Characterization of a small molecule inhibitor of melanogenesis that inhibits tyrosinase activity and scavenges nitric oxide (NO)

Background

Excessive melanin production and accumulation are characteristics of a large number of skin diseases, including melasma, and post-inflammatory hyperpigmentation. During our on-going search for new agents with an inhibitory effect on tyrosinase, we synthesized a new type of tyrosinase inhibitor, 4-(thiazolidin-2-yl)benzene-1,2-diol (MHY-794), which directly inhibits mushroom tyrosinase.

Methods

The inhibitory effect of MHY-794 on tyrosinase activity and nitric oxide (NO) scavenging activity was evaluated in cell free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of MHY-794 in vitro. HRM2 hairless mice were used to evaluate anti-melanogenic effects of MHY-794 in vivo.

Results

MHY-794 effectively inhibited mushroom tyrosinase activity in cell free system. In silico docking simulation also supported the inhibitory effects of MHY-794 on mushroom tyrosinase. MHY-794 also proved to be effective at scavenging nitric oxide (NO), which serves as an important modulator in the melanogenesis signaling pathway. In addition, MHY-794 effectively inhibited SNP (NO donor)-induced melanogenesis by directly inhibiting tyrosinase and diminishing NO-mediated melanogenesis signaling in B16 melanoma cells. The anti-melanogenic effects of MHY-794 were further confirmed in HRM2 hairless mice. Ultraviolet light (UV) significantly up-regulated NO-mediated melanogenesis signaling in HRM2 hairless mice, but MHY-794 effectively inhibited both melanogenesis and diminished UV-induced NO-signaling.

Conclusions

Our results indicate that MHY-794 is highly effective at inhibiting NO-mediated melanogenesis in vitro and in vivo by direct NO scavenging and directly inhibiting tyrosinase activity, and suggest that MHY-794 be considered a new developmental candidate for the treatment of hyper-pigmentation disorders.

General significance

MHY-794, which showed great efficacy on NO-mediated melanogenesis by direct NO scavenging as well as direct inhibition of tyrosinase catalytic activity, might be utilized for the development of a new candidate for treatment of the hyper-pigmentation disorders.     

The JNK/NFκB pathway is required to activate murine lymphocytes induced by a sulfated polysaccharide from Ecklonia cava

Background

The proven immunomodulatory and immune system activating properties of Ecklonia cava (E. cava) have been attributed to its plentiful polysaccharide content. Therefore, we investigated whether the sulfated polysaccharide (SP) of E. cava specifically activates the protein kinases (MAPKs) and nuclear factor-κB (NFκB) to incite immune responses.

Methods

To assess immune responsiveness, lymphocytes were isolated from spleens of ICR mice and cultured with SP and its inhibitors. Assays included ³H-thymidine incorporation, flow cytometry, real time polymerase chain reaction (rtPCR), enzyme linked immunosorbent assay (ELISA), intracellular cytokine assay, Western blot, and electrophoretic mobility shift assay (EMSA).

Results

SP dose-dependently increased the proliferation of lymphocytes without cytotoxicity. In particular, SP markedly enhanced the proliferation and differentiation of CD3⁺ mature T cells and CD45R/B220⁺ pan B cells. Additionally, SP increased the expression and/or production of IL-2, IgG_1a, and IgG_2b compared to that in untreated cells. The subsequent application of JNK (SP600125), NFκB (PDTC), and serine protease (TPCK) inhibitors significantly inhibited the proliferation and IL-2 production of SP-treated lymphocytes as well as the phosphorylation of JNK and IκB, the activation of nuclear NFκB p65, and binding of NFκB p65 DNA. Moreover, co-application of both JNK and NFκB inhibitors completely blocked the proliferation of lymphocytes even in the presence of SP.

Conclusion

These results suggest that SP induced T and B cell responses via both JNK and NFκB pathways.

General significance

The effect of SP on splenic lymphocyte activation was assayed here for the first time and indicated the underlying functional mechanism.     

Effect of Propofol Post-treatment on Blood–Brain Barrier Integrity and Cerebral Edema After Transient Cerebral Ischemia in Rats

