Seunghoon Lee

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.

Beneficial effects of endogenous and exogenous melatonin on neural reconstruction and functional recovery in an animal model of spinal cord injury.

Abstract:  The purpose of this study was to investigate the beneficial effects of endogenous and exogenous melatonin on functional recovery in an animal model of spinal cord injury (SCI). Eight‐week‐old male Sprague‐Dawley (SD, 250–260 g) rats were used for contusion SCI surgery. All experimental groups were maintained under one of the following conditions: 12/12‐hr light/dark (L/D) or 24:0‐hr constant light (LL). Melatonin (10 mg/kg) was injected subcutaneously for 4 wk, twice daily (07:00, 19:00). Locomotor recovery, inducible nitric oxide synthase (iNOS), glial fibrillary acidic protein gene expression, and muscle atrophy‐related genes, including muscle atrophy F‐box (MAFbx) and muscle‐specific ring‐finger protein 1 (MuRF1) gene expression were evaluated. Furthermore, autophagic signaling such as Beclin‐1 and LC3 protein expression was examined in the spinal cord and in skeletal muscle. The melatonin treatment resulted in increased hind‐limb motor function and decreased iNOS mRNA expression in the L/D condition compared with the LL condition (P < 0.05), indicating that endogenous melatonin had neuroprotective effects. Furthermore, the MAFbx, MuRF1 mRNA level, and converted LC3 II protein expression were decreased in the melatonin‐treated SCI groups under the LL (P < 0.05), possibly in response to the exogenous melatonin treatment. Therefore, it seems that both endogenous and exogenous melatonin contribute to neural recovery and to the prevention of skeletal muscle atrophy, promoting functional recovery after SCI. Finally, this study supports the benefit of endogenous melatonin and use of exogenous melatonin as a therapeutic intervention for 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.

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.

Salutary effects of melatonin combined with treadmill exercise on cartilage damage

Osteoarthritis (OA) is a major cause of disability in the adult population. The purpose of this study was to evaluate the effects of melatonin with graded dosage on extracellular matrix synthesis and cellular death in response to cartilage damage in vitro and in vivo. TNF‐α reduced the viability of primary cultured chondrocytes and extracellular matrix protein, but melatonin at concentrations of 1 μm and 1 nm restored them. Rats with knee instability induced by intra‐articular collagenase were used for the in vivo study. Joint parameters were significantly augmented in the collagenase injection‐only group but not in the melatonin‐alone or melatonin+exercise groups, as cartilage degeneration progressed. Serum TNF‐α and IL‐6 were upregulated by collagenase injection, which was attenuated by melatonin with and without exercise in the early phase. TGF‐β1 mRNA was either conserved or enhanced by melatonin with and without exercise at the early phase. In particular, melatonin combined with exercise dramatically decreased the expression of not only catabolic mediators but also cellular death markers with lower mineralization. At the advanced phase, prolonged melatonin treatment promoted mineralization through caspase‐3‐mediated chondrocyte apoptosis. However, co‐intervention induced extracellular matrix remodeling through increases in IL‐6, EPAS‐1, and MMP‐13. Reconstructed micro‐CT images showed that collagenase injection induced subchondral bone erosion, formation of parameniscal osteophytes, and reduction of trabecular bone thickness regardless of the intervention, which was minimized by combined intervention. In conclusion, we suggest that melatonin with treadmill exercise may have both preventive and synergistic effects on rescue from cartilage degeneration and is more effective in the initial phase.

Middle cerebral artery occlusion methods in rat versus mouse models of transient focal cerebral ischemic stroke.

