• Biomolecules & Therapeutics
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• v.22 no.5
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• pp.445-452
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• 2014

The purpose of this study was to investigate the therapeutic effects of DA-9801, an optimized extract of Dioscorea species, on diabetic peripheral neuropathy in a type 2 diabetic animal model. In this study, db/db mice were treated with DA-9801 (30 and 100 mg/kg, daily, p.o.) for 12 weeks. DA-9801 reduced the blood glucose levels and increased the withdrawal latencies in hot plate tests. Moreover, it prevented nerve damage based on increased nerve conduction velocity and ultrastructural changes. Decrease of nerve growth factor (NGF) may have a detrimental effect on diabetic neuropathy. We previously reported NGF regulatory properties of the Dioscorea genus. In this study, DA-9801 induced NGF production in rat primary astrocytes. In addition, it increased NGF levels in the sciatic nerve and the plasma of type 2 diabetic animals. DA-9801 also increased neurite outgrowth and mRNA expression of Tieg1/Klf10, an NGF target gene, in PC12 cells. These results demonstrated the attenuation of diabetic peripheral neuropathy by oral treatment with DA-9801 via NGF regulation. DA-9801 is currently being evaluated in a phase II clinical study.

• Biomolecules & Therapeutics
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• v.23 no.4
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• pp.320-326
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• 2015

The clinical benefits of oncogenic BRAF inhibitor therapies are limited by the emergence of drug resistance. In this study, we investigated the role of a negative regulator of the MAPK pathway, Spry2, in acquired resistance using BRAF inhibitor-resistant derivatives of the BRAF-V600E melanoma (A375P/Mdr). Real-time RT-PCR analysis indicated that the expression of Spry2 was higher in A375P cells harboring the BRAF V600E mutation compared with wild-type BRAF-bearing cells (SK-MEL-2) that are resistant to BRAF inhibitors. This result suggests the ability of BRAF V600E to evade feedback suppression in cell lines with BRAF V600E mutations despite high Spry2 expression. Most interestingly, Spry2 exhibited strongly reduced expression in A375P/Mdr cells with acquired resistance to BRAF inhibitors. Furthermore, the overexpression of Spry2 partially restored sensitivity to the BRAF inhibitor PLX4720 in two BRAF inhibitor-resistant cells, indicating a positive role for Spry2 in the growth inhibition induced by BRAF inhibitors. On the other hand, long-term treatment with PLX4720 induced pERK reactivation following BRAF inhibition in A375P cells, indicating that negative feedback including Spry2 may be bypassed in BRAF mutant melanoma cells. In addition, the siRNA-mediated knockdown of Raf-1 attenuated the rebound activation of ERK stimulated by PLX4720 in A375P cells, strongly suggesting the positive role of Raf-1 kinase in ERK activation in response to BRAF inhibition. Taken together, these data suggest that RAF signaling may be released from negative feedback inhibition through interacting with Spry2, leading to ERK rebound and, consequently, the induction of acquired resistance to BRAF inhibitors.

• Biomolecules & Therapeutics
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• v.20 no.3
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• pp.326-331
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• 2012

We recently reported that Val-SN-38, a novel valine ester prodrug of SN-38, had greatly improved the intracellular accumulation of SN-38 in MCF-7 cell line, probably through enhanced uptake via amino acid transporters. In the present study, the efficacy of Val-SN-38 was further investigated both in vitro and in vivo. It was found that the in vitro cytotoxic effect of Val-SN-38 was similar to that of SN-38. Moreover, Val-SN-38 exhibited an equal potency to that of SN-38 in survival experiments in vivo. Because these results seemed to be contrary to the previous finding, further investigation was performed to find out the underlying cause of the contradiction. As only the lactone form is known to have cytotoxic activity, the proportion of lactone in Val-SN-38 and SN-38 was determined, but no differences were found. However, it turned out that Val-SN-38 had poor stability compared with SN-38, which resulted in a decrease in beneficial efficacy for Val-SN-38. Overall, the present study showed that a valine-added prodrug approach could be advantageous provided that the stability of the compound can be ensured. We believe this is a noteworthy study that unravels the discrepancy between intracellular accumulation and efficacy of valine-added prodrug.

