Kyung Seuk Song

Baicalein induces autophagic cell death through AMPK/ULK1 activation and downregulation of mTORC1 complex components in human cancer cells

Baicalein, a flavonoid and aglycon hydrolyzed from baicalin, has anticancer properties in several human carcinomas, but its molecular mechanisms of action remain unclear. Here, we show that baicalein leads to human cancer cell death by inducing autophagy rather than apoptosis, because cell death induced by baicalein was completely reversed by suppressing the expression levels of key molecules in autophagy such as Beclin 1, vacuolar protein sorting 34 (Vps34), autophagy‐related (Atg)5 and Atg7, but not by pan‐caspase inhibitor. Our data revealed that baicalein significantly increased the number of green fluorescence protein–cytosol‐associated protein light chain 3 (GFP–LC3)‐containing puncta and LC3B‐II expression levels, which were further enhanced by chloroquine treatment. Furthermore, a luciferase‐based reporter assay showed that the ratio of RLuc–LC3wt/RLuc–LC3G120A was greatly reduced. The data suggested that baicalein induced not only autophagosome formation, but also autophagic flux. Experiments using short interfering RNAs and pharmacological inhibitors revealed that Beclin 1, Vps34, Atg5, Atg7 and UNC‐51 (Caenorhabditis elegans)‐like kinase 1 (ULK1) play pivotal roles in mediating baicalein‐induced autophagy. Moreover, baicalein activated AMP‐activated protein kinase (AMPK)α, leading to ULK1 activation through phosphorylation at Ser555, whereas both protein and mRNA levels of mammalian target of rapamycin (mTOR) and Raptor, upstream inhibitors of ULK1 and autophagy, were markedly downregulated by baicalein. Our data suggest that the anticancer effects of baicalein are mainly due to autophagic cell death through activation of the AMPK/ULK1 pathway and inhibition of mTOR/Raptor complex 1 expression. These results provide new mechanistic insights into the anticancer functions of autophagy inducers, such as baicalein, which may be used as potential therapeutics for cancer treatment.

Protective effects of alpha-pinene in mice with cerulein-induced acute pancreatitis.

AIMS Acute pancreatitis (AP) is a complicated inflammatory disease that has an unknown underlying pathogenesis. Because alpha-pinene can modulate inflammation, we examined whether alpha-pinene plays a role in AP. MAIN METHODS Alpha-pinene was administered intraperitoneally 1h prior to the first injection of cerulein. Once AP developed, cerulein, a stable cholecystokinin analog, was injected hourly over a 6-h period. Blood samples were taken 6h later to determine serum amylase and lipase levels. The pancreas and lungs were rapidly removed for morphological examination, myeloperoxidase assay, and real-time reverse transcription polymerase chain reaction. We also isolated the pancreatic acinar cells using a collagenase solution. Cell viability, and cytokine productions were measured in pancreatic acini. KEY FINDINGS Intraperitoneal administration of alpha-pinene reduced the pancreatic weight (PW) to body weight (BW) ratio and the serum levels of amylase and lipase. Alpha-pinene treatment also reduced histological damage and myeloperoxidase activity in the pancreas and lungs. Furthermore, alpha-pinene pretreatment reduced the production of pancreatic tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 during cerulein-induced AP. In vitro, alpha-pinene inhibited cerulein-induced cell death and cytokine production in isolated cerulein-treated pancreatic acinar cells. SIGNIFICANCE These findings suggest that alpha-pinene has an anti-inflammatory effect during cerulein-induced AP.

In vivo genotoxicity evaluation of lung cells from Fischer 344 rats following 28 days of inhalation exposure to MWCNTs, plus 28 days and 90 days post-exposure.