Although propofol has been reported to offer neuroprotection against cerebral ischemia injury, its impact on cerebral edema following ischemia is not clear. The objective of this investigation is to evaluate the effects of propofol post-treatment on blood–brain barrier (BBB) integrity and cerebral edema after transient cerebral ischemia and its mechanism of action, focusing on modulation of aquaporins (AQPs), matrix metalloproteinases (MMPs), and hypoxia inducible factor (HIF)-1α. Cerebral ischemia was induced in male Sprague–Dawley rats (n = 78) by occlusion of the right middle cerebral artery for 1 h. For post-treatment with propofol, 1 mg kg^?1 min^?1 of propofol was administered for 1 h from the start of reperfusion. Nineteen rats undergoing sham surgery were also included in the investigation. Edema and BBB integrity were assessed by quantification of cerebral water content and extravasation of Evans blue, respectively, following 24 h of reperfusion. In addition, the expression of AQP-1, AQP-4, MMP-2, and MMP-9 was determined 24 h after reperfusion and the expression of HIF-1α was determined 8 h after reperfusion. Propofol post-treatment significantly reduced cerebral edema (P < 0.05) and BBB disruption (P < 0.05) compared with the saline-treated control. The expression of AQP-1, AQP-4, MMP-2, and MMP-9 at 24 h and of HIF-1α at 8 h following ischemia/reperfusion was significantly suppressed in the propofol post-treatment group (P < 0.05). Propofol post-treatment attenuated cerebral edema after transient cerebral ischemia, in association with reduced expression of AQP-1, AQP-4, MMP-2, and MMP-9. The decreased expression of AQPs and MMPs after propofol post-treatment might result from suppression of HIF-1α expression.     

Interplay of reactive oxygen species,intracellular Ca2+ and mitochondrial homeostasis in the apoptosis of prostate cancer cells by deoxypodophyllotoxin

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti‐neoplastic agents of natural product origin. We previously reported anti‐proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC‐3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase‐3‐dependent pathway that is activated due to dysregulated mitochondrial function. DPT‐treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca surge, increased mitochondrial membrane potential (MMP, ΔΨ_m), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase‐3 activation, which in turn induced apoptosis. The antioxidant N‐acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca surge, on the other hand the Ca²⁺ chelator BAPTA inhibited the Ca overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca²⁺ flux. This suggested that both ROS and Ca signaling play roles in the increased MMP via Ca‐dependent and/or ‐independent mechanisms, since ΔΨ_m elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS‐ and Ca‐activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca²⁺ flux homeostasis. J. Cell. Biochem. 114: 1124–1134, 2013. © 2012 Wiley Periodicals, Inc.     

Structural modification of siRNA for efficient gene silencing

Small interfering RNA (siRNA) holds a great promise for the future of genomic medicine because of its highly sequence-specific gene silencing and universality in therapeutic target. The medical use of siRNA, however, has been severely hampered by the inherent physico-chemical properties of siRNA itself, such as low charge density, high structural stiffness and rapid enzymatic degradation; therefore, the establishment of efficient and safe siRNA delivery methodology is an essential prerequisite, particularly for systemic administration. For an efficient systemic siRNA delivery, it is a critical issue to obtain small and compact siRNA polyplexes with cationic condensing reagents including cationic polymers, because the size and surface properties of the polyplexes are major determinants for achieving desirable in vivo fate. Unfortunately, synthetic siRNA is not easily condensed with cationic polymers due to its intrinsic rigid structure and low spatial charge density. Accordingly, the loose siRNA polyplexes inevitably expose siRNA to the extracellular environment during systemic circulation, resulting in low therapeutic efficiency and poor biodistribution. In this review, we highlight the innovative approaches to increase the size of siRNA via structural modification of the siRNA itself. The attempts include several methodologies such as hybridization, chemical polymerization, and micro- and nano-structurization of siRNA. Due to its increased charge density and flexibility, the structured siRNA can produce highly condensed and homogenous polyplexes compared to the classical monomeric siRNA. As a result, stable and compact siRNA polyplexes can enhance serum stability and target delivery efficiency in vivo with desirable biodistribution. The review specifically aims to provide the recent progress of structural modification of siRNA. In addition, the article also briefly and concisely explains the improved physico-chemical properties of structured siRNA with respect to stability, condensation ability and gene silencing efficiency.     

DNA binding activity of Anabaena sensory rhodopsin transducer probed by fluorescence correlation spectroscopy

Anabaena sensory rhodopsin transducer (ASRT) is believed to be a major player in the photo-signal transduction cascade, which is triggered by Anabaena sensory rhodopsin. Here, we characterized DNA binding activity of ASRT probed by using fluorescence correlation spectroscopy. We observed clear decrease of diffusion coefficient of DNA upon binding of ASRT. The dissociation constant, K_D, of ASRT to 20?bp-long DNA fragments lied in micro-molar range and varied moderately with DNA sequence. Our results suggest that ASRT may interact with several different regions of DNA with different binding affinity for global regulation of several genes that need to be activated depending on the light illumination.     

Generation of functional antibodies for mammalian membrane protein crystallography

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Potent MCH-1 receptor antagonists from cis-1,4-diaminocyclohexane-derived indane analogs

Benzimidazole and indane are the two key fragments in our potent and selective MCH-1 receptor (MCHR1) antagonists. To identify novel linkers connecting the two fragments, we investigated diamino-cycloalkane-derived analogs and discovered highly potent antagonists with cis-1,4-diaminocyclohexane as a unique spacer in this chemical class. Structural overlay suggested that cis-1-substituted-4-aminocyclohexane functions as a bioisostere of 4-substituted-piperidine and that the active conformation adopts a U-shaped orientation.