Study on the secondary metabolites of grasshopper-derived fungi Arthrinium sp. NF2410.

Two new 2-carboxymethyl-3-hexyl-maleic anhydride derivatives, arthrianhydride A (1) and B (2), along with three known compounds 3-5, were isolated from the fermentation broth of a grasshopper-associated fungus Arthrinium sp. NF2410. The structures of new compounds 1 and 2 were determined based on the analysis of the HR-ESI-MS and NMR spectroscopic data. Furthermore, compounds 1 and 2 were evaluated on inhibitory activity against the enzyme SHP2 and both of them showed moderate inhibitory activity against SHP2.

Growth Factors, and Cytokines; Understanding the Role of Tyrosine Phosphatase SHP2 in Gametogenesis and Early Embryo Development.

Growth factors and cytokines have vital roles in germ cell development, gamete maturation, and early embryo development. Cell surface receptors are present for growth factors and cytokines to integrate with and trigger protein signaling in the germ and embryo intracellular milieu. Src-homology-2-containing phosphotyrosine phosphatase (SHP2) is a ubiquitously expressed, multifunctional protein that plays a central role in the signaling pathways involved in growth factor receptors, cytokine receptors, integrins, and G protein-coupled receptors. Over recent decades, researchers have recapitulated the protein signaling networks that influence gamete progenitor specification as well as gamete differentiation and maturation. SHP2 plays an indispensable role in cellular growth, survival, proliferation, differentiation, and migration, as well as the basic events in gametogenesis and early embryo development. SHP2, a classic cytosolic protein and a key regulator of signal transduction, displays unconventional nuclear expression in the genital organs. Several observations provided shreds of evidence that this behavior is essential for fertility. The growth factor and cytokine-dependent roles of SHP2 and its nuclear/cytoplasmic presence during gamete maturation, early embryonic development and embryo implantation are fascinating and complex subjects. This review is intended to summarize the previous and recent knowledge about the SHP2 functions in gametogenesis and early embryo development.

Regulation of autoimmune arthritis by the SHP-1 tyrosine phosphatase.

BACKGROUND: The Src homology region 2 domain-containing phosphatase-1 (SHP-1) is known to exert negative regulatory effects on immune cell signaling. Mice with mutations in the Shp1 gene develop inflammatory skin disease and autoimmunity, but no arthritis. We sought to explore the role of SHP-1 in arthritis using an autoimmune mouse model of rheumatoid arthritis. We generated Shp1 transgenic (Shp1-Tg) mice to study the impact of SHP-1 overexpression on arthritis susceptibility and adaptive immune responses. METHODS: SHP-1 gene and protein expression as well as tyrosine phosphatase activity were evaluated in spleen cells of transgenic and wild type (WT) mice. WT and Shp1-Tg (homozygous or heterozygous for the transgene) mice were immunized with human cartilage proteoglycan (PG) in adjuvant, and arthritis symptoms were monitored. Protein tyrosine phosphorylation level, net cytokine secretion, and serum anti-human PG antibody titers were measured in immune cells from WT and Shp1-Tg mice. WT mice were treated with regorafenib orally to activate SHP-1 either before PG-induced arthritis (PGIA) symptoms developed (preventive treatment) or starting at an early stage of disease (therapeutic treatment). Data were statistically analyzed and graphs created using GraphPad Prism 8.0.2 software. RESULTS: SHP-1 expression and tyrosine phosphatase activity were elevated in both transgenic lines compared to WT mice. While all WT mice developed arthritis after immunization, none of the homozygous Shp1-Tg mice developed the disease. Heterozygous transgenic mice, which showed intermediate PGIA incidence, were selected for further investigation. We observed differences in interleukin-4 and interleukin-10 production in vitro, but serum anti-PG antibody levels were not different between the genotypes. We also found decreased tyrosine phosphorylation of several proteins of the JAK/STAT pathway in T cells from PG-immunized Shp1-Tg mice. Regorafenib administration to WT mice prevented the development of severe PGIA or reduced disease severity when started after disease onset. CONCLUSIONS: Resistance to arthritis in the presence of SHP-1 overexpression likely results from the impairment of tyrosine phosphorylation (deactivation) of key immune cell signaling proteins in the JAK/STAT pathway, due to the overwhelming tyrosine phosphatase activity of the enzyme in Shp1-Tg mice. Our study is the first to investigate the role of SHP-1 in autoimmune arthritis using animals overexpressing this phosphatase. Pharmacological activation of SHP-1 might be considered as a new approach to the treatment of autoimmune arthritis.

