• Journal of Life Science
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• v.18 no.8
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• pp.1059-1065
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• 2008

KIF21A is a member of the Kinesin superfamily proteins (KIFs), which are microtubule-dependent molecular motors, anterograde axonal transporters of cargoes. Recently, congenital fibrosis of the extraocular muscles 1 (CFEOM1) has been shown to result from a small number of recurrent heterozygous missense mutations of KIF21A. CFEOM1 results from the inability of mutated KIF21A to successfully deliver cargoes to the development of the occulo-motor neuron or neuromuscular junction. Here, we used an yeast two-hybrid system to identify a protein that interacts with the WD-40 repeat domain of KIF21A and found a specific interaction with Purkinje cell protein-2 (Pcp-2), a small protein also known as L7. Pcp-2 protein bound to the WD-40 domain of KIF21A and KIF21B but not to other KIFs in yeast two-hybrid assays. In addition, this specific interaction was also observed in the glutathione S-transferase pull-down assay. An antibody to Pcp-2 specifically co-immunoprecipitated KIF21A associated with Pcp-2 from mouse brain extracts. These results suggest that Pcp-2 may be involved in the KIF21A-mediated transport as a KIF21A adaptor protein.

• Journal of Korean Neurosurgical Society
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• v.30 no.8
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• pp.963-969
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• 2001

Objectives : Cord blood stem cells have been widely used as donor cells for bone marrow transplantation recently. These cells can give rise to a variety of hematopoietic lineages to repopulate the blood. Recent observations reveal that some bone marrow cells and bone marrow stromal cells(MSCs) can grow to become either neurons or glial cells. It is, however, unclear whether or not there exists stems cells which can differentiate into neurons in the blood during the early stages of postnatal life. Methods : Human cord blood stem cells were prepared from human placenta after full term delivery. To induce neuronal differentiation of stem cells, ${\beta}$-mercaptoethanol was treated. To confirm the neuro-glial characteristics of differentiated stem cells, immunocytochemical stain for NeuN, neurofilament, glial fibrillary acidic protein(GFAP), microtubule associated protein2(MAP2) was performed. RT-PCR was performed for detecting nestin mRNA and MAP2 mRNA. Results : We showed in this experiment that neuro-glial markers(NeuN, neurofilament, MAP2, GFAP) were expressed and axon-like cytoplasmic processes are elaborated in the cultured human cord blood stem cells prepared from new born placenta after full term delivery. Nestin mRNA was also detected in fresh cord blood monocytes. Conclusions : These results suggest that human cord blood derived stem cells may be potential sources of neurons in early postnatal life.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.3
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• pp.167-172
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• 2004

The kinesin proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF5 is a heterotetrameric motor that conveys vesicles and plays an important role in neuronal function. Here, we used the yeast two-hybrid system to identify the neuronal protein(s) that interacts with the tail region of KIF5 and found a specific interaction with ${\beta}III$ spectrin. The amino acid residues between 1394 and 1774 of ${\beta}III$ spectrin were required for the interaction with KIF5C. ${\beta}III$ spectrin also bound to the tail region of neuronal KIF5A and ubiquitous KIF5B but not to other kinesin family members in the yeast two-hybrid assay. In addition, these proteins showed specific interactions, confirmed by GST pull-down assay and co-immunoprecipitation. ${\beta}III$ spectrin interacted with GST-KIF5 fusion proteins, but not with GST alone. An antibody to ${\beta}III$ spectrin specifically co-immunoprecipitated KIF5s associated with ${\beta}III$ spectrin from mouse brain extracts. These results suggest that KTF5 motor proteins transport vesicles or organelles that are coated with ${\beta}III$ spectrin.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.12
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• pp.4981-4985
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• 2015

