Download Figure 4 – Albendazole induced cell cycle arrest at G2/M phase.

Repurposing Albendazole: new potential as a chemotherapeutic agent with preferential activity
against HPV-negative head and neck squamous cell cancer
Farhad Ghasemi, Morgan Black, Nicole Pinto, Kara M Ruicci, John Yoo, Kevin Fung, Danielle MacNeil, Joe S Mymryk, John W Barrett, Anthony C Nichols
Schulich School of Medicine & Dentistry, Western University, London, ON, Canada
Abstract
Background: Albendazole is an anti-helminthic drug that has
been shown to exhibit anti-cancer properties, however its activity
in head and neck squamous cell cancer (HNSCC) was unknown.
Methods: Using a series of in vitro assays, we assessed the ability
of albendazole to inhibit proliferation in 20 HNSCC cell lines. The
HNSCC cell lines were tested for their susceptibility to a range of
albendazole doses (1nM-10μM). Cell lines that responded to
treatment were further examined for cell death, inhibition of
migration, cell cycle arrest and microtubulin distribution.
Results: Thirteen of fourteen human papillomavirus-negative
HNSCC cell lines responded to albendazole, with an average IC50
of 152 nM. In contrast, only 3 of 6 human papillomaviruspositive HNSCC cell lines responded. Albendazole treatment
resulted in apoptosis, inhibition of cell migration and cell cycle
arrest in the G2/M phase.
Figure 2 – Albendazole caused cell death and apoptosis. A)
Live/dead assays were performed with Cal33, HSC2, SCC47, 93VU-147T and WI38 cell samples that were exposed to vehicle
(DMSO-only) or 0.5 μM of albendazole (ABZ) for 24 hours (four
replicates per treatment). “(S)” marks cells lines susceptible, and
“(R)” marks cell lines not susceptible to albendazole in cell
proliferation assays. B) Cal33 cells were exposed to vehicle
(DMSO-only), 0.5 μM and 1 μM ABZ, and immunoblotted for PARP.
Staurosporine (St.) treatment was used as a positive control for
apoptosis. Presence of cleaved PARP (c-PARP) at 1 μM albendazole
treatment suggests the involvement of apoptosis pathways in cell
death.
Figure 4 – Albendazole induced cell cycle arrest at G2/M phase. Cal33 (HPV-negative),
SCC47 (HPV-positive) and WI38 (normal) cells were exposed to vehicle (DMSO-only) and 0.5
μM albendazole (ABZ) for 24 hours (3 replicates per treatment). BrdU and PI staining were
carried out prior to flow cytometry analysis. The average increase in the percentage of G2/M
phase cells in the normal WI38 cell line following albendazole treatment was significantly less
than the G2/M phase increase in the Cal33 (unpaired t-test, p<0.0001) and SCC47 (unpaired t-test,
p<0.0001) cell lines. “(S)” marks cells lines susceptible, and “(R)” marks cell lines not susceptible
to albendazole in the cell proliferation assays. * p<0.05, ** p<0.01, *** p<0.001, and NS = not
significant.
Conclusion: This study indicates that albendazole acts to inhibit
the proliferation of human papillomavirus-negative HNSCC cell
lines and warrants further study as a potential chemotherapeutic
agent for patients suffering from head and neck cancer
Introduction
• Head and neck cancer is the 6th most common cancer
diagnosis worldwide1.
• A subset of head and neck squamous cell carcinoma (HNSCC)
is associated with the human papillomavirus (HPV). Patients
with HPV-positive disease experience better outcomes2.
• Poor response of HPV-negative HNSCC patients necessitates
the development of new therapeutics3.
• Albendazole is a benzimidazole carabamate with a long history
of safe and routine use to treat parasitic infections4.
• Albendazole works by binding to tubulin, and disrupting
microtubule polymerization5.
• Accumulating evidence suggests that albendazole may have
growth inhibitory capacity against cancer cells6-9.
• We have investigated the potential activity of albendazole in
HNSCC.