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.

A new abietane diterpenoid from Ajuga ovalifolia var. calantha induces human lung epithelial A549 cell apoptosis by inhibiting SHP2.

The Src-homology 2 domain-containing phosphatase 2 (SHP2), encoded by PTPN11, has been reported oncogenic tyrosine phosphatase associated with various tumors and played critical roles in many cell signaling events. Targeting SHP2 by small molecules may be a promising way for cancer therapy. Herein, a new abietane diterpenoid, named 3-acetoxylteuvincenone G (3-AG), was isolated from the whole plants of Ajuga ovalifolia var. calantha. The structure of the new compound was elucidated by means of extensive spectroscopic analyses. Using recombinant enzyme activity assay and cellular thermal shift assay, we found that 3-AG was a selective inhibitor of SHP2. Molecular docking suggested 3-AG displayed an orientation favorable to nucleophilic attack in the catalytic domain of SHP2. 3-AG suppressed A549 cell proliferation (IC50 = 10.79 ± 0.14 µM), invasion and induced cell apoptosis through SHP2/ERK1/2 and SHP2/AKT pathways. In summary, 3-AG, a potent, selective, and efficacious SHP2 inhibitor, may be a promising small molecule to treat human lung epithelial cancer.

PTPN11 (SHP2) Is Indispensable for Growth Factors and Cytokine Signal Transduction During Bovine Oocyte Maturation and Blastocyst Development.

This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.

Cafestol Activates Nuclear Factor Erythroid-2 Related Factor 2 and Inhibits Urotensin II-Induced Cardiomyocyte Hypertrophy.

Through population-based studies, associations have been found between coffee drinking and numerous health benefits, including a reduced risk of cardiovascular disease. Active ingredients in coffee have therefore received considerable attention from researchers. A wide variety of effects have been attributed to cafestol, one of the major compounds in coffee beans. Because cardiac hypertrophy is an independent risk factor for cardiovascular events, this study examined whether cafestol inhibits urotensin II (U-II)-induced cardiomyocyte hypertrophy. Neonatal rat cardiomyocytes were exposed only to U-II (1 nM) or to U-II (1 nM) following 12-h pretreatment with cafestol (1-10 µ M). Cafestol (3-10 µ M) pretreatment significantly inhibited U-II-induced cardiomyocyte hypertrophy with an accompanying decrease in U-II-induced reactive oxygen species (ROS) production. Cafestol also inhibited U-II-induced phosphorylation of redox-sensitive extracellular signal-regulated kinase (ERK) and epidermal growth factor receptor transactivation. In addition, cafestol pretreatment increased Src homology region 2 domains-containing phosphatase-2 (SHP-2) activity, suggesting that cafestol prevents ROS-induced SHP-2 inactivation. Moreover, nuclear factor erythroid-2-related factor 2 (Nrf2) translocation and heme oxygenase-1 (HO-1) expression were enhanced by cafestol. Addition of brusatol (a specific inhibitor of Nrf2) or Nrf2 siRNA significantly attenuated cafestol-mediated inhibitory effects on U-II-stimulated ROS production and cardiomyocyte hypertrophy. In summary, our data indicate that cafestol prevented U-II-induced cardiomycyte hypertrophy through Nrf2/HO-1 activation and inhibition of redox signaling, resulting in cardioprotective effects. These novel findings suggest that cafestol could be applied in pharmacological therapy for cardiac diseases.

Induction of phosphatase shatterproof 2 by evodiamine suppresses the proliferation and invasion of human cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most common fatal carcinomas and is well known to be lack of effective treatment. Thus, novel therapeutic strategies are greatly needed. Evodiamine, a quinozole alkaloid isolated from evodia rutaecarpa Bentham, has been demonstrated to exhibit anti-tumor effects on many cancer cells. However, little is known in terms of the effects on cholangiocarcinoma. In this study, we studied whether this traditional Chinese Medicine could serve as new potential therapeutic drugs to treat CCA. We discovered that evodiamine inhibited CCA cell proliferation and induced apoptosis. Moreover, evodiamine inhibited CCA cell migration and invasion. Mechanistically, our studies demonstrated that evodiamine inhibited the activation of IL-6 -induced STAT3 signaling activation, and the inhibitory effect was likely due to the upregulation of phosphatase shatterproof 2 (SHP-2), a negative feedback regulator of IL-6/STAT3. Blockage of SHP-2 through small interference RNA (siRNA) abolished the evodiamine -induced IL-6/STAT3 signaling inhibition. Moreover, in vivo experiment showed evodiamine inhibited the tumor growth of nude mice bearing TFK-1 xenografts. In summary, our results implied evodiamine as a promising anti-cancer agent in the treatment of CCA, and the mechanism is likely due to the inhibition of IL-6/STAT3 signaling with upregulating the expression levels of SHP-2.

