Komaroviquinone-Derivatives, Revealed Anti-Tumor Effect on High-Risk Multiple Myeloma Cells In Vitro as Well as In Vivo

Background:

Recent progress in the treatment of multiple myeloma (MM) has improved its prognosis. However, the MM patients with high-risk cytogenetic abnormalities still showed significantly shorter survival. We have been focusing on komaroviquinone (KQN) which was isolated from Dracocephalum komarovi and had anti-tripanosomal activity. Since a number of anti-cancer drugs have been developed from natural products with anti-protozoal activities, we tried to examine anti-tumor effect of novel KQN-derivatives against high-risk MM cells.

Results and Discussion:

(1) Screening of KQN derivatives and the anti-myeloma activity: We screened total of 16 synthetic KQN-derivatives with regard to their ability to inhibit growth of MUM24 cells, established from a thalidomide resistant patient, harboring high-risk cytogenetic abnormalities such as t (4;14) and TP53 gene deletion. Two KQN-derivatives, GTN024 and GTN005 were found to inhibit proliferation of MUM24 (IC₅₀: 3.29 µM, 16.25 µM, respectively). When MUM24 cells were co-cultured with 5µM GTN024 and 20µM GTN005, annexin-V-positive fraction significantly increased, suggesting that GTN024 and GTN005 induced apoptosis.

(2) ROS production and anti-myeloma activity: Because GTN024 includes benzoquinone structure, we examined production of reactive oxygen species (ROS) by KQN-derivatives in MM cells. We found that GTN024 produced ROS and inhibited growth of MM cells, which was abrogated by pre-incubation with antioxidants. GTN005 does not have benzoquinone structure, and produced small amount of ROS. Its growth inhibitory activity did not change despite incubation of MUM24 cells with antioxidants. These results suggested that KQN-derivatives showed growth inhibition of MM cells via ROS production in GTN024 and ROS-independent mechanism in GTN005.

(3) Toxicity of GTN024 and GTN005: To evaluate the effect of GTNs on normal hematopoiesis, we conducted colony formation assays using murine bone marrow cells. GTN024 significantly suppressed the production of total colonies of normal hematopoietic cells in 1µM or higher dose, but GTN005 did not inhibit the production of colonies in IC₅₀ or higher dose. Therefore, GTN005 was not considered to be toxic to normal hematopoietic stem cells.

(4) Structural optimization and anti-myeloma activity: To improve the ROS-independent anti-MM effect of GTN005, we modified the benzene ring which is a common structure of KQN-derivatives. We developed two new derivatives, GTN054 and GTN057, having 1,4-hydroxy benzene or 1-hydroxy-4-acetoxy benzene, respectively. These compounds significantly inhibited proliferation of MUM24 (IC₅₀< 7µM). The results suggested that hydroxyl benzene structure was necessary for potent anti-MM effect. These two derivatives induced ROS production in MUM24 cells. However, growth inhibition did not change by pre-incubation with antioxidants. Therefore, anti-MM effects of GTN054 and GNT057 were independent of ROS production.

(5)Anti-tumor effects in vivo: Intraperitoneal injections of GTN024 (10mg/kg BW) significantly delayed the growth of subcutaneous KMS11-derived plasmacytoma in SCID xenografts. When GTN024 was injected to mice in 20mg/kg BW or lower dose, no significant organ damages or hematological toxicity was observed. In higher dose, 40mg/kg BW, hematological toxicity was not observed, but gastrointestinal obstruction occurred. By pharmacokinetic analysis of GTN024, the Cmax was 392nM at 15min after injection of GTN024 and the half-life was 23min when 20mg/kg BW was injected.

Conclusion: Our results implicated the possibility of KQN-derivatives as drugs for overcoming high-risk MM.