In Vitro Screening for Cytotoxic Effect of Pore Forming Colicin N and Its Domains on Human Cancer Cells

Main Article Content

Methawee Duangkaew
Wanatchaporn Arunmanee


Protein-based drugs have increasingly become an important segment of cancer treatment. In comparison with chemotherapy, they offer high efficacy and fewer side effects due to specifically targeting only cancer cells. Monoclonal antibodies are currently the main protein-based drugs in the market but their complexity and limitations in tumour penetration led to the development of alternative protein therapeutics such as pore-forming toxins. Colicin N (ColN), a pore-forming protein produced by E. coli, was previously found to exhibit cytotoxicity and selectivity in human lung cancer cells with promising potential for further development. Here we aimed to screen for the cytotoxicity of ColN in breast (MCF-7 and MDA-MB-231), lung (A549) and colon cancer cells (HT-29 and HCT-116) by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay with various concentrations for 72 h and to investigate the cytotoxic effect of ColN domains on cancer cells. It showed that ColN mildly mediated the decrease in cell viability except for MCF-7. The highest effect was seen in A549 and HCT-116 cells which showed 31.9% and 31.5% decrease in cell viability, respectively. The mild inhibition or promotion of cancer cell proliferation by ColN tend to be based on the cell types. Furthermore, to search for the functional domain of ColN used for cytotoxicity, full-length ColN and truncated ColN with deletion of translocating, receptor binding and pore-forming domains were also tested on HCT-116 colon cancer cells. The findings indicated that HCT-116 cells were not significantly sensitive to ColN but full length ColN caused slight decrease in cancer cell viability. The data in this study will benefit the further development of ColN for alternative protein-based cancer therapy.

Article Details

How to Cite
Methawee Duangkaew, & Wanatchaporn Arunmanee. (2022). In Vitro Screening for Cytotoxic Effect of Pore Forming Colicin N and Its Domains on Human Cancer Cells. Tropical Life Sciences Research, 33(1), 163–177.
Original Article


Arunmanee W, Ecoy G A U, Khine H E E, Duangkaew M, Prompetchara E, Chanvorachote P and Chaotham C. (2020). Colicin N mediates apoptosis and suppresses integrin-modulated survival in human lung cancer cells. Molecules 25(4): 816.

Arunmanee W, Duangkaew M, Taweecheep P, Aphicho K, Lerdvorasap P, Pitchayakorn J, Intasuk C, Jiraratmetacon R, Syamsidi A, Chanvorachote P, Chaotham C and Pornputtapong N. (2021). Resurfacing receptor binding domain of Colicin N to enhance its cytotoxic effect on human lung cancer cells. Computational and Structural Biotechnology Journal 19: 5225–5234.

Avila A D, Calderón C F, Pérez R M, Pons C, Pereda C M and Ortiz A R. (2007). Construction of an immunotoxin by linking a monoclonal antibody against the human epidermal growth factor receptor and a hemolytic toxin. Biological Research 40(2): 173–183.

Avila A D, Mateo de Acosta C and Lage A. (1989). A carcinoembryonic antigen-directed immunotoxin built by linking a monoclonal antibody to a hemolytic toxin. International Journal of Cancer 43(5): 926–929.

Bhattacharyya N P, Skandalis A, Ganesh A, Groden J and Meuth M. (1994). Mutator phenotypes in human colorectal carcinoma cell lines. Proceedings of the National Academy of Sciences 91(14): 6319.

Cascales E, Buchanan S K, Duche D, Kleanthous C, Lloubes R, Postle K, Riley M, Slatin S and Cavard D. (2007). Colicin biology. Microbiology and Molecular Biology Reviews 71(1): 158–229.

Chumchalova J and Smarda J. (2003). Human tumor cells are selectively inhibited by colicins. Folia Microbiologica 48(1): 111–115.

Dingermann T. (2008). Recombinant therapeutic proteins: production platforms and challenges. Biotechnology Journal 3(1): 90–97.

Dover L G, Evans L J A, Fridd S L, Bainbridge G, Raggett E M and Lakey J H. (2000). Colicin pore-forming domains bind to Escherichia coli trimeric porins. Biochemistry 39(29): 8632–8637.

Evans L J A, Goble M L, Hales K A and Lakey J H. (1996). Different sensitivities to acid denaturation within a family of proteins: Implications for acid unfolding and membrane translocation. Biochemistry 35(40): 13180–13185.

Gest C, Joimel U, Huang L, Pritchard L L, Petit A, Dulong C, Buquet C, Hu C Q, Mirshahi P, Laurent M, Fauvel-Lafève F, Cazin L, Vannier J P, Lu H, Soria J, Li H, Varin R and Soria C. (2013). Rac3 induces a molecular pathway triggering breast cancer cell aggressiveness: Differences in MDA-MB-231 and MCF-7 breast cancer cell lines. BMC Cancer 13: 63.

