GNF-7, a novel FLT3 inhibitor, overcomes drug resistance for the treatment of FLT3­ITD acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation accounts for a large proportion of AML patients and diagnosed with poor prognosis. Although the prognosis of FLT3-ITD AML has been greatly improved, the drug resistance frequently occurred in the treatment of FLT3 targeting drugs. GNF-7, a multitargeted kinase inhibitor, which provided a novel therapeutic strategy for overriding leukemia. In this study, we explored the antitumor activity of GNF-7 against FLT3-ITD and clinically-relevant drug resistance in FLT3 mutant AML. METHODS: Growth inhibitory assays were performed in AML cell lines and Ba/F3 cells expressing various FLT3 mutants to evaluate the antitumor activity of GNF-7 in vitro. Western blotting was used to examine the inhibitory  effect of GNF-7 on FLT3 and its downstream pathways. Molecular docking and cellular thermal shift assay (CETSA) were performed to demonstrate the binding of FLT3 to GNF-7. The survival benefit of GNF-7 in vivo was assessed in mouse models of transformed Ba/F3 cells harboring FLT3-ITD and FLT3-ITD/F691L mutation. Primary patient samples and a patient-derived xenograft (PDX) model were also used to determine the efficacy of GNF-7. RESULTS: GNF-7 inhibited the cell proliferation of Ba/F3 cells expressing FLT3-ITD and exhibited potently anti-leukemia activity on primary FLT3-ITD AML samples. Moreover, GNF-7 could bind to FLT3 protein and inhibit the downstream signaling pathway activated by FLT3 including STAT5, PI3K/AKT and MAPK/ERK. In vitro and in vivo studies showed that GNF-7 exhibited potent inhibitory activity against FLT3-ITD/F691L that confers resistant to quizartinib (AC220) or gilteritinib. Importantly, GNF-7 showed potent cytotoxic effect on leukemic stem cells, significantly extend the survival of PDX model and exhibited similar therapy effect compared with gilteritinib. CONCLUSIONS: Our results show that GNF-7 is a potent FLT3-ITD inhibitor and may become a promising lead compound applied for treating some of the clinically drug resistant patients.

Subsurface mobility of land applied greenhouse nutrient feed water and environmental implications.

Greenhouse nutrient feedwater (GNF) discharge is considered a potential contributor to eutrophication issues in Lake Erie, Ontario. Land application of GNF is an accepted legislated management response to mitigate the impact of such nutrient loads. To assess the potential environmental impacts of this management practice, field infiltration experiments were conducted at four different greenhouse operations near Leamington, Ontario. Over a three-year study, GNF was applied on agricultural land adjacent to the greenhouse operations in the fall during the first year, and along with a bromide tracer in the summer and fall in Years 2 and 3, respectively. The GNF was applied at the maximum allowable rates as defined in legislation. Chemical constituents (nutrients, metals and the conservative tracer bromide) were monitored within the soil profile matrix and pore water above the water table. The results showed that, even with the GNF being applied at the highest permissible rates, the species of interest remained within the unsaturated soil zone at low concentrations over three to six months sampling intervals. The bromide tracer test demonstrated that highly mobile species could move through permeable soils to the water table depth in a potential worst-case application scenario. However, considering the low initial concentrations, long vadose zone residence time and the low mass flux, it would appear that land application of GNF, when applied in accordance with Ontario's Regulations, is a feasible and environmentally reasonable treatment option for managing GNF.

Label-free electrochemical detection of glucose and glycated hemoglobin (HbA1c).

