Induction of Apoptosis and Anti-Tumor Effects of a Novel Pan Inhibitor of Bcl-2 and Mcl-1 Apogossypolone (ApoG2) in Multiple Myeloma Cells.

Multiple myeloma remains an incurable malignancy and overall survival has not been improved despite responses to conventional and high-dose chemotherapy. Over-expression of both Bcl-2 and Mcl-1 is frequent in multiple myeloma which renders myeloma cells resistant to apoptosis by chemotherapy, and overexpression is associated with relapse and poorer survival. Inhibition of the anti-apoptotic function of Bcl-2 family member proteins such as Bcl-2 and Mcl-1 represents an attractive new strategy for developing anticancer drugs. Apogossypolone (ApoG2) is a novel derivative of the naturally occurring polyphenolic compound gossypol which has aldehyde moieties removed and further modification to make it pharmaceutically more stable. More particularly, ApoG2 is a potent inhibitor of Mcl-1 and Bcl-2, with Ki value of 25 nM for Mcl-1, 35 nM for Bcl-2, respectively. In this study, trypan blue dye exclusion, Hoechst 33258 staining, DNA ladder formation and annexin-V-PI flow cytometric analysis were used to determine the cellular activities of ApoG2 on cell growth inhibition, cell viability, cell cycle and apoptosis. Cleavage of caspase-3 and caspase-9 was analyzed by colorimetirc assay. Xenograft model of Wus1 cells (from Peking Union Medical College Hospital) in nude mice were used to determine the antitumor activity of compounds. We found that ApoG2 resulted in a dose and time-dependent inhibition of multiple myeloma cell proliferation, with IC50 value to both U266 and Wus1 cells at 0.1 to 0.2 uM at 48 hours after treatment. ApoG2 effectively induced apoptosis of multiple myeloma cells as evidenced by typical morphological changes, transmission electron microscopy, DNA ladder formation and increase in the percentage of cells in subdiploid peak. Colorimetric assays further showed activation of both caspase-3 and caspase-9. In a parallel direct comparison study, ApoG2 was more potent than the parental compound gossypol in both growth inhibition and induction of apoptosis. Of interest, cell cycle analysis of both U266 and Wus1 cells treated with ApoG2 produced a slightly G2 arrest, increasing from 9.7% to 19.6% in U266 cells, and from 9.8% to 31.7% in Wus1 cells, respectively. This was different from gossypol which induced mainly G1 arrest. Preliminary in vivo antitumor activity of ApoG2 was examined in xenograft model of Wus1 cells in nude mice, and growth inhibition (T/C) of 32.7% and 33.4% was obtained at 60 mg/kg, and 40 mg/kg, respectively. In addition, there was no body weight loss for both treated groups in comparison with the vehicle treated mice. Our results demonstrated that a potent pan inhibitor of Bcl-2 and Mcl-1 ApoG2 had significant effect of antiproliferation and induction of apoptosis on multiple myeloma cells in vitro and in vivo. ApoG2 may represent a promising new anticancer agent with a novel molecular mechanism and warrant further investigation as a single agent or in combination for human multiple myeloma with Bcl-2/Mcl-1 overexpression.