Hao-hao Zhang1, Shan Kuang1, Ying Wang1, Xiao-xiao Sun1, Yuan Gu1, Li-hong Hu2, Qiang Yu1. 1. Department of Tumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. 2. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Abstract
AIM: To study the function and mechanism of bigelovin, a sesquiterpene lactone from the flower of Chinese herb Inula hupehensis, in regulating JAK2/STAT3 signaling and cancer cell growth. METHODS: HepG2 cells stably transfected with the STAT3-responsive firefly luciferase reporter plasmid (HepG2/STAT3 cells), and a panel of human cancer cell lines were used to identify active compounds. Cell viability was measured using MTT assay. Western blotting was used to detect protein expression and phosphorylation. Kinase assays were performed and the reaction between bigelovin and thiol-containing compounds was analyzed with LC-MS. RESULTS: Bigelovin (1-50 μmol/L) dose-dependently inhibited the IL-6-induced STAT3 activation in HepG2/STAT3 cells (IC50=3.37 μmol/L) and the constitutive STAT3 activation in A549 and MDA-MB-468 cells. Furthermore, bigelovin dose-dependently inhibited JAK2 phosphorylation in HeLa and MDA-MB-468 cells, as well as the enzymatic activity of JAK2 in vitro (IC50=44.24 μmol/L). Pretreatment of the cells with DTT (500 μmol/L) or GSH (500 μmol/L) eliminated the inhibitory effects of bigelovin on the IL-6-induced and the constitutive STAT3 activation. The results in LC-MS analysis suggested that bigelovin might react with cysteine residues of JAK2 leading to inactivation of JAK2. Bigelovin (5 and 20 μmol/L) had no effects on the signaling pathways of growth factors EGF, PDGF or insulin. Finally, bigelovin suppressed the cell viability and induced apoptosis in 10 different human cancer cell lines, particularly those with constitutively activated STAT3. CONCLUSION: Bigelovin potently inhibits STAT3 signaling by inactivating JAK2, and induces apoptosis of a variety of human cancer cells in vitro.
AIM: To study the function and mechanism of bigelovin, a sesquiterpene lactone from the flower of Chinese herb Inula hupehensis, in regulating JAK2/STAT3 signaling and cancer cell growth. METHODS: HepG2 cells stably transfected with the STAT3-responsive firefly luciferase reporter plasmid (HepG2/STAT3 cells), and a panel of humancancer cell lines were used to identify active compounds. Cell viability was measured using MTT assay. Western blotting was used to detect protein expression and phosphorylation. Kinase assays were performed and the reaction between bigelovin and thiol-containing compounds was analyzed with LC-MS. RESULTS: Bigelovin (1-50 μmol/L) dose-dependently inhibited the IL-6-induced STAT3 activation in HepG2/STAT3 cells (IC50=3.37 μmol/L) and the constitutive STAT3 activation in A549 and MDA-MB-468 cells. Furthermore, bigelovin dose-dependently inhibited JAK2 phosphorylation in HeLa and MDA-MB-468 cells, as well as the enzymatic activity of JAK2 in vitro (IC50=44.24 μmol/L). Pretreatment of the cells with DTT (500 μmol/L) or GSH (500 μmol/L) eliminated the inhibitory effects of bigelovin on the IL-6-induced and the constitutive STAT3 activation. The results in LC-MS analysis suggested that bigelovin might react with cysteine residues of JAK2 leading to inactivation of JAK2. Bigelovin (5 and 20 μmol/L) had no effects on the signaling pathways of growth factors EGF, PDGF or insulin. Finally, bigelovin suppressed the cell viability and induced apoptosis in 10 different humancancer cell lines, particularly those with constitutively activated STAT3. CONCLUSION: Bigelovin potently inhibits STAT3 signaling by inactivating JAK2, and induces apoptosis of a variety of humancancer cells in vitro.
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