Background: Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide. Targeting autophagic cell death is emerging as a novel strategy in cancer chemotherapy. Aldose reductase (AR) catalyzes the rate limiting step of the polyol pathway of glucose metabolism; besides reducing glucose to sorbitol, AR reduces lipid peroxidation-derived aldehydes and their glutathione conjugates. A complex interplay between autophagic cell death and/or survival may in turn govern tumor metastasis. This exploratory study aimed to investigate the potential role of AR inhibition using a novel inhibitor Fidarestat in the regulation of autophagy in CRC cells. Materials and Methods: For glucose depletion (GD), HT-29 and SW480 CRC cells were rinsed with glucose-free RPMI-1640, followed by incubation in GD medium +/- Fidarestat ($10{\mu}M$). Proteins were extracted by a RIPA-method followed by Western blotting ($35-50{\mu}g$ of protein; n=3). Results: Autophagic regulatory markers, primarily, microtubule associated protein light chain (LC) 3, autophagy-related gene (ATG) 5, ATG 7 and Beclin-1 were expressed in CRC cells; glyceraldehyde-3 phosphate dehydrogenase (GAPDH) was used as an internal reference. LC3 II (14 kDa) expression was relatively high compared to LC3A/B I levels in both CRC cell lines, suggesting occurrence of autophagy. Expression of non-autophagic markers, high mobility group box (HMG)-1 and Bcl-2, was comparatively low. Conclusions: GD +/- ARI induced autophagy in HT-29 and SW-480 cells, thereby implicating Fidarestat as a promising therapeutic agent for colorectal cancer; future studies with more potent ARIs are warranted to fully dissect the molecular regulatory networks for autophagy in colorectal carcinoma.

• Biomolecules & Therapeutics
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• v.30 no.5
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• pp.447-454
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• 2022

Few studies have evaluated the role of autophagy in the development of oxaliplatin (OXT) resistance in colon cancer cells. In this study, we compared the role of autophagy between SNU-C5 colon cancer cells and OXT-resistant SNU-C5 (SNU-C5/OXTR) cells. At the same concentration of OXT, the cytotoxicity of OXT or apoptosis was significantly reduced in SNU-C5/OXTR cells compared with that in SNU-C5 cells. Compared with SNU-C5 cells, SNU-C5/OXTR cells exhibited low levels of autophagy. The expression level of important autophagy proteins, such as autophagy-related protein 5 (Atg5), beclin-1, Atg7, microtubule-associated proteins 1A/1B light chain 3B I (LC3-I), and LC3-II, was significantly lower in SNU-C5/OXTR cells than that in SNU-C5 cells. The expression level of the autophagy-essential protein p62 was also lower in SNU-C5/OXTR cells than in SNU-C5 cells. In SNU-C5/OXTR cells, the production of intracellular reactive oxygen species (ROS) was significantly higher than that in SNU-C5 cells, and treatment with the ROS scavenger N-acetylcysteine restored the reduced autophagy levels. Furthermore, the expression of antioxidant-related nuclear factor erythroid 2-related factor 2 transcription factor, heme oxygenase-1, and Cu/Zn superoxide dismutase were also significantly increased in SNU-C5/OXTR cells. These findings suggest that autophagy is significantly reduced in SNU-C5/OXTR cells compared with SNU-C5 cells, which may be related to the production of ROS in OXT-resistant cells.

• Journal of Life Science
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• v.29 no.12
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• pp.1305-1313
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• 2019

