Pichia pastoris'te TP4 Peptidinin Artırılmış Üretimi ve Hepatoselüler Karsinom HUH-7 Hücrelerine Karşı Terapötik Değerlendirmesi

Abstract

Bir katyonik antimikrobiyal peptiti olan Tilapia piscidin 4 (TP4), anti-bakteriyel, yara iyileştirici ve antikanser özellikleri de dahil olmak üzere çeşitli biyolojik rolüyle tanınmaktadır. Burada, TP4 peptitinin kodon optimizasyonlu dizisi, Pichia pastoris KM71H ekspresyon sisteminde güçlü ve indüklenebilir bir promotor olan AOX1 promotoru altında pPICZA ekspresyon vektörü kullanılarak eksprese edilmiştir. Rekombinant TP4 peptiti, Ni-NTA afinite kromatografisi ile saflaştırılmıştır. TP4'ün saflaştırılmasından sonra TP4'ün antikanser aktivitesi hepatosellüler karsinoma hücrelerinde değerlendirilmiş ve altta yatan mekanizmalar belirlenmiştir. Bu çalışmada ilk kez rekombinant TP4'ün insan HUH-7 hücre hattında güçlü antikanser aktivite gösterdiği belirlenmiştir. TP4 antimikrobiyal peptiti, antikanser etkileri nedeniyle kanser hücrelerinin tedavisinde rekabetçi bir aday olarak kullanılabilir.

Keywords

• Tilapia piscidin 4
• rekombinant peptit
• Pichia pastoris
• HUH-7

Project Number

31705-TUSEB

Enhanced Production and Therapeutic Evaluation of TP4 Peptide in Pichia pastoris Against Hepatocellular Carcinoma HUH-7 Cells

Abstract

Tilapia piscidin 4 (TP4), a cationic antimicrobial peptide, is recognized for its diverse biological roles, including antibacterial, wound-healing, and anticancer properties. Herein, the codon-optimized sequence of TP4 peptide was expressed using the pPICZαA expression vector containing the AOX1 promoter, a strong and inducible promoter, in the Pichia pastoris KM71H expression system. Recombinant TP4 peptide was purified by Ni-NTA affinity chromatography. After purification, the anticancer activity of TP4 was assessed in HUH-7 hepatocellular carcinoma cells, and the underlying mechanisms were determined. In the present study, it was demonstrated for the first time that recombinant TP4 displayed strong anticancer activity in the human HUH-7 cell line. The TP4 antimicrobial peptide can be used as a competitive candidate for the treatment of cancer cells due to its anticancer effects.

Keywords

• Tilapia piscidin 4
• recombinant peptide
• Pichia pastoris
• HUH-7

Supporting Institution

This project was supported by Health Institutes of Türkiye (TUSEB) with project no. 31705.

Project Number

31705-TUSEB

Ethical Statement

There are no ethical issues regarding the publication of this study.

References

1. [1]https://gco.iarc.fr/today/en/dataviz/pie?mode=cancer&group_populations=1&cancers=39&types=1, Ziyaret tarihi: 14.03.2024.
2. [2] Chen, Z., Xie, H. et al., (2020) Recent progress in treatment of hepatocellular carcinoma, Am J Cancer Res., 10 (9) 2993-3036.
3. [3] Lien, S. and Lowman, H.B. (2003) Therapeutic peptides, Trends Biotechnol., 21(12) 556-62.
4. [4] Boohaker, R.J., Lee, M.W., Vishnubhotla, P., Perez, J. M. and Khaled A. R. (2012) The Use of Therapeutic Peptides to Target and to Kill Cancer Cells, Current Medicinal Chemistry, 19 3794-3804.
5. [5] Chauhan, S., et al., (2021) Antimicrobial peptides against colorectal cancer-a focused review Pharmacological Research.
6. [6] Peng, K.C. et al., (2012) Five Different Piscidins from Nile Tilapia, Oreochromis niloticus: Analysis of Their Expressions and Biological Functions, plosone, 7, 11.
7. [7] Mahrous, K.F. et al., (2020) Piscidin 4: Genetic expression and comparative immunolocalization in Nile tilapia (Oreochromis niloticus) following challenge using different local bacterial strains, Developmental & Comparative Immunology.
8. [8] Dyshlovoy, S.A. and Honecker, F., (2020) Marine Compounds and Cancer: Updates 2020 Marine Drugs, 18, 643.

