Yonggeun Hong

Melatonin treatment induces interplay of apoptosis, autophagy, and senescence in human colorectal cancer cells

In Asia, the incidence of colorectal cancer has been increasing gradually due to a more Westernized lifestyle. The aim of study is to determine the interaction between melatonin‐induced cell death and cellular senescence. We treated HCT116 human colorectal adenocarcinoma cells with 10 μm melatonin and determined the levels of cell death‐related proteins and evaluated cell cycle kinetics. The plasma membrane melatonin receptor, MT1, was significantly decreased by melatonin in a time‐dependent manner, whereas the nuclear receptor, RORα, was increased only after 12 hr treatment. HCT116 cells, which upregulated both pro‐apoptotic Bax and anti‐apoptotic Bcl‐xL in the early response to melatonin treatment, activated autophagic as well as apoptotic machinery within 18 hr. Melatonin decreased the S‐phase population of the cells to 57% of the control at 48 hr, which was concomitant with a reduction in BrdU‐positive cells in the melatonin‐treated cell population. We found not only marked attenuation of E‐ and A‐type cyclins, but also increased expression of p16 and p‐p21. Compared to the cardiotoxicity of Trichostatin A in vitro, single or cumulative melatonin treatment induced insignificant detrimental effects on neonatal cardiomyocytes. We found that 10 μm melatonin activated cell death programs early and induced G1‐phase arrest at the advanced phase. Therefore, we suggest that melatonin is a potential chemotherapeutic agent for treatment of colon cancer, the effects of which are mediated by regulation of both cell death and senescence in cancerous cells with minimized cardiotoxicity.

Implication of mouse Vps26b-Vps29-Vps35 retromer complex in sortilin trafficking.

The retromer complex, which mediates retrograde transport from endosomes to the trans-Golgi network, is a heteropentameric complex that contains a multifunctional cargo recognition heterotrimer consisted of the vacuolar protein sorting (Vps) subunits Vps26, Vps29, and Vps35. In mammals, there are two different isoforms of Vps26, Vps26a and Vps26b, that localize to the endosome, and to the plasma membrane, respectively. To elucidate the biological significance of the Vps26b isoform, we generated Vps26b knockout mice and studied their molecular, histological, and behavioral phenotypes. We found that the loss of Vps26b results in no significant defects in the behavior, body size, and health of the mice. Vps26b-deficient mice showed a severe reduction of Vps35 protein at cellular level and lacked the Vps26b-Vps29-Vps35 retromer complex, despite the normal presence of the Vps26a-Vps29-Vps35 retromer complex. Relatively, the amount of sortilin was increased approximately 20% in the Vps26b-deficient mice, whereas the sorLA was normal. These results suggest that mouse Vps26b-Vps29-Vps35 retromer complex is implicated in the transport of sortilin from endosomes to the trans-Golgi network (TGN).

Therapeutic Implications for Overcoming Radiation Resistance in Cancer Therapy

Ionizing radiation (IR), such as X-rays and gamma (γ)-rays, mediates various forms of cancer cell death such as apoptosis, necrosis, autophagy, mitotic catastrophe, and senescence. Among them, apoptosis and mitotic catastrophe are the main mechanisms of IR action. DNA damage and genomic instability contribute to IR-induced cancer cell death. Although IR therapy may be curative in a number of cancer types, the resistance of cancer cells to radiation remains a major therapeutic problem. In this review, we describe the morphological and molecular aspects of various IR-induced types of cell death. We also discuss cytogenetic variations representative of IR-induced DNA damage and genomic instability. Most importantly, we focus on several pathways and their associated marker proteins responsible for cancer resistance and its therapeutic implications in terms of cancer cell death of various types and characteristics. Finally, we propose radiation-sensitization strategies, such as the modification of fractionation, inflammation, and hypoxia and the combined treatment, that can counteract the resistance of tumors to IR.

Insulin/IGF Signaling-Related Gene Expression in the Brain of a Sporadic Alzheimer's Disease Monkey Model Induced by Intracerebroventricular Injection of Streptozotocin