• Biomolecules & Therapeutics
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• v.21 no.2
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• pp.114-120
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• 2013

Most patients with mutant B-Raf melanomas respond to inhibitors of oncogenic B-Raf but resistance eventually emerges. To better understand the mechanisms that determine the long-term responses of mutant B-Raf melanoma cells to B-Raf inhibitor, we used chronic selection to establish B-Raf (V600E) melanoma clones with acquired resistance to the new oncogenic B-Raf inhibitor UI-152. Whereas the parental A375P cells were highly sensitive to UI-152 ($IC_{50}$ < $0.5{\mu}M$), the resistant sub-line (A375P/Mdr) displayed strong resistance to UI-152 ($IC_{50}$ < $20{\mu}M$). Immunofluorescence analysis indicated the absence of an increase in the levels of P-glycoprotein multidrug resistance (MDR) transporter in A375P/Mdr cells, suggesting that resistance was not attributable to P-glycoprotein overexpression. In UI-152-sensitive A375P cells, the anti-proliferative activity of UI-152 appeared to be due to cell-cycle arrest at $G_0/G_1$ with the induction of apoptosis. However, we found that A375P/Mdr cells were resistant to the apoptosis induced by UI-152. Interestingly, UI-152 preferentially induced autophagy in A375P/Mdr cells but not in A375P cells, as determined by GFP-LC3 puncta/cell counts. Further, autophagy inhibition with 3-methyladenine (3-MA) partially augmented growth inhibition of A375P/Mdr cells by UI-152, which implies that a high level of autophagy may protect UI-152-treated cells from undergoing growth inhibition. Together, our data implicate high rates of autophagy as a key mechanism of acquired resistance to the oncogenic B-Raf inhibitor, in support of clinical studies in which combination therapy with autophagy targeted drugs is being designed to overcome resistance.

• Biomolecules & Therapeutics
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• v.27 no.5
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• pp.474-483
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• 2019

Vascular endothelial growth factor (VEGF) plays a pivotal role in pathologic ocular neovascularization and vascular leakage via activation of VEGF receptor 2 (VEGFR2). This study was undertaken to evaluate the therapeutic mechanisms and effects of the tetrapeptide Arg-Leu-Tyr-Glu (RLYE), a VEGFR2 inhibitor, in the development of vascular permeability and choroidal neovascularization (CNV). In cultured human retinal microvascular endothelial cells (HRMECs), treatment with RLYE blocked VEGF-A-induced phosphorylation of VEGFR2, Akt, ERK, and endothelial nitric oxide synthase (eNOS), leading to suppression of VEGF-A-mediated hyper-production of NO. Treatment with RLYE also inhibited VEGF-A-stimulated angiogenic processes (migration, proliferation, and tube formation) and the hyperpermeability of HRMECs, in addition to attenuating VEGF-A-induced angiogenesis and vascular permeability in mice. The anti-vascular permeability activity of RLYE was correlated with enhanced stability and positioning of the junction proteins VE-cadherin, ${\beta}$-catenin, claudin-5, and ZO-1, critical components of the cortical actin ring structure and retinal endothelial barrier, at the boundary between HRMECs stimulated with VEGF-A. Furthermore, intravitreally injected RLYE bound to retinal microvascular endothelium and inhibited laser-induced CNV in mice. These findings suggest that RLYE has potential as a therapeutic drug for the treatment of CNV by preventing VEGFR2-mediated vascular leakage and angiogenesis.