9. [9] Ting, C.H. and Chen, J. Y., (2018) Nile Tilapia Derived TP4 Shows Broad Cytotoxicity toward to Non-Small-Cell Lung Cancer Cells, Marine Drugs, 16, 506.
10. [10] Su, B.C. et al., (2019) Antimicrobial Peptide TP4 Induces ROS-Mediated Necrosis by Triggering Mitochondrial Dysfunction in Wild-Type and Mutant p53 Glioblastoma Cells, Cancers, 11, 171.
11. [11] Neshani, A. Et al., (2018) Extended-Spectrum Antimicrobial Activity of the Low Cost Produced Tilapia Piscidin 4 (TP4) Marine Antimicrobial Peptide, Journal of Research in Medical and Dental Science, 6 (5) 327-334.
12. [12] Zorko, M., Jerala, R. (2010) Production of recombinant antimicrobial peptides in bacteria, Methods Mol Biol., 618,61-6.
13. [13] Li, Y. (2011) Recombinant production of antimicrobial peptides in Escherichia coli: A review, Protein Expr Purif. 80:260-67.
14. [14] Hagenson, M.J. (1991) Production of recombinant proteins in the methylotrophic yeast Pichia pastoris, Bioprocess Technol., 12, 193-212.
15. [15] Cregg, J.M. (2007) Pichia Protocols Methods in Molecular Biology Series, Humana Press Inc., 389(2) 1-10.
16. [16] Huang, H.N. et al. (2020) Dietary supplementation of recombinant tilapia piscidin 4-expressing yeast enhances growth and immune response in Lates calcarifer, Aquaculture Reports, 16.
17. [17] Tai, H.M. et al. (2021) Scale-up production of and dietary supplementation with the recombinant antimicrobial peptide tilapia piscidin 4 to improve growth performance in Gallus gallus domesticus, PLOS ONE, 16(6).
18. [18] Su, B.C. (2021) Marine Antimicrobial Peptide TP4 Exerts Anticancer Effects on Human Synovial Sarcoma Cells via Calcium Overload, Reactive Oxygen Species Production and Mitochondrial Hyperpolarization, Mar. Drugs, 19, 93.
19. [19] Ting, C.H. (2016) Targeting FOSB with a cationic antimicrobial peptide, TP4, for treatment of triple-negative breast cancer, Oncotarget, 7, 26.
20. [20] Hazam, P.K. and Chen, J.Y. (2020) Therapeutic utility of the antimicrobial peptide Tilapia Piscidin 4 (TP4), Aquaculture Reports, 17.
21. [21] Karaoğlan, M., Erden-Karaoğlan, F. (2020) Effect of codon optimization and promoter choice on recombinant endo- polygalacturonase production in Pichia pastoris. Enzyme and Microbial Technology, 139, 109589.
22. [22] Wu, S. and Letchworth, G.J. (2004) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol, Biotechniques, 36,152-154.
23. [23] Schägger, H. (2006) Tricine–SDS-PAGE, Nature Protocols, 1 (1) 16-22.
24. [24] Carson, M. et al. (2007) His-tag impact on structure. Acta Crystallographica Section D” Structural Biology, 63(3) 295-301.
25. [25] Spriestersbach, A. et al. (2015) “Purification of His-Tagged Proteins” Methods in Enzymology, 559, 1-15.
26. [26] Karaoğlan, M. and Erden-Karaoğlan, F. (2021) “Extracellular Production and Purification of the β-glucanase in Pichia pastoris Expression System”. Erzincan University Journal of Science and Technology, 14 (2) 620-630.
27. [27] Günay, N., & Kuzucu, M. (2023). “Agonistic Effects of Deinoxanthin on Tamoxifen Antiproliferative Activity on HER2 Positive Breast Cancer: An In Vitro Study on MDA-MB-453”. Erzincan University Journal of Science and Technology, 16(1), 138-154.
28. [28] Karaoğlan, M. (2012) “Pichia pastoris Alkol Oksı̇daz (AOX1 ve AOX2) Genlerı̇nı̇n İnaktı̇f Edı̇lmesı̇ ve Elde Edı̇len Suşun Rekombı̇nant Proteı̇n Üretı̇mı̇nde Kullanılması” (Yüksek Lisans), Akdenı̇z Ünı̇versı̇tesı̇ Fen Bı̇lı̇mlerı̇ Enstı̇tüsü, Antalya, 15-19.
29. [29] Li, Y. et. al. (2012) “Overview on the recent study of antimicrobial peptides: Origins, functions, relative mechanisms and application” Peptides, 37,207–215.
30. [30] Zhong, C. et. al. (2020) “A Review for Antimicrobial Peptides with Anticancer Properties: Re- purposing of Potential Anticancer Agents” BIO Integration, 1(4), 156-167.
31. [31] Deslouches, B. and Peter Di, Y. (2017) “Antimicrobial Peptides with Selective Antitumor Mechanisms: Prospect for Anticancer Applications.” Oncotarget, 8(28), 46635-46651.
32. [32] Kuo, H.M. et al. (2018) “MSP-4, an Antimicrobial Peptide, Induces Apoptosis via Activation of Extrinsic Fas/FasL- and Intrinsic Mitochondria-Mediated Pathways in One Osteosarcoma Cell Line” marine drugs, 16,8.
33. [33] Portt, L. et. al. (2011) “Anti-apoptosis and cell survival: A review” Biochimica et Biophysica Acta 1813, 238–259.
34. [34] Kiraz, Y. ve ark (2016) “Major apoptotic mechanisms and genes involved in apoptosis” Tumor Biol., 37, 8471–8486