Dinaciclib decreases the levels of CDK1, cyclin B1 and Aurora A in thyroid cancer cell lines.

(A) The expression of these cell-cycle associated proteins was evaluated by Western blotting in BHP7-13, WRO82-1 and 8505C cells treated with dinaciclib (25 nM) or placebo for the indicated periods. (B) Cells were treated with dinaciclib (25 nM) or placebo for 24 h and stained with fluorescent antibodies against DAPI (blue), cyclin B1 (red) and α-tubulin (green). Cyclin B1 level was significantly reduced after treatment of dinaciclib in prophase cells of BHP7-13, WRO82-1 and 8505C. (C) Cells were treated with dinaciclib (25 nM) or placebo for 24 h and stained with fluorescent antibodies against DAPI (blue), Aurora A (red) and α-tubulin (green). Aurora A level was significantly reduced after treatment of dinaciclib in BHP7-13, WRO82-1 and 8505C cells in prophase. Scale bar, 10 μm.</p

A cyclin-dependent kinase inhibitor, dinaciclib in preclinical treatment models of thyroid cancer

<div><p>Background</p><p>We explored the therapeutic effects of dinaciclib, a cyclin-dependent kinase (CDK) inhibitor, in the treatment of thyroid cancer.</p><p>Materials and methods</p><p>Seven cell lines originating from three pathologic types of thyroid cancer (papillary, follicular and anaplastic) were studied. The cytotoxicity of dinaciclib was measured using a lactate dehydrogenase assay. The expression of proteins associated with cell cycle and apoptosis was assessed using Western blot analysis and immunofluorescence microscopy. Cell cycle distribution was measured by flow cytometry and immunofluorescence microscopy. Apoptosis and caspase-3 activity were measured by flow cytometry and fluorometric assay. Mice bearing flank anaplastic thyroid cancer (ATC) were treated with intraperitoneal injections of dinaciclib.</p><p>Results</p><p>Dinaciclib inhibited thyroid cancer cell proliferation in a dose-dependent manner. Dinaciclib had a low median-effect dose (≤ 16.0 nM) to inhibit cell proliferation in seven thyroid cancer cell lines. Dinaciclib decreased CDK1, cyclin B1, and Aurora A expression, induced cell cycle arrest in the G2/M phase, and induced accumulation of prophase mitotic cells. Dinaciclib decreased Mcl-1, Bcl-x_L and survivin expression, activated caspase-3 and induced apoptosis. <i>In vivo</i>, the growth of ATC xenograft tumors was retarded in a dose-dependent fashion with daily dinaciclib treatment. Higher-dose dinaciclib (50 mg/kg) caused slight, but significant weight loss, which was absent with lower-dose dinaciclib (40 mg/kg) treatment.</p><p>Conclusions</p><p>Dinaciclib inhibited thyroid cancer proliferation both <i>in vitro</i> and <i>in vivo</i>. These findings support dinaciclib as a potential drug for further studies in clinical trials for the treatment of patients with refractory thyroid cancer.</p></div

Dinaciclib inhibits subcutaneous xenograft growth of anaplastic thyroid cancer.

<p>(A) The therapeutic effects of dinaciclib were evaluated in mice bearing 8505C flank tumors. Daily intraperitoneal injections of lower-dose (40 mg/kg) and higher-dose (50 mg/kg) dinaciclib significantly repressed 8505C tumor growth after 6 and 4 days when compared with control mice, respectively. (B) Daily treatment of lower-dose dinaciclib did not meaningfully induce weight loss when compared with control mice during the study period. However, higher-dose dinaciclib induced significant weight loss between days 8 and 12. (C) The molecular effects of daily lower-dose dinaciclib (40 mg/kg) treatment were evaluated in 8505C tumors using Western blot analysis. ** <i>P</i> < 0.005 compared with vehicle-treated mice on day 12.</p

Dinaciclib induces cytotoxicity in thyroid cancer cells.

(A) Cytotoxicity was evaluated in cells treated with a series of six 1:1 dilutions of dinaciclib. Dose-response curves were obtained on day 4 using a LDH assay. (B) Median-effect dose (Dm) of dinaciclib on day 4 was calculated for each cell line using CompuSyn software.</p

Utility of a Histone Deacetylase Inhibitor (PXD101) for Thyroid Cancer Treatment

<div><p>Background</p><p>We evaluated the therapeutic effects of the histone deacetylase inhibitor PXD101 alone and in combination with conventional chemotherapy in treating thyroid cancer.</p> <p>Methodology/Principal Findings</p><p>We studied eight cell lines from four types of thyroid cancer (papillary, follicular, anaplastic and medullary). The cytotoxicity of PXD101 alone and in combination with three conventional chemotherapeutic agents (doxorubicin, paclitaxel and docetaxel) was measured using LDH assay. Western blot assessed expression of acetylation of histone H3, histone H4 and tubulin, proteins associated with apoptosis, RAS/RAF/ERK and PI3K/AKT/mTOR signaling pathways, DNA damage and repair. Apoptosis and intracellular reactive oxygen species (ROS) were measured by flow cytometry. Mice bearing flank anaplastic thyroid cancers (ATC) were daily treated with intraperitoneal injection of PXD101 for 5 days per week. PXD101 effectively inhibited thyroid cancer cell proliferation in a dose-dependent manner. PXD101 induced ROS accumulation and inhibited RAS/RAF/ERK and PI3K/mTOR pathways in sensitive cells. Double-stranded DNA damage and apoptosis were induced by PXD101 in both sensitive and resistant cell lines. PXD101 retarded growth of 8505C ATC xenograft tumors with promising safety. Combination therapy of PXD101with doxorubicin and paclitaxel demonstrated synergistic effects against four ATC lines <i>in</i><i>vitro</i>.</p> <p>Conclusions</p><p>PXD101 represses thyroid cancer proliferation and has synergistic effects in combination with doxorubicin and paclitaxel in treating ATC. These findings support clinical trials using PXD101 for patients with this dismal disease.</p> </div