• Biomolecules & Therapeutics
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• v.12 no.1
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• pp.43-48
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• 2004

The present study was carried out to investigate the potential acute toxicity of CKD-602 by a single intravenous dose in Sprague-Dawley rats. Ten males females were used in each test groups: a vehicle control, 34.7, 4l.7, 50.0, 60.0 and 72.0 mg/kg groups, and were given different single intravenous doses of CKD-602 to the test animals. Mortalities, clinical findings, and body weight changes were monitored for the 14-day period following the administration. At the end of l4-day observation period, all animals were sacrificed and complete gross postmortem examinations were performed. One, 1, 2, 8 and 9 cases of deaths occurred in the male dose groups of 34.7, 41.7, 50.0, 60.0 and 72.0 mg/kg, respectively, and 1, 5 and 9 cases in the female dose groups 50.0, 60.0 and 72.0 mg/kg, respectively. An increase in the incidence of clinical signs such as alopecia, skin pallor skin ulcerations, emaciation and change of fecal material was found in the both sexes of all treatment groups. A decrease or Suppression in the body weight was also observed in a dose-dependent manner. In autopsy, male and/or female rats of the treatment groups showed treatment-related gross findings such as splenomegaly, atrophy of the testis, epididymis, seminal vesicles, ovary, uterus and thymus which were dose-dependent in incidence and severity. Based on these results, it was concluded that a single intravenous injection of CKD-602 to rats caused significant toxicities in gastrointestinal, hematopoietic, and reproductive systems. The $LD_{50}$ value was 53.8 (95% confidence limit: 48.5~60.6) mg/kg for males and 60.l (95% confidence limit: 55.3~65.8) mg/kg for females. The $LD_{10}$ value was 39.9 (95% confidence limit: 3l.7~44.8) mg/kg for males and 50.3 (95% confidence limit: 40.6~54.8) mg/kg for females.

• Biomolecules & Therapeutics
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• v.27 no.3
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• pp.327-335
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• 2019

As the elderly population is increasing, Alzheimer's disease (AD) has become a global issue and many clinical trials have been conducted to evaluate treatments for AD. As these clinical trials have been conducted and have failed, the development of new theraphies for AD with fewer adverse effects remains a challenge. In this study, we examined the effects of Theracurmin on cognitive decline using 5XFAD mice, an AD mouse model. Theracurmin is more bioavailable form of curcumin, generated with submicron colloidal dispersion. Mice were treated with Theracurmin (100, 300 and 1,000 mg/kg) for 12 weeks and were subjected to the novel object recognition test and the Barnes maze test. Theracurmin-treated mice showed significant amelioration in recognition and spatial memories compared those of the vehicle-treated controls. In addition, the antioxidant activities of Theracurmin were investigated by measuring the superoxide dismutase (SOD) activity, malondialdehyde (MDA) and glutathione (GSH) levels. The increased MDA level and decreased SOD and GSH levels in the vehicle-treated 5XFAD mice were significantly reversed by the administration of Theracurmin. Moreover, we observed that Theracurmin administration elevated the expression levels of synaptic components, including synaptophysin and post synaptic density protein 95, and decreased the expression levels of ionized calcium-binding adapter molecule 1 (Iba-1), a marker of activated microglia. These results suggest that Theracurmin ameliorates cognitive function by increasing the expression of synaptic components and by preventing neuronal cell damage from oxidative stress or from the activation of microglia. Thus, Theracurmin would be useful for treating the cognitive dysfunctions observed in AD.

• Biomolecules & Therapeutics
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• v.26 no.3
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• pp.298-305
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• 2018

Rhomboid family member 2 gene (Rhbdf2) is an inactive homologue lacking essential catalytic residues of rhomboid intramembrane serine proteases. The protein is necessary for maturation of tumor necrosis factor-alpha ($TNF-{\alpha}$) converting enzyme, which is the molecule responsible for the release of $TNF-{\alpha}$. In this study, Rhbdf2 knockout (KO) mice were produced by CRISPR/CAS9. To see the effects of the failure of $TNF-{\alpha}$ release induced by Rhbdf2 gene KO, collagen-induced arthritis (CIA), which is the representative $TNF-{\alpha}$ related disease, was induced in the Rhbdf2 mutant mouse using chicken collagen type II. The severity of the CIA was measured by traditional clinical scores and histopathological analysis of hind limb joints. A rota-rod test and grip strength test were employed to evaluate the severity of CIA based on losses of physical functions. The results indicated that Rhbdf2 mutant mice showed clear alleviation of the clinical severity of CIA as demonstrated by the significantly lower severity indexes. Moreover, a grip strength test was shown to be useful for the evaluation of physical functional losses by CIA. Overall, the results showed that the Rhbdf2 gene has a significant effect on the induction of CIA, which is related to $TNF-{\alpha}$.

