title: Cucurbitacin-I (JSI-124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B leukemic cells.
abstract: BACKGROUND
  Cucurbitacin-I (JSI-124) is potent inhibitor of JAK/STAT3 signaling pathway and has anti-tumor activity in a variety of cancer including B cell leukemia. However, other molecular targets of JSI-124 beyond the JAK/STAT3 pathway are not fully understood.
  
  
  METHODS
  BJAB, I-83, NALM-6 and primary CLL cells were treated with JSI-124 as indicated. Apoptosis was measured using flow cytometry for accumulation of sub-G1 phase cells (indicator of apoptosis) and Annexin V/PI staining. Cell cycle was analyzed by FACS for DNA content of G1 and G2 phases. Changes in phosphorylation and protein expression of p38, Erk1/2, JNK, c-Jun, and XIAP were detected by Western blot analysis. STAT3 and c-Jun genes were knocked out using siRNA transfection. VEGF expression was determined by mRNA and protein levels by RT-PCR and western blotting. Streptavidin Pull-Down Assay was used to determine c-Jun binding to the AP-1 DNA binding site.
  
  
  RESULTS
  Herein, we show that JSI-124 activates c-Jun N-terminal kinase (JNK) and increases both the expression and serine phosphorylation of c-Jun protein in the B leukemic cell lines BJAB, I-83 and NALM-6. JSI-124 also activated MAPK p38 and MAPK Erk1/2 albeit at lower levels than JNK activation. Inhibition of the JNK signaling pathway failed to effect cell cycle arrest or apoptosis induced by JSI-124 but repressed JSI-124 induced c-Jun expression in these leukemia cells. The JNK pathway activation c-Jun leads to transcriptional activation of many genes. Treatment of BJAB, I-83, and NALM-6 cells with JSI-124 lead to an increase of Vascular Endothelial Growth Factor (VEGF) at both the mRNA and protein level. Knockdown of c-Jun expression and inhibition of JNK activation significantly blocked JSI-124 induced VEGF expression. Pretreatment with recombinant VEGF reduced JSI-124 induced apoptosis.
  
  
  CONCLUSIONS
  Taken together, our data demonstrates that JSI-124 activates the JNK signaling pathway independent of apoptosis and cell cycle arrest, leading to increased VEGF expression.
authors.affiliation: Manitoba Institute of Cell Biology, Winnipeg, MB, Canada.
authors.forename: Ganchimeg
authors.initials: G
authors.lastname: Ishdorj
authors.forename: James B
authors.initials: JB
authors.lastname: Johnston
authors.forename: Spencer B
authors.initials: SB
authors.lastname: Gibson
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002051:Burkitt Lymphoma / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D004789:Enzyme Activation / Q000187:drug effects; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects*; D016758:Genes, jun; D006801:Humans; D015448:Leukemia, B-Cell / Q000473:pathology*; D048670:MAP Kinase Kinase 4 / Q000037:antagonists & inhibitors / Q000502:physiology*; D009363:Neoplasm Proteins / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D016755:Proto-Oncogene Proteins c-jun / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D050796:STAT3 Transcription Factor / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014315:Triterpenes / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics
chemical_list: D009363:Neoplasm Proteins; D016755:Proto-Oncogene Proteins c-jun; D034741:RNA, Small Interfering; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D018808:Transcription Factor AP-1; D014315:Triterpenes; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D048670:MAP Kinase Kinase 4; C038106:cucurbitacin I