Jang W J, Jung S K, Vo T T L and Jeong C H. (2019). Anticancer activity of paroxetine in human colon cancer cells: Involvement of MET and ERBB3. Journal of Cellular and Molecular Medicine 23(2): 1106–1115.

Kaur S and Kaur S. (2015). Bacteriocins as potential anticancer agents. Frontiers in Pharmacology 6: 272.

Kintzing J R, Filsinger Interrante M V and Cochran J R. (2016). Emerging strategies for developing next-generation protein therapeutics for cancer treatment. Trends in Pharmacological Sciences 37(12): 993–1008.

Kohoutova D, Forstlova M, Moravkova P, Cyrany J, Bosak J, Smajs D, Rejchrt S and Bures J. (2020). Bacteriocin production by mucosal bacteria in current and previous colorectal neoplasia. BMC Cancer 20(1): 39.

Kohoutova D, Smajs D, Moravkova P, Cyrany J, Moravkova M, Forstlova M, Cihak M, Rejchrt S and Bures J. (2014). Escherichia coli strains of phylogenetic group B2 and D and bacteriocin production are associated with advanced colorectal neoplasia. BMC Infectious Diseases 14: 733.

Lagassé H A, Alexaki A, Simhadri V L, Katagiri N H, Jankowski W, Sauna Z E and KimchiSarfaty C. (2017). Recent advances in (therapeutic protein) drug development. F1000Research 6: 113.

Lancaster L E, Wintermeyer W and Rodnina M V. (2007). Colicins and their potential in cancer treatment. Blood Cells, Molecules and Diseases 38(1): 15–18.

Levenson A S and Jordan V C. (1997). MCF-7: The first hormone-responsive breast cancer cell line. Cancer Research 57(15): 3071–3078.

Liu H F, Ma J, Winter C and Bayer R. (2010). Recovery and purification process development for monoclonal antibody production. mAbs 2(5): 480–499.

Minchinton A I and Tannock I F. (2006). Drug penetration in solid tumours. Nature Reviews Cancer 6(8): 583–592.

Mutter N L, Soskine M, Huang G, Albuquerque I S, Bernardes G J L and Maglia G. (2018). Modular pore-forming immunotoxins with caged cytotoxicity tailored by directed evolution. ACS Chemical Biology 13(11): 3153–3160.

Pitchayakorn J, Intasuk C, Jiraratmetagon R, Apicho K, Lerdvorasap P, Pewnim N and Arunmanee W. (2020). Investigation into the effect of colicin N on cancer cell lines. The Proceedings of 58th Kasetsart University Annual Conference 2: 19–22.

Raguz S and Yagüe E. (2008). Resistance to chemotherapy: New treatments and novel insights into an old problem. British Journal of Cancer 99(3): 387–391.

Ridley H, Johnson C L and Lakey J H. (2010). Interfacial interactions of pore-forming colicins. In: Anderluh G and Lakey J. (Eds.). Proteins: Membrane binding and pore formation. New York: Springer Science+Business Media, LLC, 81–90.

Rodrigues N R, Rowan A, Smith M E, Kerr I B, Bodmer W F, Gannon J V and Lane D P. (1990). p53 mutations in colorectal cancer. Proceedings of the National Academy of Sciences 87(19): 7555.

Samaranayake H, Wirth T, Schenkwein D, Räty J K and Ylä-Herttuala S. (2009). Challenges in monoclonal antibody-based therapies. Annals of Medicine 41(5): 322–331.

Smarda J, Fialova M and Smarda J. (2001). Cytotoxic effects of colicins E1 and E3 on v-myb-transformed chicken monoblasts. Folia Biologica 47(1): 11–13.

Tejuca M, Anderluh G and Dalla Serra M. (2009). Sea anemone cytolysins as toxic components of immunotoxins. Toxicon 54(8): 1206–1214.

Urquhart L. (2020). Top companies and drugs by sales in 2019. Nature Reviews Drug Discovery 19(4): 228.

Vetter I R, Parker M W, Tucker A D, Lakey J H, Pattus F and Tsernoglou D. (1998). Crystal structure of a colicin N fragment suggests a model for toxicity. Structure 6(7): 863–874.

Wan L, Zeng L, Chen L, Huang Q, Li S, Lu Y, Li Y, Cheng J and Lu X. (2006). Expression, purification, and refolding of a novel immunotoxin containing humanized singlechain fragment variable antibody against CTLA4 and the N-terminal fragment of human perforin. Protein Expression and Purification 48(2): 307–313.

Yao K, Gietema J A, Shida S, Selvakumaran M, Fonrose X, Haas N B, Testa J and O’Dwyer P J. (2005). In vitro hypoxia-conditioned colon cancer cell lines derived from HCT116 and HT29 exhibit altered apoptosis susceptibility and a more angiogenic profile in vivo. British Journal of Cancer 93(12): 1356–1363.