Korean J Physiol Pharmacol.  2022 Sep;26(5):377-387. 10.4196/kjpp.2022.26.5.377.

Anti-cancer effects of fenbendazole on 5-fluorouracil-resistant colorectal cancer cells

• Park D
• Lee JH
• Yoon SP

• KMID: 2532767
• DOI: http://doi.org/10.4196/kjpp.2022.26.5.377

Abstract

Benzimidazole anthelmintic agents have been recently repurposed to overcome cancers resistant to conventional therapies. To evaluate the anti-cancer effects of benzimidazole on resistant cells, various cell death pathways were investigated in 5-fluorouracil-resistant colorectal cancer cells. The viability of wild-type and 5-fluorouracil-resistant SNU-C5 colorectal cancer cells was assayed, followed by Western blotting. Flow cytometry assays for cell death and cell cycle was also performed to analyze the anti-cancer effects of benzimidazole. When compared with albendazole, fenbendazole showed higher susceptibility to 5-fluorouracil-resistant SNU-C5 cells and was used in subsequent experiments. Flow cytometry revealed that fenbendazole significantly induces apoptosis as well as cell cycle arrest at G2/ M phase on both cells. When compared with wild-type SNU-C5 cells, 5-fluorouracilresistant SNU-C5 cells showed reduced autophagy, increased ferroptosis and ferroptosis-augmented apoptosis, and less activation of caspase-8 and p53. These results suggest that fenbendazole may be a potential alternative treatment in 5-fluorouracilresistant cancer cells, and the anticancer activity of fenbendazole does not require p53 in 5-fluorouracil-resistant SNU-C5 cells.

Keyword

Apoptosis; Colorectal cancer; Drug resistance; Fenbendazole; Fenbendazole

Figure

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  Fig. 1 Anti-cancer effects of fenbendazole in colorectal cancer cells. (A) The SNU-C5 and 5-FU-resistant SNU-C5 (SNU-C5/5-FUR) cells were mock-treated with dimethyl sulfoxide (DMSO) or treated with indicated concentrations of fenbendazole or albendazole for 3 days. The extent of cell viability was determined by MTT assay as described in Methods. Data are presented as mean ± SD. **p < 0.01 and ***p < 0.001 vs. DMSO. #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. SNU-C5. (B) The SNU-C5 and SNU-C5/5-FUR cells were mock-treated with DMSO or treated with indicated doses of fenbendazole for indicated days. Cell viability was determined as described in (A). (C) Representative images of SNU-C5 and SNU-C5/5-FUR cells treated with fenbendazole. Scale bar = 200 μm.

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  Fig. 2 Fenbendazole-induced cell cycle arrest at G2/M phase with increased p21 expression in colorectal cancer cells. (A) Cell cycle distribution in SNU-C5 and SNU-C5/5-FUR cells were assessed by flow cytometry after treatment with mock (dimethyl sulfoxide, DMSO) or indicated dose of fenbendazole for 3 days. The percentages of cells in each phase for SNU-C5 (left) and SNU-C5/5-FUR (right) cells are presented as the mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. DMSO. (B) SNU-C5 (left) and SNU-C5/5-FUR (right) cells were mock-treated with DMSO or treated with indicated dose of fenbendazole. 3 days after treatment, cells were harvested and whole cell extracts were subjected to immunoblotting using the indicated antibodies. Signal intensities of p27, p21, Cyclin B1, and C-Myc were measured by AzureSpot analysis software. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. DMSO.

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  Fig. 3 Fenbendazole-induced apoptosis via mitochondrial injury and caspase-3-poly (ADP-ribose) polymerase (PARP) pathways in colorectal cancer cells. (A) Cell death distribution was assessed by annexin V/PI staining and flow cytometry after treatment with mock (dimethyl sulfoxide, DMSO) or indicated dose of fenbendazole for 3 days. The percentages of normal, early apoptosis, late apoptosis, and necrosis in SNU-C5 (left) and SNU-C5/5-FUR (right) cells are presented as the mean ± SD. **p < 0.01 and ***p < 0.001 vs. DMSO. (B) SNU-C5 and SNU-C5/5-FUR cells were mock-treated with DMSO or treated with indicated dose of fenbendazole. 3 days after treatment, cells were harvested and whole cell extracts were subjected to immunoblotting using the anti-PARP, anti-caspase-3, and anti-cytochrome C antibodies. Signal intensity of each proteins was measured by AzureSpot analysis software. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. DMSO.