Ocimum basilicum miRNOME revisited: A cross kingdom approach.

O. basilicum is medicinally important herb having inevitable role in human health. However, the mechanism of action is largely unknown. Present study aims to understand the mechanism of regulation of key human target genes that could plausibly modulated by O. basilicum miRNAs in cross kingdom manner using computational and system biology approach. O. basilicum miRNA sequences were retrieved and their corresponding human target genes were identified using psRNA target and interaction analysis of hub nodes. Six O. basilicum derived miRNAs were found to modulate 26 human target genes which were associated `with PI3K-AKTand MAPK signaling pathways with PTPN11, EIF2S2, NOS1, IRS1 and USO1 as top 5 Hub nodes. O. basilicum miRNAs not only regulate key human target genes having a significance in various diseases but also paves the path for future studies that might explore potential of miRNA mediated cross-kingdom regulation, prevention and treatment of various human diseases including cancer.

Nitric oxide inhibits hypoxia-induced impairment of human RBC deformability through reducing the cross-linking of membrane protein band 3.

AIM: Nitric oxide (NO) prevents the decline of RBC deformability under high altitude and other ischemic and hypoxic conditions, but the clear mechanisms remain unknown. Here, we have carried out a systematic study to find the mechanisms of NO-induced regulation of RBC deformability under hypoxia. METHODS: NO levels, RBCs membrane elongation index (EI), membrane protein band 3 methemoglobin (MetHb) were determined during hypoxia (0 to 120 minutes). To validate the role of NO in regulating RBC deformability, tests were also performed with a NO donor (sodium nitroprusside) or a NO synthase inhibitor (l-nitro-arginine methylester) under 60 minutes hypoxia. RESULTS: Hypoxia for 45 minutes increased NO levels from 25.65 ± 1.95 to 35.26 ± 2.01 µmol/L, and there was a plateau after 60 minutes hypoxia. The EI did not change before 45 minutes hypoxia, but decreased from 0.567 ± 0.019 to 0.409 ± 0.042 (30 Pa) after 60 minutes hypoxia. The cross-linking of band 3 and phosphotyrosine increased after 45 minutes hypoxia. All can be alleviated by supplement NO and aggregated by inhibiting NOS. However, the MetHb was not present this trend. CONCLUSION: NO may prevent decreased of RBCs deformability through reducing the cross-linking of membrane band 3 under hypoxia; this helps microvascular perfusion of RBCs during ischemic and hypoxic disease states.

Scaffold-based novel SHP2 allosteric inhibitors design using Receptor-Ligand pharmacophore model, virtual screening and molecular dynamics.

SHP2 is a potential target for the development of novel therapies for SHP2-dependent cancers. In our research, with the aid of the 'Receptor-Ligand Pharmacophore' technique, a 3D-QSAR method was carried out to explore structure activity relationship of SHP2 allosteric inhibitors. Structure-based drug design was employed to optimize SHP099, an efficacious, potent, and selective SHP2 allosteric inhibitor. A novel class of selective SHP2 allosteric inhibitors was discovered by using the powerful 'SBP', 'ADMET' and 'CDOCKER' techniques. By means of molecular dynamics simulations, it was observed that these novel inhibitors not only had the same function as SHP099 did in inhibiting SHP2, but also had more favorable conformation for binding to the receptor. Thus, this report may provide a new method in discovering novel and selective SHP2 allosteric inhibitors.

Perturbing chondroitin sulfate proteoglycan signaling through LAR and PTPσ receptors promotes a beneficial inflammatory response following spinal cord injury.

