Fermented Perilla frutescens Ameliorates Depression-like Behavior in Sleep-Deprivation-Induced Stress Model.

Excessive stress plays a critical role in the pathogenesis of mood disorders such as depression. Fermented natural products have recently attracted attention because of their health benefits. We evaluated the antidepressant-like efficacy of fermented Perilla frutescens (FPF), and its underlying mechanisms, in sleep deprivation (SD)-induced stress mice. SD-stressed mice revealed a remarkable increase in the immobility time in both forced swimming test and tail suspension test; this increase was ameliorated by treatment with FPF at doses of 100 and 150 mg/kg. FPF treatment also reduced the level of stress hormones such as corticosterone and adrenocorticotropic hormone. Additionally, FPF increased the levels of serotonin and dopamine which were significantly decreased in the brain tissues of SD-stressed mice. The increased expression of proinflammatory cytokines, such as TNF-α and IL1ß, and the decreased expression of brain-derived neurotrophic factor (BDNF) in the stressed mice were significantly reversed by FPF treatment. Furthermore, FPF also increased phosphorylation of tropomyosin receptor kinase B (TrkB), extracellular regulated protein kinase (ERK), and cAMP response element binding protein (CREB). Among the six components isolated from FPF, protocatechuic acid and luteolin-7-O-glucuronide exhibited significant antidepressant-like effects, suggesting that they are major active components. These findings suggest that FPF has therapeutic potential for SD-induced stress, by correcting dysfunction of hypothalamic-pituitary-adrenal axis and modulating the BDNF/TrkB/ERK/CREB signaling pathway.

Assessment of mitophagy in mt-Keima Drosophila revealed an essential role of the PINK1-Parkin pathway in mitophagy induction in vivo.

Mitophagy has been implicated in mitochondrial quality control and in various human diseases. However, the study of in vivo mitophagy remains limited. We previously explored in vivo mitophagy using a transgenic mouse expressing the mitochondria-targeted fluorescent protein Keima (mt-Keima). Here, we generated mt-Keima Drosophila to extend our efforts to study mitophagy in vivo. A series of experiments confirmed that mitophagy can be faithfully and quantitatively measured in mt-Keima Drosophila. We also showed that alterations in mitophagy upon environmental and genetic perturbation can be measured in mt-Keima Drosophila. Analysis of different tissues revealed a variation in basal mitophagy levels in Drosophila tissues. In addition, we found a significant increase in mitophagy levels during Drosophila embryogenesis. Importantly, loss-of-function genetic analysis demonstrated that the phosphatase and tensin homolog-induced putative kinase 1 (PINK1)-Parkin pathway is essential for the induction of mitophagy in vivo in response to hypoxic exposure and rotenone treatment. These studies showed that the mt-Keima Drosophila system is a useful tool for understanding the role and molecular mechanism of mitophagy in vivo. In addition, we demonstrated the essential role of the PINK1-Parkin pathway in mitophagy induction in response to mitochondrial dysfunction.-Kim, Y. Y., Um, J.-H., Yoon, J.-H., Kim, H., Lee, D.-Y., Lee, Y. J., Jee, H. J., Kim, Y. M., Jang, J. S., Jang, Y.-G., Chung, J., Park, H. T., Finkel, T., Koh, H., Yun, J. Assessment of mitophagy in mt-Keima Drosophila revealed an essential role of the PINK1-Parkin pathway in mitophagy induction in vivo.

Akt3 knockdown induces mitochondrial dysfunction in human cancer cells.

Akt/PKB plays a pivotal role in cell proliferation and survival. However, the isotype-specific roles of Akt in mitochondrial function have not been fully addressed. In this study, we explored the role of Akt in mitochondrial function after stable knockdown of the Akt isoforms in EJ human bladder cancer cells. We found that the mitochondrial mass was significantly increased in the Akt1- and Akt3-knockdown cells, and this increase was accompanied by an increase in TFAM and NRF1. Akt2 knockdown did not cause a similar effect. Interestingly, Akt3 knockdown also led to severe structural defects in the mitochondria, an increase in doxorubicin-induced senescence, and impairment of cell proliferation in galactose medium. Consistent with these observations, the mitochondrial oxygen consumption rate was significantly reduced in the Akt3-knockdown cells. An Akt3 deficiency-induced decrease in mitochondrial respiration was also observed in A549 lung cancer cells. Collectively, these results suggest that the Akt isoforms play distinct roles in mitochondrial function and that Akt3 is critical for proper mitochondrial respiration in human cancer cells.

