Anticancer actions of lysosomally targeted inhibitor, LCL521, of acid ceramidase

<div><p>Acid ceramidase, which catalyzes ceramide hydrolysis to sphingosine and free fatty acid mainly in the lysosome, is being recognized as a potential therapeutic target for cancer. B13 is an effective and selective acid ceramidase inhibitor <i>in vitro</i>, but not as effective in cells due to poor access to the lysosomal compartment. In order to achieve targeting of B13 to the lysosome, we designed lysosomotropic N, N-dimethyl glycine (DMG)-conjugated B13 prodrug LCL521 (1,3-di-DMG-B13). Our previous results indicated the efficient delivery of B13 to the lysosome resulted in augmented effects of LCL521 on cellular acid ceramidase as evaluated by effects on substrate/product levels. Our current studies indicate that functionally, this translated into enhanced inhibition of cell proliferation. Moreover, there were greater synergistic effects of LCL521 with either ionizing radiation or Tamoxifen. Taken together, these results clearly indicate that compartmental targeting for the inhibition of acid ceramidase is an efficient and valuable therapeutic strategy.</p></div

LCL521 demonstrates significant effects on MCF7 cells cytotoxicity and proliferation.

<p>(<b>A</b>) <b>Viable cell analysis</b>. MCF7 cells were treated with vehicles, 0.78, 1.56, 3.125, 6.25,12.5, 25, 50, and 100μM of either B13 or LCL521 for 48h and then MTT assays were performed. The results are expressed as a percentage relative to untreated cells and are presented as means ± st dev. of single experiment with 4 time replicates. (<b>B</b>) <b>Effect of LCL521 on MCF7 cell cycle</b>. MCF7 cells were treated with vehicle or with 1, 2.5, 5, 7.5 and 10 μM LCL521 for 24h. Cells were fixed with 70% ethanol overnight before adding 500μL RNase and PI solution. Samples were kept in the dark for another 45min before the FACS analysis. (n = 4, two times experiments with duplicates in each. * <i>p</i><0.01). Representative flow cytometric analyses are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177805#pone.0177805.s003" target="_blank">S3 Fig</a>.</p

IC₅₀ values for B13 and LCL521.

<p>IC₅₀ values for B13 and LCL521.</p

Compound C blocks AICAR induced AMPK activation.

<p><b>A</b>. RAW 264.7 cells were treated with AICAR (1 mM) for different time points, and expression of phosphor-AMPKα1 (pAMPKα1) and AMPKα1 was determined by western blot. <b>B</b>. RAW 264.7 cells were treated with AICAR (1 mM) in combination with different doses of compound C (CC, 0, 1, 5, and 10 µM) for 30 min, and expression of pAMPKα1 and AMPKα1 was determined by western blot. Data are represented three to four independent experiments.</p

LCL521 represents an acute and potent inhibitor of ACDase.

<p><b>(A)</b> MCF7 cells were treated with vehicle, or with 0.1, 0.25, 0.5, 1, 1.5, 2.5, 5, and 10μM LCL521 for 1h. Cer, Sph and S1P were then extracted and quantified by LC-MS/MS. (n = 4, three times experiments with one time duplicates and two times single experiment). The actual amounts of Cer, Sph and S1P after treatment with LCL521 are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177805#pone.0177805.s001" target="_blank">S1B–S1D Fig</a> (<b>B</b>). MCF7 cells were treated with vehicle or 1μM LCL521 for 15min, 30min, 1, 2, and 5h. Sph was then extracted and quantified by LC-MS/MS. (*<i>p</i><0.05, n = 7, 3 times experiments with 2 times duplicates and 1 time triplicates).</p

Synergistic effect of LCL521 and single dose of ionizing radiation.

<p>MCF7 cells were irradiated with 0 or 2Gy using 137 Cesium irradiator. 1h after IR, each replicated treatment were further treated with vehicle or 5x 1μM LCL521. For the 5-time treatment, media were replaced every 24h with fresh media that contained either vehicle or 1μM LCL521. After that, cells were cultured for 4 weeks and then stained with crystal violet (1g/500ml formalin). Representative crystal staining is shown. Colonies count is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177805#pone.0177805.s002" target="_blank">S2 Fig</a>.</p

Compound C improves survival of endotoxemic mice.

<p>Mice (19–20 animals per group) were administrated with 20 mg/kg of body weight of LPS (filled circles), 500 mg/kg of body weight of AICAR with LPS (open triangles), 25 mg/kg of body weight of compound c (CC) with LPS (open circles), or AICAR plus CC and LPS (filled triangles) as described in Methods. The mice were monitored for lethality every 2 hour for up to 24 hours.</p

Compound C diminishes LPS induced AMPK activation.

<p><b>A</b>. RAW 264.7 cells were pretreated with vehicle or 10 µM compound C (CC) for 15 min, prior to 1 µg/mL LPS challenge. After different time points of LPS challenge, expression of pAMPKα1 and pACC was determined by western blot. <b>B</b>. RAW 264.7 cells were treated with vehicle, 1 mM AICAR, 10 µM compound C (CC), or combination of AICAR and compound C for 15 min, followed by 1 µg/mL LPS treatment for 60 min. Expression of pAMPKα1 and pACC was determined by western blot. Data are represented three to four independent experiments.</p

Compound C inhibits immune response in liver.

<p><b>A–C</b>. Mice were treated with vehicle as control, LPS (2 mg/kg of body weight), AICAR (500 mg/kg of body weight) plus LPS, compound C (CC, 25 mg/kg of body weight) plus LPS, or AICAR in combination with CC and LPS as described in Methods. 12 hours after LPS injection, the mice were sacrificed, and blood and live tissue were collected. Immunohistochemistry for CD68 (×200 magnification) (<b>A</b>), MPO activities (<b>B</b>) in liver tissue, and serum TNF levels (<b>C</b>) were determined respectively. * <i>p</i><0.05; ** <i>p</i><0.005. Data are represented as mean ± SEM of six to eight independent experiments.</p

Compound C attenuates LPS induced liver injury.

<p><b>A–B</b>. Mice were injected with vehicle as control, LPS (2 mg/kg of body weight), AICAR (500 mg/kg of body weight) plus LPS, compound C (CC, 25 mg/kg of body weight) plus LPS, or AICAR together with CC and LPS as described in Methods. 12 hours after LPS injection, the mice were sacrificed, and blood and live tissue were collected. Serum levels of alanine aminotransferase (ALT) (<b>A</b>) and aspartate aminotransferase (AST) (<b>B</b>) were measured respectively. <b>C.</b> Liver injury was determined by histological examination on HE-stained liver sections, and apoptosis and necrosis of hepatocytes were indicated by arrows (×200 magnification). * <i>p</i><0.05; ** <i>p</i><0.005. Data are represented as mean ± SEM of six to eight independent experiments.</p