5HPP-33, a Novel Tubulin-Polymerization Inhibitor Derived from Thalidomide, Directly Inhibits Proliferation and Induces Apoptosis of Human Multiple Myeloma Cells.

Abstract

Multiple myeloma is an incurable hematological malignancy despite the use of high-dose chemotherapy with hematopoietic stem cell transplantation. In addition, repeated episode of relapse may lead to refractory or resistant multiple myeloma; therefore, novel therapeutic approaches are desired in clinical settings. Recently, thalidomide has been introduced in the treatment of myeloma, and many clinical trials have since confirmed its efficacy in patients with relapsed, refractory and newly diagnosed multiple myeloma. Multiple mechanisms have been proposed for the anti-myeloma activity of thalidomide; however, its precise mechanism of action is still unclear, because thalidomide rapidly undergoes spontaneous, nonenzymatic, hydrolytic cleavage to numerous metabolites in vivo. In addition, side effects and teratogenic potential of thalidomide have often prevented its use in the direct treatment of myeloma. To address the exact anti-myeloma effect of thalidomide and develop the new derivatives without causing teratogenic effects, we have performed structural development studies of thalidomide and obtained various analogues with specific molecular properties. Among these derivatives, we report here for the first time that 2-(2,6-Diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione (5HPP-33) has most potent anti-myeloma effect with tubulin-polymerization-inhibiting activity. 5HPP-33 inhibited cellular growth of myeloma cell lines (RPMI8226, IM9, U266) and freshly isolated myeloma cells from patients in dose (0–50 μM)- and time (0–24 h)-dependent manners with an IC50 between 1–10 μM. In contrast, thalidomide itself did not inhibit cellular growth of RPMI8226 cells except exposure with high concentration (100 μM). Cultivation with 10 μM 5HPP-33 induces G2/M phase cell cycle arrest, and a strong induction of apoptosis 3 h after treatment. Immunofluorescent staining of RPMI8226 cells with anti-tubulin antibody revealed that 10 μM 5HPP-33 inhibits tubulin-polymerization. Induction of apoptosis was also confirmed in terms of both morphological changes and DNA ladder formation. Treatment with 5HPP-33 induced caspase-3 activity and PARP cleavage. Tubulin polymerization assay using microtubule protein from porcine brain revealed that 5HPP-33 showed potent tubulin-polymerization-inhibiting activity with an IC50 of 8.1 μM, comparable to that of known tubulin-polymerization inhibitors, rhizoxin and colchicines. In addition, its activity was more potent than a known thalidomide metabolite, 5-hydroxythalidomide. Interestingly, the structural requirement for activity was critical, because other analogues and derivatives of 5HPP-33 showed only slight tubulin-polimerization-inhibiting activity. To evaluate the effects of 5HPP-33 in vivo, we are currently doing the experiments to clarify the potency of tumor reduction using RPMI8226-transplanted NOD/SCID mice model. In conclusion, a novel tubulin-polymerization inhibitor, 5HPP-33, directly inhibits proliferation and induces apoptosis of myeloma cells in vitro and in vivo. In addition, one possible mechanism of growth inhibition by thalidomide might be its tubulin-polymerization inhibition activity in vivo. Taken together, 5HPP-33 is one of the promising candidates for the new therapeutic agent of multiple myeloma.