8-Epi-xanthatin induces the apoptosis of DU145 prostate carcinoma cells through signal transducer and activator of transcription 3 inhibition and reactive oxygen species generation.

Signal transducer and activator of transcription 3 (STAT3) is aberrantly activated in many human cancers. We tried to find STAT3 inhibitors from natural sources and found that Xanthium fruit extracts decreased phosphorylation of STAT3-Y705. 8-Epi-xanthatin (EXT) was isolated from the extracts. When DU145 cancer cells were treated with EXT, p-STAT3-Y705 was decreased with an IC50 of 3.2 µM. EXT decreased the expression of STAT3 target genes, such as cyclin A, cyclin D1, and BCL-2, and induced PARP cleavage, indicating apoptotic cell death. Downregulation of EXT-induced p-STAT3-Y705 was rescued by pretreating DU145 cells with antioxidants, such as N-acetyl-L-cysteine (NAC), indicating that reactive oxygen species (ROS) were involved in the EXT-induced inhibition of STAT3 activation. Furthermore, we proved the association of EXT with STAT3 protein by using a drug affinity responsive target stability (DARTS) assay and a cellular thermal shift assay (CETSA). EXT inhibited proliferation of DU145 cells with a GI50 of 6 µM and reduced tumor growth in mice xenografted with DU145 cells. Immunoblotting showed that phosphorylation of STAT3-Y705 was lower in EXT-treated tumor tissue than in control tissues. Collectively, we found that EXT binds to, and inhibits, STAT3 activation and could be a lead compound for anticancer therapy.

Corrigendum to "Activation of AMP-Activated Protein Kinase and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells".

[This corrects the article DOI: 10.1155/2013/974313.].

Jaceosidin Ameliorates Insulin Resistance and Kidney Dysfunction by Enhancing Insulin Receptor Signaling and the Antioxidant Defense System in Type 2 Diabetic Mice.

Emerging evidence has shown that flavonoids extracted from Artemisia have beneficial effects on metabolic disorders. However, whether and how jaceosidin ameliorates insulin resistance and diabetic nephropathy in type 2 diabetes mellitus is largely unknown. For 8 weeks, db/db diabetic mice were fed with or without jaceosidin. Oral jaceosidin supplementation reduced fasting blood glucose levels and insulin resistance through the upregulation of insulin receptor downstream pathways in the liver and skeletal muscles. While jaceosidin did not noticeably alter kidney filtration function, this dietary intervention contributed to attenuating the accumulation of advanced glycation end products in diabetic kidneys. The levels of VEGF-a (vascular endothelial growth factor-a) proteins in the diabetic kidneys were markedly diminished by jaceosidin treatments, which increased the expression and activity of Cu (copper) and Zn-SOD (zinc-superoxide dismutase). Therefore, it is suggested that jaceosidin supplementation elicits antidiabetic effects and treats diabetic nephropathy by augmenting insulin signaling, suppressing fibrosis, and enhancing antioxidant activity.

Ethacrynic acid inhibits STAT3 activity through the modulation of SHP2 and PTP1B tyrosine phosphatases in DU145 prostate carcinoma cells.

To identify signal transducer and activator of transcription factor 3 (STAT3) inhibitors, we generated STAT3-dependent gene expression signature by analyzing gene expression profiles of DU145 cancer cells treated with STAT3 inhibitor, piperlongumine and 2-hydroxycinnamaldehyde. Then we explored gene expression signature-based strategies using a connectivity map database and identified several STAT3 inhibitors, including ethacrynic acid (EA). EA is currently used as a diuretic drug. EA inhibited STAT3 activation in DU145 prostate cancer cells and consequently decreased the levels of STAT3 target genes such as cyclin A and MCL-1. Furthermore, EA treatment inhibited tumor growth in mice xenografted with DU145 cells and decreased p-STAT3 expression in tumor tissues. Knockdown of Src homology region 2 domain-containing phosphatase-2 (SHP2) or Protein tyrosine phosphatase 1B (PTP1B) gene expression by siRNA suppressed the ability of EA to inhibit STAT3 activation. When EA was combined with an activator of SHP2 or PTP1B, p-STAT3 expression was synergistically decreased; when EA was combined with an inhibitor of SHP2 or PTP1B, p-STAT3 expression was rescued. By using an affinity pulldown assay with biotinyl-EA, EA was shown to associate with SHP2 and PTP1B in vitro. Additionally, the drug affinity responsive target stability (DARTS) assay confirmed the direct binding of EA to SHP2 and PTP1B. SHP2 is activated by EA through active phosphorylation at Y580 and direct binding to SHP2. Collectively, our results suggest that EA inhibits STAT3 activity through the modulation of phosphatases such as SHP2 and PTP1B and may be a potential anticancer drug to target STAT3 in cancer progression.

