A synthetic Nitraria alkaloid, isonitramine protects pancreatic ß-cell and attenuates postprandial hyperglycemia.

OBJECTIVE: The extracts of Nitraria genus are composed of Nitraria alkaloids and have been used traditionally as a hypoglycemic medicine. However, the efficacy and precise mechanism of Nitraria alkaloids remain largely unknown. METHODS: Previously, we reported the total synthesis of (+)-isonitramine, one of Nitraria alkaloids. In this study, we investigated the anti-diabetic potential of isonitramine in diabetes mellitus and its underlying molecular mechanism in carbohydrate catabolism in vitro and in vivo. RESULTS: Isonitramine exerted significant inhibitory effect on α-glucosidases but not α-amylase in vitro. In zebrafish, isonitramine alleviated the streptozotocin (STZ)-induced postprandial hyperglycemia and protected the pancreatic damages against alloxan-induced oxidative stress in vivo. Also, isonitramine induced insulin without any toxicities and downregulated phosphoenolpyruvate carboxykinase (PEPCK), which catalyzes the first committed step in gluconeogenesis. CONCLUSION: Taken together, isonitramine inhibited α-glucosidase activity and PEPCK expression, while increased insulin expression, resulting in attenuating the postprandial hyperglycemia. Also, isonitramine protected the pancreas from ROS-mediated toxicities. Therefore, isonitramine may be a new drug candidate for the treatment of diabetes mellitus.

Essential Role of DNA Methyltransferase 1-mediated Transcription of Insulin-like Growth Factor 2 in Resistance to Histone Deacetylase Inhibitors.

Purpose: Histone deacetylase inhibitors (HDI) are promising anticancer therapies; however, drug resistance limits their efficacy. Here, we investigated the molecular mechanisms underlying HDI resistance, focusing on the mechanism of HDI-mediated induction of insulin-like growth factor 2 (IGF2) based on our previous study.Experimental Design: The methylation status of CCCTC-binding factor (CTCF)-binding sites in the IGF2/H19 imprinting control region (ICR) were determined by methylation-specific PCR and bisulfite sequencing. The effectiveness of single or combinatorial blockade of DNA methyltransferase 1 (DNMT1) and histone deacetylase (HDAC) was evaluated using cell viability assay and patient-derived tumor xenograft (PDX) model.Results: HDAC inhibition by vorinostat increased acetylated STAT3 (K685), resulting in transcriptional upregulation of DNMT1 DNMT1-mediated hypermethylation of CTCF-binding sites in the IGF2/H19 ICR decreased CTCF insulator activity, leading to a transcriptional upregulation of IGF2 and activation of the insulin-like growth factor 1 receptor (IGF-1R) pathway in cells with acquired or de novo vorinostat resistance. Strategies targeting DNMT1 diminished the IGF2 expression and potentiated vorinostat sensitivity in preclinical models of lung cancer with hypermethylation in the H19/IGF2 ICR. The degree of ICR hypermethylation correlated with vorinostat resistance in patient-derived lung tumors and in patients with hematologic malignancies.Conclusions: DNMT1-mediated transcriptional upregulation of IGF2 is a novel mechanism of resistance to HDIs, highlighting the role of epigenetic deregulation of IGF2 in HDI resistance and the potential value of the H19/IGF2 ICR hypermethylation and DNMT1 expression as predictive biomarkers in HDI-based anticancer therapies. Clin Cancer Res; 23(5); 1299-311. ©2016 AACR.

Activation of insulin-like growth factor receptor signaling mediates resistance to histone deacetylase inhibitors.

Histone deacetylases (HDACs) are considered promising targets in the treatment of hematologic malignancies and several types of solid tumors, including non-small cell lung cancer (NSCLC). However, the efficacy of HDAC inhibitors in solid tumors is marginal, and the mechanisms underlying resistance to HDAC inhibitors are largely unknown. Here, we demonstrate the involvement of type 1 insulin-like growth factor receptor (IGF-1R) signaling in resistance to HDAC inhibitors in NSCLC. Using MTT and soft-agar colony formation assays, we selected NSCLC cell lines that exhibited intrinsic resistance to vorinostat. Treatment with vorinostat activated IGF-1R signaling in vorinostat-resistant but not vorinostat-sensitive NSCLC cells. Other HDAC inhibitors, including trichostatin A, sodium butyrate, and depsipeptide, also activated IGF-1R signaling in vorinostat-resistant NSCLC cells. Blockade of IGF-1R signaling via IGF-1R monoclonal antibodies (mAbs) or through knockdown of IGF-1R via RNA interference sensitized vorinostat-resistant cells to HDAC inhibition. Finally, IGF-1R mAbs sensitized xenograft tumors of vorinostat-resistant cells to vorinostat treatment in vivo. These findings suggest that IGF-1R activation is generally involved in resistance to HDAC inhibitors and that targeting IGF-1R is an effective strategy for overcoming resistance to HDAC inhibitors in NSCLC.

Transduction of the MPG-tagged fusion protein into mammalian cells and oocytes depends on amiloride-sensitive endocytic pathway.

BACKGROUND: MPG is a cell-permeable peptide with proven efficiency to deliver macromolecular cargoes into cells. In this work, we examined the efficacy of MPG as an N-terminal tag in a fusion protein to deliver a protein cargo and its mechanism of transduction. RESULTS: We examined transduction of MPG-EGFP fusion protein by live imaging, flow cytometry, along with combination of cell biological and pharmacological methods. We show that MPG-EGFP fusion proteins efficiently enter various mammalian cells within a few minutes and are co-localized with FM4-64, a general marker of endosomes. The transduction of MPG-EGFP occurs rapidly and is inhibited at a low temperature. The entry of MPG-EGFP is inhibited by amiloride, but cytochalasin D and methyl-beta-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction. Overexpression of a mutant form of dynamin partially reduced the transduction of MPG-EGFP. The partial blockade of MPG-EGFP transduction by a dynamin mutant is abolished by the treatment of amiloride. MPG-EGFP transduction is also observed in the mammalian oocytes. CONCLUSION: The results show that the transduction of MPG fusion protein utilizes endocytic pathway(s) which is amiloride-sensitive and partially dynamin-dependent. Collectively, the MPG fusion protein could be further developed as a novel tool of "protein therapeutics", with potentials to be used in various cell systems including mammalian oocytes.