Anticancer activity of Pseudomonas aeruginosa derived peptide with iRGD in colon cancer therapy

Document Type : Original Article


1 Department of Microbiology and Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

2 Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

3 Department of Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

4 Department of Medical Biochemistry, Faculty of Medicine, Mashhad University of Medical, Sciences, Mashhad, Iran

5 Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

6 Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran


Objective(s): Colon cancer is well-known as a life-threatening disease. Since the current treatment modalities for this type of cancer are powerful yet face some limitations, finding novel treatments is required to achieve better outcomes with fewer side effects. Here we investigated the therapeutic potential of Azurin-p28 alone or along with iRGD (Ac-CRGDKGPDC-amide) as a tumor-penetrating peptide and 5-fluorouracil (5-FU) for colon cancer. 
Materials and Methods: Inhibitory effect of p28 with or without iRGD/5-FU was studied in CT26 and HT29, as well as the xenograft animal model of cancer. The effect of p28 alone or along with iRGD/5-FU on cell migration, apoptotic activity, and cell cycle of the cell lines was assessed. Level of the BAX and BCL2 genes, tumor suppressor genes [(p53 and collagen type-Iα1 (COL1A1), collagen type-Iα2 (COL1A2)] were assessed by quantitative RT-PCR.
Results: These findings show that using p28 with or without iRGD and 5-FU raised the level of p53 and BAX but decreased BCL2, compared with control and 5-FU groups in tissues of the tumor, which result in raising the apoptosis. 
Conclusion: It seems that p28 may be used as a new therapeutic approach in colon cancer therapy that can enhance the anti-tumor effect of 5-FU.


