• Molecules and Cells
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• 제27권1호
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• pp.67-74
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• 2009

Autophagy degrades toxic materials and old organelles, and recycles nutrients in eukaryotic cells. Whereas the studies on autophagy have been reported in other eukaryotic cells, its functioning in plants has not been well elucidated. We analyzed the roles of OsATG10 genes, which are autophagy-related. Two rice ATG10 genes - OsATG10a and OsATG10b - share significant sequence homology (about 75%), and were ubiquitously expressed in all organs examined here. GUS assay indicated that OsATG10b was highly expressed in the mesophyll cells and vascular tissue of younger leaves, but its level of expression decreased in older leaves. We identified T-DNA insertional mutants in that gene. Those osatg10b mutants were sensitive to treatments with high salt and methyl viologen (MV). Monodansylcadaverine-staining experiments showed that the number of autophagosomes was significantly decreased in the mutants compared with the WT. Furthermore, the amount of oxidized proteins increased in MV-treated mutant seedlings. These results demonstrate that OsATG10b plays an important role in the survival of rice cells against oxidative stresses.

• 대한약침학회지
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• 제22권4호
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• pp.269-278
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• 2019

Objectives: Naesohwangryeon-tang (NHT) is a type of traditional herbal formula, however, little is known about its antitumor activity. In this study, the antitumor properties of NHT was evaluated in human lung adenocarcinoma cells. Methods: To check the inhibitory effect of NHT, MTT assay was performed. Cell cycle analysis and detection of ROS production were conducted by flow cytometry. To evaluate the signaling pathway, Western blotting was conducted. Results: Our results showed that the decrease of cell proliferation by NHT stimulation occurred more significantly in A549 cells than in NCI-H460 cells. In addition, NHT-induced apoptosis was associated with the activation of caspases and production of reactive oxygen species (ROS). NHT-induced apoptosis was attenuated after pretreatments with z-VAD-fmk or N-acetylcysteine, suggesting that NHT-induced apoptosis was caspaseand ROS-dependent. Interestingly, NHT treatment led to the development of autophagic vesicular organelles and upregulation of several autophagy-related genes. The pretreatment of bafilomycin A1 decreased apoptosis slightly but increased cell viability in the presence of NHT. Conclusion: These findings indicated that NHT induces both apoptosis and cell-protective autophagy in human lung cancer cells. This data suggests that NHT might be a novel herbal drug for lung cancer.

• The Korean Journal of Physiology and Pharmacology
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• 제20권3호
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• pp.237-243
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• 2016

Oleanolic acid (OA) has a wide variety of bioactivities such as hepatoprotective, anti-inflammatory and anti-cancer activity and is used for medicinal purposes in many Asian countries. In the present study, the effect of OA on induction of autophagy in human hepatocellular carcinoma HepG2 and SMC7721 cells and the related mechanisms were investigated. MTT assay showed that OA significantly inhibited HepG2 and SMC7721 cells growth. OA treatment enhanced formation of autophagic vacuoles as revealed by monodansylcadaverine (MDC) staining. At the same time, increasing punctuate distribution of microtubule-associated protein 1 light chain 3 (LC3) and an increasing ratio of LC3-II to LC3-I were also triggered by OA incubation. In addition, OA-induced cell death was significantly inhibited by autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) pretreatment. And we found out that OA can suppress the PI3K/Akt1/mTOR signaling pathway. Furthermore, our data suggested that OA-triggered autophagy was ROS-dependent as demonstrated by elevated cellular ROS levels by OA treatment. When ROS was cleared by N-acetylcysteine (NAC), OA-induced LC3-II convertsion and cell death were all reversed. Taken together, our results suggest that OA exerts anticancer effect via autophagic cell death in hepatocellular carcinoma.

• Journal of Periodontal and Implant Science
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• 제50권5호
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• pp.291-302
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• 2020

Purpose: The objective of this study was to investigate whether phelligridin D could reduce glucose-induced oxidative stress, attenuate the resulting inflammatory response, and restore the function of human periodontal ligament cells (HPDLCs). Methods: Primary HPDLCs were isolated from healthy human teeth and cultured. To investigate the effect of phelligridin D on glucose-induced oxidative stress, HPDLCs were treated with phelligridin D, various concentrations of glucose, and glucose oxidase. Glucose-induced oxidative stress, inflammatory molecules, osteoblast differentiation, and mineralization of the HPDLCs were measured by hydrogen peroxide (H₂O₂) generation, cellular viability, alkaline phosphatase (ALP) activity, alizarin red staining, and western blot analyses. Results: Glucose-induced oxidative stress led to increased production of H₂O₂, with negative impacts on cellular viability, ALP activity, and calcium deposition in HPDLCs. Furthermore, HPDLCs under glucose-induced oxidative stress showed induction of inflammatory molecules (intercellular adhesion molecule-1, vascular cell adhesion protein-1, tumor necrosis factor-alpha, interleukin-1-beta) and disturbances of osteogenic differentiation (bone morphogenetic protein-2, and -7, runt-related transcription factor-2), cementogenesis (cementum protein-1), and autophagy-related molecules (autophagy related 5, light chain 3 I/II, beclin-1). Phelligridin D restored all these molecules and maintained the function of HPDLCs even under glucose-induced oxidative stress. Conclusions: This study suggests that phelligridin D reduces the inflammation that results from glucose-induced oxidative stress and restores the function of HPDLCs (e.g., osteoblast differentiation) by upregulating autophagy.

