Combined treatment with niclosamide and camptothecin enhances anticancer effect in U87 MG human glioblastoma cells

Abstract

Glioblastoma multiforme (GBM) is one of the deadliest cancers of the brain. Its ability to infiltrate healthy brain tissues renders it difficult to remove surgically. Furthermore, it exhibits high rates of radio- and chemoresistance, making the survival rates of patients with GBM poor. Therefore, novel effective therapies for GBM remain urgently in demand. Niclosamide is an anti-helminthic drug and recently it has been receiving attention due to its reported anticancer effects in cancer models, including GBM. Furthermore, camptothecin (CPT) is a naturally-occurring alkaloid and has been previously reported to be a potential chemotherapeutic agent by targeting the nuclear topoisomerase I. In the present study, the possible combined chemotherapeutic effects of niclosamide and CPT on the human glioblastoma cell line U87 MG was investigated by MTT assay and western blot analysis. Niclosamide exhibited synergistic activities with CPT to suppress the proliferation of U87 MG cells. Additionally, niclosamide suppressed cell proliferation and induced cell death mainly by triggering ER stress and autophagy, whilst CPT induced cell apoptosis mainly through p53-mediated mitochondrial dysfunction and activation of the MAPK (ERK/JNK) pathways. Overall, these findings suggest that co-administration of niclosamide and CPT may provide a novel therapeutic treatment strategy for GBM.

Keywords: camptothecin; cancer; chemotherapy; glioblastoma; niclosamide.

Figure 1. Niclosamide (Nicl) and CPT treatments reduce U87 MG cell viability.

U-87 MG cells were treated with the indicated concentrations of niclosamide and CPT for 48 hours. Cell viability was determined by MTT assay. Mean ± S.E.M from five replicates. ^*** p < 0.001 vs. control.

Figure 2. Niclosamide and CPT treatments induce a change in U-87 MG cell morphology and a significant reduction in cell viability.

(A) Representative images of morphology of U87 MG cells were treated with 5 μM niclosamide (Nicl), 5 μM CPT, and their combination for 24 and 48 hours. Cells were imaged by phase-contrast microscopy. (B) U87 MG cells were treated with 5 μM niclosamide (Nicl), 5 μM CPT, and their combination for 48 hours. Cell viability was determined by MTT assay. Data represent the mean ± S.E.M of at least three independent experiments. ^*** p < 0.001.

Figure 3. Niclosamide and CPT induce protein ubiquitination respectively and synergistically enhance caspase-3 and PARP cleavage.

The cell lysates were prepared from U87 cells treated with either 5 μM niclosamide (Nicl), or 5 μM CPT alone, or with combined treatment for 24 hours and resolved by SDS-PAGE and then immunoblotted with antibodies specific for ubiquitin, cleaved PARP, and cleaved caspase-3 (A). β-Actin was used as the loading control. The protein levels from Western blot were quantified by densitometry. (B) Ubiquitinated proteins. (C) Cleaved PARP and cleaved caspase-3. Data represent the mean ± S.E.M. ^* p < 0.05; ^*** p < 0.001.

Figure 4. Niclosamide enhances p53 protein and cytochrome c expression but suppresses p21 expression.

CPT strongly promotes p53, p21, and cytochrome c expression. (A) U87 MG cells were treated with either 5 μM niclosamide (Nicl), 5 μM CPT, or a combination of both for 24 hours. Thereafter, the cells were collected, and the cell lysate was prepared for Western blot analysis. The proteins were resolved by SDS-PAGE and immunoblotted with specific antibodies against p21, p53, and cytochrome c. β-Actin was used as the loading control. (B) p53 protein expression levels. (C) p21 protein expression levels. (D) Cytochrome c protein expression levels. Data represent the mean ± S.E.M of three replicates. ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001.

Figure 5. The effect of niclosamide and CPT on MAPK pathway.

(A1) Niclosamide (Nicl) inhibits ERK phosphorylation, whereas CPT induces abundant ERK phosphorylation. (A2) Densitometry analysis of p-ERK phosphorylation. (B1) Niclosamide slightly upregulates JNK phosphorylation compared with control, whereas CPT dramatically increases JNK phosphorylation. The combined treatment of the two synergistically upregulates JNK phosphorylation. (B2) Densitometric analysis of p-JNK. Total protein was isolated from U87 MG cells treated with 5 μM niclosamide, 5 μM CPT, or their combination for 24 hours. Lysates were resolved by SDS-PAGE and immunoblotted with antibodies, as indicated in the figures. ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

Figure 6. The activation of ERK by CPT has no significant effect on cell apoptosis.

U87 MG cells were pretreated with 20 μM ERK signaling pathway inhibitor PD98059 (PD) for 1 hour and followed by exposure of 5 μM CPT for 24 hours. The cells were collected, and cell lysates were prepared for Western blot analysis. Antibodies used were indicated in Figure 8. (A) Western blot profile. (B) Densitometric values for p-ERK. (C) Densitometric value for cleaved PARP. Data represent the mean ± S.E.M of three replicates. ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001.

Figure 7. Niclosamide triggers ER stress, whereas CPT inhibits ER response in U-87 MG cells.

Both niclosamide and CPT induce the autophagic response in U-87 MG cells. (A) U87 MG cells were treated with 5 μM niclosamide (Nicl), 5 μM CPT, or their combination for 24 hours, then collected for lysate preparation. Total cell lysates were resolved on SDS-PAGE and immunoblotted with antibodies specific for CHOP and LC3. β-actin was used as the loading control. (B) Relative expression levels of CHOP. (C) Relative expression of LC3II. Data represent the mean ± S.E.M of three replicates. ^*** p < 0.001.

Figure 8. Niclosamide represses cyclin D1 expression, whereas CPT markedly enhances cyclin D3 expression.

The cell lysates were prepared from U87 MG cells treated with either 5 μM niclosamide (Nicl), 5 μM CPT, or their combination for 6 and 24 hours. Samples were resolved by SDS-PAGE and immunoblotted with antibodies specific for cyclin D1 and cyclin D3. β-Actin was used as the loading control. (A) Cell lysates from cells treated for 6 hours. (B) Statistical analysis of D1 and D3 expression of 6-hour treated cells. (C) Cell lysates from cells treated for 24 hours. (D) Statistical analysis of cyclin D1 and D3 expression of 24-hour treated cells. Data represent the mean ± S.E.M of three replicates. ^* p < 0.05; ^** p < 0.001; ^*** p < 0.001.