Natural product corynoline suppresses melanoma cell growth through inducing oxidative stress.

Natural product corynoline is a unique isoquinoline alkaloid extracted from traditional Chinese medicine Corydalis bungeana Turcz, whereas its anticancer properties have not been investigated. In this study, we found that corynoline potently impairs the growth of melanoma cells, B16F10, and A375 in a concentration-dependent manner. Treatment of melanoma cells with corynoline results in G2 cell arrest accompanied by reduced cdc2 activation. Furthermore, corynoline triggers apoptosis of melanoma cells, which is associated with increased expression of Bax and cleaved caspase-3. Mechanistic study indicates that corynoline strongly induces reactive oxygen species (ROS) generation and subsequent DNA damage as evidenced by γ-H2AX accumulation. Notably, the effect of corynoline on melanoma cell cycle and apoptosis is abolished by a ROS scavenger N-acetyl cysteine (NAC), indicating a ROS-dependent mechanism. Finally, corynoline significantly inhibits in vivo B16F10 melanoma tumor growth accompanied by reduced expression of Ki-67 in tumor tissue. Taken together, our data suggest that corynoline suppresses melanoma cell growth in vitro and in vivo by inducing oxidative stress and represents a potential therapeutic agent for melanoma patients.

Celastrol is a novel selective agonist of cannabinoid receptor 2 with anti-inflammatory and anti-fibrotic activity in a mouse model of systemic sclerosis.

BACKGROUND: Increasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis. PURPOSE: The purpose of this study was to discover novel cannabinoid receptor 2 (CB2) agonist and assess the potential of CB2 activation in treating systemic sclerosis. METHODS: A gaussia princeps luciferase-based split luciferase complementation assay (SLCA) was developed for detection of the interaction between CB2 and ß-arrestin2. A library of 366 natural products was then screened as potential CB2 agonist using SLCA approach. Several GPCR functional assays, including HTRF-based cAMP assay and calcium mobilization were also utilized to evaluated CB2 activation. Bleomycin-induced experimental systemic sclerosis was used to assess the in vivo anti-fibrotic effects. Dermal thickness and collagen content were evaluated via H&E and sirius red staining. RESULTS: Celastrol was identified as a new agonist of CB2 by using SLCA. Furthermore, celastrol triggers several CB2-mediated downstream signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, and receptor desensitization in a dose-dependent manner, and it has a moderate selectivity on CB1. In addition, celastrol exhibited the anti-inflammatory properties on lipopolysaccharide (LPS) treated murine Raw 264.7 macrophages and primary macrophages. Finally, we found that celastrol exerts anti-fibrotic effects in the bleomycin-induced systemic sclerosis mouse model accompanied by reduced inflammatory conditions. CONCLUSION: Taken together, celastrol is identified a novel selective CB2 agonist using a new developed arrestin-based SLCA, and CB2 activation by celastrol reduces the inflammatory response, and prevents the development of dermal fibrosis in bleomycin-induced systemic sclerosis mouse model.

Fargesin inhibits melanin synthesis in murine malignant and immortalized melanocytes by regulating PKA/CREB and P38/MAPK signaling pathways.

BACKGROUND: Fargesin is commonly used in the treatment of allergic rhinitis, inflammation, sinusitis and headache. OBJECTIVE: The aim of the study is to investigate a new function of fargesin against melanin production and its underlying molecular mechanism. METHODS: B16F10 mouse melanoma cells, Melan-a and human epidermal melanocytes were treated with different concentrations of fargesin for the indicated time. The extracellular and cellular melanin content was detected by spectrometry at 490 nm and 405 nm, respectively. RT-qPCR and Western blot analysis were used to exam the expression of melanogenic enzymes and the activities of PKA/CREB and p38 MAPK pathway components. Zebrafish was used as an in vivo model for studying the function of fargesin in regulating melanogenesis. RESULTS: Fargesin effectively inhibited melanin production at moderate dose in mouse B16F10 melanoma cells, normal melanocyte cell lines and zebrafish. The expression of microphthalmia-associated transcription factor (MITF), its downstream melanogenic enzymes and tyrosinase activity were also strongly reduced by fargesin. Moreover, the increase of melanin production induced by UVB and forskolin could be fully reversed by fargesin treatment. Fargesin also effectively inhibited the activation of PKA/CREB and p38 MAPK as well as their interactions, which in turn is responsible for the expression of MITF and melanogenic enzymes. CONCLUSIONS: These results show that fargesin can function as an anti-melanogenic agent, at least in part, by inhibiting PKA/CREB and p38/MAPK signaling pathways. Therefore, fargesin and its derivatives may potentially be used for preventing hyperpigmentation disorders in the future.

