Targeting YAP Degradation by a Novel 1,2,4-Oxadiazole Derivative via Restoration of the Function of the Hippo Pathway.

Recent evidence has linked the dysregulation of the Hippo pathway to tumorigenesis and cancer progression due to its pivotal role in regulating the stability of the oncoprotein YAP. Based on an unexpected finding from the SAR study of a recently reported oxadiazole-based EGFR/c-Met dual inhibitor (compound 1), we identified a closely related derivative, compound 2, which exhibited cogent antitumor activities while devoid of compound 1's ability to promote EGFR/c-Met degradation. Compound 2 acted, in part, by facilitating YAP degradation through activation of its upstream kinase LATS1. However, it did not alter the phosphorylation status of MST1/2, a LATS1 kinase, suggesting an alternative mechanism for LATS1 activation. Orally administered compound 2 was effective in suppressing MDA-MB-231 xenograft tumor growth while exhibiting a satisfactory safety profile. From a therapeutic perspective, compound 2 might help foster new therapeutic strategies for cancer treatment by restoring the Hippo pathway regulatory function to facilitate YAP degradation.

1,2,4-Oxadiazole derivatives targeting EGFR and c-Met degradation in TKI resistant NSCLC.

Development of small-molecule agents with the ability to facilitate oncoprotein degradation has emerged as a promising strategy for cancer therapy. Since EGFR and c-Met are both implicated in oncogenesis and tumor progression, we initiated a screening program by using an in-house library to identify agents capable of inducing the concomitant suppression of EGFR and c-Met expression, which led to the identification of compound 1, a 1,2,4-oxadiazole derivative. Based on the scaffold of 1, we developed a series of derivatives to assess their efficacies in facilitating the downregulation of EGFR and c-Met, among which compound 48 represented the optimal agent. 48 showed equipotent antiproliferative activity against a panel of five NSCLC cell lines with different EGFR mutational status (IC50 = 0.2-0.6 µM), while the same panel exhibited differential sensitivity to different EGFR kinase inhibitors tested. Cell cycle analysis indicated that the antiproliferative activity of 48 was associated with its ability to cause G2/M arrest and, to a lesser extent, apoptosis. Western blot and RT-PCR analyses revealed that 48 facilitated the downregulation of EGFR and c-Met at the protein level. In vivo data showed that oral administration of 48 was effective in suppressing gefitinib-resistant H1975 xenograft tumor growth in nude mice, and at a suboptimal dose, could sensitize H1975 tumors to gefitinib. Based on these findings, 48 represents a promising candidate for further development to target EGFR TKI-resistant NSCLC via dual inhibition of EGFR and c-Met oncoproteins.

Loxapine, an antipsychotic drug, suppresses intracellular multiple-antibiotic-resistant Salmonella enterica serovar Typhimurium in macrophages.

BACKGROUND: The emergence of multiple-antibiotic-resistant (MAR) Salmonella has been a serious threat worldwide. Salmonella can invade into host cells and evade the attacks of host humoral defenses and antibiotics. Thus, a new antibacterial agent capable of inhibiting intracellular Salmonella is highly needed. METHODS: The anti-intracellular activity and cytotoxicity of drugs on intracellular bacteria and macrophages were assayed using intracellular CFU assay and MTT cell viability assay, respectively. The uptake of gentamicin into macrophage and the effect of autophagy inhibitor on loxapine's anti-intracellular Salmonella activity were assessed by using image-based high-content system. The expression of bacterial genes was measured by real-time PCR. The efflux pump activity of bacteria was measured by Hoechst accumulation assays. RESULTS: With our efforts, an antipsychotic drug, loxapine, was identified to exhibit high potency in suppressing intracellular MAR S. Typhimurium, Staphylococcus aureus, Shigella flexneri or Yersinia enterocolitica. Subsequent investigations indicated that loxapine's anti-intracellular bacteria activity was not associated with increased penetration of gentamicin into bacteria and macrophages. Loxapine didn't inhibit bacterial growth in broth at concentration up to 500 µM and has no effect on Salmonella's type III secretion system genes' expression. Blockage of autophagy also didn't reverse loxapine's anti-intracellular activity. Lastly, loxapine suppressed bacterial efflux pump activity in all bacteria tested. CONCLUSION: Altogether, our data suggested that loxapine might suppress intracellular bacteria through inhibiting of bacterial efflux pumps. In light of its unique activity, loxapine represents a promising lead compound with translational potential for the development of a new antibacterial agent against intracellular bacteria.