• Journal of Korean society of Dental Hygiene
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• v.23 no.4
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• pp.219-226
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• 2023

Objectives: This study aimed to investigate the effects of rhubarb extract on osteoclast differentiation in bone marrow-derived macrophages (BMMs). Osteoclasts are vital for bone resorption and remodeling. Osteoclast dysregulation can contribute to various bone-related disorders that directly affect oral health. Rhubarb, a medicinal plant with anti-inflammatory properties, has been shown to modulate bone metabolism. Methods: BMMs were isolated from the femurs and tibias of 5-week-old C57BL/6 mice and cultured in the presence of mouse macrophage colony-stimulating factor (M-CSF) for 3 days. Subsequently, BMMs were treated with M-CSF and receptor activator of nuclear factor-κB ligand (RANKL) to induce osteoclast differentiation. Results: Rhubarb extract effectively suppressed osteoclast differentiation in BMMs. Furthermore, rhubarb extract inhibited the mRNA expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK), which are essential for osteoclastogenesis. Moreover, it inhibited the RANKL-induced expression of nuclear factor of activated T cell c1 (NFATc1), a crucial transcription factor in osteoclast differentiation. Conclusions: These results suggest that rhubarb extract promotes oral health by inhibiting osteoclastogenesis in BMMs. Thus, rhubarb extract shows promise as a therapeutic agent for bone-related disorders that directly affect oral health, particularly those associated with abnormal osteoclast activity. Further research and exploration of the underlying mechanisms are warranted to fully understand their potential clinical applications.

• Journal of Ginseng Research
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• v.35 no.2
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• pp.209-218
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• 2011

Ginseng has been used as a general tonic agent to invigorate the human body as an adaptogenic agent. In a previous report, we have shown that ginseng contains a novel glycolipoprotein called gintonin. The main function of gintonin is to transiently enhance intracellular free $Ca^{2+}$ $[Ca^{2+}]_i$ levels in animal cells. The previous method for gintonin isolation included multiple steps using organic solvents. In the present report, we developed a simple method for the preparation of crude gintonin from ginseng root as well as stem and leaf, which produced a higher yield of gintonin than the previous one. The yield of gintonin was 0.20%, 0.29%, and 0.81% from ginseng root, stem, and leaf, respectively. The apparent molecular weight of gintonin isolated from stem and leaf through sodium dodecyl sulfate polyacrylamide gel electrophoresis was almost same as that from root but the compositions of amino acids, carbohydrates or lipids differed slightly between them. We also examined the effects of crude gintonin from ginseng root, stem, and leaf on endogenous $Ca^{2+}$-activated $Cl^-$ channel (CaCC) activity of Xenopus oocytes through mobilization of $[Ca^{2+}]_i$. We found that the order of potency for the activation of CaCC was ginseng root > stem > leaf. The $ED_{50}$ was $1.4{\pm}1.4$, $4.5{\pm}5.9$, and $3.9{\pm}1.1$ mg/mL for root, stem and leaf, respectively. In the present study, we demonstrated for the first time that in addition to ginseng root, ginseng stem and leaf also contain gintonin. Gintonin can be prepared from a simple method with higher yield of gintonin from ginseng root, stem, and leaf. Finally, these results demonstrate the possibility that ginseng stem and leaf could also be utilized for ginstonin preparation after a simple procedure, rather than being discarded.