2'-hydroxycinnamaldehyde inhibits cancer cell proliferation and tumor growth by targeting the pyruvate kinase M2.

It is reported that 2'-hydroxycinnamaldehyde (HCA), isolated from cinnamon, has anti-tumor effects through the modulation of multi-target molecules. In this study, we identified pyruvate kinase M2 (PKM2) as a direct target of HCA by use of biochemical methods including affinity chromatography, drug affinity responsive target stability, and cellular thermal shift assay. PKM2 is up-regulated in multiple cancer types and is considered as a potential target for cancer therapy. HCA binds directly to PKM2 and selectively decreases the phosphorylation of PKM2 at Tyr105, indicating a potential anti-proliferative effect on prostate cancer cells. As a PKM2 activator, HCA increases pyruvate kinase activity by promoting the tetrameric state of PKM2. However, HCA suppresses protein kinase activity of PKM2 by decreasing the phosphorylation at Tyr105. Moreover, this leads to a decrease of PKM2-mediated STAT3 phosphorylation at Tyr705 and a down-regulation of target genes, including MEK5 and cyclin D1. Furthermore, HCA suppresses tumor growth and the release of tumor extracellular vesicles in vivo by inhibiting the phosphorylation of PKM2. Collectively, our results suggest that HCA may be a potential anticancer agent targeting PKM2 in cancer progression.

Cinnamaldehydes in Cancer Chemotherapy.

Cinnamaldehyde and cinnamaldehyde-derived compounds are candidates for the development of anticancer drugs that have received extensive research attention. In this review, we summarize recent findings detailing the positive and negative aspects of cinnamaldehyde and its derivatives as potential anticancer drug candidates. Furthermore, we describe the in vivo pharmacokinetics and metabolism of cinnamaldehydes. The oxidative and antioxidative properties of cinnamaldehydes, which contribute to their potential in chemotherapy, have also been discussed. Moreover, the mechanism(s) by which cinnamaldehydes induce apoptosis in cancer cells have been explored. In addition, evidence of the regulatory effects of cinnamaldehydes on cancer cell invasion and metastasis has been described. Finally, the application of cinnamaldehydes in treating various types of cancer, including breast, prostate, and colon cancers, has been discussed in detail. The effects of cinnamaldehydes on leukemia, hepatocellular carcinoma, and oral cancer have been summarized briefly. Copyright © 2016 John Wiley & Sons, Ltd.

Tanshinone IIA exerts antitumor activity against vestibular schwannoma cells by inhibiting the expression of hypoxia-inducible factor-1α.

The aim of the present study was to evaluate the effect of the herbal medicine, tanshinone IIA (Tan IIA), on vestibular schwannoma (VS) cells and assess the functional targets of Tan IIA. HEI­193 cells and Nf2­/­mouse Schwann (SC4) cells were used to investigate the inhibitory effects of Tan IIA on VS. Cell viability was measured using an MTT assay and apoptosis was assessed by flow cytometry. Western blot analysis and reverse transcription quantitative polymerase chain reaction (RT­qPCR) were performed to assess the expression of hypoxia­inducible factor­1α (HIF­1α) and its signaling pathways. In addition, the effect of Tan IIA on HIF­1α transcription was determined using a luciferase reporter assay. Schwannoma cell proliferation was observed to be inhibited as the Tan IIA concentration increased under normoxic and hypoxic conditions. Furthermore, Tan IIA induced apoptosis in the HEI­193 cells and inhibited the protein expression of HIF­1α in the HEI­193 cells under hypoxia, thus repressing the transcriptional activity of HIF­1α. The present study demonstrated that HIF­1α is expressed in hypoxic VS cells and Tan IIA inhibits VS cells by suppressing the activity of HIF­1α. In conclusion, these results indicate that Tan IIA is a potential chemotherapeutic agent for the treatment of VS.