BACKGROUND: Traumatic spinal cord injury (SCI) results in upregulation of chondroitin sulfate proteoglycans (CSPGs) by reactive glia that impedes repair and regeneration in the spinal cord. Degradation of CSPGs is known to be beneficial in promoting endogenous repair mechanisms including axonal sprouting/regeneration, oligodendrocyte replacement, and remyelination, and is associated with improvements in functional outcomes after SCI. Recent evidence suggests that CSPGs may regulate secondary injury mechanisms by modulating neuroinflammation after SCI. To date, the role of CSPGs in SCI neuroinflammation remains largely unexplored. The recent discovery of CSPG-specific receptors, leukocyte common antigen-related (LAR) and protein tyrosine phosphatase-sigma (PTPσ), allows unraveling the cellular and molecular mechanisms of CSPGs in SCI. In the present study, we have employed parallel in vivo and in vitro approaches to dissect the role of CSPGs and their receptors LAR and PTPσ in modulating the inflammatory processes in the acute and subacute phases of SCI. METHODS: In a clinically relevant model of compressive SCI in female Sprague Dawley rats, we targeted LAR and PTPσ by two intracellular functionally blocking peptides, termed ILP and ISP, respectively. We delivered ILP and ISP treatment intrathecally to the injured spinal cord in a sustainable manner by osmotic mini-pumps for various time-points post-SCI. We employed flow cytometry, Western blotting, and immunohistochemistry in rat SCI, as well as complementary in vitro studies in primary microglia cultures to address our questions. RESULTS: We provide novel evidence that signifies a key immunomodulatory role for LAR and PTPσ receptors in SCI. We show that blocking LAR and PTPσ reduces the population of classically activated M1 microglia/macrophages, while promoting alternatively activated M2 microglia/macrophages and T regulatory cells. This shift was associated with a remarkable elevation in pro-regenerative immune mediators, interleukin-10 (IL-10), and Arginase-1. Our parallel in vitro studies in microglia identified that while CSPGs do not induce an M1 phenotype per se, they promote a pro-inflammatory phenotype. Interestingly, inhibiting LAR and PTPσ in M1 and M2 microglia positively modulates their inflammatory response in the presence of CSPGs, and harnesses their ability for phagocytosis and mobilization. Interestingly, our findings indicate that CSPGs regulate microglia, at least in part, through the activation of the Rho/ROCK pathway downstream of LAR and PTPσ. CONCLUSIONS: We have unveiled a novel role for LAR and PTPσ in regulating neuroinflammation in traumatic SCI. Our findings provide new insights into the mechanisms by which manipulation of CSPG signaling can promote recovery from SCI. More importantly, this work introduces the potential of ILP/ISP as a viable strategy for modulating the immune response following SCI and other neuroinflammatory conditions of the central nervous system.

Dual Allosteric Inhibition of SHP2 Phosphatase.

SHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell proliferation, differentiation, and survival. Recently, we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (1) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor, which binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies that enabled the identification of a second, distinct small molecule allosteric site. SHP244 (2) was identified as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that 2 binds and stabilizes the inactive, closed conformation of SHP2, at a distinct, previously unexplored binding site-a cleft formed at the interface of the N-terminal SH2 and PTP domains. Derivatization of 2 using structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by 1 and 2 was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies.

Omega-3 free fatty acids attenuate insulin-promoted breast cancer cell proliferation.

High insulin levels in obese people are considered as a risk factor to induce breast carcinogenesis. And consumption of fish oils which mainly contain omega-3 fatty acids is associated with a reduced risk of breast cancer. However, whether omega-3 free fatty acids (FFAs) modulate insulin signaling pathway to prevent breast cancer is poorly understood. The current study tested the hypothesis that omega-3 FFAs attenuate insulin-induced breast cancer cell proliferation and regulate insulin signaling pathway. We show here that omega-3 FFAs attenuate MCF-7 cell proliferation and Akt and Erk1/2 phosphorylation levels stimulated by insulin. Knockdown Shp2 by siRNA resulted in significantly elevated omega-3 FFAs-activated Akt phosphorylation but failed to change insulin-stimulated Akt and Erk1/2 phosphorylation. And viable cell number was not affected by either downregulation of Shp2 expression or Erk1/2 inhibitor U0126 treatment. These observations indicated that omega-3 FFAs attenuate insulin-promoted breast cancer cell proliferation and insulin-activated Akt phosphorylation.

Noonan syndrome, PTPN11 mutations, and brain tumors. A clinical report and review of the literature.