Hypnotic effect of AR-001 through adenosine A1 receptor.

Insomnia is one of the most common sleep disorders, affecting 10-15% of the global population. Because classical remedies used to treat insomnia have various side effects, new therapeutics for insomnia are attracting attention. In the present study, we found that N2-Ethyl-N4-(furan-2-ylmethyl) quinazoline-2,4-diamine (AR-001) has adenosine A1 receptor agonistic activity and exhibits hypnotic efficacy by decreasing sleep onset latency and increasing total sleep time in a pentobarbital-induced sleep model. This hypnotic effect of AR-001 was significantly inhibited by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). As a result of immunohistochemistry, AR-001 was shown to increase neural activity in the sleep-promoting region, ventrolateral preoptic nucleus (VLPO), and decrease neural activity in the wake-promoting region, basal forebrain (BF), and lateral hypothalamus (LH), and that these effects of AR-001 were significantly inhibited by DPCPX treatment. In addition, AR-001 increased adenosine A1 receptor mRNA levels in the hypothalamus. In conclusion, this study suggests that AR-001 has a hypnotic effect, at least partially, through adenosine A1 receptor and may have therapeutic potential for insomnia.

p21(WAF¹/C¹P¹) deficiency induces mitochondrial dysfunction in HCT116 colon cancer cells.

p21(WAF1/CIP1) is a critical regulator of cell cycle progression. However, the role of p21 in mitochondrial function remains poorly understood. In this study, we examined the effect of p21 deficiency on mitochondrial function in HCT116 human colon cancer cells. We found that there was a significant increase in the mitochondrial mass of p21(-/-) HCT116 cells, as measured by 10-N-nonyl-acridine orange staining, as well as an increase in the mitochondrial DNA content. In contrast, p53(-/-) cells had a mitochondrial mass comparable to that of wild-type HCT116 cells. In addition, the expression levels of the mitochondrial biogenesis regulators PGC-1α and TFAM and AMPK activity were also elevated in p21(-/-) cells, indicating that p21 deficiency induces the rate of mitochondrial biogenesis through the AMPK-PGC-1α axis. However, the increase in mitochondrial biogenesis in p21(-/-) cells did not accompany an increase in the cellular steady-state level of ATP. Furthermore, p21(-/-) cells exhibited significant proliferation impairment in galactose medium, suggesting that p21 deficiency induces a defect in the mitochondrial respiratory chain in HCT116 cells. Taken together, our results suggest that the loss of p21 results in an aberrant increase in the mitochondrial mass and in mitochondrial dysfunction in HCT116 cells, indicating that p21 is required to maintain proper mitochondrial mass and respiratory function.

Sex Differences in the Preventive Effect of Cardiovascular and Metabolic Therapeutics on Dementia.

Dementia is a clinical syndrome characterized by progressive impairment of cognitive and functional abilities. As currently applied treatments for dementia can only delay the progression of dementia and cannot fundamentally cure it, much attention is being paid to reducing its incidence by preventing the associated risk factors. Cardiovascular and metabolic diseases are well-known risk factors for dementia, and many studies have attempted to prevent dementia by treating these risk factors. Growing evidence suggests that sex-based factors may play an important role in the pathogenesis of dementia. Therefore, a deeper understanding of the differences in the effects of drugs based on sex may help improve their effectiveness. In this study, we reviewed sex differences in the impact of therapeutics targeting risk factors for dementia, such as cardiovascular and metabolic diseases, to prevent the incidence and/or progression of dementia.

Glucose deprivation is associated with Chk1 degradation through the ubiquitin-proteasome pathway and effective checkpoint response to replication blocks.

