Discovery of New Imidazo[2,1-b]thiazole Derivatives as Potent Pan-RAF Inhibitors with Promising In Vitro and In Vivo Anti-melanoma Activity.

BRAF is an important component of MAPK cascade. Mutation of BRAF, in particular V600E, leads to hyperactivation of the MAPK pathway and uncontrolled cellular growth. Resistance to selective inhibitors of mutated BRAF is a major obstacle against treatment of many cancer types. In this work, a series of new (imidazo[2,1-b]thiazol-5-yl)pyrimidine derivatives possessing a terminal sulfonamide moiety were synthesized. Pan-RAF inhibitory effect of the new series was investigated, and structure-activity relationship is discussed. Antiproliferative activity of the target compounds was tested against the NCI-60 cell line panel. The most active compounds were further tested to obtain their IC50 values against cancer cells. Compound 27c with terminal open chain sulfonamide and 38a with a cyclic sulfamide moiety showed the highest activity in enzymatic and cellular assay, and both compounds were able to inhibit phosphorylation of MEK and ERK. Compound 38a was selected for testing its in vivo activity against melanoma. Cellular and animal activities are reported.

Jowiseungchungtang Inhibits Amyloid-ß Aggregation and Amyloid-ß-Mediated Pathology in 5XFAD Mice.

Alzheimer's disease (AD) is a neurodegenerative disease, which is accompanied by memory loss and cognitive dysfunction. Although a number of trials to treat AD are in progress, there are no drugs available that inhibit the progression of AD. As the aggregation of amyloid-ß (Aß) peptides in the brain is considered to be the major pathology of AD, inhibition of Aß aggregation could be an effective strategy for AD treatment. Jowiseungchungtang (JWS) is a traditional oriental herbal formulation that has been shown to improve cognitive function in patients or animal models with dementia. However, there are no reports examining the effects of JWS on Aß aggregation. Thus, we investigated whether JWS could protect against both Aß aggregates and Aß-mediated pathology such as neuroinflammation, neurodegeneration, and impaired adult neurogenesis in 5 five familial Alzheimer's disease mutations (5XFAD) mice, an animal model for AD. In an in vitro thioflavin T assay, JWS showed a remarkable anti-Aß aggregation effect. Histochemical analysis indicated that JWS had inhibitory effects on Aß aggregation, Aß-induced pathologies, and improved adult hippocampal neurogenesis in vivo. Taken together, these results suggest the therapeutic possibility of JWS for AD targeting Aß aggregation, Aß-mediated neurodegeneration, and impaired adult hippocampal neurogenesis.

Uncaria rhynchophylla ameliorates amyloid beta deposition and amyloid beta-mediated pathology in 5XFAD mice.

One of the pathological hallmarks of Alzheimer's disease (AD) is the abnormal aggregation of amyloid beta (Aß) peptides. Uncaria rhynchophylla (UR), one of the Uncaria species, has long been used to treat neurodegenerative disease. In particular, it has been reported that UR inhibits aggregation of Aß in vitro. However, little is known about the histological effects of UR treatment on Aß pathology in AD animal models. In the present study, we investigated the effect of UR on Aß aggregation, Aß-mediated pathologies and adult hippocampal neurogenesis in the brain of 5XFAD mice. First, using the thioflavin T assay and amyloid staining, we demonstrated that UR treatment effectively inhibited Aß aggregation and accumulation in the cortex and subiculum. Second, immunofluorescence staining showed that administration of UR attenuated gliosis and neurodegeneration in the subiculum and cortex. Third, UR treatment ameliorated impaired adult hippocampal neurogenesis. The present results indicate that UR significantly alleviates Aß deposition and Aß-mediated neuropathology in the brain in 5XFAD mice, suggesting the potency of UR as a preventive and therapeutic agent for AD.

IDH2 deficiency increases the liver susceptibility to ischemia-reperfusion injury via increased mitochondrial oxidative injury.

Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) is a major producer of mitochondrial NADPH, required for glutathione (GSH)-associated mitochondrial antioxidant systems including glutathione peroxidase (GPx) and glutathione reductase (GR). Here, we investigated the role of IDH2 in hepatic ischemia-reperfusion (HIR)-associated mitochondrial injury using Idh2-knockout (Idh2-/-) mice and wild-type (Idh2+/+) littermates. Mice were subjected to either 60min of partial liver ischemia or sham-operation. Some mice were administered with 2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (mito-TEMPO, a mitochondria-targeting antioxidant). HIR induced severe histological and functional damages of liver in both Idh2+/+ mice and Idh2-/- mice and those damages were more severe in Idh2-/- mice than in wild-type littermates. HIR induces dysfunction of IDH2, leading to the decreases of NADPH level and mitochondrial GR and GPx functions, consequently resulting in mitochondrial and cellular oxidative injury as reflected by mitochondrial cristae loss, mitochondrial fragmentation, shift in mitochondrial fission, cytochrome c release, and cell death. These HIR-induced changes were greater in Idh2-/- mice than wild-type mice. The mito-TEMPO supplement significantly attenuated the aforementioned changes, and these attenuations were much greater in Idh2-/- mice when compared with wild-type littermates. Taken together, results have demonstrated that HIR impairs in the IDH2-NADPH-GSH mitochondrial antioxidant system, resulting in increased mitochondrial oxidative damage and dysfunction, suggesting that IDH2 plays a critical role in mitochondrial redox balance and HIR-induced impairment of IDH2 function is associated with the pathogenesis of ischemia-reperfusion-induced liver failure.

Anthraquinones from Morinda longissima and their insulin mimetic activities via AMP-activated protein kinase (AMPK) activation.

AMP-activated protein kinase (AMPK) activators are known to increase energy metabolism and to reduce body weight, as well as to improve glucose uptake. During for searching AMPK activators, a new anthraquinone, modasima A (10), along with eighteen known analogues (1-9 and 11-19) were isolated from an ethanol extract of the roots of Morinda longissima Y. Z. Ruan (Rubiaceae). Using the fluorescent tagged glucose analogues, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-D-glucose (2-NBDG), insulin mimetics were screened with compounds 1-19 in 3T3-L1 adipocytes. Among them, compounds 2, 8 and 10 enhanced significantly glucose uptake into adipocytes and up-regulated the phosphorylated AMPK (Thr172) whereas the glucose uptake enhancing activities of compounds 2, 8 and 10 were abrogated by treatment of compound C, an AMPK inhibitor. Taken together, these anthraquinones showed the potential action as insulin mimetic to improve glucose uptake via activation of AMPK.

Development of DH-I-180-3 loaded lipid nanoparticle for photodynamic therapy.

Photodynamic therapy (PDT) is a promising noninvasive treatment modality for cancer. Photosensitizer and specific wave length of light are the key component of PDT. DH-I-180-3, a second generation photosensitizer, was incorporated into lipid nanoparticle for simultaneous fluorescent imaging and targeting therapy. Solid lipid nanoparticle (SLN) and nanostructured lipid carriers (NLC) based on poloxamer 188 as surfactant and lecithin as co-surfactant were prepared using solvent evaporation and hot homogenization technique. Stearic acid and Capmul(®) MCM C8 were utilized as solid lipid and liquid lipid, respectively. The particle size of SLN and NLCs was around 200 nm and decreased when a part of stearic acid was replaced with Capmul(®) MCM C8. Drug loading efficacy was significantly enhanced when the percentage amount of liquid lipid increased. All the polydispersity indices of the SLN/NLCs were below 0.3, and displayed a narrow particle size distribution. Zeta potentials of all the lipid nanoparticles were below -30 mV, maintaining sufficient repulsive force and achieving enhanced physical stability. No significant change in the particle size and polydispersity index was observed from lyophilized SLN/NLCs. When the photocytotoxic effects of the formulations were evaluated in MCF-7 cells, GI 50 of SLN was less than half of DH-I-180-3 solution, and NLCs containing either 5 or 15%w/w of Capmul(®) MCM C8 exerted higher cytotoxicity than SLN. The fluorescence microscope images displayed enhanced cellular accumulation of DH-I-180-3 loaded in SLN and NLCs, which was closely correlated with the photocytotoxicity results. It was concluded that the incorporation of DH-I-180-3 into the nanoparticles enhanced their targeting efficacy and improved photocytotoxicity.

Oleanane triterpenes as protein tyrosine phosphatase 1B (PTP1B) inhibitors from Camellia japonica.