Experimental stroke research commonly employs focal cerebral ischemic rat models (Bederson et al., 1986a; Longa et al., 1989). In human patients, ischemic stroke typically results from thrombotic or embolic occlusion of a major cerebral artery, usually the middle cerebral artery (MCA). Experimental focal cerebral ischemia models have been employed to mimic human stroke (Durukan and Tatlisumak, 2007). Rodent models of focal cerebral ischemia that do not require craniotomy have been developed using intraluminal suture occlusion of the MCA (MCA occlusion, MCAO) (Rosamond et al., 2008). Furthermore, mouse MCAO models have been widely used and extended to genetic studies of cell death or recovery mechanisms (Liu and McCullough, 2011). Genetically engineered mouse stroke models are particularly useful for evaluation of ischemic pathophysiology and the design of new prophylactic, neuroprotective, and therapeutic agents and interventions (Armstead et al., 2010). During the past two decades, MCAO surgical techniques have been developed that do not reveal surgical techniques for mouse MCAO model engineering. Therefore, we compared MCAO surgical methods in rats and mice. Forty-five male Sprague-Dawley rats, weighing 240–260 g, and thirty-four male C57BL/6 mice, weighing 20–25 g, were selected for this study. 4-0 and 6-0 monofilament nylon (AILEE Co., Busan, Korea) was used in MCAO surgery. The monofilament was cut into 5-cm (for rats) or 2-cm (for mice) pieces, and the tip of the monofilament was blunted by heating or poly-L-lysine coating (Sigma-Aldrich, St. Louis, MO, USA) (outer diameter of monofilament: 0.4–0.45 mm for rats and 0.15–0.18 mm for mice). All surgical instruments and materials were autoclaved, and the surgical procedure was performed under sterile conditions. This study was approved by the Ethics Committee for Animal Care and Use at Inje University (Approval No. 2012-29), which is certified by the Korean Association of Laboratory Animal Care. The animals were anesthetized with an intraperitoneal injection of Zoletil (tiletamine + zolazepam cocktail at 40 mg/kg or ketamine at 80 mg/kg) and xylazine (10 mg/kg) (Lee et al., 2012). After anesthetic induction, the animals were placed on a heating pad on a surgical table. During the surgical procedure, the body temperature was continuously monitored with a rectal probe and maintained at 36.5–37.0°C. The surgical region was disinfected with povidone-iodine or 70% alcohol. A midline neck incision was made, and the soft tissues over the trachea were gently retracted with a retractor. The common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully isolated from the vagus nerve. Typically, the CCA bifurcates into the ECA and ICA, which flow toward the cranial and facial regions, respectively, and then the ICA bifurcates into the MCA and pterygopalatine artery (PPA). The occipital artery (OA) originates from the bifurcation point of the ECA which is placed on the side of the ICA (Figure 1A). Two closely spaced permanent knots were then placed at the distal part of the ECA (below the suprathyroid artery) to prevent the backflow of blood. A microvascular clamp was placed in the ICA and transiently proximal to the CCA junction. The tied section of the ECA was dissected using microscissors to insert the monofilament and reach the CCA junction, and a knot was placed below the arteriotomy in the ECA. The microvascular clamp placed in the ICA was removed to allow for filament insertion. The filament was carefully inserted, up to 18 to 20 mm for rats and 9 to 11 mm for mice, into the MCA from the CCA junction (Figure 1A; captured image). After confirmation of MCA blockage, the rat model allowed a blood supply from the CCA, whereas the mouse model allowed a blood supply after the occlusion period. After 60–90 minutes, the filament was carefully withdrawn until the tip was near the arteriotomy. Following removal of the filament, the knot was tightened in the ECA. When reperfusion was confirmed, the neck was sewn using surgical thread. To relieve pain and discomfort in the postoperative period, topical lidocaine gel was applied to the incision region, and the animal received 1.0 mL of normal saline subcutaneously as volume replenishment after the surgery. At 24 hours after the surgery, the animals were sacrificed and analyzed for brain infarction. All procedures had to be finished within 15 minutes, excluding the occlusion and reperfusion time (Figure 1). After 24 hours after reperfusion, infarct volume was calculated using 2% TTC staining method (Figure ​(Figure1C1C–E) (Bederson et al., 1986b; Park et al., 2012). Figure 1 Schematic representation of surgical procedure and quantification of infarct volume in subjected rodents. In summary, to develop standard and high-quality rodent models of stroke, several points should be taken in MCAO: (1) 0.40–0.45 mm outer diameter 4-0 monofilament nylon (for rats) and 0.15–0.18 mm outer diameter of 6-0 monofilament nylon (for mice) by heating or poly-L-lysine coating. (2) Thread insertion length: 18–20 mm (for rats) and 9–11 mm (for mice). (3) Operation period: maximum of 15 minutes. (4) Occlusion period: 60 minutes. (5) MCA occlusion allows CCA reperfusion for rats or bilateral CCA occlusion for mice.

