Previously, we reported that EB1089 inhibited the growth of NCI-H929 myeloma cells via cell cycle arrest and apoptosis. In the present study, we investigated whether a combined EB1089 and TGF-β1 synergistically inhibited the cell proliferation of myeloma cell lines. While TGF-β1 alone could not inhibit the proliferation of any of the tested myeloma cells, synergistic effect between EB1089 (1x10-8 M) and TGF-β1 (1 ng/ml) was observed in NCI-H929 cells. TGF-β1 intensified the decreased expression of CDK2, CDK4, CDK6 and cyclin D1 in EB1089-treated NCI-H929 cells. However, these effects did not intensify to decrease CDK2 activity of EB1089-treated NCI-H929 cells, resulting in no difference in the extent of G1 arrest between EB1089- and both agents-treated cells. Remarkably, both agents synergistically induce apoptosis of NCI-H929 cells, which was accompanied with up-regulation of Bax, degradation of PARP and Rb proteins, and loss of mitochondrial transmembrane potential (Δψm). EB1089 caused the induction of SMAD4, a mediator of TGF-β1 signaling. In addition, a combined EB1089 and TGF-β1 increased p21 and JNK/SAPK activity whereas neither EB1089 nor TGF-β1 affected p21 and JNK/SAPK activity. Taken together, these results suggest that treatment with both EB1089 and TGF-β1 synergistically inhibits the proliferation of NCI-H929 cells through apoptosis.

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