Measuring glucose together with glycated hemoglobin (HbA1c) could provide short-term and long-term information of blood glycemic condition after a single treatment. However, it has been a challenge to quantify glucose and HbA1c from single sample drop and strip of electrodes with suitable sensitivity, selectivity, and efficiency. In this paper, we demonstrated a label free & single sample drop electrochemical detection method of glucose and HbA1c by modifying carbon electrodes. Glucose oxidase and capture antibodies (C-Ab) against HbA1c were immobilized onto the first working electrode (WE1) and the second working electrode (WE2) of dual working screen-printed carbon electrodes (DWSPCEs), respectively. WE2 was modified with Gold Nano Flower. After that, 3-mercaptopropionic acid was coated as a linker. Finally, C-Ab was bonded by the linker. The relationship between gold surface area and concentration of HgAuCl4 was evaluated to optimize HbA1c incubation time. Linear calibration curves for glucose concentration (0.02-35 mM), HbA1c concentration (0.01 to 1mgml-1), and HbA1c percentage solution (0-14%) were obtained, with correlation coefficients of 0.99. Sensitivities of the biosensor for glucose and HbA1c were 0.5 µAmm-2mM-1 and 0.09 µAmm-2µg-1ml, respectively. The biosensor also showed proper stability (>93%, 45days) and selectivity (>92%). The efficiency of the proposed biosensor was also compared with those of commercial kits using whole blood samples of diabetic and normal cases. Results of this study demonstrate that this biosensor can measure both glucose and HbA1c electrochemically using label-free methods to overview glycemic conditions of blood samples using cheap and commercially viable dual carbon electrode-based biosensors.

Allosteric Inhibition of c-Abl to Induce Unfolded Protein Response and Cell Death in Multiple Myeloma.

Endoplasmic reticulum stress activates inositol-requiring enzyme 1α (IRE1α) and protein kinase, R-like endoplasmic reticulum kinase (PERK), the two principal regulators of the unfolded protein response (UPR). In multiple myeloma, adaptive IRE1α signaling is predominantly activated and regulates cell fate along with PERK. Recently, we demonstrated that GNF-2, an allosteric c-Abl inhibitor, rheostatically enhanced IRE1α activity and induced apoptosis through c-Abl conformational changes in pancreatic ß cells. Herein, we analyzed whether the pharmacological modulation of c-Abl conformation resulted in anti-myeloma effects. First, we investigated the effects of GNF-2 on IRE1α activity and cell fate, followed by an investigation of the anti-myeloma effects of asciminib, a new allosteric c-Abl inhibitor. Finally, we performed RNA sequencing to characterize the signaling profiles of asciminib. We observed that both GNF-2 and asciminib decreased cell viability and induced XBP1 mRNA splicing in primary human myeloma cells and myeloma cell lines. RNA sequencing identified the induction of UPR- and apoptosis-related genes by asciminib. Asciminib re-localized c-Abl to the endoplasmic reticulum, and its combination with a specific IRE1α inhibitor, KIRA8, enhanced cell death with the reciprocal induction of CHOP mRNA expression. Together, the allosteric inhibition of c-Abl-activated UPR with anti-myeloma effects; this could be a novel therapeutic target for multiple myeloma.

Targeting BCR-Abl in the treatment of Philadelphia-chromosome positive chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is an indolent malignant hematological disease that accounts for about 15% of all cases of leukemia. This disorder results from the formation of the Philadelphia chromosome that involves a reciprocal translocation that produces a lengthened chromosome 9 and shortened chromosome 22 - the Philadelphia chromosome. As a consequence of the translocation, the dysregulated BCR-Abl fusion oncoprotein is formed and it produces the abnormal proliferation of white blood cells. The treatment of CML with imatinib revolutionized the treatment of this disorder and led to the discovery and development of dozens of effective targeted protein kinase inhibitors. Imatinib (first generation), dasatinib, nilotinib, and bosutinib (second generation) have been FDA-approved for frontline therapy, and ponatinib (third generation) is approved for resistant disease with a T315I mutation. Each of these drugs is orally bioavailable. The BCR-Abl fusion protein lacks the physiological N-terminal myristoyl group that binds to a hydrophobic pocket in the large protein kinase lobe and inhibits enzyme activity. The absence of the myristoyl group leads to enhanced protein kinase catalytic activity. Asciminib was designed to bind to this binding pocket to reduce Abl kinase activity. Asciminib is orally effective and was FDA-approved as a third-line treatment for CML and a first-line treatment in patients with the T315I mutation. It blocks the activity of BCR-Abl by interacting with the myristate-binding site located 23 Å from the ATP-binding site and is the prototype of a type IV inhibitor. Asciminib is a so-called STAMP inhibitor that Specifically Targets the Abl Myristoyl Pocket.

Anti-cancer effects of 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one derivatives on hepatocellular carcinoma harboring FGFR4 activation.