Noonan syndrome (NS), an autosomal dominant disorder, is characterized by short stature, congenital heart defects, developmental delay, and facial dysmorphism. PTPN11 mutations are the most common cause of NS. PTPN11 encodes a non-receptor protein tyrosine phosphatase, SHP2. Hematopoietic malignancies and solid tumors are associated with NS. Among solid tumors, brain tumors have been described in children and young adults but remain rather rare. We report a 16-year-old boy with PTPN11-related NS who, at the age of 12, was incidentally found to have a left temporal lobe brain tumor and a cystic lesion in the right thalamus. He developed epilepsy 2 years later. The temporal tumor was surgically resected because of increasing crises and worsening radiological signs. Microscopy showed nodules with specific glioneuronal elements or glial nodules, leading to the diagnosis of dysembryoplastic neuroepithelial tumor (DNT). Immunohistochemistry revealed positive nuclear staining with Olig2 and pERK in small cells. SHP2 plays a key role in RAS/MAPK pathway signaling which controls several developmental cell processes and oncogenesis. An amino-acid substitution in the N-terminal SHP2 domain disrupts the self-locking conformation and leads to ERK activation. Glioneuronal tumors including DNTs and pilocytic astrocytomas have been described in NS. This report provides further support for the relation of DNTs with RASopathies and for the implication of RAS/MAPK pathways in sporadic low-grade glial tumors including DNTs. © 2017 Wiley Periodicals, Inc.

Identification of demethylincisterol A3 as a selective inhibitor of protein tyrosine phosphatase Shp2.

Shp2 is a classical non-receptor protein tyrosine phosphatase (PTP) involved in many human diseases such as Noonan syndrome and tumors, and identified as a potential therapeutic target. In order to find a potent and selective Shp2 inhibitor, we screened a diverse collection of the secondary metabolites from endophyte fungi using an in vitro enzyme assay, and finally identified a potent Shp2 inhibitor, HLP46 (demethylincisterol A3) from Pestalotiopsis sp. HLP46 was reported to have anti-tumor and anti-inflammation activity previously. We provide the first evidence that HLP46 is an inhibitor of the Shp2. HLP46 shows high selective inhibition of Shp2 over Shp1, PTP1B, Lyp, STEP, PTPRA and Cdc25b. Enzymatic kinetic analyses showed that HLP46 is a non-competitive inhibitor of Shp2. HLP46 interrupts Gab1-Shp2 association and blocked Shp2-dependent activation of the Ras/ERK signal pathway induced by EGF. Furthermore, HLP46 decreased Src activation and inhibit tumor cell migration and invasion. As expected, HLP46 has no effect on the Shp2-independent activation of ERK induced by PMA or on the activation of the PI3K/Akt pathway. We testified therapeutic efficacy targeting both Shp2 and PI3K in MCF7 cells. HLP46 does not show any synergistic inhibition with PI3K inhibitor in suppressing cell growth. Collectively, these results suggest that HLP46 is a selective Shp2 inhibitor and could inhibit Shp2-dependent cell signaling in human cells.

Chlorogenic Acid Prevents Osteoporosis by Shp2/PI3K/Akt Pathway in Ovariectomized Rats.

Cortex Eucommiae is used worldwide in traditional medicine, various constituents of Cortex Eucommiae, such as chlorogenic acid (CGA), has been reported to exert anti-osteoporosis activity in China, but the mechanism about their contribution to the overall activity is limited. The aims of this study were to determine whether chlorogenic acid can prevent estrogen deficiency-induced osteoporosis and to analyze the mechanism of CGA bioactivity. The effect of CGA on estrogen deficiency-induced osteoporosis was performed in vivo. Sixty female Sprague-Dawley rats were divided randomly among a sham-operated group and five ovariectomy (OVX) plus treatment subgroups: saline vehicle, 17α-ethinylestradiol (E2), or CGA at 9, 27, or 45 mg/kg/d. The rats' femoral metaphyses were evaluated by micro-computed tomography (µCT). The mechanism of CGA bioactivity was investigated in vitro. Bone mesenchymal stem cells (BMSCs) were treated with CGA, with or without phosphoinositide 3-kinase (PI3K) inhibitor LY294002. BMSCs proliferation and osteoblast differentiation were assessed with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and alkaline phosphatase, with or without Shp2 interfering RNA (RNAi). The results display that CGA at 27 and 45 mg/kg/day inhibited the decrease of bone mineral density (BMD) that induced by OVX in femur (p< 0.01), significantly promoted the levels of bone turnover markers, and prevented bone volume fraction (BV/TV), connectivity density (CoonD), trabecular number (Tb.N), trabecular thickness (Tb.Th) (all p< 0.01) to decrease and prevented the trabecular separation (Tb.Sp), structure model index (SMI)(both p< 0.01) to increase. CGA at 1 or 10 µM enhanced BMSC proliferation in a dose-dependent manner. CGA at 0.1 to 10 µM increased phosphorylated Akt (p-Akt) and cyclin D1. These effects were reversed by LY294002. CGA at 1 or 10 µM increased BMSC differentiation to osteoblasts (p< 0.01), Shp2 RNAi suppressed CGA-induced osteoblast differentiation by decreasing Shp2, p-Akt, and cyclin D1. This study found that CGA improved the BMD and trabecular micro-architecture for the OVX-induced osteoporosis. Therefore, CGA might be an effective alternative treatment for postmenopausal osteoporosis. CGA promoted proliferation of osteoblast precursors and osteoblastic differentiation of BMSCs via the Shp2/PI3K/Akt/cyclin D1 pathway.

