Niclosamide induces protein ubiquitination and inhibits multiple pro-survival signaling pathways in the human glioblastoma U-87 MG cell line

<div><p>Glioblastoma is the most common and lethal malignant primary brain tumor for which the development of efficacious chemotherapeutic agents remains an urgent need. The anti-helminthic drug niclosamide, which has long been in use to treat tapeworm infections, has recently attracted renewed interest due to its apparent anticancer effects in a variety of <i>in vitro</i> and <i>in vivo</i> cancer models. However, the mechanism(s) of action remains to be elucidated. In the present study, we found that niclosamide induced cell toxicity in human glioblastoma cells corresponding with increased protein ubiquitination, ER stress and autophagy. In addition, niclosamide treatment led to down-regulation of Wnt/β-catenin, PI3K/AKT, MAPK/ERK, and STAT3 pro-survival signal transduction pathways to further reduce U-87 MG cell viability. Taken together, these results provide new insights into the glioblastoma suppressive capabilities of niclosamide, showing that niclosamide can target multiple major cell signaling pathways simultaneously to effectively promote cell death in U-87 MG cells. Niclosamide constitutes a new prospect for a therapeutic treatment against human glioblastoma.</p></div

Niclosamide represses expression of β-catenin and its downstream effectors.

<p>(A) U-87 MG cells were treated with two levels of niclosamide as indicated in the figure for 24 h. Total protein isolated from cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific for β-catenin and proteins from two target genes, cyclin D1 and survivin. β-Actin was used as a loading control. (B) Time course effect of niclosamide on the expression of cyclin D1 and survivin.</p

Niclosamide treatment reduces U-87 MG cell viability.

<p>U-87 MG cells were treated with the indicated concentrations of niclosamide for 24 h. Cell viability was determined by MTS assay. Data represent the mean ± S.E.M of at least three independent experiments. (***) <i>p</i><0.001.</p

Niclosamide inhibits STAT3 expression.

<p>Total protein was isolated from U-87 MG cells treated with 5 μM niclosamide at the times indicated in the figure. Lysates were resolved by SDS-PAGE and immunoblotted with either P-STAT3 or STAT3-specific antibodies. β-Actin was used as loading control.</p

Niclosamide promotes protein ubiquitination and apoptosis in U-87 MG cells.

<p>(A) U-87 MG cells were treated with 5 μM niclosamide and cells were collected at the indicated time points. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibody specific to ubiquitin. (B) Total cell lysates were isolated from U-87 MG cells treated with the indicated concentrations of niclosamide for 24 h, resolved by SDS-PAGE and then immunoblotted with antibody specific for ubiquitin and PARP, an apoptotic protein. β-Actin was utilized as a loading control. The image is representative of at least three independent experiments. Relative expression levels of ubiquitinated proteins and cleaved PARP protein from cells with and without niclosamide treatment were quantified by densitometry (C and D, respectively). Data represent the mean ± S.E.M of at least three independent experiments. (***) <i>p</i><0.01 and (*) <i>p</i><0.05, respectively; NS = no significant difference.</p

Niclosamide triggers ER stress and the autophagic response in U-87 MG cells.

<p>(A) U-87 MG cells were treated with 5 μM niclosamide and collected at the indicated time points. Total cell lysates were resolved on SDS-PAGE and immunoblotted with antibodies specific for CHOP and LC3. (B) U-87 MG cells were treated with 2.5 or 5 μM niclosamide and total protein was isolated from lysed cells after 24 h. Lysates were resolved on SDS-PAGE and immunoblotted with LC3-specific antibody. (C) Representative image of U-87 MG cells incubated with 5 μM niclosamide for 24 h and then stained with MDC (0.05 mM). Fluorescence particles in the cytoplasm indicate autophagic vacuoles.</p

Niclosamide suppresses AKT and ERK expression in U-87 MG cells.

<p>U-87 MG cells were treated with 5 μM niclosamide and cells were collected at time points indicated in the figure. Total protein was isolated from treated cells, resolved by SDS-PAGE and immunoblotted with specific antibodies against P-AKT, AKT, P-ERK and ERK. β-Actin was used as a loading control.</p

Schematic representation of niclosamide-mediated molecular mechanisms within human glioblastoma U-87 MG cell line.

<p>Niclosamide simultaneously inhibited multiple pro-survival signal transduction pathways and it activated other major cellular responses that inhibit cell proliferation and survival and facilitate cell death. Green lines represent activation/promotion of molecular pathways and red lines represent inhibition of cell signaling.</p