Hepatocellular carcinoma (HCC) is disease with a high mortality rate and limited treatment options. Alterations of fibroblast growth factor receptor 4 (FGFR4) has been regarded as an oncogenic driver for HCC and a promising target for HCC therapeutics. Herein, we report that GNF-7, a multi-targeted kinase inhibitor, and its derivatives including SIJ1263 (IC50 < 1 nM against FGFR4) are highly potent FGFR4 inhibitors and are capable of strongly suppressing proliferation of HCC cells and Ba/F3 cells transformed with wtFGFR4 or mtFGFR4. Compared with known FGFR4 inhibitors, both GNF-7 and SIJ1263 possess much higher (up to 100-fold) anti-proliferative activities via FGFR signaling blockade and apoptosis on HCC cells. Especially, SIJ1263 is 80-fold more potent (GI50 = 24 nM) on TEL-FGFR4 V550E Ba/F3 cells than BLU9931, which suggests that SIJ1263 would be effective for overriding drug resistance. In addition, both substances strongly suppress migration/invasion and colony formation of HCC cells. It is worth noting that SIJ1263 is superior to GNF-7 with regards to the fact that activities of SIJ1263 are higher than those of GNF-7 in all assays performed in this study. Collectively, this study provides insight into designing highly potent FGFR4 inhibitors capable of potentially overcoming drug-resistance for the treatment of HCC patients.

Novel Small Molecules Capable of Blocking mtRAS-Signaling Pathway.

RAS mutants are involved in approximately 30% of all human cancers and have been regarded as undruggable targets owing to relatively smooth protein surface and obscure binding pockets. In our previous study, we have demonstrated that GNF-7, a multi-targeted kinase inhibitor, possesses potent anti-proliferative activity against Ba/F3 cells transformed with NRAS-G12D. Based on our further analysis using Ba/F3 cells transformed with mtRAS, we discovered a series of pyrimido[4,5-d]pyrimidin-2-one analogues as mtRAS-signaling pathway blockers. In addition, our efforts expanded the assessment to cancer cells with mtRAS, which revealed that these substances are also capable of strongly suppressing the proliferation of various cancer cells harboring KRAS-G12D (AsPC-1), KRAS-G12V (SW480, DU-145), KRAS-G12C (H358), KRAS-G13D (MDA-MB-231), KRAS-Q61L (HT-29), and NRAS-Q61L (OCI-AML3). We herein report novel and potent mtRAS-signaling pathway blockers, SIJ1795 and SIJ1772, possessing 2 to 10-fold increased anti-proliferative activities compared to those of GNF-7 on cancer cells harboring mtRAS as well as on Ba/F3 cells transformed with mtRAS. Both SIJ1795 and SIJ1772 attenuate phosphorylation of RAS downstream molecules (AKT and MEK) and induce apoptosis and G0/G1 cell cycle arrest on cancer cells with mtRAS. Moreover, both substances substantially suppress the migration, invasion, and colony formation of cancer cells harboring mtRAS. Taken together, this study led us to identification of SIJ1795 and SIJ1772 capable of strongly inhibiting mtRAS-signaling pathway on cancer cells harboring mtRAS.

Porcupine inhibitors: Novel and emerging anti-cancer therapeutics targeting the Wnt signaling pathway.

Porcupine is a constituent of the 19 membered Wnt family with diverse biological features such as cell differentiation, cell proliferation, cell migration, apoptosis, etc. Porcupine is a membrane-bound o-acyltransferase family protein that modulates Wnt protein through palmitoylation to allow it to depart the secretory pathway and activate cellular responses. Inhibition of Porcupine prevents palmitoylation of Wnt ligands which in turn blocks the transport of Wnt to the extracellular membrane, thus prevents the immoderate production of ß-catenin which helps to control the aberrant cell growth. Clinically, Porcupine inhibitors have shown their potential in treating majorly colorectal cancer, pancreatic cancer, hepatocellular carcinoma, head and neck cancer etc. Till date, none of the Porcupine inhibitors have been in the market and only four molecules, LGK974, ETC159, CGX1321 and RXC004 have reached the Phase I clinical trial. Present review gives a comprehensive insight on Porcupine as a novel drug target for the treatment of cancer as well as recent update on many novel heterocyclic Porcupine inhibitors with their chemical structures and pharmacology. Their physico chemical properties were also predicted using SwissADME server. Major concerns during their development have also been summarised which may throw some light for the future development of novel Porcupine inhibitors for the treatment of cancer.

