Isoalantolactone exerts anti-melanoma effects via inhibiting PI3K/AKT/mTOR and STAT3 signaling in cell and mouse models.

BACKGROUND AND AIM: Although the anti-cancer activity of isoalantolactone (IATL) has been extensively studied, the anti-melanoma effects of IATL are still unknown. Here, we have investigated the anti-melanoma effects and mechanism of action of IATL. MTT and crystal violet staining assays were performed to detect the inhibitory effect of IATL on melanoma cell viability. Apoptosis and cell cycle arrest induced by IATL were examined using flow cytometry. The molecular mechanism of IATL was explored by Western blotting, confocal microscope analysis, molecular docking, and cellular thermal shift assay (CETSA). A B16F10 allograft mouse model was constructed to determine the anti-melanoma effects of IATL in vivo. The results showed that IATL exerted anti-melanoma effects in vitro and in vivo. IATL induced cytoprotective autophagy in melanoma cells by inhibiting the PI3K/AKT/mTOR signaling. Moreover, IATL inhibited STAT3 activation both in melanoma cells and allograft tumors not only by binding to the SH2 domain of STAT3 but also by suppressing the activity of its upstream kinase Src. These findings demonstrate that IATL exerts anti-melanoma effects via inhibiting the STAT3 and PI3K/AKT/mTOR signaling pathways, and provides a pharmacological basis for developing IATL as a novel phytotherapeutic agent for treating melanoma clinically.

A traditional prescription comprising Astragali Radix and Schisandra chinensis Fructus induces apoptosis and protective autophagy in hepatocellular carcinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatocellular carcinoma (HCC) poses a growing challenge to global health efforts. The 5-year survival rate of HCC patients is still dismal. A traditional prescription Qi-Wei-Wan (QWW) comprising Astragali Radix and Schisandra chinensis Fructus has traditionally been used for HCC treatment according to traditional Chinese medicine theory, but the pharmacological basis is not clear. AIM OF THE STUDY: This study aims to investigate the anti-HCC effects of an ethanolic extract of QWW (hereafter, QWWE) and the mechanism of action. MATERIALS AND METHODS: An UPLC-Q-TOF-MS/MS method was developed to control the quality of QWWE. Two human HCC cell lines (HCCLM3 and HepG2) and a HCCLM3 xenograft mouse model were employed to investigate the anti-HCC effects of QWWE. The anti-proliferative effect of QWWE in vitro was determined by MTT, colony formation and EdU staining assays. Apoptosis and protein levels were examined by flow cytometry and Western blotting, respectively. Nuclear presence of signal transducer and activator of transcription 3 (STAT3) was examined by immunostaining. Transient transfection of pEGFP-LC3 and STAT3C plasmids was performed to assess autophagy and determine the involvement of STAT3 signaling in QWWE's anti-HCC effects, respectively. RESULTS: We found that QWWE inhibited the proliferation of and triggered apoptosis in HCC cells. Mechanistically, QWWE inhibited the activation of SRC and STAT3 at Tyr416 and Tyr705, respectively; inhibited the nuclear translocation of STAT3; lowered Bcl-2 protein levels, while increased Bax protein levels in HCC cells. Over-activating STAT3 attenuated the cytotoxic and apoptotic effects of QWWE in HCC cells. Moreover, QWWE induced autophagy in HCC cells by inhibiting mTOR signaling. Blocking autophagy with autophagy inhibitors (3-methyladenine and chloroquine) enhanced the cytotoxicity, apoptotic effect and the inhibitory effect on STAT3 activation of QWWE. Intragastric administration of QWWE at 10 mg/kg and 20 mg/kg potently repressed tumor growth and inhibited STAT3 and mTOR signaling in tumor tissues, but did not significantly affect mouse body weight. CONCLUSION: QWWE exhibited potent anti-HCC effects. Inhibiting the STAT3 signaling pathway is involved in QWWE-mediated apoptosis, while blocking mTOR signaling contributes to QWWE-mediated autophagy induction. Blockade of autophagy enhanced the anti-HCC effects of QWWE, indicating that the combination of an autophagy inhibitor and QWWE might be a promising therapeutic strategy for HCC management. Our findings provide pharmacological justifications for the traditional use of QWW in treating HCC.