Melatonin combined with exercise cannot alleviate cerebral injury in a rat model of focal cerebral ischemia/reperfusion injury

Previous studies have demonstrated that melatonin combined with exercise can alleviate secondary damage after spinal cord injury in rats. Therefore, it is hypothesized that melatonin combined with exercise can also alleviate ischemic brain damage. In this study, adult rats were subjected to right middle cerebral artery occlusion after receiving 10 mg/kg melatonin or vehicle subcutaneously twice daily for 14 days. Forced exercise using an animal treadmill was performed at 20 m/min for 30 minutes per day for 6 days prior to middle cerebral artery occlusion. After middle cerebral artery occlusion, each rat received melatonin combined with exercise, melatonin or exercise alone equally for 7 days until sacrifice. Interestingly, rats receiving melatonin combined with exercise exhibited more severe neurological deficits than those receiving melatonin or exercise alone. Hypoxia-inducible factor 1α mRNA in the brain tissue was upregulated in rats receiving melatonin combined with exercise. Similarly, microtubule associated protein-2 mRNA expression was significantly upregulated in rats receiving melatonin alone. Chondroitin sulfate proteoglycan 4 (NG2) mRNA expression was significantly decreased in rats receiving melatonin combined with exercise as well as in rats receiving exercise alone. Furthermore, neural cell loss in the primary motor cortex was significantly reduced in rats receiving melatonin or exercise alone, but the change was not observed in rats receiving melatonin combined with exercise. These findings suggest that excessive intervention with melatonin, exercise or their combination may lead to negative effects on ischemia/reperfusion-induced brain damage.

Establishment of a surgically induced cryptorchidism canine recipient model for spermatogonial stem cell transplantation.

Transplantation of spermatogonial stem cells (SSCs) in experimental animal models has been used to study germ line stem cell biology and to produce transgenic animals. The species-specific recipient model preparation is important for the characterization of SSCs and the production of offspring. Here, we investigated the effects of surgically induced cryptorchidism in dog as a new recipient model for spermatogonial stem cell transplantation. Artificially unilateral or bilateral cryptorchidism was induced in ten mature male dogs by surgically returning the testis and epididymis to the abdominal cavity. The testes and epididymides were collected every week after the induction of artificial cryptorchidism (surgery) for one month. To determine the effect of surgical cryptorchidism, the seminiferous tubule diameter was measured and immunohistochemistry using PGP9.5 and GATA4 antibodies was analyzed. The diameters of the seminiferous tubules of abdominal testes were significantly reduced compared to those of the scrotal testes. Immunohistochemistry results showed that PGP9.5 positive undifferentiated spermatogonia were significantly reduced after surgical cryptorchidism induction, but there were no significant changes in GATA-4 positive sertoli cells. To evaluate the testis function recovery rate, orchiopexy was performed on two dogs after 30 days of bilateral cryptorchidism. In the orchiopexy group, SCP3 positive spermatocytes were detected, and spermatogenesis was recovered 8 weeks after orchiopexy. In this study, we provided optimum experimental conditions and time for surgical preparation of a recipient canine model for SSC transplantation. Additionally, our data will contribute to recipient preparation by using surgically induced cryptorchidism in non-rodent species.

Dose-dependent effects of busulfan on dog testes in preparation for spermatogonial stem cell transplantation

Successful male germ cell transplantation requires depletion of the host germ cells to allow efficient colonization of the donor spermatogonial stem cells. Although a sterilizing drug, busulfan, is commonly used for the preparation of recipient models before transplantation, the optimal dose of this drug has not yet been defined in dogs. In this study, 1-year-old mongrel dogs were intravenously injected with three different concentrations of busulfan (10, 15, or 17.5 mg/kg). Four weeks after busulfan treatment, no fully matured spermatozoa were detected in any of the busulfan-treated groups. However, small numbers of PGP9.5-positive spermatogonia were detected in all treatment groups, although no synaptonemal complex protein-3-positive spermatocytes were detected. Of note, acrosin-positive spermatids were not detected in the dogs treated with 15 or 17.5 mg/kg busulfan, but were detected in the other group. Eight weeks after busulfan treatment, the dogs treated with 10 mg/kg busulfan fully recovered, but those in the other groups did not. PGP9.5-positive spermatogonia were detected in the 10 mg/kg group, and at a similar level as in the control group, but these cells were rarely detected in the 15 and 17.5 mg/kg groups. These results suggest that a dose of 15-17.5 mg/kg is optimal for ablative treatment with busulfan to prepare the recipient dogs for male germ cell transplantation. At least eight weeks should be allowed for recovery. The results of this study might facilitate the production of recipient dogs for male germ cell transplantation and can also contribute to studies on chemotherapy.

Ascorbic Acid Increases Demethylation in Somatic Cell Nuclear Transfer Embryos of the pig (Sus scrofa)

Objective Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT). Methods Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3) in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR. Results Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1, Sox2, and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid. Conclusion Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.