The first small molecules capable of strongly suppressing proliferation of cancer cells harboring BRAF class I/II/III mutations.

BRAF mutants are categorized into three classes according to dependency on RAS signaling and RAF dimerization-dependency. Class I BRAF V600 mutants (RAS-independent monomer) are sensitive to vemurafenib. In contrast, both class II mutants (RAS-independent dimer) and class III mutants (RAS-dependent heterodimer) are insensitive to vemurafenib. It is not likely that BRAF inhibitors capable of inhibiting all classes of BRAF mutants are currently available. Herein, we report GNF-7 and its novel derivative, SIJ1227 as the first BRAF inhibitors capable of inhibiting all classes of BRAF mutants. Compared with vemurafenib and PLX8394, both GNF-7 and SIJ1227 possess much more strong anti-proliferative activities on melanoma (A375 and C8161) and lung cancer cells (H1755 and H1666) harboring BRAF V600E (class I mutant), BRAF G464E/G469A (class II mutant) and BRAF G466V (class III mutant), respectively. Also, both GNF-7 and SIJ1227 are capable of inhibiting more strongly colony formation than vemurafenib and PLX8394 in 3D soft agar assay using C8161 melanoma cells. In addition, GNF-7 and SIJ1227 suppress more strongly migration/invasion of these cancer cells than vemurafenib and PLX8394. Taken together, both GNF-7 and SIJ1227 are much superior to vemurafenib and PLX8394 in terms of capability to inhibit all classes of BRAF mutants.

The Bcr-Abl inhibitor GNF-7 inhibits necroptosis and ameliorates acute kidney injury by targeting RIPK1 and RIPK3 kinases.

Necroptosis is a form of programmed, caspase-independent cell death that is involved in various pathologic disorders such as ischemia/reperfusion injury, acute kidney injury and inflammatory bowel diseases. Identification of necroptosis inhibitors has great therapeutic potential for the treatment of necroptosis-associated diseases. In this study, we identified that the Bcr-Abl inhibitor GNF-7 was a potent inhibitor of necroptosis. GNF-7 inhibited necroptosis in both human and mouse cells, while not protecting cells from apoptosis. Drug affinity responsive target stability assay (DARTS) demonstrated that it binded with RIPK1 and RIPK3. GNF-7 inhibited RIPK1 and RIPK3 kinase activities and thus disrupted RIPK1-RIPK3 necrosome complex formation. In vivo, GNF-7 ameliorated both cisplatin- and ischemia/reperfusion-induced AKI. Orally administration of GNF-7 attenuated renal cell necrosis and reduced pro-inflammatory responses in mouse models of AKI. Taken together, our study shows that GNF-7 is a novel necroptosis inhibitor and has great potential for the treatment of acute renal inflammatory disorders by targeting both RIPK1 and RIPK3 kinases.

Imatinib and GNF-5 Exhibit an Inhibitory Effect on Growth of Hepatocellar Carcinoma Cells by Downregulating S-phase Kinase-associated Protein 2.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is one of the leading causes of cancer-related deaths worldwide. Imatinib and GNF-5 are breakpoint cluster region-Abelson murine leukemia tyrosine kinase inhibitors which have been approved for the treatment of chronic myeloid leukemia and various solid tumors. However, the effect and underlying mechanisms of imatinib and GNF-5 in HCC remain poorly defined. In this study, we investigated the anticancer activity and underlying mechanisms of imatinib and GNF-5 in HepG2 human hepatocarcinoma cells. Cell proliferation and anchorage-independent colony formation assays were done to evaluate the effects of imatinib and GNF-5 on the growth of HepG2 cells. The cell cycle was assessed by flow cytometry and verified by immunoblot analysis. Gene overexpression and knockdown assays were conducted to evaluate the function of S-phase kinase-associated protein 2 (Skp2). Imatinib and GNF-5 significantly inhibited the growth of HepG2 cells. Imatinib and GNF-5 induced G0/G1 phase cell cycle arrest by downregulating Skp2 and upregulating p27 and p21. Overexpression of Skp2 reduced the effect of imatinib and GNF-5 on HepG2 cells. Knockdown of Skp2 suppressed the proliferation and induced G0/G1 phase arrest. Furthermore, knockdown of Skp2 enhanced the effect of imatinib and GNF-5 on growth of HepG2 cells. In conclusion, imatinib and GNF-5 effectively suppress HepG2 cell growth by inhibiting Skp2 expression. Skp2 promotes the cell proliferation and reverse G0/G1 phase cell cycle arrest and it represents a potential therapeutic target for HCC treatment.