Gracillin exerts anti-melanoma effects in vitro and in vivo: role of DNA damage, apoptosis and autophagy.

BACKGROUND: Melanoma is an aggressive cancer. Gracillin has been reported to treat various types of cancer, such as colorectal and lung cancer. However, there is a paucity of research on the anti-melanoma effects of gracillin. PURPOSE: The aim of this study was to assess the anti-melanoma effects and mechanisms of action of gracillin in vitro and in vivo. METHODS: Cell viability was detected using MTT and crystal violet staining assays. Cell proliferation was examined by EdU staining assays. Cell cycle arrest and apoptosis were analyzed by flow cytometry. Autophagic flux was monitored under a confocal microscope. Protein levels were determined by immunoblotting. LY294002 and rapamycin (Rapa) were used to determine the involvement of PI3K/AKT/mTOR signaling in gracillin-mediated autophagy. Signal transducer and activator of transcription 3 (STAT3) was overactivated to explore the contribution of the STAT3 signaling pathway in the anti-melanoma effects of gracillin. A B16F10 allograft mouse model was developed to evaluate the anti-melanoma effects of gracillin in vivo. RESULTS: We demonstrated that in melanoma cells, gracillin inhibited proliferation, induced G0/G1 phase cell cycle arrest, evoked apoptosis, and triggered autophagic cell death. Gracillin induced DNA damage in melanoma cells. Moreover, it suppressed the phosphorylation/activation of PI3K, AKT, mTOR, and 4E-BP1 in melanoma cells. Inhibiting PI3K/AKT and mTOR activity using LY294002 and Rapa, respectively, increased the protein level of LC3B-II in gracillin-treated melanoma cells. Furthermore, gracillin downregulated the protein levels of p-JAK2 (Tyr1007/1008), p-Src (Tyr416), and p-STAT3 (Tyr705) in melanoma cells. Over-expression of STAT3 in A375 cells significantly mitigated the cytotoxic and apoptotic effects of gracillin. In vivo studies showed that gracillin (1 mg/kg or 8 mg/kg, administered intraperitoneally for 16 consecutive days) suppressed B16F10 tumor growth and Src/STAT3 and AKT/mTOR signaling in tumors. No overt toxicity was observed in mice. CONCLUSION: Induction of DNA damage, inhibition of PI3K/AKT/mTOR signaling and suppression of STAT3 signaling are involved in gracillin-mediated cell cycle arrest, autophagic cell death and apoptosis, respectively, in melanoma cells. These findings provide novel insights into the anti-melanoma molecular mechanisms of gracillin, and suggest a potential role of gracillin in melanoma management.

Exploring the Effective Components and Mechanism of Action of Japanese Ardisia in the Treatment of Autoimmune Hepatitis Based on Network Pharmacology and Experimental Verification.

Japanese Ardisia is widely used as a hepatoprotective and anti-inflammatory agent in China. However, the active ingredients in Japanese Ardisia and their potential mechanisms of action in the treatment of autoimmune hepatitis (AIH) are unknown. The pharmacodynamic substance and mechanism of action of Japanese Ardisia in the treatment of AIH were investigated using network pharmacology and molecular docking technology in this study. Following that, the effects of Japanese Ardisia were evaluated using the concanavalin A (Con A)-induced acute liver injury rat model. The active ingredients and targets of Japanese Ardisia were searched using the Traditional Chinese Medicine Systems Pharmacology database, and hepatitis-related therapeutic targets were identified through GeneCards and Online Mendelian Inheritance in Man databases. A compound-target network was then constructed using Cytoscape software, and enrichment analysis was performed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Molecular docking technology was used to simulate the docking of key targets, and the AIH rat model was used to validate the expression of key targets. Nineteen active chemical components and 143 key target genes were identified. GO enrichment analysis revealed that the treatment of AIH with Japanese Ardisia mainly involved DNA-binding transcription factor binding, RNA polymerase II-specific DNA transcription factor binding, cytokine receptor binding, receptor-ligand activity, ubiquitin-like protein ligase binding, and cytokine activity. In the KEGG enrichment analysis, 165 pathways were identified, including the lipid and atherosclerotic pathway, IL-17 signaling pathway, TNF signaling pathway, hepatitis B pathway, and the AGE-RAGE signaling pathway in diabetic complications. These pathways may be the key to effective AIH treatment with Japanese Ardisia. Molecular docking showed that quercetin and kaempferol have good binding to AKT1, IL6, VEGFA, and CASP3. Animal experiments demonstrated that Japanese Ardisia could increase the expression of AKT1 and decrease the expression of CASP3 protein, as well as IL-6, in rat liver tissues. This study identified multiple molecular targets and pathways for Japanese Ardisia in the treatment of AIH. At the same time, the effectiveness of Japanese Ardisia in treating AIH was verified by animal experiments.

