Gui-Xin Chou

Involvement of p38 MAPK signaling pathway in the anti-melanogenic effect of San-bai-tang, a Chinese herbal formula, in B16 cells

AIM OF THE STUDY San-bai-tang (SBT), a Chinese herbal formula, is traditionally used as a skin whitener in China. In our previous screening assays, SBT was identified as an effective tyrosinase inhibitor. In this study, we aim to investigate the anti-melanogenic effect and mechanisms of SBT in B16 cells. MATERIALS AND METHODS Cell viability was examined by the MTT assay. Cellular tyrosinase activity and melanin content were determined using spectrophotographic methods. Protein expression was analyzed by immunoblotting. RESULTS SBT inhibited tyrosinase activity with an IC(50) of 215.6 ± 10.3 μg/ml, and decreased cellular melanin content with an IC(50) of 254.8 ± 14.5 μg/ml at 48 h. MTT assay demonstrated that 48-h SBT (50-400 μg/ml) treatment did not show obvious cytotoxicity. Immunoblot analysis showed that SBT (100, 200 or 400 μg/ml) treatment for 48 h down-regulated the expression levels of phosphorylated-p38, MITF, tyrosinase, TRP-1 and TRP-2 in a dose-dependent manner. CONCLUSIONS SBT inhibited melanogenesis in B16 cells, and suppression of p38 MAPK signaling pathway contributed to the anti-melanogenic effect of SBT by down-regulating the expression of MITF and melanogenic enzymes. These novel findings demonstrated the anti-melanogenic effect and mechanisms of SBT, and provide pharmacological basis for the traditional use of SBT.

Flavonoids, apigenin and icariin exert potent melanogenic activities in murine B16 melanoma cells

We aimed to screen for melanogenic agents among 35 botanical compounds. The compounds were first assessed with regard to their effects on tyrosinase activity in B16 cells. At 100 μM, 13 compounds showed tyrosinase activity-enhancing effects, ranging from 2.6 to 372.8% activation. Five of them showed more than 50% enhancement and were further tested for their EC(50) values. Compared with 8-Methoxypsoralen, an effective tyrosinase activator with an EC(50) of 7.26 μM, 3 compounds exhibited smaller EC(50) values (apigenin, 0.45 μM; hyperosid, 0.92 μM; and icariin, 1.01 μM for enhancing tyrosinase activity). The 3 compounds significantly increased cellular melanin contents without affecting cell proliferation. Compared with 8-Methoxypsoralen (EC(50), 35.94 μM for stimulating pigmentation), apigenin (EC(50), 17.46 μM) and icariin (EC(50), 32.77 μM) showed better melanogenic activity, while hyperosid (EC(50), 70.4 μM) was less potent. Western blot analysis demonstrated that the 3 compounds could differentially increase the expression levels of tyrosinase, and tyrosinase-related proteins 1 and 2. Together these data suggest that apigenin and icariin exert potent melanogenic activities through, at least in part, upregulating the protein expression levels of melanogenic enzymes in B16 cells. Thus, further investigations are merited to ascertain their potential application in treating hypopigmentation disorders.

Effects of Sesquiterpenes Isolated From Largehead Atractylodes Rhizome on Growth, Migration, and Differentiation of B16 Melanoma Cells

The aims of this study were to isolate sesquiterpene compounds from the largehead atractylodes rhizome (LAR) and to investigate their effects on B16 cancer cells. A total of 8 sesquiterpenes from LAR were identified, of which eudesm-4 (15), 7-diene-9α, 11-diol (7) was isolated for the first time. All 8 compounds inhibited growth of B16 cells, and atractylenolide I (AT-I), atractylenolide II (AT-II), and atractylenolactam (ATR) were the most potent, with IC50 values of 76.46, 84.02, and 54.88 μΜ, respectively. Monomer lactone or lactam structures in the 8 compounds appeared to be critical for their antiproliferative activities. In addition, AT-I, AT-II, and ATR could induce cell differentiation and inhibit cell migration. Western blot analysis indicated that 2 of the compounds, AT-I and AT-II, could inactivate ERK, where all 3 inhibited AKT activation, suggesting that Ras/ERK and PI3K/AKT signaling pathways are involved in the action mechanisms of the LAR sesquiterpene compounds.

Inhibition of the STAT3 signaling pathway contributes to apigenin-mediated anti-metastatic effect in melanoma.

Signal transducer and activator of transcription 3 (STAT3) signaling is constantly activated in human melanoma, and promotes melanoma metastasis. The dietary flavonoid apigenin is a bioactive compound that possesses low toxicity and exerts anti-metastatic activity in melanoma. However, the anti-metastasis mechanism of apigenin has not been fully elucidated. In the present study, we showed that apigenin suppressed murine melanoma B16F10 cell lung metastasis in mice, and inhibited cell migration and invasion in human and murine melanoma cells. Further study indicated that apigenin effectively suppressed STAT3 phosphorylation, decreased STAT3 nuclear localization and inhibited STAT3 transcriptional activity. Apigenin also down-regulated STAT3 target genes MMP-2, MMP-9, VEGF and Twist1, which are involved in cell migration and invasion. More importantly, overexpression of STAT3 or Twist1 partially reversed apigenin-impaired cell migration and invasion. Our data not only reveal a novel anti-metastasis mechanism of apigenin but also support the notion that STAT3 is an attractive and promising target for melanoma treatment.