Abstract Despite their useful physico-chemical properties, carbon nanotubes (CNTs) continue to cause concern over occupational and human health due to their structural similarity to asbestos. Thus, to evaluate the toxic and genotoxic effect of multi-wall carbon nanotubes (MWCNTs) on lung cells in vivo, eight-week-old rats were divided into four groups (each group = 25 animals), a fresh air control (0 mg/m3), low (0.17 mg/m3), middle (0.49 mg/m3), and high (0.96 mg/m3) dose group, and exposed to MWCNTs via nose-only inhalation 6 h per day, 5 days per week for 28 days. The count median length and geometric standard deviation for the MWCNTs determined by TEM were 330.18 and 1.72 nm, respectively, and the MWCNT diameters ranged from 10 to 15 nm. Lung cells were isolated from five male and five female rats in each group on day 0, day 28 (only from males) and day 90 following the 28-day exposure. The total number of animals used was 15 male and 10 female rats for each concentration group. To determine the genotoxicity of the MWCNTs, a single cell gel electrophoresis assay (Comet assay) was conducted on the rat lung cells. As a result of the exposure, the olive tail moments were found to be significantly higher (p < 0.05) in the male and female rats from all the exposed groups when compared with the fresh air control. In addition, the high-dose exposed male and middle and high-dose exposed female rats retained DNA damage, even 90 days post-exposure (p < 0.05). To investigate the mode of genotoxicity, the intracellular reactive oxygen species (ROS) levels and inflammatory cytokine levels (TNF-α, TGF- β, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-γ) were also measured. For the male rats, the H2O2 levels were significantly higher in the middle (0 days post-exposure) and high- (0 days and 28 days post-exposure) dose groups (p < 0.05). Conversely, the female rats showed no changes in the H2O2 levels. The inflammatory cytokine levels in the bronchoalveolar lavage (BAL) fluid did not show any statistically significant difference. Interestingly, the short-length MWCNTs deposited in the lung cells were persistent at 90 days post-exposure. Thus, exposing lung cells to MWCNTs with a short tube length may induce genotoxicity.

Modulation of Atg5 expression by globular adiponectin contributes to autophagy flux and suppression of ethanol-induced cell death in liver cells

Globular adiponectin (gAcrp) protects liver cells from ethanol-induced apoptosis via induction of autophagy. However, the underlying mechanisms are unknown. The present study aims to investigate the potential role of autophagy-related protein 5 (Atg5), an essential Atg for the elongation of autophagosomes, in suppression of ethanol-induced cytotoxicity by gAcrp. Here, we demonstrated that suppression of Atg5 expression by ethanol was restored by pretreatment with gAcrp both in primary rat hepatocytes and human hepatoma cell line (HepG2). Moreover, ethanol-induced accumulation of p62 (sequestosome1), a marker of autophagic flux, was restored by gAcrp treatment, implying that gAcrp modulates autophagic flux in liver cells. Further, Atg5 silencing prevented p62 degradation by gAcrp, suggesting that Atg5 plays a critical role in induction of autophagic flux by gAcrp. Interestingly, gene silencing of Atg5 by siRNA abrogated restoration of autophagosome formation by gAcrp in ethanol-treated cells. Finally, protection of liver cells by gAcrp from ethanol-induced apoptosis was also significantly attenuated by knocking-down of Atg5 expression, suggesting an important role of Atg5 in autophagy induction and cellular apoptosis modulated by gAcrp. Taken together, our data demonstrated that Atg5 expression, at least in part, is implicated in gAcrp-induced autophagy and subsequent anti-apoptotic effects in ethanol-treated liver cells.

Evaluation of in vitro and in vivo genotoxicity of single-walled carbon nanotubes.

