetin on H2O2-mediated cytotoxicity, including cell cycle arrest, apoptosis and mitochondrial dysfunction, were greatly offset when HO-1 activity was artificially inhibited. Therefore, our results indicate that fisetin as an Nrf2 activator effectively abrogated oxidative stress-mediated damage in C2C12 myoblasts.

Phloroglucinol Inhibits Oxidative-Stress-Induced Cytotoxicity in C2C12 Murine Myoblasts through Nrf-2-Mediated Activation of HO-1.

Phloroglucinol is a class of polyphenolic compounds containing aromatic phenyl rings and is known to have various pharmacological activities. Recently, we reported that this compound isolated from Ecklonia cava, a brown alga belonging to the family Laminariaceae, has potent antioxidant activity in human dermal keratinocytes. In this study, we evaluated whether phloroglucinol could protect against hydrogen peroxide (H2O2)-induced oxidative damage in murine-derived C2C12 myoblasts. Our results revealed that phloroglucinol suppressed H2O2-induced cytotoxicity and DNA damage while blocking the production of reactive oxygen species. We also found that phloroglucinol protected cells from the induction of apoptosis associated with mitochondrial impairment caused by H2O2 treatment. Furthermore, phloroglucinol enhanced the phosphorylation of nuclear factor-erythroid-2 related factor 2 (Nrf2) as well as the expression and activity of heme oxygenase-1 (HO-1). However, such anti-apoptotic and cytoprotective effects of phloroglucinol were greatly abolished by the HO-1 inhibitor, suggesting that phloroglucinol could increase the Nrf2-mediated activity of HO-1 to protect C2C12 myoblasts from oxidative stress. Taken together, our results indicate that phloroglucinol has a strong antioxidant activity as an Nrf2 activator and may have therapeutic benefits for oxidative-stress-mediated muscle disease.

Effect of parasitic infection on muscular function of dystrophin gene (Dmd) deficient mouse.

BACKGROUND: Previous studies have reported many cases of Trichinella spiralis (T. spiralis) infection in normal skeletal muscle but there is little research on T. spiralis infection in abnormal muscle tissue. OBJECTIVE: To identify the effect of T. spiralis infection on muscular dystrophy, this study compared aspects of infection between normal (C57BL/10) and dystrophin-deficient Duchenne muscular dystrophy (DMD) mdx mice. METHOD: Infection rate was found to be lower in mdx mice than in C57BL/10 mice at early stages of infection; however, infection and inflammation in mdx mice persisted at later stages of infection while the infection rate and inflammation in C57BL/10 mice decreased gradually. The inflammation area was proportional to the degree of infection in both groups. Muscle strength was measured by the time of latency to fall in the wire-hanging test. Hanging time was shorter in the infected group than in the uninfected group in both C57BL/10 and mdx mice. RESULTS: Muscle strength was also reduced in mdx mice compared with C57BL/10 mice in both the un-infected and infected groups. The muscle intracellular cytokines TGF-ß and IL-6 were continuously expressed from early stage to late-stage infection. IL-10 was strongly expressed at the early stage of infection but decreased as the infection progressed. TNF-α expression remained stable from early to late-stage infection in mdx mice, while TNF-α was elevated only during early-stage infection in C57BL/10 mice. The degree of muscle damage was significantly higher in mdx mice than in C57BL/10 mice because of the high level of serum creatine kinase (CK). CONCLUSION: These results suggest that mdx mice continued in infection and inflammation until the late stages of disease, which was in contrast to the C57BL/10 mice that recovered to some extent in the late stage of infection. In addition, that dystrophin-deficient mice are not suitable for T. spiralis infection compared to normal mice, and the degree of inflammation may be worse in mdx mice.

Phloroglucinol Attenuates DNA Damage and Apoptosis Induced by Oxidative Stress in Human Retinal Pigment Epithelium ARPE-19 Cells by Blocking the Production of Mitochondrial ROS.