A Bcr-Abl Inhibitor GNF-2 Attenuates Inflammatory Activation of Glia and Chronic Pain.

GNF-2 is an allosteric inhibitor of Bcr-Abl. It was developed as a new class of anti-cancer drug to treat resistant chronic myelogenous leukemia. Recent studies suggest that c-Abl inhibition would provide a neuroprotective effect in animal models of Parkinson's disease as well as in clinical trials. However, the role of c-Abl and effects of GNF-2 in glia-mediated neuroinflammation or pain hypersensitivity has not been investigated. Thus, in the present study, we tested the hypothesis that c-Abl inhibition by GNF-2 may attenuate the inflammatory activation of glia and the ensuing pain behaviors in animal models. Our results show that GNF-2 reduced lipopolysaccharide (LPS)-induced nitric oxide and pro-inflammatory cytokine production in cultured glial cells in a c-Abl-dependent manner. The small interfering ribonucleic acid (siRNA)-mediated knockdown of c-Abl attenuated LPS-induced nuclear factor kappa light chain enhancer of activated B cell (NF-κB) activation and the production of pro-inflammatory mediators in glial cell cultures. Moreover, GNF-2 administration significantly attenuated mechanical and thermal hypersensitivities in experimental models of diabetic and inflammatory pain. Together, our findings suggest the involvement of c-Abl in neuroinflammation and pain pathogenesis and that GNF-2 can be used for the management of chronic pain.

Ni-Co Selenide Nanosheet/3D Graphene/Nickel Foam Binder-Free Electrode for High-Performance Supercapacitor.

Transition-metal selenide electrodes have recently attracted increasing interest in supercapacitors resulting from their superior electrochemical performance, lower-cost, and environmental friendliness. Herein, we report a novel bimetallic Ni-Co selenide nanosheet/three-dimensional (3D) graphene/nickel foam binder-free electrode (NiCo2.1Se3.3 NSs/3D G/NF) prepared via chemical vapor deposition followed by a simple two-step hydrothermal process in this paper. The NiCo2.1Se3.3 NSs array vertically on 3D G/NF with a uniform and stable structure without using any chemical binders. This novel electrode is flexible, highly conductive, and exhibits an excellent specific capacitance of ∼742.4 F g-1 at 1 mA cm-2. Furthermore, with a 10-fold increase to 10 mA cm-2, it still retains 471.78 F g-1 and a high cycling stability of ∼83.8% of the initial retention after 1000 cycles at 10 mA cm-2, demonstrating that NiCo2.1Se3.3 NSs/3D G/NF binder-free electrode has potential for energy storage application in high-performance supercapacitor fields.

Imidazolopiperazines Kill both Rings and Dormant Rings in Wild-Type and K13 Artemisinin-Resistant Plasmodium falciparum In Vitro.

Artemisinin (ART) resistance has spread through Southeast Asia, posing a serious threat to the control and elimination of malaria. ART resistance has been associated with mutations in the Plasmodium falciparum kelch-13 (Pfk13) propeller domain. Phenotypically, ART resistance is defined as delayed parasite clearance in patients due to the reduced susceptibility of early ring-stage parasites to the active metabolite of ART dihydroartemisinin (DHA). Early rings can enter a state of quiescence upon DHA exposure and resume growth in its absence. These quiescent rings are referred to as dormant rings or DHA-pretreated rings (here called dormant rings). The imidazolopiperazines (IPZ) are a novel class of antimalarial drugs that have demonstrated efficacy in early clinical trials. Here, we characterized the stage of action of the IPZ GNF179 and evaluated its activity against rings and dormant rings in wild-type and ART-resistant parasites. Unlike DHA, GNF179 does not induce dormancy. We show that GNF179 is more rapidly cidal against schizonts than against ring and trophozoite stages. However, with 12 h of exposure, the compound effectively kills rings and dormant rings of both susceptible and ART-resistant parasites within 72 h. We further demonstrate that in combination with ART, GNF179 effectively prevents recrudescence of dormant rings, including those bearing pfk13 propeller mutations.