Single-walled carbon nanotubes (SWCNTs) have extensive potential industrial applications due to their unique physical and chemical properties; yet this also increases the chance of human and environment exposure to SWCNTs. Due to the current lack of hazardous effect information on SWNCTs, a standardized genotoxicity battery test was conducted to clarify the genetic toxicity potential of SWCNTs (diameter: 1–1.2 nm, length: ∼20 μm) according to Organization for Economic Cooperation and Development test guidelines 471 (bacterial reverse mutation test), 473 (in vitro chromosome aberration test), and 474 (in vivo micronuclei test) with a good laboratory practice system. The test results showed that the SWCNTs did not induce significant bacterial reverse mutations at 31.3–500 μg/plate in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 or in Escherichia coli strain WP2uvrA, with and without a metabolic activation system. Furthermore, the in vitro chromosome aberration test showed no significant increase in structural or numerical chromosome aberration frequencies at SWCNT dose levels of 12.5–50 μg/ml in the presence and absence of metabolic activation. However, dose-dependent cell growth inhibition was found at all the SWCNT dose levels and statistically significant cytotoxic effects observed at certain concentrations in the presence and absence of metabolic activation. Finally, the SWCNTs did not evoke significant in vivo micronuclei frequencies in the polychromatic erythrocytes of an imprinting control region mice at 25–100 mg/kg. Thus, according to the results of the present study, the SWCNTs were not found to have a genotoxic effect on the in vitro and in vivo test systems.

Globular adiponectin modulates expression of programmed cell death 4 and miR-21 in RAW 264.7 macrophages through the MAPK/NF-κB pathway.

MicroRNA‐21 and programmed cell death 4 (PDCD4), a downstream target of miR‐21, mediate diverse physiological responses. Here we demonstrate that globular adiponectin (gAcrp) modulates expression of miR‐21 and PDCD4 in RAW 264.7 macrophages. These effects were abrogated by inhibitors of ERK1/2, JNK or NF‐κB. Conditioned media collected from gAcrp‐stimulated RAW 264.7 macrophages caused similar effects as direct gAcrp treatment, showing the paracrine effect of gAcrp. These data indicate that gAcrp modulates the miR‐21/PDCD4 axis through the ERK and JNK/NF‐κB pathways in RAW 264.7 macrophages and further suggest that the miR‐21/PDCD4 axis may be a novel target mediating adiponectin‐induced biological responses.

Multiwall Carbon Nanotube-Induced DNA Damage and Cytotoxicity in Male Human Peripheral Blood Lymphocytes

Carbon nanotubes (CNTs) have been introduced recently as a novel carrier system for both small and large therapeutic molecules. Biotin-functionalized single-wall CNTs have been conjugated with the anticancer agent taxoid using a cleavable linker, and multiwall carbon nanotubes (MWCNTs) conjugated with iron nanoparticles have been efficiently loaded with doxorubicin. 1,2 The MWCNTs are effective transporters for biological macromolecules and drugs to target cells and tissues, thereby attracting the attention of the biomedical industry. 3 –7 Administrating MWCNTs for medical application invariably involves intravenous administration and ultimate contact with human peripheral blood lymphocytes (HPBLs), yet toxicological studies on the effect of MWCNTs on HPBLs are lacking. Accordingly, this study evaluated the cytotoxic and genotoxic effects of MWCNTs on healthy male HPBLs. Healthy male HPBLs were treated with MWCNTs at 3 different concentrations (12.5, 25, and 50 μg/mL) for 48 hours. Under these conditions, the MWCNTs induced significant cell growth retardation, DNA damage, and cytotoxicity. The MWCNT-treated HPBLs also exhibited an increased intracellular reactive oxygen species level during the experimental period, which leads to cell damage and death, proliferation inhibition, DNA damage, and an inflammatory response.

Opuntia humifusa ameliorated cerulein-induced acute pancreatitis.