Artocarpus altilis (Parkinson) Fosberg Extracts and Geranyl Dihydrochalcone Inhibit STAT3 Activity in Prostate Cancer DU145 Cells.

Artocarpus altilis (Parkinson) Fosberg has traditionally been used in Indonesia for the treatment of liver cirrhosis, hypertension, and diabetes. In many other countries, it is used for the treatment of malaria, yellow fever, and dengue fever. It has been reported that A. altilis extracts have antiatherosclerotic and cytoprotective effects, but its molecular targets in tumor cells are not yet fully understood. The A. altilis extracts and the partially purified fraction have been shown to inhibit STAT3 activity and the phosphorylation of STAT3 in a dose-dependent manner. To identify the active components, a bioassay-guided isolation of the partially purified fraction resulted in the identification of a geranyl dihydrochalcone, CG901. Its chemical structure was established on the basis of spectroscopic evidence and comparison with published data. The partially purified fraction and the isolated a geranyl dihydrochalcone, CG901, down-regulated the expression of STAT3 target genes, induced apoptosis in DU145 prostate cancer cells via caspase-3 and PARP degradation, and inhibited tumor growth in human prostate tumor (DU145) xenograft initiation model. These results suggest that A. altilis could be a good natural source and that the isolated compound will be a potential lead molecule for developing novel therapeutics against STAT3-related diseases, including cancer and inflammation.

The natural compound cantharidin induces cancer cell death through inhibition of heat shock protein 70 (HSP70) and Bcl-2-associated athanogene domain 3 (BAG3) expression by blocking heat shock factor 1 (HSF1) binding to promoters.

Heat shock factor 1 (HSF1) enhances the survival of cancer cells under various stresses. The knock-out of HSF1 impairs cancer formation and progression, suggesting that HSF1 is a promising therapeutic target. To identify inhibitors of HSF1 activity, we performed cell-based screening with a library of marketed and experimental drugs and identified cantharidin as an HSF1 inhibitor. Cantharidin is a potent antitumor agent from traditional Chinese medicine. Cantharidin inhibited heat shock-induced luciferase activity with an IC50 of 4.2 µm. In contrast, cantharidin did not inhibit NF-κB luciferase reporter activity, demonstrating that cantharidin is not a general transcription inhibitor. When the HCT-116 colorectal cancer cells were exposed to heat shock in the presence of cantharidin, the induction of HSF1 downstream target proteins, such as HSP70 and BAG3 (Bcl-2-associated athanogene domain 3), was suppressed. HSP70 and its co-chaperone BAG3 have been reported to protect cells from apoptosis by stabilizing anti-apoptotic Bcl-2 family proteins. As expected, treating HCT-116 cancer cells with cantharidin significantly decreased the amounts of BCL-2, BCL-xL, and MCL-1 protein and induced apoptotic cell death. Chromatin immunoprecipitation analysis showed that cantharidin inhibited the binding of HSF1 to the HSP70 promoter and subsequently blocked HSF1-dependent p-TEFb recruitment. Therefore, the p-TEFb-dependent phosphorylation of the C-terminal domain of RNA polymerase II was blocked, arresting transcription at the elongation step. Protein phosphatase 2A inhibition with PP2CA siRNA or okadaic acid did not block HSF1 activity, suggesting that cantharidin inhibits HSF1 in a protein phosphatase 2A-independent manner. We show for the first time that cantharidin inhibits HSF1 transcriptional activity.

Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells.