• International Journal of Oral Biology
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• 제39권2호
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• pp.65-73
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• 2014

Chios Gum Mastic (CGM) is a natural resin extracted from the leaves of Pistacia lentiscus, a plant endemic to the Greek island of Chios. It has been used by traditional healers, and it has antibacterial, antifungal properties, and therapeutic benefits for the skin. The CGM reduces the formation of dental plaque and bacterial growth in oral saliva, and recent studies have demonstrated the role of antioxidant activity of CGM. Although CGM has been widely investigated, its protective effect against oxidative-damage to keratinocytes, as well as the relationship between CGM and autophagy, has not been investigated. The aim of this study was to assess the protective effect of CGM against $H_2O_2$-induced oxidative stress and to evaluate the autophagic features induced by CGM in human keratinocytes. The pretreatment with CGM significantly reduced apoptosis in $H_2O_2$-exposed HaCaT cells. It promoted the degradation of caspase-3, caspase-8, and caspase-9; and it induced the formation of the processed PARP. The treatment with CGM caused an increase in vesicle formation compared to control group. The level of p62 was reduced and the conversion of LC3-I to LC3-II was increased in CGM treated HaCaT cells. Also, the treatment with CGM increased cleavage of ATG5-ATG12 complex. In summary, CGM helps the cells to survive under stressful conditions by preventing apoptosis and enhancing autophagy. Besides, the present investigation provides evidence to support the antioxidant potential of CGM in vitro and opens up a new horizon for future experiments.

• 한국발생생물학회지:발생과생식
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• 제25권4호
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• pp.213-223
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• 2021

Controlled ovarian hyperstimulation (COH) is routinely used in the in vitro fertilization and embryo transfer (IVF-ET) cycles to increase the number of retrieved mature oocytes. However, the relationship between repeated COH and ovarian function is still controversial. Therefore, we investigated whether repeated ovarian stimulation affects ovarian aging and function, including follicular development, autophagy, and apoptosis in follicles. Ovarian hyperstimulation in mice was induced by intraperitoneal injection with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Mice subjected to ovarian stimulation once were used as a control group and 10 times as an experimental group. Repeated injections with PMSG and hCG significantly reduced the number of primary follicles compared to a single injection. The number of secondary and antral follicles increased slightly, while the number of corpus luteum increased significantly with repeated injections. On the other hand, repeated injections did not affect apoptosis in follicles associated with follicular atresia. The expression of autophagy-related genes Atg5, Atg12, LC3B, and Beclin1, cell proliferation-related genes mTOR, apoptosis-related genes Fas, and FasL was not significantly different between the two groups. In addition, the expression of the aging-related genes Dnmt1, Dnmt3a, and AMH were also not significantly different. In this study, we demonstrated that repeated ovarian stimulation in mice affects follicular development, but not autophagy, apoptosis, aging in ovary. These results suggest that repetition of COH in the IVF-ET cycle may not result in ovarian aging, such as a decrease in ovarian reserve in adult women.

• Biomolecules & Therapeutics
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• 제30권1호
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• pp.80-89
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• 2022

The targeting of DNA methylation in cancer using DNA hypomethylating drugs has been well known to sensitize cancer cells to chemotherapy and immunotherapy by affecting multiple pathways. Herein, we investigated the combinational effects of DNA hypomethylating drugs and ionizing radiation (IR) in human sarcoma cell lines both in vitro and in vivo. Clonogenic assays were performed to determine the radiosensitizing properties of two DNA hypomethylating drugs on sarcoma cell lines we tested in this study with multiple doses of IR. We analyzed the effects of 5-aza-dC or SGI-110, as DNA hypomethylating drugs, in combination with IR in vitro on the proliferation, apoptosis, caspase-3/7 activity, migration/invasion, and Western blotting using apoptosis- or autophagy-related factors. To confirm the combined effect of DNA hypomethylating drugs and IR in our in vitro experiment, we generated the sarcoma cells in nude mouse xenograft models. Here, we found that the combination of DNA hypomethylating drugs and IR improved anticancer effects by inhibiting cell proliferation and by promoting synergistic cell death that is associated with both apoptosis and autophagy in vitro and in vivo. Our data demonstrated that the combination effects of DNA hypomethylating drugs with radiation exhibited greater cellular effects than the use of a single agent treatment, thus suggesting that the combination of DNA hypomethylating drugs and radiation may become a new radiotherapy to improve therapeutic efficacy for cancer treatment.

