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

NPM knockdown induces multipolar spindles.

<p>HeLa cells were transfected with siRNA for luciferase (control) or NPM. (A) After 48 h, the whole cell lysates were analyzed by western blotting with antibody against NPM (left) and the band intensities were quantified (right). (B) Immunofluorescence with anti-NPM (green) and anti-γ-tubulin (red) antibody was performed (left). White arrows indicate centrosomes. At least 50 mitotic cells were counted in three independent experiments. The ratio of cells with multipolar spindles under the indicated conditions was quantified (below). (C) Representative multinucleated cell under the indicated conditions. (D) After 72 h, caspase-9 activity was determined using luminescence-based assay. (E) At 48 h after siRNA transfection, the cells were treated with 0–2.5 µM TC11 and then incubated for a further 72 h. Cell viability was determined by means of WST-1 assay in three independent experiments. An asterisk denotes a statistically significant difference according to Student’s <i>t</i>-test (P<0.05). Bar; 10 µm.</p

IC₅₀ values (µM) of thalidomide derivatives for inhibiting proliferation of multiple myeloma cell lines.

<p>IC₅₀ values (µM) of thalidomide derivatives for inhibiting proliferation of multiple myeloma cell lines.</p

IC₅₀ values of TC11 derivatives for inhibiting proliferation of KMS34 cells.

<p>KMS34 cells (1×10⁴ cells/well) in 96-well plate were incubated with 0–50 µM of indicated compound for 72 h. Then cell viability was determined with WST-1 assay.</p

Monomeric NPM interacts with TC11.

<p>(A) The domain structure of NPM and the amino acid sequence predicted by NPM clone 1–183, which was identified as encoding a binding protein of TC11 by mRNA display using a cDNA library prepared from KMS34 cells. The underlined part indicates the region (1–183 a. a.) identified by mRNA display selection. The sequence indicated by the solid line corresponds to that encoded by the ORF of NPM cDNA. (B) Recombinant NPM_1–183 was expressed in <i>E. coli</i> and fractionated into monomeric and oligomeric forms. Then, each fraction was subjected to 10% SDS-PAGE followed by CBB staining (left). A representative biosensorgram of NPM binding on SA sensor chips with immobilized biotinylated-TC11 is shown. The <i>K</i>_D values were determined (right).</p

TC11 inhibits tumor cell growth and induces apoptosis <i>in vivo</i>.

<p>KMS34 (1.2×10⁷ cells) were injected intraperitoneally into lcr/scid mice and plasmacytoma was established. When the size of the tumor had reached 50 mm³ (day 1), 100 µL of TC11 (20 mg/kg) or vehicle alone (10% DMSO, 1% Tween 80-saline) was injected intraperitoneally into a mouse twice with a 3-day interval (i.e., injections were done on days 1, 2, 4, 5, 7, 8, 10, 11, 13 and 14). (A) The width and length of the plasmacytoma were measured and tumor volume was calculated (n = 7). (B) Sections were stained with hematoxylin and eosin (HE). Cells with aggregated chromatin are indicated by white arrows. (C) Apoptotic cell death was detected by immunohistochemical staining with anti-single-stranded DNA antibody. Apoptotic cells are indicated by white arrows. The plot on the right side shows that the difference in the number of ssDNA-positive cells between the vehicle and TC11 groups was statistically significant (Student’s <i>t</i>-test, P<0.002). For this plot, the number of ss DNA-positive cells was counted in two fields per tumor in five tumors (10 fields in all) in each group.</p

TC11 induces apoptosis of multiple myeloma cells in a caspase-dependent manner.

<p>(A) HeLa or KMS34 cells were treated with 0–50 µM TC11 or thalidomide for 24 h. The whole cell lysates were analyzed by Western blotting with anti-PARP antibody. (B) KMS34 cells were treated with 0, 5 or 25 µM TC11 for 6 h. The whole cell lysates were analyzed by Western blotting with anti-caspase-3, 8 or 9 antibody, respectively. (C) KMS34 cells were treated with 0–50 µM TC11 or staurosporin A for 6 h. After DNA extraction, 1% agarose gel electrophoresis was performed. (D) KMS34 cells were treated with 50 µM thalidomide or TC11. After 96 h, cells were stained with FITC-coupled annexin V and propidium iodide, and induction of apoptosis was evaluated by flow cytometry.</p

Schematic representation of <i>in vitro</i> selection of TC11-binding protein using mRNA display.

<p>(A) The chemical structure of biotinylated TC11. (B) A cDNA library derived from KMS34 cells was transcribed, ligated with PEG-Puro spacer (1) and <i>in vitro</i> translated (2) to form a protein-mRNA conjugates library. The library is injected into micro fluidic chip on which TC11 is immobilized (3) and unbound molecules were washed away. The bound molecules were eluted and their mRNA portion is amplified by RT-PCR (4). The resulted DNA can be used for the next of round and analyzed by cloning and sequencing. (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038878#s4" target="_blank">Materials and Methods</a>.).</p

TC11 increases spindle multipolarity, resulting in multinucleation.

<p>HeLa cells were treated with 0–20 µM TC11 for 6 h. Then, immunofluorescence staining of γ-tubulin was performed. (A) Representative mitotic cell under the indicated conditions. (B) Mitotic cells with multipolar spindles were counted under the indicated conditions. At least 100 mitotic cells were counted per sample in three independent experiments. An asterisk denotes a statistically significant difference according to Student’s <i>t</i>-test (P<0.05). White arrows indicate centrosomes. (C) HeLa cells were treated with 5 µM TC11 for 24 h, followed by immunofluorescence staining with anti-γ-tubulin antibody (green). Nucleus was stained with DAPI (blue). Bar; 10 µm.</p