Here, antitumor mechanism of cinnamaldehyde derivative CB-PIC was elucidated in human SW620 colon cancer cells. CB-PIC significantly exerted cytotoxicity, increased sub-G1 accumulation, and cleaved PARP with apoptotic features, while it enhanced the phosphorylation of AMPK alpha and ACC as well as activated the ERK in hypoxic SW620 cells. Furthermore, CB-PIC suppressed the expression of HIF1 alpha, Akt, and mTOR and activated the AMPK phosphorylation in hypoxic SW620 cells. Conversely, silencing of AMPK α blocked PARP cleavage and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPK α in hypoxic SW620 cells, implying cross-talk between ERK and AMPK α . Furthermore, cotreatment of CB-PIC and metformin enhanced the inhibition of HIF1 α and Akt/mTOR and the activation of AMPK α and pACC in hypoxic SW620 cells. In addition, CB-PIC suppressed the growth of SW620 cells inoculated in BALB/c athymic nude mice, and immunohistochemistry revealed that CB-PIC treatment attenuated the expression of Ki-67, CD34, and CAIX and increased the expression of pAMPK α in CB-PIC-treated group. Interestingly, CP-PIC showed better antitumor activity in SW620 colon cancer cells under hypoxia than under normoxia, since it may be applied to chemoresistance. Overall, our findings suggest that activation of AMPK α and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells.

Natural flavone jaceosidin is a neuroinflammation inhibitor.

Jaceosidin is a naturally occurring flavone with pharmacological activity. Jaceosidin, as one of the major constituents of the medicinal herbs of the genus Artemisia, has been shown to exert anticancer, anti-oxidative, anti-inflammatory, and immunosuppressive effects. This study was undertaken to determine the effect of jaceosidin on microglia and neuroinflammation. Microglia are the innate immune cells in the central nervous system, and they play a central role in the initiation and maintenance of neuroinflammation. We report that jaceosidin inhibits inflammatory activation of microglia, reducing nitric oxide (NO) production and proinflammatory cytokine expression. IC50 for NO inhibition was 27 ± 0.4 µM. The flavone also attenuated microglial neurotoxicity in the microglia/neuroblastoma co-culture. Systemic injection of jaceosidin ameliorated neuroinflammation in the mouse model of experimental allergic encephalomyelitis. These results indicate that plant flavone jaceosidin is a microglial inhibitor with anti-neuroinflammation activity.

Cryptotanshinone and tanshinone IIA enhance IL-15-induced natural killer cell differentiation.

Natural killer (NK) cells are a subset of lymphocytes crucial for innate and adaptive immune responses. Here we show a stimulatory effect of cryptotanshinone (CTS) and tanshinone IIA (TS), isolated from Salvia miltiorrhiza Bunge, on the differentiation of NK cells. In the presence of IL-15, tanshinones increased NK cell maturation, NK cell differentiation and the expression of several transcription factors, including Id2, GATA3, T-bet, and Ets-1. Additionally, tanshinones increased p38 MAPK phosphorylation during NK cell differentiation. Furthermore, the p38 inhibitor SB203580 blocked the developmental effects of the tanshinones and suppressed Id2, T-bet, and Ets-1 expression during NK cell differentiation. These results suggest that tanshinones significantly increased IL-15-induced NK cell differentiation via enhancing the p38 phosphorylation and the expression of transcription factors.

Effects of obovatol on GSH depleted glia-mediated neurotoxicity and oxidative damage.