Chk1 plays a key role in the DNA replication checkpoint and in preserving genomic integrity. Previous studies have shown that reduced Chk1 function leads to defects in the checkpoint response and is closely associated with tumorigenesis. Here, we report that glucose deprivation caused the degradation of Chk1 protein without perturbing cell cycle progression. The induction of Chk1 degradation in response to glucose deprivation was observed in various cancer cell lines and in normal human fibroblasts. Therefore, it appears to be a universal phenomenon in mammalian cells. A specific proteasome inhibitor blocked glucose deprivation-induced Chk1 degradation. Ubiquitination of Chk1 was detected, indicating that the proteasome-ubiquitin pathway mediates Chk1 degradation upon glucose deprivation. Mechanistic studies have demonstrated that ATR-dependent phosphorylation of Chk1 at the Ser317 and Ser345 sites is not required, suggesting that the molecular mechanism for Chk1 degradation upon glucose deprivation is distinct from genotoxic stress-induced degradation. Under conditions of glucose deprivation, the cells manifested a defective checkpoint response to replication stress, camptothecin or hydroxyurea. The forced expression of Myc-Chk1 partially rescued the defective response to the replication block upon glucose deprivation. Taken together, our results indicate that glucose deprivation induces ubiquitin-mediated Chk1 degradation and defective checkpoint responses, implying its potential role in genomic instability and tumor development.

Melatonin suppresses doxorubicin-induced premature senescence of A549 lung cancer cells by ameliorating mitochondrial dysfunction.

Melatonin is an indolamine that is synthesized in the pineal gland and shows a wide range of physiological functions. Although the anti-aging properties of melatonin have been reported in a senescence-accelerated mouse model, whether melatonin modulates cellular senescence has not been determined. In this study, we examined the effect of melatonin on anticancer drug-induced cellular premature senescence. We found that the doxorubicin (DOX)-induced senescence of A549 human lung cancer cells and IMR90 normal lung cells was substantially inhibited by cotreatment with melatonin in a dose-dependent manner. Mechanistically, the DOX-induced G2/M phase cell cycle arrest and the decrease in cyclinB and cdc2 expression were not affected by melatonin. However, the DOX-induced increase in intracellular levels of ROS, which is necessary for premature senescence, was completely abolished upon melatonin cotreatment. In addition, the reduction in mitochondrial membrane potential that occurs upon DOX treatment was inhibited by melatonin. An aberrant increase in mitochondrial respiration was also significantly suppressed by melatonin, indicating that melatonin ameliorates the mitochondrial dysfunction induced by DOX treatment. The treatment of A549 cells with luzindole, a potent inhibitor of melatonin receptors, failed to prevent the effects of melatonin treatment on mitochondrial functions and premature senescence in cells also treated with DOX; this suggests that melatonin suppresses DOX-induced senescence in a melatonin receptor-independent manner. Together, these results reveal that melatonin has an inhibitory effect of melatonin on premature senescence at the cellular level and that melatonin protects A549 cells from DOX-induced senescence. Thus, melatonin might have the therapeutic potential to prevent the side effects of anticancer drug therapy.

Luteolin-7-O-Glucuronide Improves Depression-like and Stress Coping Behaviors in Sleep Deprivation Stress Model by Activation of the BDNF Signaling.

Stress exposure is a major risk factor for mental disorders such as depression. Because of the limitations of classical antidepressants such as side effects, low efficacy, and difficulty in long-term use, new natural medicines and bioactive molecules from plants with greater safety and efficacy have recently attracted attention. Luteolin-7-O-glucuronide (L7Gn), a bioactive molecule present in Perilla frutescens, is known to alleviate severe inflammatory responses and oxidative stress in macrophages. However, its antistress and antidepressant effects have not been elucidated. The present study aims to explore the antidepressant the effect of L7Gn on stress-induced behaviors and the underlying mechanism in a mouse sleep deprivation (SD) model. L7Gn treatment improved depression-like and stress coping behaviors induced by SD stress, as confirmed by the tail suspension test and forced swimming test. Furthermore, L7Gn treatment reduced the blood corticosterone and hippocampal proinflammatory cytokine levels which were increased by SD stress, and L7Gn also increased the mRNA and protein levels of hippocampal brain-derived neurotrophic factor (BDNF) which were reduced by SD stress. Additionally, treatment with L7Gn resulted in increases in the phosphorylation of tropomyosin-related kinase B (TrkB), extracellular signal-regulated kinase (ERK), and cAMP response element-binding protein (CREB), which are downstream molecules of BDNF signaling. These findings suggest that L7Gn have therapeutic potential for SD-induced stress, via activating the BDNF signaling.

