Evaluation Of Immunostimulant/Cytotoxic Activity Of Human Breast Cancer Prepared By Different Antigen Preparation Methods With Adjuvants Combination

Year 2022, , 96 - 110, 27.05.2022

Murat Ihlamur , Hümeyra Başarı , Yağmur Zengin , Emrah Şefik Abamor

https://doi.org/10.29233/sdufeffd.940806

Cited By: 6

Abstract

In last decades, immunotherapies became more and more significant as a cancer treatment method. Many in vivo and in vitro studies have been conducted in the literature in order to determine the appropriate diagnostic and treatment methods for cancer. During the development of therapeutic vaccines, determination of vaccine concentration which is the beginning step of in vivo studies, is critical for the healthy progression of the studies. Breast cancer which occurs by uncontrolled proliferation as a result of exposure of healthy cells in breast tissue to factors such as DNA damage, UV, radiation, carcinogens and, various chemicals has a huge portion among the cancer types. In this study, for the first time, antigens generated by two different antigen preparation methods (autoclaving, freezing-thawing) using AU-565 / MCF-7 cell lines. Then, antigens derived from human breast cancer cell lines were applied alone and with different adjuvants (Polyoxidonium, Freund’s) to the J774 macrophage cell culture system and the L929 fibroblast cell line in order to investigate immunostimulant activity and cytotoxicity via Griess reagent and MTT test respectively. At the end of the 48th hour, the cell viability well will be examined and the most appropriate antigen-adjuvant combination and its concentration for vaccine preparation were determined. Among the antigen preparation methods, it was determined that the freeze-thaw method was more effective than the autoclave method, and the antigens prepared by the freeze-thaw method from the AU-565 and MCF-7 cell lines showed the highest immunostimulatory activity at a concentration of 160 µg/ml. Among the adjuvants, the immunostimulant effect of Freund's adjuvant was found to be higher. In the light of these results, the optimal vaccine concentration against breast cancer and the viability analysis of these concentrations were determined. All in all, it is thought that antibodies obtained from in vivo studies using the most appropriate antigen preparation method and the most appropriate vaccine concentration can be used in the diagnosis and treatment of breast cancer.

Keywords

Breast Cancer, Vaccine, AU-565, MCF-7, Antigen

Farklı Antijen Hazırlama Metotları İle Hazırlanan İnsan Meme Kanseri Hücre Lizatlarının Adjuvan Kombinasyonları İle İmmünostimülan/Sitotoksik Aktivitelerinin İncelenmesi

Abstract

Meme kanseri, meme dokusundaki sağlıklı hücrelerin DNA hasarları, genetik mutasyonlar, UV, radyasyon, kanserojenler ve çeşitli kimyasal maddeler gibi faktörlere maruz kalması sonucunda kontrolsüz çoğalarak oluşmaktadır. Uygun teşhiş ve tedavi yöntemlerini belirlemek amacıyla literatürde birçok in vivo ve in vitro çalışma gerçekleştirilmiştir. In vivo çalışmaların başlangıç basamağı olan aşı konsantrasyonlarının belirlenmesi, çalışmaların sağlıklı ilerleyebilmesi için kritik bir aşamadır. Bu çalışmada ilk kez AU-565 / MCF-7 hücre hatları kullanılarak iki farklı antijen hazırlama yöntemi (otoklavlama, dondurma-çözdürme) ile antijenler oluşturulmuştur. Daha sonra insan meme kanseri hücre dizilerinden elde edilen antijenler, sırasıyla Griess reaktifi ve MTT testi ile immünostimülan aktivite ve sitotoksisiteyi araştırmak için J774 makrofaj hücre kültür sistemine ve L929 fibroblast hücre hattına tek başına ve farklı adjuvanlarla (Polyoxidonium, Freund's) uygulandı. 48. saatin sonunda her kuyudaki hücre canlılık oranı incelenip bu sonuçlar ışığında meme kanserine karşı oluşturulan en uygun aşı konsantrasyonu ve bu konsantrasyonların canlılık analizleri belirlenmiştir. Antijen hazırlama metodları arasında dondurma-çözdürme metodunun otoklavlama metoduna göre daha etkili olduğu, AU-565 ve MCF-7 hücre hattından dondurma-çözdürme metodu ile hazırlanan antijenlerin 160 µg/ml konsantrasyonda en yüksek immünostimülan etkinlik gösterdiği belirlenmiştir. Adjuvanlar arasında ise Freund’s adjuvanının immünostimülan etkisinin daha yüksek olduğu tespit edilmiştir. Bu sonuçlar doğrultusunda en uygun antijen hazırlama metodu ve belirlenen en uygun aşı konsantrasyonu kullanılarak yapılan in vivo çalışmalardan elde edilecek antikorların meme kanserine karşı tanı ve tedavide kullanılmasının mümkün olabileceği düşünülmektedir.

