Cucurbitacin I (JSI-124) Has Potent Anti-Myeloma Effects Independent of Its Inhibition of JAK2-Induced STAT3 Activation.

Abstract

Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. JSI-124 is a plant natural product identified previously as Cucurbitacin I, isolated from various plant families such as the Cucurbitaceae and Cruciferae and has been recently described as a specific inhibitor of Janus Kinase-2 (JAK-2)/signal transducer and activator of transcription-3 (STAT3). Based on the critical role of the IL-6/STAT3 pathway in MM we studied the effects of JSI-124 on different MM cells in vitro. Different human myeloma cell lines including MM1.S, IM9, OPM-2, RPMI-8226, ARH77 in addition to the murine 5TGM myeloma cell lines were incubated with increasing concentrations of JSI-124. The impact of JSI-124 on cell proliferation, cell cycle and induction of apoptosis was studied using [3H]-Thymidine incorporation, cell counting, flow cytometry and Annexin/PI staining and caspase 3, 8 and 9 activation. JSI-124 was able to inhibit proliferation and induce apoptosis in several MM cell lines, including MM1.S, IM9, OPM-2, RPMI-8226, ARH77, U266 and 5TGM in a dose- and time-dependent manner. JSI-124 lead to activation of caspase 3, 8 and 9 indicating involvement of both extrinsic and intrinsic apoptotic pathways. JSI-124-mediated induction of apoptosis was independent of JAK2/STAT3 inhibition as MM1.S and 5TGM cells, which lack constitutive STAT3 (Tyr705) activation were equally sensitive to JSI-124 compared to U266 cells which show a constitutive STAT3 Tyr705 phosphorylation. However, JSI-124 treatment was able to abrogate IL-6 and bone marrow stroma (BMSC)-induced STAT3 (Tyr705) activation in MM1.S cells. In addition we were able to observe JSI-124 dependent inhibition of constitutive STAT3 (Ser727) activation in MM cell lines. To further delineate the mechanism underlying its anti-myeloma effects we studied the impact of JSI-124 treatment on NF-κB, MAPK and PI3K pathways. Indeed, JSI-124 treatment resulted in inhibition of p-p65, p-MEK1,2 and p-Akt underscoring the effect of JSI-124 on STAT3-independent signaling. Our results indicate that JSI-124 is a powerful direct inhibitor of myeloma cells blocking constitutive and IL-6/BMSC-dependent STAT3 activation in addition to STAT3 independent signaling pathways. JSI-124 might therefore serve as a potent novel anti-myeloma agent targeting both myeloma cells and its bone marrow microenvironment. Further studies are warranted to evaluate the in vivo efficacy of JSI-124 and identify the STAT3 independent pathways contributing to myeloma cell growth and induction of apoptosis.