Nek6 suppresses the premature senescence of human cancer cells induced by camptothecin and doxorubicin treatment.

Cellular senescence plays an important role in tumor suppression. The mitotic kinase Nek6 has recently been shown to be overexpressed in various cancers and has been implicated in tumorigenesis. Previously, we reported that the down-regulation of Nek6 expression was required for p53-induced senescence. In this study, we examined the effect of Nek6 overexpression on the premature senescence of cancer cells induced by the anticancer drugs camptothecin (CPT) and doxorubicin (DOX). We found that CPT- and DOX-induced morphology changes and increases in senescence-associated ß-galactosidase staining were significantly inhibited in EJ human bladder cancer cells and H1299 human lung cancer cells overexpressing HA-Nek6. DOX-induced G2/M cell cycle arrest and the reduction in cyclin B and cdc2 levels after DOX treatment were significantly reduced by Nek6 overexpression. In addition, an increase in the intracellular levels of ROS in response to DOX was also inhibited in cells overexpressing Nek6. These results suggest that the increased expression of Nek6 renders cancer cells resistant to premature senescence, and targeting Nek6 could be an efficient strategy for cancer treatment.

DNA damage induces down-regulation of PEPCK and G6P gene expression through degradation of PGC-1alpha.

Hepatic gluconeogenesis plays a crucial role in glucose homeostasis. Although it is well established that various cellular processes are modulated by DNA damage, whether the DNA damage signaling pathway regulates gluconeogenesis has not yet been studied. In this study, we found that mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P), key enzymes for gluconeogenesis, were dramatically decreased upon IR- and UV-irradiation. PEPCK and G6P promoter activities were also suppressed by IR- and UV-irradiation, suggesting that PEPCK and G6P gene transcription are down-regulated upon DNA damage. We also found that the protein level of PGC-1α, which is a critical transcription factor for PEPCK gene expression, is decreased upon UV-irradiation. The decreased PGC-1α protein level was abolished by MG132, a potent proteasome inhibitor, suggesting that PGC-1α is degraded through the ubiquitin-proteasome pathway upon UV-irradiation. These results reveal a novel link between glucose metabolism and the DNA damage signaling pathway and suggest a possible role for PEPCK and G6P in the DNA damage response.

Aß1-40-Induced Platelet Adhesion Is Ameliorated by Rosmarinic Acid through Inhibition of NADPH Oxidase/PKC-δ/Integrin αIIbß3 Signaling.

In platelets, oxidative stress reportedly increases platelet adhesion to vessels, thus promoting the vascular pathology of various neurodegenerative diseases, including Alzheimer's disease (AD). Recently, it has been shown that ß-amyloid (Aß) can increase oxidative stress in platelets; however, the underlying mechanism remains elusive. In the present study, we aimed to elucidate the signaling pathway of platelet adhesion induced by Aß1-40, the major form of circulating Aß, through Western blotting, immunofluorescence confocal microscopy, and fluorescence-activated cell sorting analysis. Additionally, we examined whether rosmarinic acid (RA), a natural polyphenol antioxidant, can modulate these processes. Our results show that Aß1-40-induced platelet adhesion is mediated through NADPH oxidase/ROS/PKC-δ/integrin αIIbß3 signaling, and these signaling pathways are significantly inhibited by RA. Collectively, these results suggest that RA may have beneficial effects on platelet-associated vascular pathology in AD.

Effect of Caffeine Consumption on the Risk for Neurological and Psychiatric Disorders: Sex Differences in Human.