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  Fig. 4 Fenbendazole-induced autophagy via beclin-1 in colorectal cancer cells. Effect of fenbendazole on the expression of autophagy-related proteins in SNU-C5 and SNU-C5/5-FUR cells was detected by immunoblotting. SNU-C5 and SNU-C5/5-FUR cells were mock-treated with dimethyl sulfoxide (DMSO) or treated with indicated dose of fenbendazole for 3 days. Total protein was collected, and immunoblotting analysis was performed for light chain 3 (LC3) (I/II), Beclin-1, Atg16L1, Atg3, Atg5, Atg12 (detected Atg12-5 complex also), and Atg7. GAPDH was used for a loading control. Band density was analyzed by AzureSpot analysis software, and results are expressed as the mean ± SD. *p < 0.05, **p < 0.01 vs. DMSO.

• 

  Fig. 5 Fenbendazole-induced ferroptosis via glutathione peroxidase 4 (GPX4) in colorectal cancer cells. (A) Effect of fenbendazole on the expression of ferroptosis-related proteins in SNU-C5 and SNU-C5/5-FUR cells was detected by immunoblotting. SNU-C5 and SNU-C5/5-FUR cells were mock-treated with dimethyl sulfoxide (DMSO) or treated with indicated dose of fenbendazole for 3 days. Total protein was collected, and immunoblotting analysis was performed for GPX4, high mobility group box 1 (HMGB1), SCL7A11, ferritin heavy chain (FTH1), ferroportin (FPN), and transferrin receptor (TfRC). GAPDH was used for a loading control. Band density was analyzed by AzureSpot analysis software, and results are expressed as the mean ± SD. *p < 0.05, **p < 0.01 vs. DMSO. (B) The SNU-C5 and SNU-C5/5-FUR cells were treated with fenbendazole alone or with fenbendazole in combination with indicated doses of ferrostatin-1 or deferoxamine mesylate (DFOM) for 3 days. The extent of cell viability was determined by MTT assay. Data are presented as mean ± SD.

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  Fig. 6 Fenbendazole-induced necroptosis in colorectal cancer cells. Effect of fenbendazole on the expression of necroptosis-related proteins in SNU-C5 and SNU-C5/5-FUR cells was detected by immunoblotting. SNU-C5 and SNU-C5/5-FUR cells were mock-treated with dimethyl sulfoxide (DMSO) or treated with indicated dose of fenbendazole for 3 days. Total protein was collected, and immunoblotting analysis was performed for phospho-receptor-interacting protein kinase (pRIP), RIP, pRIP3, RIP3, phosphor-mixed lineage kinase domain-like protein (pMLKL), MLKL, and caspase-8. GAPDH was used for a loading control. Band density was analyzed by AzureSpot analysis software, and results are expressed as the mean ± SD. *p < 0.05, **p < 0.01 vs. DMSO.

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  Fig. 7 Fenbendazole-induced p53 activation in colorectal cancer cells. SNU-C5 and SNU-C5/5-FUR cells were mock-treated with dimethyl sulfoxide (DMSO) or treated with indicated dose of fenbendazole. 3 days after treatment, cells were harvested and whole cell extracts were subjected to immunoblotting using the indicated antibodies. Signal intensity of each proteins was measured by AzureSpot analysis software. Data are presented as the mean ± SD. *p < 0.05 vs. DMSO.

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  Fig. 8 Schematic representation of cell death pathways in colorectal cancer (CRC) cells following fenbendazole treatment. Fenbendazole induces G2/M arrest and apoptosis in both (A) 5-FU-sensitive SNU-C5 and (B) 5-FU-resistant SNU-C5 (SNU-C5/5-FUR) CRC cells. In SNU-C5 cells, fenbendazole is presumed to activate p53-mediated apoptosis by increasing p53 expression. In SNU-C5/5-FUR cells, fenbendazole triggers apoptosis without affecting p53 expression, whereas fenbendazole enhances ferroptosis by inhibiting the expression of GPX4 and SLC7A11. Therefore, although fenbendazole has anti-cancer effects on both 5-FU-sensitive and resistant CRC cells, the mechanism of action appears to be different. That is, fenbendazole promotes cell death by activating p53-mediated apoptosis in SNU-C5 cells, whereas by both enhancing p53-independent apoptosis and ferroptosis-augmented apoptosis in SNU-C5/5-FUR cells. PARP, poly (ADP-ribose) polymerase; HMGB1, high mobility group box 1; GPX4, glutathione peroxidase 4; LC3, light chain 3.

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