Screening of Chinese herbal medicines for antityrosinase activity in a cell free system and B16 cells

AIM OF THE STUDY Tyrosinase inhibitors are becoming increasingly important in controlling skin hyperpigmentation. We aimed to screen 50 extracts from traditional Chinese medicines (TCM) for tyrosinase activity-inhibiting agents. MATERIALS AND METHODS The 50 herbal extracts were prepared from 32 herbs and 18 TCM formulas, which are used as folk skin whiteners in China and have not been investigated for their skin-whitening mechanisms. Each herb and formula was extracted with 30% ethanol and water, respectively, and followed by column chromatography for isolating bioactive substances such as saponins, flavonoids and alkaloids for the antityrosinase activity study. Every extract was tested using the cell free mushroom tyrosinase inhibitory assay at 2 mg/ml for the single herb extracts and 1mg/ml for formula extracts. Extracts showing greater than 50% inhibition against mushroom tyrosinase activity were further examined by cellular tyrosinase assay in mouse B16 cells. The cytotoxicity in B16 cells was measured by methyl thiazolyl tetrazolium bromide (MTT) assay. RESULTS In the cell-free assay, 10 out of the 50 extracts demonstrated more than 50% inhibition against mushroom tyrosinase activity. These 10 extracts were further assessed by cellular tyrosinase assay, and 6 showed>50% inhibition with IC(50) values <1 mg/ml. The 6 extracts are from 3 herbs namely Ampelopsis japonica, Lindera aggregata, and Polygonatum odoratum, and 3 formulas namely Qian-wang-hong-bai-san, Qiong-yu-gao, and San-bai-tang. As compared with vitamin C, these 6 extracts showed similar or greater ratio of cell growth IC(50) to cellular tyrosinase IC(50). As compared with arbutin, extract from Ampelopsis japonica, Lindera aggregata, Qian-wang-hong-bai-san, or San-bai-tang had a similar, although extract from Polygonatum odoratum or Qiong-yu-gao had a greater, IC(50) value against murine tyrosinase activity. CONCLUSIONS From the screening assays we identified three Chinese medicinal herbs and three TCM formulas that have appreciable antityrosinase activity. Further studies are warranted to develop them as skin-whitening agents with convenient dosage forms or to identify active constituents from the extracts as useful leads for the development of skin whiteners.

Atractylenolide II induces G1 cell-cycle arrest and apoptosis in B16 melanoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE Atractylenolide II (AT-II) is a sesquiterpene compound isolated from the dried rhizome of Atractylodes macrocephala (Baizhu in Chinese), which is traditionally prescribed for melanoma treatment by Chinese medicine practitioners. Our previous study showed that AT-II can inhibit B16 cells proliferation. Here we investigate the mechanistic basis for the anti-proliferative activity of AT-II in B16 melanoma cells. MATERIALS AND METHODS Cell viability was examined by MTT assay. Cell cycle distribution and apoptosis were determined by flow cytometry. Protein expression was determined by Western blotting. RESULTS AT-II treatment for 48 h dose-dependently inhibited cell proliferation with an IC(50) of 82.3 μM, and induced G1 phase cell cycle arrest. Moreover, treatment with 75 μM AT-II induced apoptosis. These observations were associated with the decrease of the expression of Cdk2, phosphorylated-Akt, phosphorylated-ERK and Bcl-2, the increase of the expression of phosphorylated-p38, phosphorylated-p53, p21, p27, and activation of caspases-8, -9 and -3. In addition, a chemical inhibitor of p53, PFTα, significantly decreased AT-II-mediated growth inhibition and apoptosis. CONCLUSIONS We demonstrated that the G1-arresting and apoptotic effects of AT-II in B16 cells involve p38 activation as well as ERK and Akt inactivation, and the cytotoxic/apoptotic effects of AT-II are potentially p53 dependent. These findings provided chemical and pharmacological basis for the traditional application of Baizhu in melanoma treatment.

Activation of p38 MAPK pathway contributes to the melanogenic property of apigenin in B16 cells

Abstract:  We investigated the involvement of MAPK pathways in the melanogenic effect of apigenin in B16 cells. Apigenin treatment for 48 h dose (5–20 μm)‐dependently up‐regulated protein expression levels of microphthalmia‐associated transcription factor (MITF) and melanogenic enzymes including tyrosinase, tyrosinase‐related protein‐1 (TRP‐1) and TRP‐2 and enhanced the phosphorylation of p38 MAPK, without affecting the phosphorylation of JNK or ERK MAPK. Treatment with 10 μm apigenin time (6‐48 h)‐dependently elevated the protein expressions of p‐p38, MITF and melanogenic enzymes. Moreover, PD169316, a selective inhibitor of p38 kinase, suppressed the stimulatory effects of apigenin on tyrosinase activity and melanin synthesis, which were accompanied by decreased MITF protein expression. In conclusion, apigenin increased melanogenesis in B16 cells, at least in part, by activating the p38 MAPK pathway. The novel findings of this study shed light on the molecular mechanisms underlying the melanogenic activity of apigenin and suggest that apigenin/its derivatives may be potentially used for treating hypopigmentation disorders.

