AccScience Publishing / EJMO / Volume 7 / Issue 4 / DOI: 10.14744/ejmo.2023.32682
RESEARCH ARTICLE

The Cytotoxicity Effect of Recombinant Arazyme on Breast and Ovarian Cancer Cells

Roohollah Zarei Koosha1# Abdolamir Ghadaksaz2# Zoleikha Goleij1# Ghazaleh Amjadi2 Hamid Sedighian1 Saber Mehdizadeh3 Abbas Ali Imani Fooladi1
Show Less
1 Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
2 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
3 Department of Immunology, School of Medicine University of Medical Sciences, Tehran, Iran
EJMO 2023, 7(4), 318–325; https://doi.org/10.14744/ejmo.2023.32682
Submitted: 20 October 2023 | Accepted: 23 November 2023 | Published: 29 December 2022
© 2022 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution -Noncommercial 4.0 International License (CC-by the license) ( https://creativecommons.org/licenses/by-nc/4.0/ )
Abstract

Objectives: Investigating new approaches to obtain an effective therapeutic agent for treating life-threatening cancers is critical. The current study aimed to assess the anti-tumor effect of the recombinant arazyme of Serratia proteamaculans on ovarian and breast cancer in vitro.

Methods: The cytotoxic effects of r-arazyme against MCF-7 and SKOV3 cells were evaluated using MTT and lactate dehydrogenase assays. Potential apoptosis induction by r-arazyme was assessed using the Annexin V/PI kit. The Matrigel invasion test was used to evaluate the ability of r-arazyme to reduce cell invasion. In addition, an adhesion assay was performed. RT-PCR was used to measure the expression of genes involved in angiogenesis, apoptosis, and metastasis.

Results: R-arazyme showed a high cytotoxic effect against MCF-7 and SKOV3 cells in a dose-dependent manner. In addition, r-arazyme has great apoptosis-inducing potential in both cells via the activation of caspase-3 and elevation of the BAX/BCL-2 ratio. R-arazyme significantly decreased the expression levels of the angiogenesis-related genes VEGFR-1 and VEGFR-2 and inhibited both cell adhesion and invasion.

Conclusion: R-arazyme may eventually play an essential role in the development of effective therapies against ovarian and breast cancers, thereby reducing the overall morbidity and death caused by cancer. 

Keywords
Apoptosis
breast cancer
ovarian cancer
Conflict of interest
None declared.
References