Chrysoeriol suppresses hyperproliferation of rheumatoid arthritis fibroblast-like synoviocytes and inhibits JAK2/STAT3 signaling.

BACKGROUND: Fibroblast-like synoviocytes (FLS) have cancer cell-like characteristics, such as abnormal proliferation and resistance to apoptosis, and play a pathogenic role in rheumatoid arthritis (RA). Hyperproliferation of RA-FLS that can be triggered by the activation of interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling destructs cartilage and bone in RA patients. Chrysoeriol is a flavone found in medicinal herbs such as Chrysanthemi Indici Flos (the dried capitulum of Chrysanthemum indicum L.). These herbs are commonly used in treating RA. Chrysoeriol has been shown to exert anti-inflammatory effects and inhibit STAT3 signaling in our previous studies. This study aimed to determine whether chrysoeriol inhibits hyperproliferation of RA-FLS, and whether inhibiting STAT3 signaling is one of the underlying mechanisms. METHODS: IL-6/soluble IL-6 receptor (IL-6/sIL-6R)-stimulated RA-FLS were used to evaluate the effects of chrysoeriol. CCK-8 assay and crystal violet staining were used to examine cell proliferation. Annexin V-FITC/PI double staining was used to detect cell apoptosis. Western blotting was employed to determine protein levels. RESULTS: Chrysoeriol suppressed hyperproliferation of, and evoked apoptosis in, IL-6/sIL-6R-stimulated RA-FLS. The apoptotic effect of chrysoeriol was verified by its ability to cleave caspase-3 and caspase-9. Mechanistic studies revealed that chrysoeriol inhibited activation/phosphorylation of Janus kinase 2 (JAK2, Tyr1007/1008) and STAT3 (Tyr705); decreased STAT3 nuclear level and down-regulated protein levels of Bcl-2 and Mcl-1 that are transcriptionally regulated by STAT3. Over-activation of STAT3 significantly diminished anti-proliferative effects of chrysoeriol in IL-6/sIL-6R-stimulated RA-FLS. CONCLUSIONS: We for the first time demonstrated that chrysoeriol suppresses hyperproliferation of RA-FLS, and suppression of JAK2/STAT3 signaling contributes to the underlying mechanisms. This study provides pharmacological and chemical justifications for the traditional use of chrysoeriol-containing herbs in treating RA, and provides a pharmacological basis for developing chrysoeriol into a novel anti-RA agent.

A proprietary herbal drug Young Yum Pill ameliorates chronic fatigue syndrome in mice.

BACKGROUND: Chronic fatigue syndrome (CFS) is a complex disease with few effective and safe therapies. Young Yum Pill (YYP), a proprietary herbal drug, has been used to relieve CFS-like symptoms. The pharmacological basis of this application of YYP is unknown. PURPOSE: This study aimed to investigate the pharmacological effects and mechanisms of action of YYP in a mouse model of CFS. STUDY DESIGN AND METHODS: A food restriction and exhaustive swimming-induced mouse CFS model was used to evaluate the effects of YYP. Lymphocyte proliferation was assessed by MTT assays. T-lymphocyte subsets were analyzed by flow cytometry. Serum biochemical parameters were determined using commercial kits. Protein levels were measured by immunoblotting. RESULTS: Intragastric administration of YYP (2.85, 5.70, 11.40 g/kg) daily for 21 consecutive days significantly prolonged swimming time and diminished body weight loss of CFS mice. Mechanistic investigations revealed that YYP increased thymus and spleen indices of CFS mice, enhanced proliferation of lipopolysaccharide- or concanavalin A-stimulated spleen lymphocytes, and increased CD3+CD4+ and CD3+CD8+ T-cells in the spleen. YYP increased glycogen content in gastrocnemius muscle and liver, and lowered levels of triglyceride, lactic acid and urea nitrogen in sera of CFS mice. YYP suppressed the elevation of serum level of malondialdehyde, the increase of activities of lactic dehydrogenase and creatine phosphokinase, and the decrease of activity of the serum antioxidant enzyme superoxide dismutase in CFS mice. Moreover, YYP upregulated protein level of activated AMPK in gastrocnemius muscle and liver of CFS mice. CONCLUSIONS: YYP ameliorates CFS by reversing metabolic changes, reducing oxidative damage, and improving some immune function parameters in mice. This study provides pharmacological justifications for the use of YYP in treating fatigue, including CFS.