Protein tyrosine phosphatase 1B (PTP1B) plays a key role in metabolic signaling, thereby making it an exciting drug target for type 2 diabetes and obesity. Besides, there is substantial evidence that shows its overexpression is involved in breast cancer, which suggests that selective PTP1B inhibition might be effective in breast cancer treatment. As part of our continuous research on PTP1B inhibitors from medicinal plants, four oleanane-type triterpenes were isolated from an EtOAc-soluble extract of fruit peels of Camellia japonica (Theaceae), together with 6 previously known compounds of this class. Their structures were determined on the basis of spectroscopic data analysis (UV, IR, (1)H and (13)CNMR, HMBC, HSQC, NOESY, and MS). All isolates were evaluated for their inhibitory effects on PTP1B, as well as their cytotoxic effects against human breast cancer cell lines MCF7, MCF7/ADR, and MDA-MB-231. Several compounds with OH-3 or/and COOH-28 functionalities showed strong PTP1B inhibitory activity (IC50 values ranging from 3.77±0.11 to 6.40±0.81 µM) as well as significant cytotoxicity (IC50 values ranging from 0.51±0.05 to 13.55±1.44 µM).

Aglycon of rhizochalin from the Rhizochalina incrustata induces apoptosis via activation of AMP-activated protein kinase in HT-29 colon cancer cells.

Rhizochalin is a two-headed sphingolipid-like compound isolated from the sponge Rhizochalina incrustata. It has been reported that rhizocalin and its derivates have a chemopreventive and chemotherapeutic effect. However, the molecular mechanism of these effects is not understood. Here, we demonstrate that aglycon of rhizochalin (AglRhz) from the Rhizochalina incrustata induces AMP-activated protein kinase (AMPK) phosphorylation, and thereby inhibits mammalian target of rapamycin (mTOR)-p70S6 kinase-extracellular signal-regulated kinase (ERK) signaling and activator protein 1 (AP-1) activity via phosphorylation of Raptor in HT-29 cells. In addition, AglRhz induced activation of caspase-3 and poly(ADP-ribose) polymerase (PARP), and DNA fragmentation in HT-29 cells, leads to induction of apoptosis as well as suppression of tumorigenicity of HT-29 cells. Notably, AglRhz inhibits insulin-like growth factor (IGF)-1-induced AP-1 activity and cell transformation in JB6 Cl41 cells. Overall, our findings identify AMPK as an important target protein for mediating the anti-tumor properties of AglRhz in HT-29 colon cancer cells and have important implication for sponges, the most important marine source, in colon cancer.

p-HPEA-EDA, a phenolic compound of virgin olive oil, activates AMP-activated protein kinase to inhibit carcinogenesis.

Phenolic constituents of virgin olive oil are reported to have antitumor activity. However, the underlying molecular mechanisms and specific target proteins of virgin olive oil remain to be elucidated. Here, we report that dialdehydic form of decarboxymethyl ligstroside aglycone (p-HPEA-EDA), a phenolic compound of virgin olive oil, inhibits tumor promoter-induced cell transformation in JB6 Cl41 cells and suppress cyclooxygenase-2 (COX-2) and tumorigenicity by adenosine monophosphate-activated protein kinase (AMPK) activation in HT-29 cells. p-HPEA-EDA inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of extracellular signal-regulated kinases 1/2 and p90RSK in JB6 Cl41 cells, resulting in the inhibition of cell proliferation, activator protein-1 transactivation and cell transformation promoted by TPA. Moreover, p-HPEA-EDA strongly inhibited the cell viability and COX-2 expression by activation of AMPK activity in HT-29 cells, resulted from depletion of intracellular adenosine triphosphate. p-HPEA-EDA-induced activation of caspase-3 and poly-adenosine diphosphate-ribose polymerase, phosphorylation of p53 (Ser15) and DNA fragmentation in HT-29 cells, leading to apoptosis. Importantly, p-HPEA-EDA suppressed the colony formation of HT-29 cells in soft agar. In contrast, Compound C, an AMPK inhibitor, and Z-DEVD-FMK, a caspase-3 inhibitor, blocked the p-HPEA-EDA-inhibited colony formation in HT-29 cells. In vivo chorioallantoic membrane assay also showed that p-HPEA-EDA-inhibited tumorigenicity of HT-29 cells. These findings revealed that targeted activation of AMPK and inhibition of COX-2 expression by p-HPEA-EDA contribute to the chemopreventive and chemotherapeutic potential of virgin olive oil against colon cancer cells.

24-hydroxyursolic acid from the leaves of the Diospyros kaki (Persimmon) induces apoptosis by activation of AMP-activated protein kinase.