1. DeSantis C, Siegel R, Sauer A, Miller K, Fedewa S, Alcaraz K, et al. Progress and opportunities in reducing racial disparities. CA Cancer J Clin 2016;66:290–308.
2. Shah R, Rosso K, Nathanson SD. Pathogenesis, prevention, diagnosis and treatment of breast cancer. World J Clin Oncol 2014;5:283.
3. Wald M. Exogenous proteases confer a significant chemopreventive effect in experimental tumor models. Integr Cancer Ther 2008;7:295–310.
4. Bersanetti PA, Park HY, Bae KS, Son KH, Shin DH, Hirata IY, et al. Characterization of arazyme, an exocellular metalloprotease isolated from Serratia proteamaculans culture medium. Enzyme Microb Technol 2005;37:574–81.
5. Kwak JY, Lee KE, Shin DH, Maeng JS, Park DS, Oh HW, et al. Biochemical and genetic characterization of arazyme, an extracellular metalloprotease produced from Serratia proteamaculans HY-3. J Microbiol Biotechnol 2007;17:761–8.
6. Park JK, Jeong DH, Park HY, Son KH, Shin DH, Do SH, et al. Hepatoprotective effect of Arazyme on CCl4-induced acute hepatic injury in SMP30 knock-out mice. Toxicology 2008;246:132–42.
7. Hauck G. Proceedings: Vitalmicroscopic investigations of the effects of thrombin, a snake venom enzyme and histamin effect on the mesenteric microvasculature of rabbit and cat. Arzneimittel-Forschung 1976;26:1233.
8. Wols RL, Bond JS. Phe5 (4-nitro)-bradykinin: A chromogenic substrate for assay and kinetics of the metalloendopeptidase meprin. Anal Biochem 1990;191:314–20.
9. Pereira FV, Ferreira-Guimaraes CA, Paschoalin T, Scutti JA, Melo FM, Silva LS, et al. A natural bacterial-derived product, the metalloprotease arazyme, inhibits metastatic murine melanoma by inducing MMP-8 cross-reactive antibodies. PloS One 2014;9:e96141.
10. Amjadi G, Parivar K, Fazlollah Mousavi S, Imani Fooladi AA. Anti-cancer effects of recombinant arazyme from Serratia Proteomaculans. J BUON 2020;25:531–42. 
11. Babavalian H, Latifi AM, Shokrgozar MA, Bonakdar S, Tebyanian H, Shakeri F. Cloning and expression of recombinant human platelet-derived growth factor-BB in Pichia Pink. Cell Mol Biol 2016;62:45–51.
12. Behrouz B, Amirmozafari N, Khoramabadi N, Bahroudi M, Legaee P, Mahdavi M. Cloning, expression, and purification of pseudomonas aeruginosa flagellin, and characterization of the elicited anti-flagellin antibody. IRCMJ 2016;18:e28271.
13. Goudarzi G, Sattari M, Roudkenar MH, Montajabi-Niyat M, Zavaran-Hosseini A, Mosavi-Hosseini K. Cloning, expression, purification, and characterization of recombinant flagellin isolated from Pseudomonas aeruginosa. Biotechnol Lett 2009;31:1353–60.
14. Korpi F, Irajian G, Mahadavi M, Motamedifar M, Mousavi M, Laghaei P, et al. Active immunization with recombinant pila protein protects against pseudomonas aeruginosa infection in a mouse burn wound model. Available at:https://www. researchgate.net/publication/282048156_Active_Immunization_with_Recombinant_PilA_protein_Protects_Against_ Pseudomonas_aeruginosa_Infection_in_a_Mouse_Burn_ Wound_Model. Accessed Dec 22, 2023.
15. Tebyanian H, Mirhosseiny SH, Kheirkhah B, Hassanshahian M. Isolation and identification of mycoplasma synoviae from suspected ostriches by polymerase chain reaction, in kerman province, Iran. Jundishapur J Microbiol 2014;7:e19262.
16. Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. Cancer Res 1987;47:936–42.
17. Hayon T, Dvilansky A, Shpilberg O, Nathan I. Appraisal of the MTT-based assay as a useful tool for predicting drug chemosensitivity in leukemia. Leuk Lymphoma 2003;44:1957–62.
18. Amjadi G, Parivar K, Mousavi SF, Fooladi AAI. Effect of metalloprotease arazyme on the expression of MMP2 and MMP9 genes in metastasis of colon and ovarian cancer cell lines. Thrita 2019;8:e100004.
19. Moghadamtousi SZ, Karimian H, Rouhollahi E, Paydar M, Fadaeinasab M, Kadir HA. Annona muricata leaves induce G1 cell cycle arrest and apoptosis through mitochondria-mediated pathway in human HCT-116 and HT-29 colon cancer cells. J Ethnopharmacol 2014;156:277–89.
20. Maleki F, Sadeghifard N, Hosseini HM, Bakhtiyari S, Goleij Z, Behzadi E, et al. Growth-inhibitory effects of TGFαL3-SEB chimeric protein on colon cancer cell line. Biomed Pharmacother 2019;110:190–6.
21. Fan H, Li X, Wang W, Lai Q, Tang X, Gao D, et al. Effects of NMDAreceptor antagonist on the expressions of Bcl-2 and bax in the subventricular zone of neonatal rats with hypoxia–ischemia Brain damage. Cell Biochem Biophys 2015;73:323–330.
22. Heidari M, Arab S, Noroozi-Aghideh A, Tebyanian H, Latifi A. Evaluation of the substitutions in 212, 342 and 215 amino acid positions in binding site of organophosphorus acid anhydrolase using the molecular docking and laboratory analysis. Bratisl Lek Listy 2019;120:139–43.
23. Kumar S, Sharma VK, Yadav S, Dey S. Antiproliferative and apoptotic effects of black turtle bean extracts on human
breast cancer cell line through extrinsic and intrinsic pathway. Chem Cent J 2017;11:1–10.
24. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 2001;25:402–8.
25. Hajrezaie M, Paydar M, Zorofchian Moghadamtousi S, Hassandarvish P, Gwaram NS, Zahedifard M, et al. A Schiff Basederived copper (II) complex is a potent inducer of apoptosis in colon cancer cells by activating the intrinsic pathway. Sci World J 2014;2014:540463.
26. Moghadamtousi SZ, Kadir HA, Paydar M, Rouhollahi E, Karimian H. Annona muricata leaves induced apoptosis in A549 cells through mitochondrial-mediated pathway and involvement of NF-κB. BMC Complement Altern Med 2014;14:1–13.
27. Elmore S. Apoptosis: A review of programmed cell death. Toxicol Pathol 2007;35:495–516.
28. Mohammad RM, Muqbil I, Lowe L, Yedjou C, Hsu HY, Lin LT, et al. Broad targeting of resistance to apoptosis in cancer. Semin Cancer Biol 2015;35(Suppl):78–103.
29. Ouyang L, Shi Z, Zhao S, Wang FT, Zhou TT, Liu B, et al. Programmed cell death pathways in cancer: A review of apoptosis, autophagy and programmed necrosis. Cell Prolif 2012;45:487–98.
30. Findley HW, Gu L, Yeager AM, Zhou M. Expression and regulation of Bcl-2, Bcl-xl, and Bax correlate with p53 status and sensitivity to apoptosis in childhood acute lymphoblastic leukemia. Blood 1997;89:2986–93. 
31. Lee JY, Jee SB, Park WY, Choi YJ, Kim B, Kim YH, et al. Tumor suppressor protein p53 promotes 2-methoxyestradiol-induced activation of Bak and Bax, leading to mitochondriadependent apoptosis in human colon cancer HCT116 cells. J Microbiol Biotechnol 2014;24:1654–63.
32. Tang N, Ma L, Lin XY, Zhang Y, Yang DL, Wang EH, et al. Expression of PHF20 protein contributes to good prognosis of NSCLC and is associated with Bax expression. Int J Clin Exp Pathol 2015;8:12198.
33. Bashyam MD. Understanding cancer metastasis: An urgent need for using differential gene expression analysis. Cancer 2002;94:1821–9.
34. Leake I. Understanding the routes of metastasis in colorectal cancer. Nat Rev Gastroenterol Hepatol 2014;11:270. 
35. Lu J, Zhang K, Nam S, Anderson RA, Jove R, Wen W. Novel angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling. Carcinogenesis 2010;31:481–8.

Share
Back to top
Eurasian Journal of Medicine and Oncology, Electronic ISSN: 2587-196X Print ISSN: 2587-2400, Published by AccScience Publishing