The anti-inflammatory effects of an ethanolic extract of the rhizome of Atractylodes lancea, involves Akt/NF-κB signaling pathway inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried rhizome of Atractylodes lancea (Thumb.) DC. (Compositae) has been prescribed in folk medicine for the management of various inflammatory conditions such as rheumatic diseases, gastritis and hepatitis. However, the molecular mechanisms underlying the beneficial properties of this herb remain elusive. AIM OF THE STUDY: In this study, we investigated the anti-gastritis activities of Al-EE (an ethanolic extract of the herb) and explored the mechanism of action. MATERIALS AND METHODS: An ethanolic extract of the Atractylodes lancea (Thumb.) DC. (Compositae) rhizome, Al-EE, was prepared with ethanol (95%) and quality controlled using HPLC analysis. To determine the in vivo effects of this extract, we utilised a HCl/EtOH-induced gastritis rat model. In vitro assays were carried out using a lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cell model. MTT assays were used to examine cell viability, while Griess assays were carried out to measure nitric oxide (NO) production. Messenger RNA expression was examined by real-time PCR. Prostaglandin E2 (PGE2) production was examined using ELISA assays. To examine protein expression and enzymatic activities, we employed western blot analysis. Nuclear transcription factor (NF)-κB activity was determined by Luciferase reporter assays. RESULTS: The content of atractylenolide (AT)-1 and AT-2 in Al-EE was 0.45% and 5.07% (w/w), respectively (Supplementary Fig. 1). Al-EE treatment suppressed the production of NO and PGE2, reduced the mRNA expression of inducible NO synthase (iNOS), cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)-α, while also reducing the protein levels of iNOS and COX-2 in RAW264.7 macrophage cells. Furthermore, Al-EE inhibited the nuclear protein levels of NF-κB (p65) and NF-κB-driven luciferase reporter gene activity in RAW264.7 macrophage cells. Critically, intra-gastric injection of Al-EE (25 mg/kg) attenuated HCl/EtOH-induced gastric damage in SD rats, while the phosphorylation of Akt and IκBα was suppressed by Al-EE in vitro and in vivo. CONCLUSION: In summary, Al-EE has significant anti-gastritis effects in vivo and in vitro, which can be associated with the inhibition of the Akt/IκBα/NF-κB signalling pathway. This mechanistic finding provides a pharmacological basis for the use of the A. lancea rhizome in the clinical treatment of various inflammatory conditions.

A two-herb formula inhibits hyperproliferation of rheumatoid arthritis fibroblast-like synoviocytes.

Fibroblast-like synoviocytes (FLS) play a pathogenic role in rheumatoid arthritis (RA). STAT3 signaling is activated in FLS of RA patients (RA-FLS), which in turn causes RA-FLS hyperproliferation. RL is a traditional remedy for treating inflammatory diseases in China. It comprises Rosae Multiflorae Fructus and Lonicerae Japonicae Flos. A standardized ethanolic extract of RL (RLE) has been shown to exert anti-arthritic effects in collagen-induced arthritis (CIA) rats. Some constituents of RLE were reported to inhibit JAK2/STAT3 signaling in rat FLS. Here, we determined whether RLE inhibits FLS hyperproliferation, and explored the involvement of STAT3 signaling in this inhibition. In joints of CIA rats, RLE increased apoptotic FLS. In IL-6/sIL-6R-stimulated RA-FLS, RLE reduced cell viability and evoked cell apoptosis. In synovial tissues of CIA rats, RLE lowered the protein level of phospho-STAT3. In IL-6/sIL-6R-stimulated RA-FLS, RLE inhibited activation/phosphorylation of STAT3 and JAK2, decreased the nuclear localization of STAT3, and downregulated protein levels of Bcl-2 and Mcl-1. Over-activation of STAT3 diminished RLE's anti-proliferative effects in IL-6/sIL-6R-stimulated RA-FLS. In summary, RLE inhibits hyperproliferation of FLS in rat and cell models, and suppression of STAT3 signaling contributes to the underlying mechanisms. This study provides further pharmacological groundwork for developing RLE as a modern anti-arthritic drug.