Plant natural product plumbagin presents potent inhibitory effect on human cytochrome P450 2J2 enzyme.

BACKGROUND: Cytochrome P450 2J2 (CYP2J2) is not only highly expressed in many kinds of human tumors, but also promotes tumor cell growth via regulating the metabolism of arachidonic acids. CYP2J2 inhibitors can significantly reduce proliferation, migration and promote apoptosis of tumor cells by inhibiting epoxyeicosatrienoic acids (EETs) biosynthesis. Therefore screening CYP2J2 inhibitors is a significant way for the development of anti-cancer drug. PURPOSE: The aim of this study was to identify a new CYP2J2 inhibitor from fifty natural compounds obtained from plants. STUDY DESIGN: CYP2J2 inhibitor was screened from a natural compounds library and further the inhibitory manner and mechanism were evaluated. Its cytotoxicity against HepG2 and SMMC-7721 cell lines was also estimated. METHODS: The inhibitory effect was evaluated in rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant CYP2J2 (rCYP2J2), using astemizole as a probe substrate and inhibitory mechanism was illustrated through molecular docking. The cytotoxicity was detected using SRB. RESULTS: In all candidates, plumbagin showed the strongest inhibitory effect on the CYP2J2-mediated astemizole O-demethylation activity. Further study revealed that plumbagin potently inhibited CYP2J2 activity with IC50 value at 3.82 µM, 3.37 µM and 1.17 µM in RLMs, HLMs and rCYP2J2, respectively. Enzyme kinetic studies showed that plumbagin was a mixed-type inhibitor of CYP2J2 in HLMs and rCYP2J2 with Ki value of 1.88 µM and 0.92 µM, respectively. Docking data presented that plumbagin interacted with CYP2J2 mainly through GLU 222 and ALA 223. Moreover, plumbagin showed strongly cytotoxic effects on hepatoma cell lines, such as HepG2 and SMMC-7721, with lower toxicity on rat primary hepatocytes. Plumbagin had no effect on the protein expression of CYP2J2 in HepG2 and SMMC-7721, while down-regulated the mRNA level of anti-apoptosis protein Bcl-2. CONCLUSION: This study found out a new CYP2J2 inhibitor plumbagin from fifty natural compounds. Plumbagin presented a potential of anti-cancer pharmacological activity.

S100a8/NF-κB signal pathway is involved in the 800-nm diode laser-induced skin collagen remodeling.

The 800-nm diode laser is widely used for hair removal and also promotes collagen synthesis, but the molecular mechanism by which dermis responses to the thermal damage induced by the 800-nm diode laser is still unclear. Ten 2-month-old mice were irradiated with the 800-nm diode laser at 20, 40, and 60 J/cm(2), respectively. Skin samples were taken for PCR, Western blot analysis, and histological study at day 3 or 30 after laser irradiation. The expression of S100a8 and its two receptors (advanced glycosylation end product-specific receptor, RAGE and toll-like receptor 4, TRL4) was upregulated at day 3 after laser treatments. P-p65 levels were also elevated, causing the increase of cytokine (tumor necrosis factor, TNF-α and interleukin 6, IL-6) and MMPs (MMP1a, MMP9). At day 30, PCR and Western blot analysis showed significant increase of type I and III procollagen in the dermis treated with laser. Importantly, skin structure was markedly improved in the laser-irradiated skin compared with the control. Thus, it seemed that S100a8 upregulation triggered NF-κB signal pathway through RAGE and TLR4, responding to laser-induced dermis wound healing. The involvement of the NF-κB pathway in MMP gene transcription promoted the turnover of collagen in the skin, accelerating new collagen synthesis.

Daidzein stimulates collagen synthesis by activating the TGF-ß/smad signal pathway.

BACKGROUND/OBJECTIVES: The objective of this study was to investigate the effects of daidzein on collagen metabolism and its underlying mechanism in cultured skin fibroblast and nude mouse skin. METHODS: Skin fibroblasts were exposed to different concentrations of daidzein (0.5-50 µg/mL) for 24 h or 48 h, respectively. Female nude mice were treated topically with 200 µg/mL daidzein once a day for 6 weeks. Cell viability and cell cycle were determined by MTT and flow cytometer. The transcriptional activity of collagen type I was evaluated and the expression of procollagen, matrix metalloproteinase-1 (MMP1) and MMP2 were measured by real-time polymerase chain reaction. A Western blot analysis was applied to detect the levels of phosphorylated-Smad2 and Smad3. RESULTS: In the daidzein-treated cells the expression of type I procollagen increased markedly while the expressions of MMP1, and MMP2 was significantly inhibited. Additionally, the mouse skin showed more collagen deposition after daidzein treatment. The levels of transforming growth factor (TGF)-ß, phosphorylated-smad2 and smad3 were also higher in the daidzein treated skin fibroblasts than in the controls. CONCLUSIONS: The results showed that daidzein treatment can increase skin collagen synthesis and inhibit collagen degradation in vitro and in vivo. It seems that TGF-ß/smad signalling pathways play an important role in daidzein-induced collagen accumulation.