Phloroglucinol, a phenolic compound, is known to possess a potent antioxidant ability. However, its role in retinal cells susceptible to oxidative stress has not been well elucidated yet. Thus, the objective of this study was to evaluate whether phloroglucinol could protect against oxidative damage in cultured human retinal pigment epithelium ARPE-19 cells. For this purpose, ARPE-19 cells were stimula ted with hydrogen peroxide (H2O2) to mimic oxidative stress. Cell viability, cytotoxicity, apoptosis, reactive oxygen species (ROS) generation, mitochondrial function, DNA damage, and autophagy were then assessed. Our results revealed that phloroglucinol ameliorated cell viability, cytotoxicity, and DNA damage in H2O2-exposued ARPE-19 cells and blocked production of ROS. Phloroglucinol also counteracted H2O2-induced apoptosis by reducing Bax/Bcl-2 ratio, blocking activation of caspase-3, and inhibiting degradation of poly (ADP-ribose) polymerase. H2O2 caused mitochondrial impairment and increased expression levels of mitophagy markers such as PINK1and PARKIN known to be associated with mitochondrial ROS (mtROS) generation and cytosolic release of cytochrome c. However, these changes were significantly attenuated by phloroglucinol. Mito-TEMPO, a selective mitochondrial antioxidant, further enhanced the protective effect of phloroglucinol against dysfunctional mitochondria. Furthermore, H2O2 induced autophagy, but not when ARPE-19 cells were pretreated with phloroglucinol, meaning that autophagy by H2O2 contributed to the pro-survival mechanism and that phloroglucinol protected ARPE-19 cells from apoptosis by blocking autophagy. Taken together, these results suggest that phloroglucinol can inhibit oxidative stress-induced ARPE-19 cell damage and dysfunction by protecting DNA damage, autophagy, and subsequent apoptosis through mitigation of mtROS generation. Thus, phloroglucinol might have therapeutic potential to prevent oxidative stress-mediated damage in RPE cells.

Induction of apoptosis through inactivation of ROS-dependent PI3K/Akt signaling pathway by platycodin D in human bladder urothelial carcinoma cells.

Platycodin D (PD) is a triterpenoid saponin, a major bioactive constituent of the roots of Platycodon grandiflorum, which is well known for possessing various pharmacological properties. However, the anti-cancer mechanism of PD in bladder cancer cells remains poorly understood. In the current study, we investigated the effect of PD on the growth of human bladder urothelial carcinoma cells. PD treatment significantly reduced the cell survival of bladder cancer cells associated with induction of apoptosis and DNA damage. PD inhibited the expression of inhibitor of apoptosis family members, activated caspases, and induced cleavage of poly (ADP-ribose) polymerase. PD also increased the release of cytochrome c into the cytoplasm by disrupting the mitochondrial membrane potential while upregulating the expression ratio of Bax to Bcl-2. The PD-mediated anti-proliferative effect was significantly inhibited by pre-treatment with a pancaspase inhibitor, but not by an inhibitor of necroptosis. Moreover, PD suppressed the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, and the apoptosis-inducing effect of PD was further enhanced by a PI3K inhibitor. In addition, PD increased the accumulation of reactive oxygen species (ROS), whereas N-acetyl cysteine (NAC), an ROS inhibitor, significantly attenuated the growth inhibition and inactivation of the PI3K/Akt/mTOR signaling caused by PD. Furthermore, NAC significantly suppressed apoptosis, DNA damage, and decreased cell viability induced by PD treatment. Collectively, our findings indicated that PD blocked the growth of bladder urothelial carcinoma cells by inducing ROS-mediated inactivation of the PI3K/Akt/mTOR signaling.

Effect of human endogenous retrovirus-K env gene knockout on proliferation of ovarian cancer cells.