A two-herb formula inhibits STAT3 signaling and exerts anti-melanoma effects in cell and animal models.

ETHNOPHARMACOLOGICAL RELEVANCE: Malignant melanoma is a fatal cancer. Signal transducer and activator of transcription 3 (STAT3) has been proposed as a therapeutic target of melanoma. An herbal formula Huai-Hua-San (HHS) comprising Sophorae Flos (SF) and Gardeniae Fructus (GF) is traditionally used for treating cancers including melanoma, but the pharmacological basis is unknown. AIMS OF THIS STUDY: This study aimed to investigate the anti-melanoma effects of an ethanolic extract of HHS (HHSE), and explore the involvement of STAT3 signaling in the effects. MATERIALS AND METHODS: An UPLC-TOF/MS method was developed to control the quality of HHSE. A B16F10 allograft mouse model and three melanoma cell lines (B16F10, A375 and A2058) were used to determine the anti-melanoma effects of HHSE. Dacarbazine (DTIC) and Stattic were used as positive controls. Cell viability was detected using MTT and crystal violet staining assays. Cell apoptosis was analyzed by flow cytometry after the cells were stained with Annexin-V/PI. Cell invasive ability was examined using the transwell assay. Protein levels were determined by Western blotting. RESULTS: The contents of crocin I, crocin II, quercetin and kaempferol in HHSE were 0.59%, 0.98%, 4.66% and 1.15%, respectively. A clinically relevant dose of HHSE (0.1 g/kg/day, i.g. for 15 consecutive days) significantly suppressed B16F10 tumor growth in mice. HHSE dose-dependently reduced cell viability and dampened invasion of, and induced apoptosis in, melanoma cells. Mechanistic studies revealed that HHSE inhibited the phosphorylation/activation of STAT3 in B16F10 allografts and in cultured melanoma cells. In cell models, HHSE also inhibited the phosphorylation of STAT3 upstream kinases, JAK2 (Tyr1007/1008) and Src (Tyr416), lowered STAT3 nuclear levels, and down-regulated the protein levels of STAT3-targeted molecules. Over-activation of STAT3 in A375 cells significantly attenuated the cytotoxic effects of HHSE. CONCLUSIONS: HHSE exhibits anti-melanoma effects in cell and mouse models. Inhibition of STAT3 signaling contributes to the anti-melanoma mechanisms of HHSE. Our findings lay a groundwork for developing HHSE as a modern agent for melanoma management, and provide pharmacological justifications for the traditional use of HHS in treating melanoma.

Chrysoeriol ameliorates TPA-induced acute skin inflammation in mice and inhibits NF-κB and STAT3 pathways.