Main Subjects

1. Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. Cancer J Clin 2023;73:17-48
2. Cao C, Yan TD, Black D, Morris DL. A systematic review and meta-analysis of cytoreductive surgery with perioperative intraperitoneal chemotherapy for peritoneal carcinomatosis of colorectal origin. Ann Surg Oncol 2009;16:2152-2165.
3. Koppe MJ, Boerman OC, Oyen WJ, Bleichrodt RP. Peritoneal carcinomatosis of colorectal origin: Incidence and current treatment strategies. Ann Surg 2006;243:212-222.
4. Portilla AG, Cendoya I, De Tejada IL, Olabarria I, de Lecea CM, Magrach L, et al. Peritoneal carcinomatosis of colorectal origin. Current treatment. Review and update. Rev Esp Enferm Dig 2005;97:716-737.
5. Lemoine L, Sugarbaker P, Van der Speeten K. Pathophysiology of colorectal peritoneal carcinomatosis: Role of the peritoneum. World J Gastroenterol 2016;22:7692-7707.
6. Sugarbaker PH. Improving oncologic outcomes for colorectal cancer at high risk for local-regional recurrence with novel surgical techniques. Expert Rev Gastroent 2016;10:205-213.
7. Symeonidis D, Christodoulidis G, Koukoulis G, Spyridakis M, Tepetes K. Colorectal cancer surgery in the elderly: Limitations and drawbacks. Tech Coloproctol 2011;15:47-50.
8. Groza D, Gehrig S, Kudela P, Holcmann M, Pirker C, Dinhof C, et al. Bacterial ghosts as adjuvant to oxaliplatin chemotherapy in colorectal carcinomatosis. OncoImmunology 2018;7:e1424676-e1424689.
9. Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients. Cancer Gene Ther 2003;10:737-744.
10. Heimann DM, Rosenberg SA. Continuous intravenous administration of live genetically modified Salmonella typhimurium in patients with metastatic melanoma. J Immunother 2003;26:179-180.
11. Yu K-H, Zhang C, Berry GJ, Altman RB, Ré C, Rubin DL, et al. Predicting non-small cell lung cancer prognosis by fully automated microscopic pathology image features. Nat Commun 2016;7:12474-12483.
12. Nauts HC, Swift WE, Coley BL. The treatment of malignant tumors by bacterial toxins as developed by the late William B. Coley, MD, reviewed in the light of modern research. Cancer Res 1946;6:205-216.
13. Gontero P, Bohle A, Malmstrom P-U, O’Donnell MA, Oderda M, Sylvester R, et al. The role of bacillus Calmette-Guérin in the treatment of non–muscle-invasive bladder cancer. Eur Urol Suppl 2010;57:410-429.
14. Zlotta AR, Fleshner NE, Jewett MA. The management of BCG failure in non-muscle-invasive bladder cancer: An update. Can Urol Assoc J 2009;3:S199-205.
15. J Boohaker R, W Lee M, Vishnubhotla P, LM Perez J, R Khaled A. The use of therapeutic peptides to target and to kill cancer cells. Curr Med Chem 2012;19:3794-3804.
16. Karpiński TM, Adamczak A. Anticancer activity of bacterial proteins and peptides. Pharmaceutics 2018;10:54-79.
17. Anson ML, Edsall JT. Interactions of antimicrobial peptides with bacterial membranes and membrane components. Adv Protein Chem Struct Biol 1949;1:374-390.
18. Marqus S, Pirogova E, Piva TJ. Evaluation of the use of therapeutic peptides for cancer treatment. J Biomed Sci 2017;24:21-35.
19. Fialho AM, Bernardes N, Chakrabarty AM. Exploring the anticancer potential of the bacterial protein azurin. Aims Microbiol 2016;2:292-303.
20. Yaghoubi A, Khazaei M, Avan A, Hasanian SM, Cho WC, Soleimanpour S. p28 bacterial peptide, as an anticancer agent. Front Oncol 2020;10:1303-1312.
21. Beattie CW, Yamada T, Gupta TKD. Compositions and methods to prevent cancer by stabilizing p53 through non MDM2-mediated pathways. Google Patents; 2017.
22. Garizo AR, Bernardes N, Chakrabarty AM, Fialhoa AM. The anticancer potential of the bacterial protein azurin and its derived peptide p28. Clin Microbiol Rev 2019:319-328.
23. Mehta RR, Yamada T, Taylor BN, Christov K, King ML, Majumdar D, et al. A cell penetrating peptide derived from azurin inhibits angiogenesis and tumor growth by inhibiting phosphorylation of VEGFR-2, FAK and Akt. Angiogenesis 2011;14:355-369.
24. Sugahara KN TT, Karmali PP, Kotamraju VR, Agemy L, Greenwald DR, Ruoslahti E. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs. Science 2010;328:1031–1035.
25. Teesalu T SK, Kotamraju VR, Ruoslahti E. C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration. PNAS 2009;106:16157-16162.
26. Yamada T, Gupta TKD, Beattie CW. p28-mediated activation of p53 in G2-M phase of the cell cycle enhances the efficacy of DNA damaging and antimitotic chemotherapy. Cancer Res 2016;76:2354-2365.
27. Hamilton AM, Aidoudi-Ahmed S, Sharma S, Kotamraju VR, Foster PJ, Sugahara KN, et al. Nanoparticles coated with the tumor-penetrating peptide iRGD reduce experimental breast cancer metastasis in the brain. J Mol Med 2015;93:991-1001.
28. Marjaneh RM, Rahmani F, Hassanian SM, Rezaei N, Hashemzehi M, Bahrami A, et al. Phytosomal curcumin inhibits tumor growth in colitis‐associated colorectal cancer. J Cell Physiol 2018;233:6785-6798.
29. Carbone L, Carbone ET, Yi EM, Bauer DB, Lindstrom KA, Parker JM, et al. Assessing cervical dislocation as a humane euthanasia method in mice. J Am Assoc Lab Anim Sci 2012;51:352-356.
30. Amerizadeh F, Rezaei N, Rahmani F, Hassanian SM, Moradi‐Marjaneh R, Fiuji H, et al. Crocin synergistically enhances the antiproliferative activity of 5‐flurouracil through Wnt/PI3K pathway in a mouse model of colitis‐associated colorectal cancer. J Cell Biochem 2018;119:10250-10261.
