Abstract

BackgroundCucurbitacin-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.MethodsBJAB, 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.ResultsHerein, 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.ConclusionsTaken together, our data demonstrates that JSI-124 activates the JNK signaling pathway independent of apoptosis and cell cycle arrest, leading to increased VEGF expression.

Highlights

  • 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

  • Our results indicated that JSI-124 treatment induced activation of Jun N-terminal kinase (JNK) signaling pathway leading to increased Vascular Endothelial Growth Factor (VEGF) expression that is independent of STAT3

  • JSI-124 induced activation of c-Jun N-terminal kinase (JNK) and c-Jun in B leukemic cell lines mitogen activated protein kinase (MAPK) are serine/threonine-specific protein kinases that respond to extracellular stimuli and regulate various cellular activities such as gene expression, mitosis, differentiation, proliferation and cell survival/apoptosis [9,10]

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Summary

Introduction

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. JSI-124 is a selective dual inhibitor of phospho-JAK2 and phospho-STAT3 in human breast cancer, lung cancer, neuroblastoma, and growth factor (CTGF), as demonstrated in wild type and STAT3 knock-out mouse embryonic fibroblasts [8]. Far, these different activities of JSI-124 has not been investigated in parallel, and the specificity of the compound as an inhibitor of STAT3 signaling has not been defined in relation to the effects of the drug on other signaling pathways. These genes regulate many functions including cell survival, cell death, and angiogenesis [10]

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