Caffeine occurs naturally in various foods, such as coffee, tea, and cocoa, and it has been used safely as a mild stimulant for a long time. However, excessive caffeine consumption (1~1.5 g/day) can cause caffeine poisoning (caffeinism), which includes symptoms such as anxiety, agitation, insomnia, and gastrointestinal disorders. Recently, there has been increasing interest in the effect of caffeine consumption as a protective factor or risk factor for neurological and psychiatric disorders. Currently, the importance of personalized medicine is being emphasized, and research on sex/gender differences needs to be conducted. Our review focuses on the effect of caffeine consumption on several neurological and psychiatric disorders with respect to sex differences to provide a better understanding of caffeine use as a risk or protective factor for those disorders. The findings may help establish new strategies for developing sex-specific caffeine therapies.

Impact of Sleep Disorder as a Risk Factor for Dementia in Men and Women.

Sleep is an essential physiological process, especially for proper brain function through the formation of new pathways and processing information and cognition. Therefore, when sleep is insufficient, this can result in pathophysiologic conditions. Sleep deficiency is a risk factor for various conditions, including dementia, diabetes, and obesity. Recent studies have shown that there are differences in the prevalence of sleep disorders between genders. Insomnia, the most common type of sleep disorder, has been reported to have a higher incidence in females than in males. However, sex/gender differences in other sleep disorder subtypes are not thoroughly understood. Currently, increasing evidence suggests that gender issues should be considered important when prescribing medicine. Therefore, an investigation of the gender-dependent differences in sleep disorders is required. In this review, we first describe sex/gender differences not only in the prevalence of sleep disorders by category but in the efficacy of sleep medications. In addition, we summarize sex/gender differences in the impact of sleep disorders on incident dementia. This may help understand gender-dependent pathogenesis of sleep disorders and develop therapeutic strategies in men and women.

UV light induces premature senescence in Akt1-null mouse embryonic fibroblasts by increasing intracellular levels of ROS.

Akt/PKB plays a pivotal role in cell survival and proliferation. Previously, we reported that UV-irradiation induces extensive cell death in Akt2(-/-) mouse embryonic fibroblasts (MEFs) while Akt1(-/-) MEFs show cell cycle arrest. Here, we find that Akt1(-/-) MEFs exhibit phenotypic changes characteristics of senescence upon UV-irradiation. An enlarged and flattened morphology, a reduced cell proliferation and an increased senescence-associated beta-galactosidase (SA beta-gal) staining indicate that Akt1(-/-) MEFs undergo premature senescence after UV-irradiation. Restoring Akt1 expression in Akt1(-/-) MEFs suppressed SA beta-gal activity, indicating that UV-induced senescence is due to the absence of Akt1 function. Notably, levels of ROS were rapidly increased upon UV-irradiation and the ROS scavenger NAC inhibits UV-induced senescence of Akt1(-/-) MEFs, suggesting that UV light induces premature senescence in Akt1(-/-) MEFs by modulating intracellular levels of ROS. In conjunction with our previous work, this indicates that different isoforms of Akt have distinct function in response to UV-irradiation.

Cooperation between p21 and Akt is required for p53-dependent cellular senescence.

Cellular senescence has been implicated in normal aging, tissue homeostasis, and tumor suppression. Although p53 has been shown to be a central mediator of cellular senescence, the signaling pathway by which it induces senescence remains incompletely understood. In this study, we have shown that both Akt and p21 are required to induce cellular senescence in response to p53 expression. In a p53-induced senescence model, we found that Akt activation was essential for inducing a cellular senescence phenotype. Surprisingly, Akt inhibition did not abolish p53-induced cell cycle arrest, but it suppressed the increase in intracellular reactive oxygen species (ROS) levels. The results of the cell cycle and morphological analysis suggest that p53 induced quiescence, not senescence, following Akt inhibition. Conversely, the inhibition of p21 induction abolished cell cycle arrest but did not affect the p53-induced increase in ROS levels. Additionally, p21 and Akt separately controlled cell cycle arrest and ROS levels, respectively, during H-Ras-induced senescence in human normal fibroblasts. The mechanistic analysis revealed that Akt increased ROS levels through NOX4 induction, and increased Akt-dependent NF-κB binding to the NOX4 promoter is responsible for NOX4 induction upon p53 expression. We further showed that Akt activation upon p53 expression is mediated by mammalian target of rapamycin complex 2. In addition, p53-mediated IL6 and IL8 induction was abrogated by Akt inhibition, suggesting that Akt activation is also required for the senescence-associated secretory phenotype. Collectively, these results suggest that p53 simultaneously controls multiple pathways to induce cellular senescence through p21 and Akt.