Development of Potent Adenosine Monophosphate Activated Protein Kinase (AMPK) Activators.

Previously, we reported the identification of a thiazolidinedione-based adenosine monophosphate activated protein kinase (AMPK) activator, compound 1 (N-[4-({3-[(1-methylcyclohexyl)methyl]-2,4-dioxothiazolidin-5-ylidene}methyl)phenyl]-4-nitro-3-(trifluoromethyl)benzenesulfonamide), which provided a proof of concept to delineate the intricate role of AMPK in regulating oncogenic signaling pathways associated with cell proliferation and epithelial-mesenchymal transition (EMT) in cancer cells. In this study, we used 1 as a scaffold to conduct lead optimization, which generated a series of derivatives. Analysis of the antiproliferative and AMPK-activating activities of individual derivatives revealed a distinct structure-activity relationship and identified 59 (N-(3-nitrophenyl)-N'-{4-[(3-{[3,5-bis(trifluoromethyl)phenyl]methyl}-2,4-dioxothiazolidin-5-ylidene)methyl]phenyl}urea) as the optimal agent. Relative to 1, compound 59 exhibits multifold higher potency in upregulating AMPK phosphorylation in various cell lines irrespective of their liver kinase B1 (LKB1) functional status, accompanied by parallel changes in the phosphorylation/expression levels of p70S6K, Akt, Foxo3a, and EMT-associated markers. Consistent with its predicted activity against tumors with activated Akt status, orally administered 59 was efficacious in suppressing the growth of phosphatase and tensin homologue (PTEN)-null PC-3 xenograft tumors in nude mice. Together, these findings suggest that 59 has clinical value in therapeutic strategies for PTEN-negative cancer and warrants continued investigation in this regard.

[Protective Effects of Ginkgolide N Against Glutamate-Induced Injury in PC12 Cells].

OBJECTIVE: To study the protective effects of ginkgolide N against glutamte-induced injury in PC12 cells and its mechanisms. METHODS: The injury model was established by treating PC12 cells with glutamate, and PC12 cells were treated with different concentrations of ginkgolide N with ginkgolide B as control. The cells activity was analyzed by MTT assay. The apoptosis of PC12 cells were examined by acridine orange( AO) staining, the reactive oxygen species and mitochondrial membrane potential of PC12 cells were examined by flow cytometry. Western blot method was used to examine the expression of Cleaved Caspase-3 protein. RESULTS: Ginkgolides N of 2-8 µgmol/L inhibited PC12 cells apoptosis and ROS accumulation induced by glutamate,stabilized membrane potential of damaged PC12, and reduced the expression of Cleaved Caspase-3 protein. CONCLUSION: Ginkgolide N has a protective effect on PC12 cells injury induced by glutamate, and the mechanism may be associated with reducing ROS generation, stabilizing membrane potential and inhibiting the expression of Cleaved Caspase-3 protein.

Targeting the Warburg effect with a novel glucose transporter inhibitor to overcome gemcitabine resistance in pancreatic cancer cells.