31. Marjaneh RM, Rahmani F, Hassanian SM, Rezaei N, Hashemzehi M, Bahrami A, et al. Phytosomal curcumin inhibits tumor growth in colitis-associated colorectal cancer. J Cell Physiol 2018;233:6785-6798.
32. Elmi S, Sallam NA, Rahman MM, Teng X, Hunter AL, Moien-Afshari F, et al. Sulfaphenazole treatment restores endothelium-dependent vasodilation in diabetic mice. Curr Vasc Pharmacol 2008;48:1-8.
33. Kuwana T, Bouchier-Hayes L, Chipuk JE, Bonzon C, Sullivan BA, Green DR, et al. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol Cell 2005;17:525-35.
35. Kuwana T, Bouchier-Hayes L, Chipuk JE, Bonzon C, Sullivan BA, Green DR, et al. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol Cell 2005;17:525-535.
36. Wang J-L, Liu D, Zhang Z-J, Shan S, Han X, Srinivasula SM, et al. Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc Natl Acad Sci USA 2000;97:7124-7129.
37. Niazi S, Purohit M, Niazi JH. Role of p53 circuitry in tumorigenesis: A brief review. Eur J Med Chem 2018;158:7-24.
38. Valente JF, Queiroz JA, Sousa F. p53 as the focus of gene therapy: Past, present and future. Curr Drug Targets 2018;19:1801-1817.
39. Yamada T, Goto M, Punj V, Zaborina O, Kimbara K, Gupta TD, et al. The bacterial redox protein azurin induces apoptosis in J774 macrophages through complex formation and stabilization of the tumor suppressor protein p53. Infect Immun 2002;70:7054-7062.
40. Punj V, Bhattacharyya S, Saint-Dic D, Vasu C, Cunningham EA, Graves J, et al. Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer. Oncogene 2004;23:2367-2378.
41. Huang Y, Zhou J, Cheng X, Su Z. Deciphering the Interactions between an Anticancer Bacteriocin and the P53 DNA Binding Domain. Biophys J 2018;114:222a.
42. Sheibani N, Newman P, Frazier W. Thrombospondin-1, a natural inhibitor of angiogenesis, regulates platelet-endothelial cell adhesion molecule-1 expression and endothelial cell morphogenesis. Mol Biol Cell 1997;8:1329-1341.
43. Wu J, Sheibani N. Modulation of VE‐cadherin and PECAM‐1 mediated cell–cell adhesions by mitogen‐activated protein kinases. J Cell Biochem 2003;90:121-137.
44. Koga Y, Pelizzola M, Cheng E, Krauthammer M, Sznol M, Ariyan S, et al. Genome-wide screen of promoter methylation identifies novel markers in melanoma. Gene Res 2009;19:1462-1470.
45. Mori K, Enokida H, Kagara I, Kawakami K, Chiyomaru T, Tatarano S, et al. CpG hypermethylation of collagen type I α 2 contributes to proliferation and migration activity of human bladder cancer. Int J Oncol 2009;34:1593-1602.
46. Shin K, Lim A, Zhao C, Sahoo D, Pan Y, Spiekerkoetter E, et al. Hedgehog signaling restrains bladder cancer progression by eliciting stromal production of urothelial differentiation factors. Cancer cell 2014;26:521-533.
47. Shin K, Lee J, Guo N, Kim J, Lim A, Qu L, et al. Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder. Nature 2011;472:110-114.
48. Zou X, Feng B, Dong T, Yan G, Tan B, Shen H, et al. Up-regulation of type I collagen during tumorigenesis of colorectal cancer revealed by quantitative proteomic analysis. Proteomics 2013;94:473-485.
49. Liang Y, Diehn M, Bollen AW, Israel MA, Gupta N. Type I collagen is overexpressed in medulloblastoma as a component of tumor microenvironment. J Neurooncol 2008;86:133-141.
50. Gao F, Li M, Xiang R, Zhou X, Zhu L, Zhai Y. Expression of CLDN6 in tissues of gastric cancer patients: Association with clinical pathology and prognosis. Onc Let 2019;17:4621-4625.
51. Lin P, Tian P, Pang J, Lai L, He G, Song Y, et al. Clinical significance of COL1A1 and COL1A2 expression levels in hypopharyngeal squamous cell carcinoma. Onc Let 2020;20:803-809.
52. Mestdagt M, Polette M, Buttice G, Noël A, Ueda A, Foidart JM, et al. Transactivation of MCP‐1/CCL2 by β‐catenin/TCF‐4 in human breast cancer cells. Int J Cancer 2006;118:35-42.
53. Yoshidome H, Kohno H, Shida T, Kimura F, Shimizu H, Ohtsuka M, et al. Significance of monocyte chemoattractant protein-1 in angiogenesis and survival in colorectal liver metastases. Int J Cancer 2009;34:923-930.
54. Xu M, Wang S, Qi Y, Chen L, Frank JA, Yang XH, et al. Role of MCP‐1 in alcohol‐induced aggressiveness of colorectal cancer cells. Mole carcin 2016;55:1002-1011.
55. Haabeth OAW, Lorvik KB, Hammarström C, Donaldson IM, Haraldsen G, Bogen B, et al. Inflammation driven by tumour-specific Th1 cells protects against B-cell cancer. Nature 2011;2:1-12.
56. Haabeth OAW, Lorvik KB, Yagita H, Bogen B, Corthay A. Interleukin-1 is required for cancer eradication mediated by tumor-specific Th1 cells. OncoImmunology 2016;5:e1039763-1039773.
57. North R, Neubauer RH, Huang J, Newton RC, Loveless SE. Interleukin 1-induced, T cell-mediated regression of immunogenic murine tumors. Requirement for an adequate level of already acquired host concomitant immunity. J Exp Med 1988;168:2031-2043.
58. Warso M, Richards J, Mehta D, Christov K, Schaeffer C, Bressler LR, et al. A first-in-class, first-in-human, phase I trial of p28, a non-HDM2-mediated peptide inhibitor of p53 ubiquitination in patients with advanced solid tumours. Br J Cancer 2013;108:1061-1070.
59. Lulla RR, Goldman S, Yamada T, Beattie CW, Bressler L, Pacini M, et al. Phase I trial of p28 (NSC745104), a non-HDM2-mediated peptide inhibitor of p53 ubiquitination in pediatric patients with recurrent or progressive central nervous system tumors: A Pediatric Brain Tumor Consortium Study. J Neurooncol 2016;18:1319-1325.