Licochalcone (LC), isolated from the roots of Glycyrrhiza inflata has multiple pharmacological effects including anti-inflammatory and anti-tumor activities. To date, Licochalcone C (LCC) has induced apoptosis and inhibited cell proliferation in oral and bladder cancer cells, but lung cancer has not yet been studied. In addition, no study reported LCC-induced autophagy in cancer until now. The present study was designed to investigate the effect of LCC on gefitinib-sensitive and -resistant lung cancer cells and elucidate the mechanism of its action. The 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay data showed that LCC significantly inhibited cell viability in non-small cell lung cancer (NSCLC) HCC827 (gefitinib-sensitive) and HCC827GR (gefitinib-resistant) cell lines. Interestingly, Annexin V/7-aminoactinomycin D double staining and cell cycle analysis showed an apoptosis rate within about 20% at the highest concentration of LCC. LCC induced G2/M arrest by reducing the expression of the cell cycle G2/M related proteins cyclin B1 and cdc2 in NSCLC cell lines. Treatment of LCC also induced autophagy by increasing the expression of the autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3) and the protein autophagy-related gene 5 involved in the autophagy process. In addition, LCC increased the production of reactive oxygen species (ROS), and the cell viability was partially restored by treatment with the ROS inhibitor N-acetyl-L-cysteine. In western blotting analysis, the expression of cdc2 was increased and LC3 was decreased by the simultaneous treatment of NAC and LCC. These results indicate that LCC may contribute to anti-tumor effects by inducing ROS-dependent G2/M arrest and autophagy in NSCLC. In conclusion, LCC treatment may be useful as a potential therapeutic agent against NSCLC.

• Journal of Korean Neurosurgical Society
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• v.64 no.5
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• pp.705-715
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• 2021

Objective : Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3-asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI. Methods : Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9-10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×10⁵ cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining. Results : Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups. Conclusion : Our results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.

• Animal cells and systems
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• v.5 no.4
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• pp.327-331
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• 2001

There is a critical developmental period during which brain sexual differentiation proceeds irreversibly under the influence of gonadal hormone. Recently, kinesin superfamily-associated protein 3 (KAP3) gene expressed during the 'critical period' of rat brain differentiation was identified by us (Choi and Lee, 1999). KAP3 functions as a microtubule-based motor that transports membranous organelles anterogradely in cells, including neurons (Yamazaki et al., 1996). mRNA level of KAP3 gene markedly increased before the initiation of puberty. Neonatal treatment of estrogen clearly inhibited the prepubertal increase in KAP3 mRNA level (Choi and Lee, 1999). In the present study, we aimed to investigate the effects of polychlorinated biphenyls (PCBs), as endocrine disruptors (EDs) on the expression of KAP3 gene during the 'critical period' of rat brain development. In our data, PCBs significantly decreased the expression of KAP3 gene in the fetal (day 17) and the neonatal (day 6 after birth in) male and female rat brains. The body weight and the breeding ability were significantly decreased in the PCBs-exposed rats compared with the control. These results showed that PCBs affect the transcriptional level of brain sexual differentiation related gene, KAP3, in the fetal and the neonatal rat brains. The maternal exposure to the PCBs may lead to toxic response in embryonic brain sexual differentiation and breeding ability after sexual maturation. This study indicates that KAP3 gene may be useful as a gene marker to analyze the molecular mechanism of toxic response in the animal brain development and sexual maturation exposed to PCBs.

• The Journal of the Korea Contents Association
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• v.9 no.4
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• pp.355-363
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• 2009

Increased oxidative stress by abnormal mitochondrial function can damage cell signal transduction and gene expression, and induce insulin resistance or diabetes. Autophagy, however, improve insulin resistance by clearance of malfunctioning mitochondria. Exercise also recovers the muscle dysfunction and degeneration by activating mitochondrial biogenesis. As it seems that exercise and autophagy might act as an orchestrated network to induce mitochondrial biogenesis, we investigated whether autophagy is involved in AMPK signal pathway stimulated by exercise or AICAR to increase mitochondrial biogenesis. And it showed that PGC-1 and mtTFA, but not autophagy marker LC3 mRNA expression were significantly increased by 6 hr of acute exercise. On the other hand, PGC-1 and mtTFA mRNA expression were upregulated by AICAR treatment to C2C12 myotube. However these genes were not inhibited by LC3 siRNA transfection. These results provide the evidence that autopahgy affects on mitochondrial biogenesis through different signal pathway from AMPK signal transduction.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.3
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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.