The natural alkaloid Jerantinine B has activity in acute myeloid leukemia cells through a mechanism involving c-Jun.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogenous hematological malignancy with poor long-term survival. New drugs which improve the outcome of AML patients are urgently required. In this work, the activity and mechanism of action of the cytotoxic indole alkaloid Jerantinine B (JB), was examined in AML cells. METHODS: We used a combination of proliferation and apoptosis assays to assess the effect of JB on AML cell lines and patient samples, with BH3 profiling being performed to identify early effects of the drug (4 h). Phosphokinase arrays were adopted to identify potential driver proteins in the cellular response to JB, the results of which were confirmed and extended using western blotting and inhibitor assays and measuring levels of reactive oxygen species. RESULTS: AML cell growth was significantly impaired following JB exposure in a dose-dependent manner; potent colony inhibition of primary patient cells was also observed. An apoptotic mode of death was demonstrated using Annexin V and upregulation of apoptotic biomarkers (active caspase 3 and cleaved PARP). Using BH3 profiling, JB was shown to prime cells to apoptosis at an early time point (4 h) and phospho-kinase arrays demonstrated this to be associated with a strong upregulation and activation of both total and phosphorylated c-Jun (S63). The mechanism of c-Jun activation was probed and significant induction of reactive oxygen species (ROS) was demonstrated which resulted in an increase in the DNA damage response marker γH2AX. This was further verified by the loss of JB-induced C-Jun activation and maintenance of cell viability when using the ROS scavenger N-acetyl-L-cysteine (NAC). CONCLUSIONS: This work provides the first evidence of cytotoxicity of JB against AML cells and identifies ROS-induced c-Jun activation as the major mechanism of action.

Low-dose Bisphenol A Activates Cyp11a1 Gene Expression and Corticosterone Secretion in Adrenal Gland via the JNK Signaling Pathway.

Certain commonly used compounds that interfere with the functions of the endocrine system are classified as endocrine-disrupting chemicals (EDCs). Bisphenol A (BPA) is an EDC that is widely used in food containers. BPA levels in human sera are commonly observed to be approximately 1-100 nM. Compared with the effects of BPA on the gonads, its effects on the adrenal gland are poorly understood. To investigate the influence of BPA on steroidogenesis, we examined the activity of the steroidogenic gene Cyp11a1 and its regulatory pathways in mouse Y1 adrenal cortex cells. Treatment with BPA at < 100 µM did not cause cell death. However, increased promoter activity and protein expression of Cyp11a1 were induced by low doses of BPA (10-1000 nM). Moreover, BPA induced c-Jun phosphorylation, and a specific inhibitor of c-Jun N-terminal kinase (JNK) significantly suppressed BPA-induced steroidogenesis. Thus, treatment of adrenal cells with low doses of BPA activated Cyp11a1 and increased corticosterone production through the JNK/c-Jun signaling pathway. Identical results were observed in rats after BPA injection. The abnormal induction of hormone synthesis by BPA in the adrenal gland might be linked to human metabolic defects and neuropsychiatric disorders.

High glucose induces vascular endothelial growth factor production in human synovial fibroblasts through reactive oxygen species generation.

BACKGROUND: Diabetes is an independent risk factor of osteoarthritis (OA). Angiogenesis is essential for the progression of OA. Here, we investigated the intracellular signaling pathways involved in high glucose (HG)-induced vascular endothelial growth factor (VEGF) expression in human synovial fibroblast cells. METHODS: HG-mediated VEGF expression was assessed with qPCR and ELISA. The mechanisms of action of HG in different signaling pathways were studied using Western blotting. Knockdown of proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of c-Jun to the VEGF promoter. RESULTS: Stimulation of OA synovial fibroblasts (OASF) with HG induced concentration- and time-dependent increases in VEGF expression. Treatment of OASF with HG increased reactive oxygen species (ROS) generation. Pretreatment with NADPH oxidase inhibitor (APO or DPI), ROS scavenger (NAC), PI3K inhibitor (Ly294002 or wortmannin), Akt inhibitor, or AP-1 inhibitor (curcumin or tanshinone IIA) blocked the HG-induced VEGF production. HG also increased PI3K and Akt activation. Treatment of OASF with HG increased the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the VEGF promoter. CONCLUSIONS: Our results suggest that the HG increases VEGF expression in human synovial fibroblasts via the ROS, PI3K, Akt, c-Jun and AP-1 signaling pathway. GENERAL SIGNIFICANCE: We link high glucose on VEGF expression in osteoarthritis.

ß2-adrenergic receptors inhibit the expression of collagen type II in growth plate chondrocytes by stimulating the AP-1 factor Jun-B.

The sympathetic nervous system can regulate both osteoblast and chondrocyte growth and activity through ß(2)-adrenergic receptors (ß(2)-AR). We have shown previously that ß(2)-AR activate both adenylyl cyclase and mitogen-activated protein kinases ERK1/2 in growth plate chondrocytes prepared from ribs of embryonic E18.5 mice. Here we examined ß(2)-AR inhibition of collagen type II (Col II) expression in growth plate chondrocytes and the molecular pathways involved. Stimulation of ß(2)-AR by isoproterenol inhibited Col II mRNA and protein levels by ∼50% beginning at 2 h, with both remaining suppressed over 24 h. This inhibition was blocked by propranolol and inhibitors of either MEK1 or PKA. Isoproterenol stimulated an AP-1-luciferase reporter and increased the expression of AP-1 factors c-Fos, Fra-1, Fra-2, c-Jun, and Jun-B but had no effect on Jun-D. Stimulation of AP-1 activity was blocked by inhibitors of MEK1 or PKA. siRNA inhibition of AP-1 factors showed that depletion of only Jun-B attenuated isoproterenol-mediated inhibition of Col II. Transfection with jun-B or c-fos showed selective inhibition of Col II mRNA and a Col II luciferase reporter construct by jun-B. Isoproterenol as well as jun-B overexpression in the chondrocytes also inhibited the expression of Sox-6 mRNA and protein, and depletion of Jun-B abrogated ß(2)-AR inhibition of Sox-6. Collectively, these findings demonstrate regulation of chondrocyte differentiation through ß(2)-AR mediated by ERK1/2 and PKA stimulation of the AP-1 factor Jun-B that inhibits the expression of Sox-6 and Col II.