Figure 3 - Albendazole impaired cell migration. Scratch assays
were performed with 4 HPV-positive and 4 HPV-negative cell lines,
exposed to various concentrations of albendazole, and imaged at 0,
2, 5, 8 and 12 hours post-treatment. Albendazole significantly
reduced the rate of cell migration in cell lines that were susceptible
in IC50 analysis, and led to no significant changes in migration rate
in the non-susceptible cell lines (ANCOVA analysis, ns=not
significant, ** p<0.01, *** p<0.001).
Results
Figure 5 – Immunofluorescence shows changes to α-tubulin distribution of Cal33 cells after
exposure to various concentrations of albendazole for 24 hours. Experimental treatments
included vehicle (DMSO), 0.1 μM, 0.5 μM and 1 μM albendazole for 24 hours. α-tubulin was
visualized using IF (green) and nuclei were stained with DAPI (blue). 40x fluorescence
microscopy was used to assess changes in cellular morphology and tubulin assembly. A dosedependent disruption in the distribution of α-tubulin was observed.
Discussion and Conclusion
Figure 1. Albendazole inhibits the cell growth of HNSCC cell lines, with
preferential activity in HPV-negative cell lines. A) HPV-negative, HPV-positive
and normal cell lines were tested against increasing doses of albendazole in
order to calculate IC50 values. Cell lines that did not reach 50% viability at the
maximum dose (10 µM) are denoted as NS (not susceptible). B) Mean potency of
albendazole for HPV-negative, HPV-positive and normal cell lines. This potency
trend suggests that albendazole demonstrates preferential activity in HPVnegative cell lines (unpaired t-test; p<0.05). * p<0.05 and *** p<0.001.
• Repurposing available drugs, with established safety profiles, is an attractive alternative to highly
costly targeted cancer therapies10,11.
• Albendazole had a potent anti-proliferative effect in our panel of HNSCC, with preferential activity
against HPV-negative cell lines.
• Growth inhibition was associated with cell death in the sensitive lines, likely involving apoptosis
based on observed PARP cleavage.
• Flow cytometry analysis demonstrated that albendazole treatment arrested HNSCC cells in the
G2/M phase, potentially due to the drugs ability to inhibit tubulin polymerization. Cells are most
sensitive to radiation during the G2/M phase of the cell cycle, hence, albendazole may have
potential as a radiosensitizer in HNSCC treatment6,12.
• The low bioavailability of the orally administered drug may serve as a potential limiting factor for its
use13. Investigations with intravenous delivery, nanoparticle assemblies and solid-dispersion
techniques offer potential solutions to increase plasma concentration of administered
albendazole14-16.
Grant Support:
This study was partially funded by a Canadian
Institute Health Research grants MOP 142491
to JSM and ACN, and MOP 340674 to ACN.
Conflicts of Interest:
Authors declare no conflict of interest.
References
1. Jemal et al. 2011 Ca Cancer J Clin 61:69.
2. Fakhry et al. 2008 J Natl Cancer Inst. 100:261.
3. Nichols et al. 2013 Curr Oncol 20:212.
4. Horton 2000 Parasitology 121:S113.
5. Lacey 1990 Parasitol Today 6:112.
6. Patel et al. 2011 Radiat Oncol 6:160
7. Pourgholami et al. 2001 Cancer Lett 165:43
8. Pourgholami et al. 2005 Cancer Chemother
Pharmacol 55:425
9. Pourgholami et al. 2006 Clin Cancer Res
12:1928
10. Light et al. 2013 Cancer 119:3900
11.Gupta et al. 2013 Trends Pharmacol Sci
34:508
12. Pawlik and Keyomarsi 2004 Radiat Oncol
59:928
13. Galtier et al. 1991 J Pharm Sci 30:705
14. Liu et al. 2013 Biomed Mater Res 101B:998
15. Kohri et al. 1999 J Pharm Pharmacol 51:159
16. Panwar et al. 2010 Int J Nanomedicine 5:101