Investigation of the 1064 nm Q-switched Nd:YAG laser on collagen expression in an animal model.

BACKGROUND AND PURPOSE: The objective of this study was to evaluate the changes of collagen expression and its possible molecular mechanism in the rat skin induced by 1064 nm Q-switched Nd:YAG laser treatments. METHODS: The dorsal skin of Sprague-Dawley (SD) rats was irradiated with the 1064 nm laser at fluences of 0, 0.6, 1.5, and 2.5 J/cm2, respectively. Then biochemical analysis was used to quantify hydroxyproline content in the skin. The mRNA expressions of procollagen, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) were analyzed by using reverse transcription-polymerase chain reaction (RT-PCR). The activities of mitogen-activated protein kinase (MAPK) family members were detected by Western blot analysis. RESULTS: The 1064 nm laser treatments led to a marked increase in collagen content in a dose-dependent manner. The expression of types I and III collagen, TIMP1 and TIMP2, in the skin was markedly upregulated, whereas the expression of MMP2 and MMP3 was significantly decreased after laser treatments. Both extracellular signal-related kinase (Erk)1/2 and JNK MAPK pathways were activated by the 1064 nm laser irradiation. CONCLUSIONS: The 1064 nm laser irradiation could markedly increase collagen synthesis and inhibit collagen degradation. The activation of Erk1/2 and JNK MAPK seems to play a role in collagen production in the rat skin, induced by the 1064 nm laser.

The 800-nm diode laser irradiation induces skin collagen synthesis by stimulating TGF-ß/Smad signaling pathway.

The 800-nm diode laser is used clinically for hair removal and leg vein clearance. However, the effects of the laser on skin collagen synthesis have not been established. This study aims to research whether the 800-nm laser can be used for non-ablative rejuvenation and its possible mechanism by using an animal model. Eight 2-month-old rats were irradiated with the 800-nm diode laser at 20, 40, and 60 J/cm(2), respectively. Skin samples were taken for histological study and dermal thickness measurement at day 30 after laser irradiation. The expression of procollagen type I, III, IV, transforming growth factor-ß (TGF-ß), Smad2, 3, 4, and phosphorylated-Smad2, 3 in the rat skin was analyzed 24 h after completing all laser treatments by using RT-PCR and Western blot. Immunohistochemistry was performed to evaluate the content of type I collagen in the skin at day 30 after laser irradiation. The 800-nm diode laser treatments markedly improved the histological structure and increased dermal thickness compared to the non-irradiated controls. Laser irradiation at 40 J/cm(2) significantly up-regulated the expression of procollagen type I and IV, TGF-ß and Smad2, 3, 4. The p-Smad2 and p-Smad3 levels were also enhanced in the laser-irradiated skin. The 800-nm laser is effective in improving skin structure and inducing skin new collagen expression. New collagen synthesis induced by the 800-nm laser was mediated by TGF-ß/Smad signaling pathway. Thus, it seemed that the 800-nm laser could be used for non-ablative rejuvenation in the future.

Effects of the 532-nm and 1,064-nm Q-switched Nd:YAG lasers on collagen turnover of cultured human skin fibroblasts: a comparative study.

Cultured human skin fibroblasts were irradiated twice successively with the 1.5 J/cm(2) of 532-nm and 1,064-nm lasers, respectively. The mRNA of procollagen, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), heat-shock protein 70 (Hsp70), interleukin-6 (IL-6) and transforming growth factor beta (TGF-beta) were analyzed at 24 and 48 h post-irradiation by using RT-PCR. Both lasers significantly increased the expression of type I and III procollagen, TIMP1, and TIMP2, but decreased MMP1 and MMP2 expression. The 1,064-nm laser initiated TGF-beta expression while the 532-nm laser elicited the increase of Hsp70 and IL-6. The increase/decrease rates of procollagen, TIMPs and MMPs for the 1,064-nm laser were higher than that of the 532-nm laser. Thus, both lasers effectively accelerated collagen synthesis and inhibited collagen degradation. Collagen synthesis induced by the 1,064-nm laser might be partly due to the upregulation of TGF-beta expression, while the increase of Hsp70 and IL-6 might be partly responsible for collagen synthesis stimulated by the 532-nm laser. With the parameters used in this study, the 1,064-nm infrared laser is more effective in promoting the beneficial molecular activities than the 532-nm visible laser.