Gemcitabine resistance remains a significant clinical challenge. Here, we used a novel glucose transporter (Glut) inhibitor, CG-5, as a proof-of-concept compound to investigate the therapeutic utility of targeting the Warburg effect to overcome gemcitabine resistance in pancreatic cancer. The effects of gemcitabine and/or CG-5 on viability, survival, glucose uptake and DNA damage were evaluated in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cell lines. Mechanistic studies were conducted to determine the molecular basis of gemcitabine resistance and the mechanism of CG-5-induced sensitization to gemcitabine. The effects of CG-5 on gemcitabine sensitivity were investigated in a xenograft tumor model of gemcitabine-resistant pancreatic cancer. In contrast to gemcitabine-sensitive pancreatic cancer cells, the resistant Panc-1 and Panc-1(GemR) cells responded to gemcitabine by increasing the expression of ribonucleotide reductase M2 catalytic subunit (RRM2) through E2F1-mediated transcriptional activation. Acting as a pan-Glut inhibitor, CG-5 abrogated this gemcitabine-induced upregulation of RRM2 through decreased E2F1 expression, thereby enhancing gemcitabine-induced DNA damage and inhibition of cell survival. This CG-5-induced inhibition of E2F1 expression was mediated by the induction of a previously unreported E2F1-targeted microRNA, miR-520f. The addition of oral CG-5 to gemcitabine therapy caused greater suppression of Panc-1(GemR) xenograft tumor growth in vivo than either drug alone. Glut inhibition may be an effective strategy to enhance gemcitabine activity for the treatment of pancreatic cancer.

Vancomycin activates σ(B) in vancomycin-resistant Staphylococcus aureus resulting in the enhancement of cytotoxicity.

The alternative transcription factor σ(B) is responsible for transcription in Staphylococcus aureus during the stress response. Many virulence-associated genes are directly or indirectly regulated by σ(B). We hypothesized that treatment with antibiotics may act as an environmental stressor that induces σ(B) activity in antibiotic-resistant strains. Several antibiotics with distinct modes of action, including ampicillin (12 µg/ml), vancomycin (16 or 32 µg/ml), chloramphenicol (15 µg/ml), ciprofloxacin (0.25 µg/ml), and sulfamethoxazole/trimethoprim (SXT, 0.8 µg/ml), were investigated for their ability to activate this transcription factor. We were especially interested in the stress response in vancomycin-resistant S. aureus (VRSA) strains treated with vancomycin. The transcription levels of selected genes associated with virulence were also measured. Real-time quantitative reverse transcription PCR was employed to evaluate gene transcription levels. Contact hemolytic and cytotoxicity assays were used to evaluate cell damage following antibiotic treatment. Antibiotics that target the cell wall (vancomycin and ampicillin) and SXT induced σ(B) activity in VRSA strains. Expression of σ(B)-regulated virulence genes, including hla and fnbA, was associated with the vancomycin-induced σ(B) activity in VRSA strains and the increase in cytotoxicity upon vancomycin treatment. These effects were not observed in the sigB-deficient strain but were observed in the complemented strain. We demonstrate that sub-minimum inhibitory concentration (sub-MIC) levels of antibiotics act as environmental stressors and activate the stress response sigma factor, σ(B). The improper use of antibiotics may alter the expression of virulence factors through the activation of σ(B) in drug-resistant strains of S. aureus and lead to worse clinical outcomes.

Enhancement of an electroporation system for gene delivery using electrophoresis with a planar electrode.

In this paper a new electroporation (EP) system is developed, which includes an EP microchip and a logic circuit, which combined with electrophoresis (ES), can provide site-specific enhancement of gene concentration. In this ES-EP microchip, an arc planar electrode provides the ES function for DNA attraction, and interdigitated array electrodes provide appropriate electric fields for the EP on the chip surface. In addition, the adherent cells can be manipulated in situ without detachment of the ES-EP microchip, which performs the "Lab on a chip". Experimental results have shown that the efficiency of gene transfection with an attracting-electric field (35.89%) becomes much higher than that without an attracting-electric field (16.62%). Cell numbers as low as 10(4) cells, and DNA as little as 4 microg are sufficient for evaluating the phenotypic effects following the over-expression of the introduced genes on the ES-EP microchip. The proposed system has the advantages of portability, cost-effectiveness, a high transfection rate and ease of operation.