Keywords

Meme Kanseri, Aşı, AU-565, MCF-7, Antijen

References

• [1] F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L.A. Torre, and A. Jemal, “Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries,” CA Cancer J Clin., 68 (6), 394-424, 2018.
• [2] I.A. Cree, “Cancer biology,” Methods Mol Biol., 731, 1-11, 2011.
• [3] O. Ginsburg, C.H. Yip, A. Brooks, A. Cabanes, M. Caleffi, J. A. Dunstan Yataco, B. Gyawali, V. McCormack, M. McLaughlin de Anderson, R. Mehrotra, A. Mohar, R. Murillo, L.E Pace, E. D. Paskett, A. Romanoff, A. F. Rositch, J. R. Scheel, M. Schneidman, K. Unger-Saldaña, V. Vanderpuye, T. Y. Wu, S. Yuma, A. Dvaladze, C. Duggan, and B. O. Anderson, “Breast cancer early detection: A phased approach to implementation,” Cancer, 126, 2379-2393, 2020.
• [4] X. Dai, H. Cheng, and Z. Bai, J. Li, “Breast Cancer Cell Line Classification and Its Relevance with Breast Tumor Subtyping,” J. Cancer, 8 (16), 3131-3141, 2017.
• [5] J. J. Gao, and S. M. Swain, “Luminal A Breast Cancer and Molecular Assays: A Review,” Oncologist, 23(5), 556-565, 2018.
• [6] F. Schettini, T. Pascual, B. Conte, N. Chic, F. Brasó-Maristany, P. Galván, O. Martínez, B. Adamo, M. Vidal, M. Muñoz, A. Fernández-Martinez, C. Rognoni, G. Griguolo, V. Guarneri, P. F. Conte, M. Locci, J. C. Brase, B. Gonzalez-Farre, P. Villagrasa, S. De Placido, R. Schiff, J. Veeraraghavan, M. F. Rimawi, C. K. Osborne, S. Pernas, C. M. Perou, L. A. Carey and A. Prat, “HER2-enriched subtype and pathological complete response in HER2-positive breast cancer: A systematic review and meta-analysis,” Cancer Treat Rev., 84, 101965, 2020.
• [7] Ş. Comşa, A. M. Cimpean, and M. Raıca, “The Story of MCF-7 Breast Cancer Cell Line: 40 years of Experience in Research,” Anticancer Research, 35 (6), 3147-3154, 2015.
• [8] H. P. Hughes, F. Van Knapen, H. J. Atkinson, A. H. Balfour, and D. L. Lee, “A new soluble antigen preparation of Toxoplasma gondii and its use in serological diagnosis,” Clin. Exp. Immunol., 49 (1), 239-246, 1982.
• [9] C. A. da Costa, V. P. de Toledo, O. enaro, P. Williams, and W. Mayrink, “Montenegro skin test-evaluation of the composition and stability of the antigen preparation,” Mem. Inst. Oswaldo Cruz, 91 (2), 4-193, 1996.
• [10] J. C. Aguilar and E. G. Rodríguez, “Vaccine adjuvants revisited,” Vaccine, 25 (19), 3752-62, 2007.
• [11] S. M. Sivakumar, M. M. Safhi, M. Kannadasan, and N. Sukumaran, “Vaccine adjuvants-Current status and prospects on controlled release adjuvancity,” Saudi Pharm. J., 19 (4), 197-206, 2011.
• [12] A. M. Dvorak and H. F. Dvorak, “Structure of Freund's complete and incomplete adjuvants. Relation of adjuvanticity to structure,” Immunology, 1974. 27(1): p. 99-114.
• [13] S. V. Dambaeva, D. V. Mazurov, N. M. Golubeva, V. A. D'yakonova, B. V. Pinegin, and R. M. Khaitov, “Effect of polyoxidonium on the phagocytic activity of human peripheral blood leukocytes,” Russ J Immunol., 8 (1), 53-60, 2003.
• [14] N. S. Bryan and M. B. Grisham, “Methods to detect nitric oxide and its metabolites in biological samples,” Free Radical Biology and Medicine, 43 (5), 645-657, 2007.
• [15] K. Sangdee and P. Thummabenjapone, “Evaluation of Antigen Preparation Methods for Polyclonal Antibody Production against Streptomyces spp.,” British Microbiology Research Journal, 2, 137-145, 2012.
• [16] W. P. Tansey, “Freeze-thaw lysis for extraction of proteins from Mammalian cells,” CSH protocols, 2006.
• [17] K. L. Manchester, “Use of UV methods for measurement of protein and nucleic acid concentrations,” Biotechniques, 20 (6), 968-70, 1996.
• [18] I. Guevara, J. Iwanejko, A. Dembińska-Kieć, J. Pankiewicz, A. Wanat, P. Anna, I. Gołabek, S. Bartuś, M. Malczewska-Malec, and A. Szczudlik, “Determination of nitrite/nitrate in human biological material by the simple Griess reaction,” Clin Chim Acta., 22, 274 (2), 177-88, 1998.
• [19] A. Thakur, H. Kaur, and S. Kaur, “Studies on the protective efficacy of freeze thawed promastigote antigen of Leishmania donovani along with various adjuvants against visceral leishmaniasis infection in mice,” Immunobiology, 220 (9), 1031-8, 2015.