Earlier studies indicate that obovatol (OBO), isolated from a medicinal herb Magnolia obovata, has anti-inflammatory and anti-oxidative properties. Depletion of glutathione (GSH) in glial cells with the γ-glutamylcysteine synthase inhibitor D,L-buthionine-S,R-sulfoximine (BSO) is known to produce oxidative stress which, in turn, induces these cells to secrete inflammatory cytokines and other neurotoxic substances. In the present study, we investigated the ability of OBO to protect SH-SY5Y neuroblastoma cells from this effect. Human microglia, astrocytes and their surrogate THP-1 and U373 cell lines were activated by treatment with BSO. Such treatment depleted their intracellular GSH and increased levels of damage to DNA, lipids and proteins (8-OHdG, lipid peroxide, protein carbonyls and 3-nitrotyrosine), and activated the inflammatory pathways P38 MAP kinase and NFκB. These are accompanied by release of proinflammatory factors such as TNFα, IL-6 and nitric oxide. Their conditioned media were toxic to SH-SY5Y cells. All these effects were attenuated by pre-treatment with OBO. Prior treatment of SH-SY5Y cells with OBO also attenuated THP-1 or U373 conditioned media neurotoxicity and also reduced oxidative damage produced by treatment with hydrogen peroxide or BSO. Prior treatment with OBO potentiated survival of SH-SY5Y cells exposed to conditioned medium from BSO-treated THP-1, U373 cells, microglia and astrocytes. The data indicate that OBO could be anti-inflammatory, anti-oxidative and neuroprotective, and be an effective agent for inhibiting pathogenesis in neurological diseases such as Alzheimer disease, Parkinson disease and amyotrophic lateral sclerosis in which glial-mediated neuroinflammation and oxidative stress are thought to contribute to disease progression.

Antiplatelet activity of obovatol, a biphenolic component of Magnolia Obovata, in rat arterial thrombosis and rabbit platelet aggregation.

AIM: Thrombosis occurs in the coronary arteries via the activation of platelets, and leads to acute myocardial infarction and sudden death. Obovatol, a major biphenolic component of Magnolia Obovata leaves, displays anti-inflammatory and acyl Co-A cholesterol acyltrasferase inhibitory effects. The purpose of this study was to determine the effects of obovatol on thrombus formation in vivo and platelet activation in vitro and ex vivo. METHODS: We investigated the antiplatelet and antithrombotic activities of obovatol in rat carotid arterial thrombosis in vivo along with platelet aggregation in vitro and ex vivo. Its possible cellular mechanism of antiplatelet activity was investigated by testing PLC-γ2 activation, arachidonic acid cascade, calcium mobilization and granule secretion. RESULTS: Oral administration of obovatol prevented carotid thrombosis, but also significantly inhibited collagen-induced platelet aggregation. Obovatol did not change coagulation times, such as activated partial thromboplastin time and prothrombin time, indicating that the antithrombotic effect of obovatol might be due to antiplatelet activity rather than anticoagulation activity. Obovatol inhibited in vitro collagen- and arachidonic acid-induced rabbit platelet aggregation in a concentration-dependent manner (1-10 µM), with IC(50) values of 2.4 ± 0.8 and 4.8 ± 0.9 µM, respectively. Obovatol blocked collagen-mediated phospholipase C-γ2 phosphorylation, cytoplasmic calcium mobilization, arachidonic acid liberation and serotonin secretion. CONCLUSION: Obovatol has a potent antithrombotic effect, which may be due to antiplatelet activity. The antiplatelet activity of obovatol is mediated by inhibition of PLC-γ2 phosphorylation. Thus, obovatol may be a potential candidate to treat cardiovascular disease.

2'-Hydroxycinnamaldehyde targets low-density lipoprotein receptor-related protein-1 to inhibit lipopolysaccharide-induced microglial activation.

2'-Hydroxycinnamaldehyde (HCA) isolated from the stem bark of Cinnamomum cassia and its derivative 2'-benzoyloxycinnamaldehyde (BCA) were reported to have anti-angiogenic, anti-proliferative, and anti-inflammatory effects in several human cancer cells and RAW 264.7 macrophage cells. However, effects of HCA/BCA on the neuroinflammation have not been investigated. In the present study, a potential anti-neuroinflammatory effect of HCA/BCA was assessed in lipopolysaccharide (LPS)-stimulated microglial cultures and microglia/neuroblastoma cocultures. Nitric oxide production, inflammatory gene expression, and signaling pathways were investigated. HCA/BCA significantly decreased the production of nitric oxide and tumor necrosis factor-alpha (TNF-α) in microglial cells. HCA/BCA also attenuated the expression of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) and TNF-α at mRNA level via blockade of ERK, JNK, p38 MAPK, and NF-κB activation. Moreover, HCA/BCA was neuroprotective by reducing microglia-mediated neuroblastoma cell death in a microglia-neuroblastoma co-culture. Affinity chromatography and LC-MS/MS analysis identified low-density lipoprotein receptor-related protein 1 (LRP1) as a potential molecular target of HCA in microglial cells. Based on the studies using the receptor-associated protein (RAP) that blocks a ligand binding to LRP1 and the siRNA-mediated LRP1 gene silencing, we were able to conclude that HCA inhibited LPS-induced microglial activation via LRP1. Our results suggest that HCA/BCA be anti-inflammatory and neuroprotective in the CNS by targeting LRP1, and may have a therapeutic potential against neuroinflammatory diseases.