We reported previously that the intracerebroventricular streptozotocin (icv-STZ)-treated cynomolgus monkey showed regionally specific glucose hypometabolism in FDG-PET imaging, similar to that observed in the early stages of sporadic Alzheimers disease (sAD). However, further pathological analyses of this model at the molecular level are needed to validate it as a feasible model for sAD. Two cynomolgus monkeys were injected with 2 mg/kg STZ into the cerebellomedullary cistern at day 1, 7 and 14. Two control monkeys were given normal saline. At 5 months after injection, the expression levels of genes encoding 9 upstream molecules in insulin/insulin-like growth factor (IGF) signaling and markers for 4 cell-type populations in the frontal cortex, hippocampus, posterior cingulate, precuneus, and occipital cortex of control and icv-STZ treated cynomolgus monkeys were examined. Real-time quantitative PCR analyses demonstrated that the overall mRNA expression of insulin/IGF signaling-related genes was mainly impaired in the anterior part of the cerebrum, frontal cortex, and hippocampus, similar to the early stage of sAD. The changes were accompanied by the loss of oligodendrocytes and neurons. The posterior part of the cerebrum did not show degenerative alterations. The present study provides important fundamental information on the icv-STZ monkey model for sAD. These results may help guide future studies using this model for the investigation of pathological mechanisms and the development of drugs for sAD.

The effects of smartphone use on upper extremity muscle activity and pain threshold

[Purpose] The purpose of this study was to determine whether muscle activity and pressure-induced pain in the upper extremities are affected by smartphone use, and to compare the effects of phone handling with one hand and with both hands. [Subjects] The study subjects were asymptomatic women 20–22 years of age. [Methods] The subjects sat in a chair with their feet on the floor and the elbow flexed, holding a smartphone positioned on the thigh. Subsequently, the subjects typed the Korean anthem for 3 min, one-handed or with both hands. Each subject repeated the task three times, with a 5-min rest period between tasks to minimize fatigue. Electromyography (EMG) was used to record the muscle activity of the upper trapezius (UT), extensor pollicis longus (EPL), and abductor pollicis (AP) during phone operation. We also used a dolorimeter to measure the pressure-induced pain threshold in the UT. [Results] We observed higher muscle activity in the UT, AP, and EPL in one-handed smartphone use than in its two-handed use. The pressure-induced pain threshold of the UT was lower after use of the smartphone, especially after one-handed use. [Conclusion] Our results show that smartphone operation with one hand caused greater UT pain and induced increased upper extremity muscle activity.

Beneficial effects of melatonin combined with exercise on endogenous neural stem/progenitor cells proliferation after spinal cord injury.

Endogenous neural stem/progenitor cells (eNSPCs) proliferate and differentiate into neurons and glial cells after spinal cord injury (SCI). We have previously shown that melatonin (MT) plus exercise (Ex) had a synergistic effect on functional recovery after SCI. Thus, we hypothesized that combined therapy including melatonin and exercise might exert a beneficial effect on eNSPCs after SCI. Melatonin was administered twice a day and exercise was performed on a treadmill for 15 min, six days per week for 3 weeks after SCI. Immunohistochemistry and RT-PCR analysis were used to determine cell population for late response, in conjunction with histological examination and motor function test. There was marked improvement in hindlimb function in SCI+MT+Ex group at day 14 and 21 after injury, as documented by the reduced size of the spinal lesion and a higher density of dendritic spines and axons; such functional improvements were associated with increased numbers of BrdU-positive cells. Furthermore, MAP2 was increased in the injured thoracic segment, while GFAP was increased in the cervical segment, along with elevated numbers of BrdU-positive nestin-expressing eNSPCs in the SCI+MT+Ex group. The dendritic spine density was augmented markedly in SCI+MT and SCI+MT+Ex groups. These results suggest a synergistic effect of SCI+MT+Ex might create a microenvironment to facilitate proliferation of eNSPCs to effectively replace injured cells and to improve regeneration in SCI.

Beneficial effects of melatonin on stroke-induced muscle atrophy in focal cerebral ischemic rats

Muscle atrophy is the result of two opposing conditions that can be found in pathological or diseased muscles: an imbalance in protein synthesis and degradation mechanisms. Thus, we investigated whether exogenous melatonin could regulate muscle components in stroke-induced muscle atrophy in rats. Comparing muscle phenotypes, we found that long-term melatonin administration could influence muscle mass. Muscle atrophy-related genes, including muscle atrophy F-box (MAFbx) and muscle ring finger 1 (MuRF1) were significantly down-regulated in melatonin-administered rats in the gastrocnemius. However, only MAFbx at the mRNA level was attenuated in the soleus of melatonin-administered rats. Insulin-like growth factor-1 receptor (IGF-1R) was significantly over-expressed in melatonin-administered rats in both the gastrocnemius and soleus muscles. Comparing myosin heavy chain (MHC) components, in the gastrocnemius, expression of both slow- and fast-type isoforms were significantly enhanced in melatonin-administered rats. These results suggest that long-term exogenous melatonin-administration may have a prophylactic effect on muscle atrophy through the MuRF1/MAFbx signaling pathway, as well as a potential therapeutic effect on muscle atrophy through the IGF-1-mediated hypertrophic signaling pathway in a stroke animal model.