Isolation of anticancer constituents from flos genkwa (Daphne genkwa Sieb.et Zucc.) through bioassay-guided procedures.

BackgroundFlos Genkwa (yuanhua in Chinese), the dried flower buds of Daphne genkwa Sieb.et Zucc. (Thymelaeaceae), is a traditional Chinese medicinal herb mainly used for diuretic, antitussive, expectorant, and anticancer effects. However, systematic and comprehensive studies on Flos Genkwa and its bioactivity are limited.ResultsAfter confirmation of the anti-tumor activity, the 95% ethanolic extract was subjected to successive solvent partitioning to petroleum ether, dichloromethane, n-butanol, and water soluble fractions. Each fraction was tested using the same biological activity model, and the dichloromethane fraction had the highest activity. The dichloromethane fraction was subjected to further chromatographic separation for the isolation of compounds 1–13. Among the 13 compounds, the diterpene esters (compounds 10–13) showed anticancer activity, whereas the flavonoids, lignanoids, and peptides showed moderate activity. Compound 13 was a new daphnane diterpenoid, which was named genkwanin VIII.The preliminary antitumor mechanism of yuanhuacine was studied by protein expression and cell cycle analysis in MCF-7 cancer cells.ConclusionThe present investigation tends to support the traditional use of Flos Genkwa for treating cancer. Through bioassay-guided fractionation and isolation techniques, the CH2Cl2 fraction was determined as the active fraction of the flower buds of D. genkwa, and the anti-tumor activity was ascribable to the compounds 10–13.

Inhibition of the p38 and PKA signaling pathways is associated with the anti-melanogenic activity of Qian-wang-hong-bai-san, a Chinese herbal formula, in B16 cells.

ETHNOPHARMACOLOGICAL RELEVANCE Qian-wang-hong-bai-san (QW), a Chinese herbal formula, is traditionally used as a skin whitening agent in China. AIM OF STUDY In our previous screening assays, QW was identified as an effective tyrosinase inhibitor. In this study, we aim to investigate the underlying mechanism of the anti-melanogenic effect of QW in B16 cells. MATERIALS AND METHODS Cytotoxicity of QW in B16 cell line was examined by MTT assay. Cellular tyrosinase activity was determined based on the melanin content measured at 475 nm with a microplate spectrophotometer. Protein expression was analyzed by Western blotting and quantified by Quantity One. RESULTS QW dose-dependently inhibited tyrosinase activity and decreased melanin content at 48 h without significant cytotoxicity in B16 cells. Western blot analysis showed that QW treatment down-regulated the expression levels of phospho-p38, phospho-CREB, MITF, tyrosinase, TRP-1 and TRP-2 in a dose-dependent manner. At the same time, QW treatment for 48 h inhibited IBMX-induced elevation of cellular melanin content and tyrosinase activity. However, the attenuation of IBMX-mediated up-regulations of phospho-CREB and phospho-PKA was readily observed with 60 min of QW treatment. CONCLUSIONS The anti-melanogenic activity of QW in B16 melanoma cells can be attributed, at least in part, to the inhibition of the p38 MAPK and PKA signaling pathways. These findings shed new light on the molecular mechanisms of the skin-whitening property of QW.

Inhibition of STAT3 signalling contributes to the antimelanoma action of atractylenolide II

Our previous studies showed that atractylenolide II (AT‐II) has antimelanoma effects in B16 melanoma cells. In this study, we investigated the involvement of STAT3 signalling in the antimelanoma action of AT‐II. Daily administration of AT‐II (12.5, 25 mg/kg, i.g.) for 14 days significantly inhibited tumor growth in a B16 xenograft mouse model and inhibited the activation/phosphorylation of STAT3 and Src in the xenografts. In B16 and A375 cells, AT‐II (20, 40 μm) treatment for 48 h dose‐dependently reduced protein expression levels of phospho‐STAT3, phospho‐Src, as well as STAT3‐regulated Mcl‐1 and Bcl‐xL. Overexpression of a constitutively active variant of STAT3, STAT3C in A375 cells diminished the antiproliferative and apoptotic effects of AT‐II. These data suggest that inhibition of STAT3 signalling contributes to the antimelanoma action of AT‐II. Our findings shed new light on the mechanism of action underlying the antimelanoma effects of AT‐II and provide further pharmacological basis for developing AT‐II as a novel melanoma chemopreventive/chemotherapeutic agent.