Pim-selective inhibitor DHPCC-9 reveals Pim kinases as potent stimulators of cancer cell migration and invasion

<p>Abstract</p> <p>Background</p> <p>Pim family kinases are small constitutively active serine/threonine-specific kinases, elevated levels of which have been detected in human hematopoietic malignancies as well as in solid tumours. While we and others have previously shown that the oncogenic Pim kinases stimulate survival of hematopoietic cells, we now examined their putative role in regulating motility of adherent cancer cells. For this purpose, we inhibited Pim kinase activity using a small molecule compound, 1,10-dihydropyrrolo[2,3-<it>a</it>]carbazole-3-carbaldehyde (DHPCC-9), which we had recently identified as a potent and selective inhibitor for all Pim family members.</p> <p>Results</p> <p>We now demonstrate that the Pim kinase inhibitor DHPCC-9 is very effective also in cell-based assays. DHPCC-9 impairs the anti-apoptotic effects of Pim-1 in cytokine-deprived myeloid cells and inhibits intracellular phosphorylation of Pim substrates such as Bad. Moreover, DHPCC-9 slows down migration and invasion of cancer cells derived from either prostate cancer or squamocellular carcinoma patients. Silencing of Pim expression reduces cell motility, while Pim overexpression enhances it, strongly suggesting that the observed effects of DHPCC-9 are dependent on Pim kinase activity. Interestingly, DHPCC-9 also abrogates NFATc-dependent migration of cancer cells, implying that NFATc factors mediate at least part of the pro-migratory effects of Pim kinases.</p> <p>Conclusions</p> <p>Altogether, our data indicate that DHPCC-9 is not only a powerful tool to investigate physiological effects of the oncogenic Pim family kinases, but also an attractive molecule for drug development to inhibit invasiveness of Pim-overexpressing cancer cells.</p

Tricyclic Benzo[cd]azulenes Selectively Inhibit Activities of Pim Kinases and Restrict Growth of Epstein-Barr Virus-Transformed Cells

Peer reviewe

Benzo[<i>cd</i>]azulenes inhibit Pim-1-dependent cell survival.

<p>(<b>A</b>) FDCP1 cell lines stably expressing neomycin (FD/Neo) or Pim-1 (FD/Pim44) were cultured for 24 h with or without IL-3 in the presence of DMSO or 5 µM inhibitors, after which cell viability was analyzed by the MTT assay. Graph represents means and standard deviations from two independent experiments with duplicate samples. Statistically significant differences between inhibitor-treated cells as compared to DMSO-treated control cells have been marked with asterisks. (<b>B</b>) Cells grown in the absence of IL-3 were stained with Trypan blue and live cells were counted at the indicated time-points. Points represent means and standard deviations from triplicate determinations from one of two similar experiments. (<b>C–D</b>) Cells were cultured for 24 h without IL-3 in the presence of increasing concentrations of either <b>1a</b> (<b>C</b>) or <b>2f</b> (<b>D</b>). Cell viability was analysed by the MTT assay and EC₅₀ values were determined. Points represent means and standard deviations from three independent experiments with duplicate samples.</p

Benzo[<i>cd</i>]azulene 1a decreases cancer cell migration without affecting cell viability or Pim protein levels.

<p>(<b>A–B</b>) PC-3 cells were cultured on 24-well plates, treated with either 0.1% DMSO (Ctrl) or 10 µM <b>1a</b> and scratched with a sterile 200 µL pipette tip. Pictures were taken at indicated time-points and analyzed. Shown are representative pictures from each time-point. The graph represents means and standard deviations from triplicate samples. (<b>C</b>) MTT assay was used to study the effects of DMSO and <b>1a</b> on PC-3 cell viability. Shown are means and standard deviations from duplicate samples from one of two similar experiments. (<b>D</b>) Western blotting with antibodies specific for each Pim family member was carried out with samples of PC-3 cells that had been treated for 24 h with either 0.1% DMSO (Ctrl) or 10 µM <b>1a</b>. Shown is a representative picture from two independent experiments. (<b>E–F</b>) Wound healing assays were performed with the UT-SCC-12A cell line similarly to the PC-3 cell line. Shown are representative pictures from indicated time-points. The graph represents means and standard deviations from triplicate samples.</p

Selectivity of benzo[<i>cd</i>]azulenes against recombinant kinases.

<p>Kinase assays were carried out at 10 µM concentrations of benzo[<i>cd</i>]azulenes dissolved in DMSO. Residual activity of the kinases is shown.</p>*<p>marks for residual activity ≤50%.</p

Synthetic steps in the preparation of new benzo[<i>cd</i>]azulenes.

<p>Synthetic steps in the preparation of new benzo[<i>cd</i>]azulenes.</p

Benzo[<i>cd</i>]azulenes prevent proliferation of lymphoblastoid cell lines.

<p>LCL cell lines were cultured for up to 9 days in optimal density on 6-well plates and treated with either 0.1% DMSO (Ctrl) or 10 µM <b>1a</b>. Viable cells were counted by Trypan blue staining. Shown are means and standard deviations from two parallel samples in one representative experiment.</p

Efficacy of benzo[<i>cd</i>]azulenes as Pim-1 inhibitors <i>in vitro</i> and in cell-based assays.

1<p>Prepared according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055409#pone.0055409-Aumller1" target="_blank">[28]</a>.</p>2<p>Prepared according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055409#pone.0055409-Aumller2" target="_blank">[29]</a>.</p>3<p>Prepared according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055409#pone.0055409-Kiriazis1" target="_blank">[30]</a>.</p><p>Residual <i>in vitro</i> activity of Pim-1 was determined in the presence of 10 µM concentrations of benzo[<i>cd</i>]azulenes dissolved in DMSO. Data were calculated as the percentage of Pim-1 autophosphorylation as compared with DMSO-treated controls. Residual cellular viabilities were determined by the MTT assay from FD/Neo and FD/Pim44 cells that had been cultured for 24 h without IL-3 in the presence of 0.1% DMSO or 5 µM inhibitors. Data were calculated as the percentage of viable cells in treated cultures as compared with DMSO-treated controls from at least two independent experiments with duplicate samples. N.D. means that viability was not determined.</p