Extracellular domain of V-set and immunoglobulin domain containing 1 (VSIG1) interacts with sertoli cell membrane protein, while its PDZ-binding motif forms a complex with ZO-1

V-set and immunoglobulin domain containing 1 (VSIG1) is a newly discovered member of the junctional adhesion molecule (JAM) family; it is encoded by a gene located on human chromosome X and preferentially expressed in a variety of cancers in humans. Little is known about its physiological function. To determine the role(s) of VSIG1 in mammalian spermatogenesis, we first generated a specific antibody against mouse VSIG1 and examined the presence and localization of the protein in tissues. RTRCR and Western blot analysis of the mouse tissues indicated that VSIG1 was specifically expressed in the testis. Furthermore, the results of our trypsinization and biotinylation assays strongly support the assumption that VSIG1 is localized on the testicular germ cell surface. In order to determine whether VSIG1 is capable of participation in homotypic interactions, we performed a GST-pull down assay by using recombinant GST-fusion and Histagging proteins. The pull-down assay revealed that each GST-fusion Ig-like domain shows homotypic binding. We further show that mVSIG1 can adhere to the Sertoli cells through its first Ig-like domain. To identify the protein that interacted with cytoplasmic domain, we next performed co-immunoprecipitation analysis. This analysis showed that ZO-1, which is the central structural protein of the tight junction, is the binding partner of the cytoplasmic domain of mouse VSIG1. Our findings suggest that mouse VSIG1 interacts with Sertoli cells by heterophilic adhesion via its first Ig-like domain. In addition, its cytoplasmic domain is critical for binding to ZO-1.

Melatonin treatment combined with treadmill exercise accelerates muscular adaptation through early inhibition of CHOP‐mediated autophagy in the gastrocnemius of rats with intra‐articular collagenase‐induced knee laxity

The purpose of this study was to determine the effects of melatonin intervention on gastrocnemius remodeling in rats with collagenase‐induced knee instability. Type VII collagenase was injected into the right knee to induce joint laxity with cartilage destruction. Melatonin (MT; 10 mg/kg) injection was performed twice daily subcutaneously, and treadmill exercise (Ex; 11 m/min) was conducted for 1 hr/day at a frequency of 5 days/wk for 4 wks. The gastrocnemius mass, which was reduced with collagenase injection only (Veh), was increased with collagenase injection with melatonin treatment with and without exercise in the early phase, and the mass in both limbs was significantly different in the Veh compared with the MT group. However, there was an increase in the relative muscle weight to body weight ratio in the Veh group at the advanced stage. Insulin‐like growth factor receptor (IGF‐IR) was downregulated in the Veh group, whereas IGF‐IR was upregulated in the MT and MT + Ex groups. Joint laxity induced enhancement of autophagic proteolysis (LC3 II) in the muscle, which was recovered to values similar to those in the normal control group (Con) compared with those in the MT and MT + Ex groups. Although intra‐articular collagenase increased the total C/EBP homology protein (CHOP) levels at 1 wk and decreased them at 4 wks in the Veh group, CHOP in the nucleus was upregulated continuously. Prolonged melatonin treatment with and without exercise intervention suppressed nuclear localization of ATF4 and CHOP with less activation of caspase‐3, at the advanced phase. Moreover, the interventions promoted the expression of myosin heavy chain (MHC) isoforms under the control of myogenin. Concomitant with a beneficial effect of melatonin with and without exercise, step length of the saline‐injected limb and the collagenase‐injected supporting side was maintained at values similar to those in control rats. Taken together, the findings demonstrate that melatonin with and without exercise accelerate remodeling of the gastrocnemius through inhibition of nuclear CHOP in rats with collagenase‐induced knee instability.

Comparison of surgical methods of transient middle cerebral artery occlusion between rats and mice.

Rodent models of focal cerebral ischemia that do not require craniotomy have been developed by intraluminal suture middle cerebral artery occlusion (MCAo). Mouse MCAo models have been widely used and extended to genetic studies of cell death or recovery mechanisms. Therefore, we compared surgery-related parameters and techniques between such rats and mice. In rodent MCAo models, has to be considered body temperature during the operative period, as well as the need for the use of a standardized tip in terms of the outer diameter of probes. Induction of focal cerebral ischemia was measured by neurological dysfunction parameters. Our methods could induce stable moderate-severity ischemic brain injury models and histological alteration at 24 hr after MCAo surgery. Moreover approximately 80% (rats) and 85% (mice) survival ratios were shown indicating with model engineering success. Finally, we described and compared major parameters between rats and mice, including probe size, thread insert length, operation and occlusion periods, and differences in the procedures.