[Effect of triptolide on airway remodeling and the expression of phosphoinositide 3-kinases in asthmatic rats].

AIM: To explore the effect of Triptolide on airway remodeling and the expression of Phosphoinositide 3-Kinases in asthmatic rats. METHODS: 40 rats were randomly divided into 5 groups (n = 8): (1) Control group; (2) Asthmatic 4 weeks group; (3) Asthmatic 6 weeks group; (4) Therapeutic 4 weeks group; (5) Therapeutic 6 weeks group. The airway resistance and eosinophilic inflammation of airway wall were observed. The airway wall thickness (WA/Pi), the bronchial smooth muscle thickness (smooth muscle area/Pi) and the number of bronchial smooth muscle nucleus (N/Pi) were measured by image analysis system. The expression of PI3K protein and mRNA were determined by immunohistochemical staining and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: (1) The expression of PI3K p85alpha protein and mRNA in asthmatic 4 weeks group and asthmatic 6 weeks group were significantly higher than control group, respectively (P < 0.01). The above-mentioned parameters of therapeutic 6 weeks group were significantly lower than those of asthmatic 4 weeks group, asthmatic 6 weeks group and therapeutic 4 weeks group, respectively (P < 0.01, P < 0.01 P < 0.05). (2) The WA/Pi, the smooth muscle area/Pi and the N/Pi of asthmatic 4 weeks group and asthmatic 6 weeks group were significantly higher than control group, respectively (P < 0.01). The above-mentioned parameters of therapeutic 6 weeks group were significantly lower than those of asthmatic 4 weeks group, asthmatic 6 weeks group and therapeutic 4 weeks group, respectively (P < 0.01). (3) The airway resistance of asthmatic 4 weeks group and asthmatic 6 weeks group were significantly higher than the control group, respectively (P < 0.01). The above-mentioned parameters of therapeutic 6 weeks group were significantly lower than those of asthmatic 4 weeks group, asthmatic 6 weeks group and therapeutic 4 weeks group, respectively (P < 0.01, P < 0.01, P < 0.05). CONCLUSION: The proliferation of airway smooth muscle is a remarkable character of airway remodeling in asthma. The PI3K signal pathway may be involved in the process. Triptolide may reduce AHR and decrease the proliferation of ASMCs by inhibiting the expression of PI3K. It may have potential therapeutic effects in the asthmatic airway remodeling.

Enhancement of an electroporation system for gene delivery using electrophoresis with planar electrodes.

We developed a new electroporation system, including a microchip and a logic circuit, and combining with the function of electrophoresis, which can site-specific enhancement of the gene concentration. We have demonstrated that the electroporation microchip could enhance and target in vitro gene transfection for cell lines. In this micro-device, the outer electrodes could provide the electrophoresis function for DNA attraction, and the inner electrodes could provide appropriate electric fields for the electroporation on the chip surface. The electrostatic force can be designed into specific regions, where the DNA plasmids are attracted to provide the region-targeting function. This study successfully demonstrates that the electrostatic force can attract DNA plasmids to the cell surface and highly enhance the gene delivery. Experimental results showed that the efficiency of gene transfection with an attracting-electric field become much higher than that without an attracting-electric field. Furthermore, the adherent cells could be manipulated in situ without detachment by this EP microchip. The system has several advantages of portable, cost-effective, high transfection rate and easy operation.

Optimizing the atmospheric sampling sites using fuzzy mathematic methods.

A new approach applying fuzzy mathematic theorems, including the Primary Matrix Element Theorem and the Fisher Classification Method, was established to solve the optimization problem of atmospheric environmental sampling sites. According to its basis, an application in the optimization of sampling sites in the atmospheric environmental monitoring was discussed. The method was proven to be suitable and effective. The results were admitted and applied by the Environmental Protection Bureau (EPB) of many cities of China. A set of computer software of this approach was also completely compiled and used.