BACKGROUND: Among various human endogenous retroviruses (HERVs), the HERV-K (HML-2) group has been reported to be highly related to cancer. In pancreatic cancer cells, shRNA-mediated downregulation of HERV-K env RNA decreases cell proliferation and tumor growth through the RAS-ERK-RSK pathway; in colorectal cancer, CRISPR-Cas9 knockout (KO) of the HERV-K env gene affects tumorigenic characteristics through the nupr-1 gene. OBJECTIVE: The effect of HERV-K env KO has not been studied in ovarian cancer cell lines. In this study, we analyzed the tumorigenic characteristics of ovarian cancer cell lines, including cell proliferation, migration, and invasion, and the expression patterns of related proteins after CRISPR-Cas9 KO of the HERV-K env gene. METHODS: The HERV-K env gene KO was achieved using the CRISPR-Cas9 system in ovarian cancer cell lines SKOV3 and OVCAR3. Tumorigenic characteristics including cell proliferation, migration, and invasion were analyzed, and related protein expression was investigated by western blot analysis. RESULTS: The expression of the HERV-K env gene in KO cells was significantly reduced at RNA and protein levels, and tumorigenic characteristics including cell proliferation, migration, and invasion were significantly reduced. In HERV-K env KO SKOV3 cells, the expression of the RB protein was significantly up-regulated and the cyclin B1 protein level was significantly reduced. In contrast, in HERV-K env KO OVCAR3 cells, the level of phospho-RB protein was significantly reduced, but other protein levels were not changed. CONCLUSION: The results of this study showed that HERV-K env gene KO affects cell proliferation, invasion, and migration of ovarian cells through RB and Cyclin B1 proteins, but the specific regulation pattern can differ by cell line.

Downregulated pol-miR-140-3p induces the expression of the kinesin family member 5A against Streptococcus parauberis infection in olive flounder.

MicroRNAs (miRNAs) are small non-coding RNAs that participate in various biological and cellular processes by regulating target gene expression. miRNAs are also known to play vital roles in the pathogenesis of various diseases, including infections, as well as the disease progression and defense responses. In this study, we examined the expression levels of pol-miR-140-3p and its target gene, kinesin family member 5A (KIF5A), in association with the Streptococcus parauberis (S. parauberis) infection, a major bacterial pathogen that causes streptococcosis in olive flounder (Paralichthys olivaceus). KIF5A is a heavy chain isoform of kinesin-1, which is known to be brain-specific, and this study is the first examination of KIF5A expression related to the regulation of miRNA in olive flounder (named PoKIF5A). There were significant differences in expression levels between infected and healthy olive flounder as the expression of pol-miR-140-3p in the infected fish was lower than that in the control, while the expression of PoKIF5A was higher in the infected fish than in the healthy controls. These contradictory results suggest that downregulated pol-miR-140-3p induces the expression of PoKIF5A against S. parauberis infection in olive flounder.

Protection against Oxidative Stress-Induced Apoptosis by Fermented Sea Tangle (Laminaria japonica Aresch) in Osteoblastic MC3T3-E1 Cells through Activation of Nrf2 Signaling Pathway.

The purpose of the present study was to explore the efficacy of fermented extract of sea tangle (Laminaria japonica Aresch, FST) with Lactobacillus brevis on DNA damage and apoptosis in hydrogen peroxide (H2O2)-stimulated osteoblastic MC3T3-E1 cells and clarify related signaling pathways. Our results showed that exposure to FST significantly improved cell viability, inhibited apoptosis, and suppressed the generation of reactive oxygen species (ROS) in H2O2-stimulated cells. In addition, H2O2 triggered DNA damage in MC3T3-E1 cells was markedly attenuated by FST pretreatment. Moreover, H2O2-induced mitochondrial dysfunctions associated with apoptotic events, including loss of mitochondrial membrane potential (MMP), decreased Bcl-2/Bcl-2 associated x-protein (Bax) ratio, and cytosolic release of cytochrome c, were reduced in the presence of FST. FST also diminished H2O2-induced activation of caspase-3, which was associated with the ability of FST to protect the degradation of poly (ADP-ribose) polymerase. Furthermore, FST notably enhanced nuclear translocation and phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2) in the presence of H2O2 with concomitant upregulation of heme oxygenase-1 (HO-1) expression. However, artificial blockade of this pathway by the HO-1 inhibitor, zinc protoporphyrin IX, greatly abolished the protective effect of FST against H2O2-induced MC3T3-E1 cell injury. Taken together, these results demonstrate that FST could protect MC3T3-E1 cells from H2O2-induced damage by maintaining mitochondrial function while eliminating ROS along with activation of the Nrf2/HO-1 antioxidant pathway.