Coronavirus S protein-induced fusion is blocked prior to hemifusion by Abl kinase inhibitors.

Enveloped viruses gain entry into host cells by fusing with cellular membranes, a step that is required for virus replication. Coronaviruses, including the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and infectious bronchitis virus (IBV), fuse at the plasma membrane or use receptor-mediated endocytosis and fuse with endosomes, depending on the cell or tissue type. The virus spike (S) protein mediates fusion with the host cell membrane. We have shown previously that an Abelson (Abl) kinase inhibitor, imatinib, significantly reduces SARS-CoV and MERS-CoV viral titres and prevents endosomal entry by HIV SARS S and MERS S pseudotyped virions. SARS-CoV and MERS-CoV are classified as BSL-3 viruses, which makes experimentation into the cellular mechanisms involved in infection more challenging. Here, we use IBV, a BSL-2 virus, as a model for studying the role of Abl kinase activity during coronavirus infection. We found that imatinib and two specific Abl kinase inhibitors, GNF2 and GNF5, reduce IBV titres by blocking the first round of virus infection. Additionally, all three drugs prevented IBV S-induced syncytia formation prior to the hemifusion step. Our results indicate that membrane fusion (both virus-cell and cell-cell) is blocked in the presence of Abl kinase inhibitors. Studying the effects of Abl kinase inhibitors on IBV will be useful in identifying the host cell pathways required for coronavirus infection. This will provide an insight into possible therapeutic targets to treat infections by current as well as newly emerging coronaviruses.

Anti-growth Effects of Imatinib and GNF5 via Regulation of Skp2 in Human Hepatocellular Carcinoma Cells.

BACKGROUND: Human hepatocellular carcinoma (HCC) is a common liver tumor and the main cause of cancer-related death. Tyrosine kinase inhibitors, such as imatinib and GNF5 which were developed to treat chronic myelogenous leukemia, regulate the progression of various cancers. The aim of this study was to confirm the anti-tumor activity of tyrosine kinase inhibitors through regulation of S-phase kinase-associated protein 2 (Skp2), an important oncogenic factor in various cancer cells, in human hepatocarcinoma SK-HEP1 cells. METHODS: Cell viability and colony formation assays were conducted to evaluate the effects of imatinib, GNF5 and GNF2 on the growth of SK-HEP1 cells. Using immunoblot analysis, we assessed change of the activation of caspases, PARP, Akt, mitogen-activated protein kinases, and Skp2/p27/p21 pathway by imatinib and GNF5 in SK-HEP1 cells. Using sh-Skp2 HCC cells, the role of Skp2 in the effects of imatinib and GNF5 was evaluated. RESULTS: Imatinib and GNF5 significantly inhibited the growth of SK-HEP1 cells. Treatment of imatinib and GNF5 decreased Skp2 expression and Akt phosphorylation, and increased the expression of p27, p21, and active-caspases in SK-HEP1 cells. In sh-Skp2 HCC cells, cell growth and the expression of Skp2 were inhibited by more than in the mock group treated with imatinib and GNF5. CONCLUSIONS: These results suggest that the anti-growth activity of tyrosine kinase inhibitors may be associated with the regulation of p27/p21 and caspases through Skp2 blockage in HCC cells.