Obovatol attenuates microglia-mediated neuroinflammation by modulating redox regulation.

BACKGROUND AND PURPOSE: Obovatol isolated from the medicinal herb Magnolia obovata exhibits a variety of biological activities. Here, the effect of obovatol and its mechanism of action on microglial activation, neuroinflammation and neurodegeneration were investigated. EXPERIMENTAL APPROACH: In microglial BV-2 cells stimulated with lipopolysaccharide (LPS), we measured nitric oxide (NO) and cytokine production, and activation of intracellular signalling pathways by reverse transcription-polymerase chain reaction and Western blots. Cell death was assayed in co-cultures of activated microglia (with bacterial LPS) and neurons and in LPS- induced neuroinflammation in mice in vivo. KEY RESULTS: Obovatol inhibited microglial NO production with an IC50 value of 10 mM. Obovatol also inhibited microglial expression of proinflammatory cytokines and inducible nitric-oxide synthase, which was accompanied by the inhibition of multiple signalling pathways such as nuclear factor kappa B, signal transducers and activators of transcription 1, and mitogen-activated protein kinases. In addition, obovatol protected cultured neurons from microglial toxicity and inhibited neuroinflammation in mice in vivo. One molecular target of obovatol in microglia was peroxiredoxin 2 (Prx2), identified by affinity chromatography and mass spectrometry. Obovatol enhanced the reactive oxygen species (ROS)-scavenging activity of Prx2 in vitro, thereby suppressing proinflammatory signalling pathways of microglia where ROS plays an important role. CONCLUSIONS AND IMPLICATIONS: Obovatol is not only a useful chemical tool that can be used to investigate microglial signalling, but also a promising drug candidate against neuroinflammatory diseases. Furthermore, our results indicate that Prx2 is a novel drug target that can be exploited for the therapeutic modulation of neuroinflammatory signalling.

Obovatol from Magnolia obovata inhibits vascular smooth muscle cell proliferation and intimal hyperplasia by inducing p21Cip1.

AIMS: Obovatol is isolated from Magnolia obovata leaves and this active component has various pharmacological properties such as anti-oxidant, anti-platelet, anti-fungal and anti-inflammatory activities. In the present study, we investigated the inhibitory effects of obovatol on in vitro vascular smooth muscle cell (VSMC) proliferation and in vivo neointimal formation in a rat carotid artery injury model. METHODS AND RESULTS: Obovatol (1-5 microM) exerted concentration-dependent inhibition on platelet-derived growth factor (PDGF)-BB-induced rat VSMC proliferation, without exhibiting any cellular toxicity or apoptosis, as determined by cell count, [3H]thymidine incorporation and Annexin-V-binding analyses. Treatment with obovatol blocked the cell cycle in G1 phase by down-regulating the expression of cyclins and CDKs, and selectively up-regulating the expression of p21Cip1, a well-known CDK inhibitor. Effects of perivascular delivery of obovatol were assessed 14 days after injury. The angiographic mean luminal diameters of the obovatol-treated groups (100 microg and 1 mg: 0.78+/-0.06 and 0.77+/-0.07AU, respectively) were significantly larger than that of the control group (0.58+/-0.07AU). The obovatol-treated groups (100 microg and 1mg: 0.14+/-0.04 and 0.09+/-0.03 mm2, respectively) showed significant reduction in neointimal formation versus the control group (0.17+/-0.02 mm2). Immunohistochemical staining demonstrated strong expression of p21Cip1 in the neointima of the obovatol-treated groups. CONCLUSIONS: These data suggest that obovatol inhibits VSMC proliferation by perturbing cell cycle progression, possibly due to activation of p21Cip1 pathway. These results also show that obovatol may have potential as an anti-proliferative agent for the treatment of restenosis and atherosclerosis.