• Molecules and Cells
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• 제45권6호
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• pp.403-412
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• 2022

Hypoxia leads to significant cellular stress that has diverse pathological consequences such as cardiovascular diseases and cancers. MicroRNAs (miRNAs) are one of regulators of the adaptive pathway in hypoxia. We identified a hypoxia-induced miRNA, miR-34c, that was significantly upregulated in hypoxic human umbilical cord vein endothelial cells (HUVECs) and in murine blood vessels on day 3 of hindlimb ischemia (HLI). miR-34c directly inhibited BCL2 expression, acting as a toggle switch between apoptosis and autophagy in vitro and in vivo. BCL2 repression by miR-34c activated autophagy, which was evaluated by the expression of LC3-II. Overexpression of miR-34c inhibited apoptosis in HUVEC as well as in a murine model of HLI, and increased cell viability in HUVEC. Importantly, the number of viable cells in the blood vessels following HLI was increased by miR-34c overexpression. Collectively, our findings show that miR-34c plays a protective role in hypoxia, suggesting a novel therapeutic target for hypoxic and ischemic diseases in the blood vessels.

• The Korean Journal of Physiology and Pharmacology
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• 제27권1호
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• pp.1-8
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• 2023

Hypothyroidism alone can lead to myocardial fibrosis and result in heart failure, but traditional hormone replacement therapy does not improve the fibrotic situation. Hydrogen sulfide (H₂S), a new gas signaling molecule, possesses anti-inflammatory, antioxidant, and anti-fibrotic capabilities. Whether H₂S could improve hypothyroidism-induced myocardial fibrosis are not yet studied. In our study, H₂S could decrease collagen deposition in the myocardial tissue of rats caused by hypothyroidism. Furthermore, in hypothyroidism-induced rats, we found that H₂S could enhance cystathionine-gamma-lyase (CSE), not cystathionine β-synthase (CBS), protein expressions. Finally, we noticed that H₂S could elevate autophagy levels and inhibit the transforming growth factor-β1 (TGF-β1) signal transduction pathway. In conclusion, our experiments not only suggest that H₂S could alleviate hypothyroidism-induced myocardial fibrosis by activating autophagy and suppressing TGF-β1/SMAD family member 2 (Smad 2) signal transduction pathway, but also show that it can be used as a complementary treatment to conventional hormone therapy.

• Nutrition Research and Practice
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• 제18권2호
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• pp.210-222
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• 2024

BACKGROUND/OBJECTIVES: Chronic renal failure (CRF) is a complex pathological condition that lacks a cure. Certain Chinese medicines, such as melittin, a major component in bee venom, have shown efficacy in treating CRF patients. On the other hand, the mechanisms underlying the therapeutic effects of melittin are unclear. MATERIALS/METHODS: A 5/6 nephrectomy model (5/6 Nx) of renal failure was established on rats for in vivo assays, and mouse podocyte clone 5 (MPC5) mouse podocyte cells were treated with angiotensin II (AngII) to establish an in vitro podocyte damage model. The 24-h urine protein, serum creatinine, and blood urea nitrogen levels were evaluated after one, 2, and 4 weeks. Hematoxylin and eosin staining, Masson staining, and periodic acid-Schiff staining were used to examine the pathological changes in kidney tissues. A cell counting kit 8 assay was used to assess the cell viability. Reverse transcription polymerase chain reaction and Western blot were used to assess the mRNA and protein levels in the cells, respectively. RESULTS: In the rat 5/6 Nx, melittin reduced the 24-h urinary protein excretion and the serum creatinine and blood urea nitrogen levels. Furthermore, the renal pathology was improved in the melittin-treated 5/6 Nx rats. Melittin promoted podocin, nephrin, Beclin 1, and the LC3II/LC3I ratio and inhibited phosphorylated mammalian target of rapamycin (mTOR)/mTOR in 5/6 Nx-induced rats and AngII-induced MPC5 mouse podocyte cells. Moreover, inhibiting autophagy with 3-MA weakened the effects of melittin on podocin, nephrin, and the LC3II/LC3I ratio in podocytes. CONCLUSION: Melittin may offer protection against kidney injury, probably by regulating podocyte autophagy. These results provide the theoretical basis for applying melittin in CRF therapy.