Anti-growth Effects of Imatinib and GNF5 via Regulation of Skp2 in Human Hepatocellular Carcinoma Cells

BACKGROUND: Human hepatocellular carcinoma (HCC) is a common liver tumor and the main cause of cancer-related death. Tyrosine kinase inhibitors, such as imatinib and GNF5 which were developed to treat chronic myelogenous leukemia, regulate the progression of various cancers. The aim of this study was to confirm the anti-tumor activity of tyrosine kinase inhibitors through regulation of S-phase kinase-associated protein 2 (Skp2), an important oncogenic factor in various cancer cells, in human hepatocarcinoma SK-HEP1 cells. METHODS: Cell viability and colony formation assays were conducted to evaluate the effects of imatinib, GNF5 and GNF2 on the growth of SK-HEP1 cells. Using immunoblot analysis, we assessed change of the activation of caspases, PARP, Akt, mitogen-activated protein kinases, and Skp2/p27/p21 pathway by imatinib and GNF5 in SK-HEP1 cells. Using sh-Skp2 HCC cells, the role of Skp2 in the effects of imatinib and GNF5 was evaluated. RESULTS: Imatinib and GNF5 significantly inhibited the growth of SK-HEP1 cells. Treatment of imatinib and GNF5 decreased Skp2 expression and Akt phosphorylation, and increased the expression of p27, p21, and active-caspases in SK-HEP1 cells. In sh-Skp2 HCC cells, cell growth and the expression of Skp2 were inhibited by more than in the mock group treated with imatinib and GNF5. CONCLUSIONS: These results suggest that the anti-growth activity of tyrosine kinase inhibitors may be associated with the regulation of p27/p21 and caspases through Skp2 blockage in HCC cells.

Aromaticity of graphene nanoflakes in a new way: fragment analysis by combination of the nucleus-independent chemical shifts and the anisotropy of current induced density.

A graphene nanoflake (GNF) is a polycyclic aromatic hydrocarbon (PAH) with a huge two-dimensional π-conjugated carbon material in which a central benzene ring is surrounded by identical benzene-type rings through infinite alternant method. In this paper, we explore the structure-aromaticity relationship of the GNFs and the GNFs with hollow sites (GNFHs) by combining the nucleus-independent chemical shifts (NICS) with the anisotropy of the current induced density (ACID). Firstly, the benzene is a typical aromatic molecule (NICS = -9.671 ppm), GNFs 1-6 is darned with benzene and the corresponding GNFHs 1'-6'. Secondly, the NICS values of GNFs 1-6 alternately vary: -1.214 (1) > -13.847 (2) < -2.662 (3) > -14.530 (4) < -3.932 (5) > -13.978 (6) ppm, the GNFs (2, 4, 6) with even fragments of annulene have larger aromaticity than that of GNFs (1, 3, 5) with odd fragments of annulene. Significantly, the NICS values of GNFs 1-6 can also be fragment analyzed by the NICS values and ACID of benzene and corresponding GNFHs 1'-6'. The NICS values for GNFs (2, 4, 6) can be roughly estimated by the NICS value of benzene minus the NICS value of the GNFHs (2', 4', 6'), respectively. The NICS values for GNFs (1, 3, 5) can be roughly estimated by the NICS value of the GNFHs (1', 3', 5') minus the NICS value of benzene, respectively. We hope that the present work can provide a simple and reliable method for the rational design of the GNF with aromaticity, which may be used to understand the origin of the graphene nanoflake aromatic properties.

Novel factors modulating human ß-cell proliferation.

ß-Cell dysfunction in type 1 and type 2 diabetes is accompanied by a progressive loss of ß-cells, and an understanding of the cellular mechanism(s) that regulate ß-cell mass will enable approaches to enhance hormone secretion. It is becoming increasingly recognized that enhancement of human ß-cell proliferation is one potential approach to restore ß-cell mass to prevent and/or cure type 1 and type 2 diabetes. While several reports describe the factor(s) that enhance ß-cell replication in animal models or cell lines, promoting effective human ß-cell proliferation continues to be a challenge in the field. In this review, we discuss recent studies reporting successful human ß-cell proliferation including WS6, an IkB kinase and EBP1 inhibitor; harmine and 5-IT, both DYRK1A inhibitors; GNF7156 and GNF4877, GSK-3ß and DYRK1A inhibitors; osteoprotegrin and Denosmab, receptor activator of NF-kB (RANK) inhibitors; and SerpinB1, a protease inhibitor. These studies provide important examples of proteins and pathways that may prove useful for designing therapeutic strategies to counter the different forms of human diabetes.