Dinaciclib accumulates cells in G2/M phase and inhibits mitotic progression in prophase.

(A) Cell cycle analysis was undertaken by evaluating the DNA content using flow cytometry in BHP7-13 cells treated with placebo or dinaciclib (25 nM) for 24 h. (B) Statistical analyses revealed that dinaciclib (25 nM) significantly arrested cells in the G2/M phase at 24 h in all seven thyroid cancer cell lines. (C) The percentage of thyroid cancer cells in mitosis was assessed after treatment with placebo or dinaciclib (25 nM) for 24 h. Cells were stained with DAPI and chromosome features were evaluated using immunofluorescence confocal microscopy. Mitotic index was assessed with a minimum of 352 cells counted from at least ten different fields for each condition. Dinaciclib significantly increased the proportion of cells in mitosis in three thyroid cancer cell lines. (D) The distribution of cells in mitosis was determined by counting a minimum of 38 mitotic cells from ten different fields by confocal microscopy for each condition. Quantification analyses revealed 100% of mitotic cells were in prophase by the treatment of dinaciclib (25 nM) for 24 h. ** P < 0.005 compared with vehicle-treated cells.</p

Dinaciclib decreases Mcl-1, Bcl-x_L and survivin levels, activates caspase-3 and induces apoptosis in thyroid cancer cells.

<p>(A) Western blot analysis was performed in cells treated with dinaciclib (25 nM) or vehicle for the indicated time. The levels of Mcl-1, Bcl-x_L and survivin were evaluated in BHP7-13, WRO82-1 and 8505C cells. (B) Caspase-3 activity was detected using fluorometric assay kit in cells treated with dinaciclib (25 nM) or vehicle for 24 h. (C) Statistical analyses of early apoptotic cells determined by flow cytometry to detect Annexin V-positive and PI-negative staining showed dinaciclib significantly induced early apoptosis at 24 h in BHP7-13, WRO82-1 and 8505C cells. (D) Sub-G1 apoptosis was detected by measuring the DNA content using flow cytometry in cells treated with dinaciclib (25 nM) or vehicle for 72 h. Dinaciclib increased the proportions of sub-G1 cells in all seven thyroid cancer cell lines. ** <i>P</i> < 0.005 compared with vehicle-treated cells.</p

PXD101 accumulates ROS and inhibits RAS/RAF/ERK and PI3K/AKT/mTOR signaling pathways in sensitive cells, and induces DNA damage in all cell lines.

<p>A, DCFH-DA fluorescence was used to measure ROS by flow cytometry in thyroid cancer cells treated with PXD101 for 16 hours. Increasing doses of PXD101 produced more ROS in WRO82-1 and 8505C, but this effect was not observed in BHP7-13. B, two cell lines represent the ability of PXD101 to accumulate ROS. C, Immunoblot shows PXD101 repressed p-ERK1/2 (Thr202/Tyr204), p-AKT (Ser473), p-4E-BP1 (Thr37/46) and p-S6 ribosomal protein (Ser235/236) in 8505C. The inhibitory effects were not observed when low dose of PXD101 (0.625 µmol/L) applied in BHP7-13 and WRO82-1. D, increasing doses of PXD101 enhanced degradation of KU70, KU80 and RAD51, and enhanced expression of p-H2AX (Ser139), RAD52 and ERCC1 in BHP7-13, WRO82-1 and 8505C.</p

PXD101 induces cytotoxicity and increases acetylation of histone H3, histone H4 and tubulin in thyroid cancer cells.

<p>A, dose-response curves were obtained on day 4 from cells treated with a series of six 1:1 dilutions of PXD101. B, Dm of PXD101 on day 4 was calculated using CompuSyn software for each cell line. Among seven follicular cell-derived thyroid cancer lines, ATC cell lines (8305C, 8505C, KAT18 and KAT4C) had the lowest Dm, followed by well-differentiated follicular (WRO82-1) and papillary (BHP7-13) cancer, and follicular undifferentiated thyroid cancer (FRO81-2). Medullary thyroid cancer cells (TT) also had low Dm. C, PXD101 induced acetylation of histone H3 and histone H4 in a dose-dependent manner. PXD101 also increased acetylation of tubulin in BHP7-13, WRO82-1 and 8505C.</p

PXD101 induces apoptosis in BHP7-13, WRO82-1 and 8505C.

<p>A, apoptosis was analyzed by measurements of DNA content using flow cytometry at 48 and 72 hours. Increasing doses of PXD101 induced higher proportion of sub-G1 cells in all cell lines. B, immunoblot showed PXD101 degraded executioner caspase-3 in three cell lines.</p