There are multiple lines of evidence that persimmon extract and its constituents have potent antitumor activity against human cancer cells. However, the molecular mechanisms of 24-hydroxyursolic acid, a triterpenoid found in persimmon, on antitumor activities are not yet understood. Here, we demonstrate that 24-hydroxyursolic acid inhibited cell proliferation, strongly activated AMP-activated protein kinase (AMPK) and mediated critical anticancer effects by inhibition of cyclooxygenase (COX-2) expression in HT-29 cells. In addition, 24-hydroxyursolic acid induced cellular apoptosis by activation of poly(ADP-ribose) polymerase (PARP), caspase-3, and phosphorylation of p53 at Ser15. It also strongly induced DNA fragmentation in HT-29 cells and thereby significantly inhibited colony formation of HT-29 cells in soft agar. In addition, 24-hydroxyursolic acid blocked the EGF-induced ERKs phosphorylation and led to the inhibition of AP-1 activity and cell transformation in JB6 CL41 cells. Collectively, these findings are the first to reveal a molecular basis for the anticarcinogenic action of 24-hydroxyursolic acid and might account for the reported chemopreventive and chemotherapic effects of persimmon extracts.

Primary structure of myostracal prism soluble protein (MPSP) in oyster shell, Crassostrea gigas.

Soluble protein (MPSP, myostracal prism soluble protein) obtained from myostracum in oyster shell (Crassostrea gigas) was characterized using biochemical and molecular biological techniques. From an analysis of secondary protein structure, it was shown that beta-structure was predominant in MPSP. And via in vitro assays, the relation of MPSP to biomineral phase and morphology was studied. SDS-PAGE revealed one major protein band of 20 kDa. An amino acid sequence of 160 amino acids was deduced for myostracum by characterization of the complementary DNA encoding the protein. The deduced protein was composed of a high proportion of Gly and Asp, typifying a calcium-binding protein for shell formation, and a relatively high proportion of Val, Ala and Ile, typifying an adhesive protein. In contrast to prevailing expectations, (Gly-Asp)n-type sequence motifs exist in MPSP, demanding a revision of previous theories of protein-mineral interactions. The cDNA sequence of myostracum is elucidated for the first time.

Tanshinones inhibit mast cell degranulation by interfering with IgE receptor-mediated tyrosine phosphorylation of PLCgamma2 and MAPK.

Recently we have isolated four active components from Tanshen (the root of Salvia miltiorrhiza Bunge, Labiatae) responsible for the anti-allergic activities. In this study, the molecular mechanism of action of tanshinones for the inhibition of mast cell degranulation was investigated by testing their effects on the signaling components of the high affinity IgE receptor FcepsilonRI. Activation of FcepsilonRI produced immediate tyrosine phosphorylation of Syk, mitogen-activated protein kinase extracellular signal-regulated kinase, ERK1/ERK2 (p44, p42), and phospholipase Cgamma2 (PLCgamma2). 5,16-Dihydrotanshinone-I possessed the strongest inhibitory effects on mast cell degranulation and markedly reduced FcepsilonRI-mediated tyrosine phosphorylation of ERK and PLCgamma2. This suggests that tanshinones possibly exert their anti-allergic activities by affecting FcepsilonRI-mediated tyrosine phosphorylation of ERK and PLCgamma2. Abbreviations. FcepsilonRI:high affinity IgE receptor ERK:extracellular signal regulated kinase PLC: phospholipase C

Molecular mechanisms of inhibitory activities of tanshinones on lipopolysaccharide-induced nitric oxide generation in RAW 264.7 cells.

The effects of four tanshinones isolated from Tanshen (the root of Salvia miltiorrhiza Bunge, Labiatae) were tested for their inhibition of nitric oxide production in macrophage cells, and the underlying molecular mechanisms studied. Of the four tanshinones used, 15, 16-dihydrotanshinone-I, tanshinone-IIA and cryptotanshinone, but not tanshinone I, demonstrated significant inhibition of the LPS-induced nitric oxide production in RAW 264.7 cells, with calculated IC50 values of 5, 8, and 1.5 microM, respectively. Tanshinones exerted inhibitory activities on the LPS-induced nitric oxide production only when applied concurrently with LPS, and tanshinone-IIA and cryptotanshinone were found to inhibit LPS-induced NF-kappaB mobilization and extracellular-regulated kinase (ERK) activation, respectively. These results suggest that tanshinones inhibit LPS-induced nitric oxide generation by interfering with the initial stage of LPS-induced expression of certain genes. NF-kappaB and ERK could be the molecular targets for tanshinones for the inhibition of LPS-induced nitric oxide production in macrophage cells.