Proteome profile of spleen in rock bream (Oplegnathus fasciatus) naturally infected with rock bream iridovirus (RBIV).

BACKGROUND: Rock bream iridovirus (RBIV) is one of the most dangerous pathogens that causes the highest mortality in the aquaculture of rock bream (Oplegnathus fasciatus). Even though RBIV infection leads to huge economic loss, proteome studies on RBIV-infected rock bream have not been conducted to provide information about the differential protein expression pattern by the host protection system. OBJECTIVE: The purpose of this study was to investigate the protein expression patterns in spleens of rock bream olive after infection by RBIV or mixed infection by RBIV and bacteria. METHODS: Depending on the infection intensity and sampling time point, fish were divided into five groups: uninfected healthy fish at week 0 as the control (0C), heavily infected fish at week 0 (0H), heavily mixed RBIV and bacterial infected fish at week 0 (0MH), uninfected healthy fish at week 3 (3C), and lightly infected fish at week 3 (3L). Proteins were extracted from the spleens of infected rock bream. We used 2-DE analysis with LC-MS/MS to investigate proteome changes in infected rock bream. RESULTS: The results of the LC-MS/MS analyses showed different protein expression profiles after infection. Proteins related to oxygen transport and energy generation, such as hemoglobin, beta-globin, and ATP synthase, were mostly expressed in the infected spleen. Whereas proteins involved in structure and cell movement, such as tubulin, myosin, actin binding proteins, and intermediate filament proteins, were down-regulated in the infected spleens. The protein expression profiles between infection by RBIV and mixed infection by RBIV and bacteria showed similar patterns. CONCLUSIONS: Our results indicated that infection by RBIV or mixed infection by RBIV and bacteria triggered energy generation and oxygen-transport, but cell migration and constructional changes in the spleen were extremely decreased.

Nargenicin A1 attenuates lipopolysaccharide-induced inflammatory and oxidative response by blocking the NF-κB signaling pathway.

Inflammation caused by the excessive production of pro-inflammatory mediators and cytokines in abnormally activated macrophages promotes the initiation and progression of many diseases along with oxidative stress. Previous studies have suggested that nargenicin A1, an antibacterial macrolide isolated from Nocardia sp. may be a potential treatment for inflammatory responses and oxidative stress, but the detailed mechanisms are still not well studied. In this study, we investigated the inhibitory effect of nargenicin A1 on inflammatory and oxidative stress in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and zebrafish (Danio rerio) models. Our results indicated that nargenicin A1 treatment significantly inhibited LPS-induced release of pro-inflammatory mediators including nitric oxide (NO) and prostaglandin E2, which was associated with decreased inducible NO synthase and cyclooxygenase-2 expression. In addition, nargenicin A1 attenuated the LPS-induced expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and monocyte chemotactic protein-1, reducing their extracellular secretion. Nargenicin A1 also suppressed LPS-induced generation of reactive oxygen species. Moreover, nargenicin A1 abolished the LPS-mediated nuclear translocation of nuclear factor-kappa B (NF-κB) and the degradation of inhibitor IκB-α, indicating that nargenicin A1 exhibited anti-inflammatory effects by inhibiting the NF-κB signaling pathway. Furthermore, nargenicin A1 showed strong protective effects against NO and ROS production in LPS-injected zebrafish larvae. In conclusion, our findings suggest that nargenicin A1 ameliorates LPS-induced anti-inflammatory and antioxidant effects by downregulating the NF-κB signaling pathway, and that nargenicin A1 can be a potential functional agent to prevent inflammatory- and oxidative-mediated damage.