The inhibition of Nek6 function sensitizes human cancer cells to premature senescence upon serum reduction or anticancer drug treatment.

The induction of premature senescence in cancer cells was proposed as an effective cancer treatment strategy. In this paper, we show that the inhibition of Nek6 expression by Nek6 siRNA-mediated knockdown or the overexpression of a dominant negative form of Nek6 (Nek6KM) induced premature senescence as well as cell death under reduced serum conditions in multiple cancer cell lines, including both p53 wild-type and p53 mutant/null backgrounds. Moreover, cancer cells expressing Nek6KM exhibited significantly increased premature senescence upon treatment with the anticancer drugs doxorubicin (DOX) and camptothecin (CPT). Significantly, the overexpression of Nek6KM also inhibited tumor growth and promoted premature senescence in vivo in a xenograft mouse model. Taken together, our results further confirm that Nek6 plays an important role in the premature senescence of cancer cells, suggesting that Nek6 may be a potential therapeutic target for human cancers.

Loss of Med1/TRAP220 promotes the invasion and metastasis of human non-small-cell lung cancer cells by modulating the expression of metastasis-related genes.

Med1/TRAP220 is an essential component of the TRAP/Mediator complex. In this study, we present a novel function of Med1 in human non-small-cell lung cancer (NSCLC) progression. We found that the loss of Med1 expression was strongly associated with increased rates of invasion and metastasis in NSCLC patients. Consistent with lung cancer patient data, the knockdown of Med1 in NSCLC cell lines led to an increase in cell migration and invasion. Med1-depleted cells displayed an increase in metastasis in a xenograft tumor model and in an in vivo metastasis assay. Moreover, a microarray analysis revealed that the mRNA levels of the metastasis-related genes uPAR, ID2, ID4, PTP4A1, PKP3, TGM2, PLD1, TIMP2, RGS2, and HOXA4 were altered upon Med1 knockdown. Collectively, these results suggest that the loss of Med1 increases the invasive potential of human NSCLC cells by modulating the expression of metastasis-related genes.

Nek6 overexpression antagonizes p53-induced senescence in human cancer cells.

Nek6 is an NIMA-related kinase that plays a critical role in mitotic cell cycle progression. Recent studies have shown that Nek6 is upregulated in various human cancers, but the function of Nek6 in tumorigenesis is largely unknown. Here, we examined the role of Nek6 in cellular senescence. Our data revealed that Nek6 expression is decreased both in both the replicative senescence of human normal fibroblasts and premature senescence induced by p53 expression in EJ human bladder cancer cells and H1299 human lung cancer cells. Interestingly, the enforced expression of Nek6 in EJ and H1299 cells completely suppresses p53-induced senescence, whereas the expression of kinase-dead Nek6 did not affect p53-induced senescence. Mechanistic studies revealed that cell cycle arrest in the G1 and G2/M phases, as well as the reduction of cyclin B and cdc2 protein level upon p53 expression were significantly reduced by Nek6 overexpression. In addition, p53-induced increases in intracellular levels of ROS were also inhibited in cells overexpressing Nek6. These results suggest that the downregulation of Nek6 expression is required for the onset of p53-induced cellular senescence and imply a possible role of Nek6 in tumorigenesis.

Nek6 is involved in G2/M phase cell cycle arrest through DNA damage-induced phosphorylation.

Nek6 is a recently identified NIMA-related kinase that is required for mitotic cell cycle progression. In the present study, we examined the role of Nek6 in the DNA damage response. We found that Nek6 is phosphorylated upon IR and UV irradiation through the DNA damage checkpoint in vivo. Nek6 is also directly phosphorylated by the checkpoint kinases Chk1 and Chk2 in vitro. Notably, Nek6 activation during mitosis is completely abolished by IR and UV irradiation. Moreover, the ectopic expression of Nek6 overrides DNA damage-induced G(2)/M arrest. These results suggest that Nek6 is a novel target of the DNA damage checkpoint and that the inhibition of Nek6 activity is required for proper cell cycle arrest in the G(2)/M phase upon DNA damage.