Fenben is a broad spectrum benzimidazole anthelmintic used against parasitic organisms of gastrointestinal tract including giardia, roundworms, hookworms, whipworms, the tapeworm genus Taenia, pinworms and aelurostrongylus. It is also known to have anti-inflammatory and immunomodulatory properties and has been shown to have anti-neoplastic activity. However, its mechanisms of action in cancer cells remain unknown.

Previous studies showed that benzimidazole compounds such as fenbendazole (FZ) and metronidazole inhibited cell growth in human neoplasms. Additionally, the antiparasitic agent mebendazole, another drug in this class, reduced tumor growth in mice. This suggests that these agents may have potential as an anti-cancer therapy.

We previously reported that FZ disrupts microtubule structures in human cancer cells and induces cell death. We also demonstrated that it prevents cellular proliferation by inhibiting the expression of GLUT transporters and hexokinase II, two key glycolytic enzymes in cancer cells. Furthermore, we found that it binds to the tubulin protein, blocking its ability to promote microtubule polymerization and altering their organization. In addition, fenbendazole prevents mitochondrial membrane potential collapse, a process that enables the accumulation of pro-oxidative reactive oxygen species and causes mitochondrial dysfunction.

In the present study, we further characterized the effect of FZ on cell viability by using a viability assay with varying concentrations of the drug and in hypoxia. Incubation of cancer cells with FZ in severe hypoxia significantly augmented the cell viability inhibitory effect, which increased with increasing drug doses. This result was consistent with the survival curves obtained in the same cells as in normal culture, demonstrating that FZ exerts a direct anti-proliferative effect on cancer cells in severe hypoxia.

A significant fraction of the tubulin in FZ treated cells was acetylated, a modification that is associated with microtubule stability. To investigate whether the acetylation of tubulin could be responsible for the inhibitory effect of FZ, NSCLC H460 and A549 cells were treated with various amounts of FZ for 24 h and cell extracts were analysed by Western blot using Ac-a-tubulin specific antibody. While treatment with colchicine, nocodazole and taxol induced marked reduction of acetylated tubulin, FZ had a less pronounced effect on tubulin acetylation.

We further investigated the mechanism by which FZ causes mitochondrial dysfunction and apoptosis. HeLa cells were incubated with different concentrations of FZ and then stained with a red mitochondrial marker Mitotracker. Cells were analyzed by fluorescence microscopy and cell lysates were analyzed for the levels of cytosolic p53 protein by Western blot. Dose dependent FZ treatment significantly increased the nuclear accumulation of WT p53 in NSCLC cells and this correlated well with the enhanced apoptotic activity. This is in agreement with our earlier findings that transcriptionally active p53 is involved in augmenting cell death following mild disruption of the microtubule network caused by FZ. fenben