BACKGROUND: Chrysoeriol is a flavone found in diverse dietary and medicinal herbs such as Lonicerae Japonicae Flos (the dried flower bud or newly bloomed flower of Lonicera japonica Thunb.). These herbs are commonly used for treating inflammatory diseases. Herbal extracts containing chrysoeriol have been shown to have anti-inflammatory effects and inhibit nuclear factor-kappa B (NF-κB) signaling. Some of these extracts can inhibit signal transducers and activators of transcription 3 (STAT3) signaling in cancer cells. PURPOSE: This study aimed to determine whether chrysoeriol has anti-inflammatory effects and whether NF-κB and STAT3 pathways are involved in the effects. STUDY DESIGN AND METHODS: A TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ear edema mouse model and LPS-stimulated RAW264.7 cells were used to evaluate the effects of chrysoeriol. Griess reagent was used to measure the production of nitric oxide (NO). Western blot and enzyme-linked immunosorbent assays were employed to detect protein levels. RT-qPCR analyses were used to detect mRNA levels. Haematoxylin and eosin (H&E) staining was employed to examine the pathological conditions in animal tissues. RESULTS: In the mouse model, chrysoeriol ameliorated acute skin inflammation, evidenced by reduced ear thickness, ear weight and number of inflammatory cells in inflamed ear tissues. The compound lowered protein levels of phospho-p65 (Ser536), phospho-STAT3 (Tyr705), inducible nitric oxide synthases (iNOS), cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), IL-1ß and tumor necrosis factor α (TNF-α) in mouse swollen ears. In LPS-stimulated RAW264.7 cells, chrysoeriol also lowered levels of these proteins. In addition, chrysoeriol decreased the production of NO and prostaglandin E2; inhibited the phosphorylation of inhibitor of κB (Ser32), p65 (Ser536) and Janus kinase 2 (Tyr1007/1008); decreased nuclear localization of p50, p65 and STAT3; and down-regulated mRNA levels of pro-inflammatory cytokines IL-6, IL-1ß and TNF-α that are transcriptionally regulated by NF-κB and STAT3 in the cell model. CONCLUSION: We for the first time demonstrated that chrysoeriol ameliorates TPA-induced ear edema in mice, and that inhibition of JAK2/STAT3 and IκB/p65 NF-κB pathways are involved in the anti-inflammatory effects of chrysoeriol. This study provides chemical and pharmacological justifications for the use of chrysoeriol-containing herbs in treating inflammatory diseases, and provides pharmacological groundwork for developing chrysoeriol as a novel anti-inflammatory agent.

MiR-let-7a/f-CCR7 signaling is involved in the anti-metastatic effects of an herbal formula comprising Sophorae Flos and Lonicerae Japonicae Flos in melanoma.

BACKGROUND: Metastasized melanoma is extremely difficult to treat. Activation of C-C chemokine receptor type 7 (CCR7) has been linked to melanoma metastasis. CCR7 can be directly regulated by miR-let-7. We have previously shown that an ethanolic extract of an herbal formula comprising Sophorae Flos and Lonicerae Japonicae Flos (SLE) inhibits melanoma cell migration and invasion. PURPOSE: In this study, we determined whether SLE suppresses melanoma metastasis, and whether regulation of miR-let-7a/f-CCR7 signaling is involved in the effect. STUDY DESIGN AND METHODS: Small RNA sequencing was conducted to compare miRNA expression profiles of B16F10 tumors dissected from SLE-treated or untreated mice. Western blot and RT-qPCR analyses were employed to examine protein and miRNA levels, respectively. A B16F10 melanoma lung metastasis mouse model was used to evaluate the effects of SLE on melanoma metastasis. MiR-let-7a/f-knockdown and CCR7-overexpression cell models were used to investigate the involvement of miR-let-7a/f-CCR7 signaling in the anti-metastatic effects of SLE. RESULTS: It was found that SLE upregulated levels of miR-let-7a/f in B16F10 melanoma tissues. SLE significantly elevated levels of miR-let-7a/f, lowered the protein level of CCR7, inhibited the phosphorylation of CCR7 downstream molecules p38 and JNK in B16F10 and A375 melanoma cells. SLE inhibited B16F10 melanoma lung metastasis in mice. SLE upregulated levels of miR-let-7a/f, and lowered protein levels of CCR7, MMP-2, MMP-9, phospho-p38 (Thr180/Tyr182) and phospho-JNK (Thr183/Tyr185) in melanoma-invaded lung tissues. Knockdown of miR-let-7a/f diminished the effects of SLE on CCR7 signaling in, and invasion of, melanoma cells. Overexpression of CCR7 lessened the effects of SLE in inhibiting the phosphorylation of p38 and JNK in, and the invasive capability of, melanoma cells. CONCLUSION: We for the first time demonstrated that SLE inhibits melanoma metastasis in mice, and that regulation of the miR-let-7a/f-CCR7 pathway contributes to the anti-metastatic mechanisms of SLE. These findings provide a pharmacological basis for developing SLE as a modern agent for treating metastatic melanoma. Additionally and importantly, this study suggests that regulating the miR-let-7a/f-CCR7 pathway is a novel strategy for controlling melanoma metastasis.