Obovatol isolated from Magnolia obovata enhances pentobarbital-induced sleeping time: Possible involvement of GABAA receptors/chloride channel activation.

This study aimed to investigate the effects of obovatol isolated from Magnolia obovata on pentobarbital-induced sleeping behaviors and to determine whether these effects were mediated by GABA(A) receptors/chloride channel activation, using a western blot technique and Cl(-) sensitive fluorescence probe. GABA(A) receptors subunits expression and chloride influx were investigated in cultured cerebellar granule cells. Obovatol (0.05, 0.1, and 0.2 mg/kg) prolonged the sleeping time induced by pentobarbital (42 mg/kg). In addition, obovatol (20 and 50 microM) significantly increased Cl(-) influx in the primary cultured cerebellar granule cells. Moreover, obovatol increased the expression of GABA(A) receptor alpha-, beta-, and gamma-subunits. However, it had no effect on the abundance of the expression of glutamic acid decarboxylase (GAD), suggesting that obovatol might not activate GAD. These results suggest that obovatol potentiates pentobarbital-induced sleeping time through the GABA(A) receptors/chloride channel activation.

Cryptotanshinone inhibits constitutive signal transducer and activator of transcription 3 function through blocking the dimerization in DU145 prostate cancer cells.

Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in most human solid tumors and is involved in the proliferation, angiogenesis, immune evasion, and antiapoptosis of cancer cells, researchers have focused on STAT3 as a target for cancer therapy. We screened for natural compounds that inhibit the activity of STAT3 using a dual-luciferase assay. Cryptotanshinone was identified as a potent STAT3 inhibitor. Cryptotanshinone rapidly inhibited STAT3 Tyr705 phosphorylation in DU145 prostate cancer cells and the growth of the cells through 96 hours of the treatment. Inhibition of STAT3 Tyr705 phosphorylation in DU145 cells decreased the expression of STAT3 downstream target proteins such as cyclin D1, survivin, and Bcl-xL. To investigate the cryptotanshinone inhibitory mechanism in DU145 cells, we analyzed proteins upstream of STAT3. Although phosphorylation of Janus-activated kinase (JAK) 2 was inhibited by 7 micromol/L cryptotanshinone at 24 hours, inhibition of STAT3 Tyr705 phosphorylation occurred within 30 minutes and the activity of the other proteins was not affected. These results suggest that inhibition of STAT3 phosphorylation is caused by a JAK2-independent mechanism, with suppression of JAK2 phosphorylation as a secondary effect of cryptotanshinone treatment. Continuing experiments revealed the possibility that cryptotanshinone might directly bind to STAT3 molecules. Cryptotanshinone was colocalized with STAT3 molecules in the cytoplasm and inhibited the formation of STAT3 dimers. Computational modeling showed that cryptotanshinone could bind to the SH2 domain of STAT3. These results suggest that cryptotanshinone is a potent anticancer agent targeting the activation STAT3 protein. It is the first report that cryptotanshinone has antitumor activity through the inhibition of STAT3.

Farnesyl protein transferase and tumor cell growth inhibitory activities of lipiferolide isolated from Liriodendron tulipifera.

The methanolic extract of the leaves of Liriodendron tulipifera was found to show inhibitory activity towards farnesyl protein transferase (FPTase). Bioassay-guided fractionation of the methanolic extract resulted in the isolation of lipiferolide, an inhibitor of FPTase. This compound inhibited the FPTase activity in a dose-dependent manner, and showed cell growth inhibitory activity against several tumor cells.