Objective The aim of this study was to evaluate the effects of Opuntia humifusa (OH) on cerulein-induced acute pancreatitis (AP). Methods Acute pancreatitis was induced via intraperitoneal injection of cholecystokinin analog cerulein (50 &mgr;g/kg). In the OH pretreatment group, OH was administered intraperitoneally (100, 250, or 500 mg/kg) 1 hour before first cerulein injection. In the posttreatment group, OH was administered intraperitoneally (500 mg/kg) 1 hour after the first cerulein injection. Furthermore, we isolated the pancreatic acinar cells using collagenase method, then investigated the acinar cell viability, cytokine productions, and the regulating mechanisms. Results The both pretreatment and posttreatment of OH treatment attenuated the severity of AP, as shown by the histology of the pancreas and lung, and inhibited neutrophil infiltration; serum amylase and lipase activities; proinflammatory cytokine expression such as interleukin 1, interleukin 6, and tumor necrosis factor &agr;; and cell death including apoptosis and necrosis. Furthermore, OH inhibited the activation of c-Jun N-terminal kinases. Conclusions These results suggest that OH reduces the severity of AP by inhibiting acinar cell death through c-Jun N-terminal kinases.

Physico-chemical characterization-based safety evaluation of nanocalcium

Nano- and microcalcium provided from the KFDA were compared in terms of physico-chemical properties. Calcium samples were tested using EF-TEM and X-ray diffractometry to check for size/morphology and crystal formation, respectively. Two samples of nano- and microcalcium were selected for further evaluation by FE-SEM, DLS (nano-size, 200-500nm; agglomerate, >5 μm; micro-size, 1.5-30 μm), and electron spin resonance. Both samples were heterogeneous in size, existed as single crystal and aggregated form, and did not generate reactive oxygen species. The specific surface area of nano- and microcalcium measured by N2 Brunauere Emmette Teller method was 12.90±0.27 m(2)/g and 1.12±0.19 m(2)/g, respectively. Inductively coupled plasma optical emission spectrometry analysis revealed the release of 2-3 times more calcium ion from nano- compared to microcalcium at pH 5 and 7. Genotoxicity and acute single-dose and repeated-dose 14-day oral toxicity testing in SD rats performed to evaluate the safety of nanocalcium did not reveal toxicity. However, long-term monitoring will be required for an unequivocal conclusion. A nanocalcium dose of 1 g/kg is recommended as the maximum dose for repeated dose 13-week oral toxicity. Further studies could provide details of toxicity of nanocalcium on the repeated dose 13-week oral toxicity test.

Gene expression profiling of kidneys from Sprague–Dawley rats following 12-week inhalation exposure to silver nanoparticles

Abstract The specific properties of silver nanoparticles (AgNPs), such as antimicrobial activity and electrical conductivity, allow them to be used in many fields. However, their expanding application is also raising health, environmental and safety concerns. Previous in vivo AgNP toxicity studies have indicated a gender-different accumulation of silver in the kidneys, with 2–3 times more silver in female kidneys compared to male kidneys. However, no other studies have further addressed this gender difference. Accordingly, the current study investigated the gender-dependent effect of AgNPs on the kidney gene level based on toxicogenomic studies of kidneys obtained from rats exposed to AgNPs via inhalation for 12 weeks. When compared with the fresh air control, the silver nanoparticle-exposed kidneys included 104 genes with a more than 1.3-fold expression increase. For the male rat kidneys exposed to a low or high dose of silver nanoparticles, 96 genes exhibited expression changes, where six genes changed with both the low and high dose; four increased and two decreased. Meanwhile, for the female rat kidneys exposed to a low or high dose of silver nanoparticles, 66 genes exhibited expression changes, where 11 genes changed with both the low and high dose; nine increased and two decreased. Gender-dependent gene expression changes of more than 2-fold were linked to 163 genes, with 79 genes in the male kidneys and 84 genes in the female kidneys, plus gender-dependent gene expression changes of more than 5-fold were linked to 21 genes. However, no genes involved in apoptosis or the cell cycle were activated by the 12-week silver nanoparticle inhalation exposure. Overall, the male rat kidneys showed a higher expression of genes involved in xenobiotic metabolism, while the female rat kidneys showed a higher expression of genes involved in extracellular signaling.