Arnicolide D exerts anti-melanoma effects and inhibits the NF-κB pathway.

BACKGROUND: Melanoma is a lethal cancer. NF-κB has been validated as a molecular target for melanoma treatment. Current therapies for melanoma have limitations. Novel targeted therapeutics are needed. Arnicolide D (Ar-D), a sesquiterpene lactone isolated from the dried whole plant of Centipeda minima (L.) A. Br. et Aschers., has been reported to inhibit NF-κB activity in colorectal cancer cells. PURPOSE: To investigate the anti-melanoma effects of Ar-D in vitro and in vivo; and to determine whether Ar-D inhibits the NF-κB pathway in melanoma cells. METHODS: A B16F10 allograft mouse model and two melanoma cell lines (A375 and B16F10) were used to investigate the anti-melanoma effects of Ar-D in vivo and in vitro. Dacarbazine was used as a positive control. Cell viability was assessed by MTT and crystal violet staining assays. Cell cycle arrest and apoptosis were analyzed by flow cytometry. Protein levels were determined by Immunoblotting. RESULTS: In vivo assays showed that the average tumor weight in Ar-D-treated group (4 mg/kg, i.p, 15 days) was reduced by 53.7%, when compared with the control group. In vitro studies demonstrated that Ar-D reduced cell viability, induced G2/M cell cycle arrest and apoptosis, elevated levels of cell cycle regulatory proteins p53 and p21, and lowered levels of G2/M checkpoint proteins Cdc2 and Cyclin B1 in melanoma cells. Mechanistically, Ar-D inhibited the activity of IKKα/ß, the degradation of IκBα, and the phosphorylation and expression of NF-κB p65 in melanoma cells. CONCLUSION: Ar-D has anti-melanoma effects, and inhibition of the IKK/IκBα/NF-κB p65 pathway is involved in the effects.

Schisandra fruits for the management of drug-induced liver injury in China: A review.

BACKGROUND: With increasing use of pharmaceuticals, drug-induced liver injury (DILI) has become a significant therapeutic challenge to physicians all over the world. Drugs based on Schisandra fruits (SF for short, the fruits of Schisandra chinensis or Schisandra sphenanthera) or synthetic analogues of schisandrin C, are commonly prescribed for treating DILI in China. PURPOSE: This review summarizes the literature regarding the application of SF-derived drugs in patients with DILI and current understanding of mechanisms underlying the protective effects of SF against liver injury. METHODS: Keywords related to drug-induced liver injury and Schisandra fruits were searched in the following databases: Pubmed, Cochrane Library, Google Scholar, LiverTox, China National Knowledge Infrastructure (CNKI), Chinese Scientific Journal database (VIP), and Wanfang database. All studies, published in English or Chinese, were included. Clinical study exclusion criteria: if patients received other Chinese herbal medicines in a study, the study will not be included in this review. RESULTS: Clinical studies have shown that SF-derived drugs are effective in inhibiting drug-induced elevation of serum levels of alanine aminotransferase, aspartate transaminase and total bilirubin. Cellular and animal studies have demonstrated that crude SF extracts, lignan compounds found in SF, and SF-derived drugs are effective in protecting the liver against xenobiotic-induced injury. Regulation of cytochrome P450 enzyme activity, anti-oxidation, anti-inflammation and acceleration of liver regeneration are involved in the hepatoprotective mechanisms of SF. CONCLUSION: SF-derived drugs are effective in ameliorating DILI in China. To verify the clinical efficacy of these drugs, high-quality clinical studies are needed.

A TCM formula comprising Sophorae Flos and Lonicerae Japonicae Flos alters compositions of immune cells and molecules of the STAT3 pathway in melanoma microenvironment.