Plasiatine, an Unprecedented Indole-Phenylpropanoid Hybrid from Plantago asiatica as a Potent Activator of the Nonreceptor Protein Tyrosine Phosphatase Shp2.

Plasiatine (1), isolated from the seeds of Plantago asiatica, is an unprecedented indole analogue linked to a phenylpropanoid moiety via a carbon bond that builds up a novel heteromeric construction with a C19N2 scaffold. Its structure was determined by spectroscopic data and computational evidence. Notably, experimental assay demonstrated that 1 significantly enhanced the activity of the nonreceptor protein tyrosine phosphatase Shp2 in vitro in a concentration-dependent manner with an EC50 value of 0.97 µM, and activated phosphorylation of ERK, a known target of Shp2. Moreover, plasiatine (1) promoted hepatocellular HepG2 cells migration. Molecular docking suggested that plasiatine (1) binds to the catalytic cleft of Shp2. These results identified plasiatine (1) as the first small molecule Shp2 activator, and it warrants further investigation as a novel pharmaceutical tool to study the function of Shp2 in tumorigenesis.

Azacitidine and Sorafenib Therapy in a Pediatric Patient With Refractory Acute Myeloid Leukemia With Monosomy 7 and Somatic PTPN11 Mutation.

Monosomy 7 is a well-documented cytogenetic aberration in pediatric acute myeloid leukemia (AML) and may occur in combinations with molecular abnormalities including PTPN11 mutation. PTPN11 mutations contribute to leukemogenesis through upregulation of Ras pathway signaling. We present the case of a 3-year-old female with AML with monosomy 7 and somatic PTPN11 mutation who was refractory to conventional AML chemotherapy but responded to a novel regimen of azacitidine and sorafenib followed by stem cell transplantation. Combination therapy with azacitidine and sorafenib may be an effective therapeutic strategy for patients with AML with Ras pathway abnormalities.

Genetic alterations in endometrial cancer by targeted next-generation sequencing.

Many genetic factors play important roles in the development of endometrial cancer. The aim of this study was to investigate genetic alterations in the Taiwanese population with endometrial cancer. DNA was extracted from 10 cases of fresh-frozen endometrial cancer tissue. The exomes of cancer-related genes were captured using the NimbleGen Comprehensive Cancer Panel (578 cancer-related genes) and sequenced using the Illumina Genomic Sequencing Platform. Our results revealed 120 variants in 99 genes, 21 of which were included in the Oncomine Cancer Research Panel used in the National Cancer Institute Match Trial. The 21 genes comprised 8 tumor suppressor candidates (ATM, MSH2, PIK3R1, PTCH1, PTEN, TET2, TP53, and TSC1) and 13 oncogene candidates (ALK, BCL9, CTNNB1, ERBB2, FGFR2, FLT3, HNF1A, KIT, MTOR, PDGFRA, PPP2R1A, PTPN11, and SF3B1). We identified a high frequency of mutations in PTEN (50%) and genes involved in the endometrial cancer-related molecular pathway, which involves the IL-7 signaling pathway (PIK3R1, n=1; AKT2, n=1; FOXO1, n=1). We report the mutational landscape of endometrial cancer in the Taiwanese population. We believe that this study will shed new light on fundamental aspects for understanding the molecular pathogenesis of endometrial cancer and may aid in the development of new targeted therapies.