A Novel Phthalimide Derivative,TC11, Has Preclinical Effects on High-Risk Myeloma Cells and Osteoclasts

Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the C_max was 2.1 μM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.     

Transformation of trans-Golgi Network During the Cell Cycle in a Green Alga, Botryococcus braunii

trans -Golgi network (TGN), and the changes in its structure and behavior throughout the cell cycle of a unicellular green alga, Botryococcus braunii, were examined with deep-etching replicas and in cryo-fixed/freeze-substituted specimens. In interphase cells, the TGN consisted of a hemispherically shaped cisterna (TGN-cisterna) with regularly distributed pores on the surface and a tubular network (TGN-tubules) with clathrin-coated vesicles. The TGNs changed their structure drastically throughout the cell cycle. The TGN-cisterna disappeared from the beginning of nuclear division to the completion of the cell wall, in contrast that TGN-tubules with the clathrin-coated vesicles were always observed. The TGN-tubules produced at least five other kinds of vesicles depending on the stage of the cell cycle: 200-nm vesicles with fibrillar substances and multivesicular bodies in interphase, 180–240 nm vesicles during cell division, and 400–450 nm vesicles containing fibrils and small masses of electron-dense substances, and 200-nm vesicles containing electron-dense spherical substances just after cell division. During cell wall formation, TGN-tubules were small and had only a few clathrin-coated vesicles. After cell wall formation, TGN-tubules grew and a TGN-cisterna with pores appeared again. Received 19 October 1998/ Accepted in revised form 1 March 1999     

Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice

We have isolated phytochrome B (phyB) and phyC mutants from rice (Oryza sativa) and have produced all combinations of double mutants. Seedlings of phyB and phyB phyC mutants exhibited a partial loss of sensitivity to continuous red light (Rc) but still showed significant deetiolation responses. The responses to Rc were completely canceled in phyA phyB double mutants. These results indicate that phyA and phyB act in a highly redundant manner to control deetiolation under Rc. Under continuous far-red light (FRc), phyA mutants showed partially impaired deetiolation, and phyA phyC double mutants showed no significant residual phytochrome responses, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Interestingly, the phyB phyC double mutant displayed clear R/FR reversibility in the pulse irradiation experiments, indicating that both phyA and phyB can mediate the low-fluence response for gene expression. Rice is a short-day plant, and we found that mutation in either phyB or phyC caused moderate early flowering under the long-day photoperiod, while monogenic phyA mutation had little effect on the flowering time. The phyA mutation, however, in combination with phyB or phyC mutation caused dramatic early flowering.     

Phytotoxic substances with allelopathic activity may be central to the strong invasive potential of Brachiaria brizantha

The grass Brachiaria brizantha, native to eastern Africa, becomes naturalized and dominant quickly in the non-native areas. It was hypothesized that phytotoxic chemical interaction between this plant and native plants may play an important role in the invasion of B. brizantha. However, no potent phytotoxic substance has been reported in this species. Therefore, we investigated possible allelopathic activity and searched for phytotoxic substances with allelopathic activity in B. brizantha. An aqueous methanol extract of B. brizantha inhibited the growth of roots and shoots of garden cress (Lepidium sativum), lettuce (Lactuca sativa), timothy (Phleum pratense) and ryegrass (Lolium multiflorum) seedlings. The extract was purified by several chromatographic runs and three allelopathically active substances were isolated and identified by spectral analysis as (6R,9R)-3-oxo-α-ionol, (6R,9S)-3-oxo-α-ionol and 4-ketopinoresinol. (6R,9R)-3-Oxo-α-ionol and (6R,9S)-3-oxo-α-ionol inhibited root and shoot growth of garden cress at concentrations greater than 30 and 10 μM, respectively. The activity of (6R,9S)-3-oxo-α-ionol was 5.3- to 6.2-fold that of (6R,9R)-3-oxo-α-ionol. The stereochemistry of the hydroxyl group at position C-9 may be important for the inhibitory activities of those compounds. 4-Ketopinoresinol inhibited root and shoot growth of garden cress at concentrations greater than 30 μM. The growth inhibitory activity of (6R,9S)-3-oxo-α-ionol was the greatest and followed by 4-ketopinoresinol and (6R,9R)-3-oxo-α-ionol. These results suggest that those phytotoxic substances may contribute to the allelopathic effect caused by B. brizantha and may be involved in the invasion of B. brizantha.     

A phthalimide derivative that inhibits centrosomal clustering is effective on multiple myeloma

Despite the introduction of newly developed drugs such as lenalidomide and bortezomib, patients with multiple myeloma are still difficult to treat and have a poor prognosis. In order to find novel drugs that are effective for multiple myeloma, we tested the antitumor activity of 29 phthalimide derivatives against several multiple myeloma cell lines. Among these derivatives, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3- dione (TC11) was found to be a potent inhibitor of tumor cell proliferation and an inducer of apoptosis via activation of caspase-3, 8 and 9. This compound also showed in vivo activity against multiple myeloma cell line KMS34 tumor xenografts in ICR/SCID mice. By means of mRNA display selection on a microfluidic chip, the target protein of TC11 was identified as nucleophosmin 1 (NPM). Binding of TC11 and NPM monomer was confirmed by surface plasmon resonance. Immunofluorescence and NPM knockdown studies in HeLa cells suggested that TC11 inhibits centrosomal clustering by inhibiting the centrosomal-regulatory function of NPM, thereby inducing multipolar mitotic cells, which undergo apoptosis. NPM may become a novel target for development of antitumor drugs active against multiple myeloma.