A traditional Chinese medicine (TCM) formula (SL) comprising Sophorae Flos and Lonicerae Japonicae Flos was used for treating melanoma in ancient China. We have previously shown that an ethanolic extract of SL (SLE) possesses anti-melanoma effects and suppresses STAT3 signaling in vitro and in vivo. STAT3 has been linked to the development of melanoma immunosuppressive microenvironment. In this work, we investigated whether SLE inhibits melanoma growth by reprogramming the tumor microenvironment in mouse and co-culture cell models. In B16F10 melanoma-bearing mice, we found that intragastric administration of SLE (1.2 g/kg) dramatically inhibited tumor growth. This observation was associated with the downregulation of protein levels of phospho-STAT3 (Tyr 705) and STAT3-regulated immunosuppressive cytokines, and mRNA levels of STAT3-targeted genes involved in tumor growth and immune evasion. We also observed increased Th, Tc and dendritic cells in the melanomas and spleens in SLE-treated mice compared to that in control mice. In a co-culture system composed of B16F10 cells and mouse primary splenic lymphocytes, it was found that SLE not only inhibited STAT3 activation in B16F10 cells, but also downregulated mRNA levels of STAT3-targeted genes in the splenic lymphocytes. In this co-culture setting, SLE decreased the levels of STAT3-regulated immunosuppressive cytokines, increased the percentages of Th, Tc and dendritic cells as well. Furthermore, effects of SLE on STAT3 phosphorylation, cytokine levels and immune cell subtype percentages were significantly weaker in the B16STAT3C cells (stable cells harboring a constitutively active STAT3 variant STAT3C)/splenic lymphocytes co-culture system than in the B16V cells (cells stably transfected with the empty vector)/splenic lymphocytes co-culture system, indicating that STAT3 over-activation diminishes SLE's effects. In summary, our findings indicate that reprograming the immune microenvironment, partially mediated by inhibiting STAT3 signaling, contributes to the anti-melanoma mechanisms of SLE. This study provides further pharmacological groundwork for developing SLE as a modern agent for melanoma prevention/treatment, and supports the notion that reprograming immunosuppressive microenvironment is a viable anti-melanoma strategy.

Young Yum pill inhibits inflammatory mediators and nuclear factor-kappa B signaling in lipopolysaccharide-stimulated RAW 264.7 macrophages.

OBJECTIVE: To investigate the effect of Young Yum pill (YYP) on inflammatory mediators in cultured RAW 264.7 cells and elucidate the nuclear factor-kappa B (NF-κB)-related mechanism behind the action. METHODS: YYP was extracted with 95% ethanol Lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were used to evaluate the effect of YYP on inflammatory mediators. Production of nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess test and enzyme-linked immunosorbent assay, respectively. The levels of genes and proteins involved in the generation of inflammatory mediators were examined using real-time polymerase chain reaction and Western blotting, respectively. RESULTS: YYP dose-dependently suppressed LPS-induced production of NO, PGE2 and tumor necrosis factor-α (TNF-α), and elevation of mRNA and protein levels of inducible NO synthase and cyclooxygenase-2 in RAW 264.7 macrophages. These observations were associated with decreased NF-κB p65 phosphorylation and nuclear localization, enhanced Akt (protein kinase B) phosphorylation, as well as reduced inhibitor of κB (IκB) α degradation and IκB kinase α/ß phosphorylation. CONCLUSION: The present study demonstrated an inhibitory effect of YYP on the NF-κB-regulated inflammatory mediators NO, PGE2 and TNF-α in LPS-stimulated RAW 264.7 macrophages, providing a pharmacological basis for the use of YYP in treating inflammatory disorders.

[Synthesis of biodiesel from crude oil by immobilized lipase].

We used immobilized lipase from Candida sp. 99-125 to produce fatty acid methyl esters (FAMEs) from crude oil and methanol. We studied the effects of phospholipids on activity of immobilized lipase, reaction velocity, stability of immobilized lipase and the stability of immobilized lipase in crude and refined oil. Results showed that the activity of the lipase immersed in petroleum ether with 1% phospholipids dropped more quickly than the lipase in petroleum ether without phospholipids. When soybean oil was used without phospholipids as material, the FAMEs yield of 15 min was 26.2%, whereas the yield decreased to 12.4% when there were 5% phospholipids in the soybean oil. However when the phospholipids content was below 1%, the stability of the lipase did not change obviously. The lipase was stable when used to catalyze crude soybean oil and crude jatropha oil, after 10 cycles the FAMEs yield was still above 70%. This lipase showed great potential for industrial production of biodiesel from crude oil.