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01.08.2021 | Original Paper

ARA-linker-TGFαL3: a novel chimera protein to target breast cancer cells

verfasst von: Abdolamir Ghadaksaz, Abbas Ali Imani Fooladi, Hamideh Mahmoodzadeh Hosseini, Taher Nejad Satari, Mohsen Amin

Erschienen in: Medical Oncology | Ausgabe 8/2021

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Abstract

Targeted cancer therapies based on overexpressed receptors and the fractions containing immunotoxins and bacterial metabolites are one of the well-known methods to overcome the chemotherapy resistance of cancer cells. In this paper, we designed ARA-linker-TGFαL3, using Arazyme, a Serratia proteamaculans metabolite, and a third loop segment of TGFα to target EGFR-expressing breast cancer cells. After cloning in pET28a (+), the expression of recombinant protein was optimized in Escherichia coli strain BL21 (DE3). MDA-MB-468 (EGFR positive) and MDA-MB-453 (EGFR negative) breast cancer cell lines were employed. Also, the chemotherapeutic drug, Taxotere (Docetaxel), was employed to compare cytotoxicity effects. Cell ELISA assessed the binding affinity of recombinant proteins to the receptor, and the cytotoxicity was detected by MTT and lactate dehydrogenase release assays. The interfacing with cancer cell adhesion was evaluated. Furthermore, the induction of apoptosis was examined utilizing flow cytometric analysis, and caspase-3 activity assay. Moreover, RT-PCR was conducted to study the expression of apoptosis (bax, bcl2, and casp3), angiogenesis (vegfr2), and metastasis (mmp2 and mmp9) genes. ARA-linker-TGFαL3 revealed a higher binding affinity, cytotoxicity, and early apoptosis induction in MDA-MB-468 cells compared to the effects of Arazyme while both recombinant proteins showed similar effects on MDA-MB-453. In addition, the Taxotere caused the highest cytotoxicity on cancer cells through induction of late apoptosis. Meanwhile, the expression of angiogenesis and metastasis genes was decreased in both cell lines after treatment with either ARA-linker-TGFαL3 or Arazyme. Our in vitro results indicated the therapeutic effect of ARA-linker-TGFαL3 on breast cancer cells.

Chen Z, Xu L, Shi W, et al. Trends of female and male breast cancer incidence at the global, regional, and national levels, 1990–2017. Breast Cancer Res Treat. 2020;180(2):481–90. https://doi.org/10.1007/s10549-020-05561-1.CrossRefPubMed

Waks AG, Winer EP. Breast cancer treatment. JAMA. 2019;321(3):316–316. https://doi.org/10.1001/jama.2018.19323.CrossRefPubMed

Singh SK, Singh S, Lillard JW Jr, et al. Drug delivery approaches for breast cancer. Int J Nanomed. 2017;12:6205. https://doi.org/10.2147/IJN.S140325.CrossRef

Mahmoodzadeh Hosseini H, Ali Imani Fooladi A, Soleimanirad J, et al. Exosome/staphylococcal enterotoxin B, an anti tumor compound against pancreatic cancer. J BUON Off J Balk Union Oncol. 2014;19(2):440–8.

Commander H, Whiteside G, Perry C. Vandetanib. Drugs. 2011;71(10):1355–65. https://doi.org/10.2165/11595310-000000000-00000.CrossRefPubMed

Litzenburger BC, Creighton CJ, Tsimelzon A, et al. High IGF-IR activity in triple-negative breast cancer cell lines and tumor grafts correlates with sensitivity to anti-IGF-IR therapy. Clin Cancer Res. 2011;17(8):2314–27. https://doi.org/10.1158/1078-0432.ccr-10-1903.CrossRefPubMed

Milanezi F, Carvalho S, Schmitt FC. EGFR/HER2 in breast cancer: a biological approach for molecular diagnosis and therapy. Expert Rev Mol Diagn. 2008;8(4):417–34. https://doi.org/10.1586/14737159.8.4.417.CrossRefPubMed

Wykosky J, Fenton T, Furnari F, et al. Therapeutic targeting of epidermal growth factor receptor in human cancer: successes and limitations. Chin J Cancer. 2011;30(1):5. https://doi.org/10.5732/cjc.010.10542.CrossRefPubMedPubMedCentral

Garrett TP, McKern NM, Lou M, et al. Crystal structure of a truncated epidermal growth factor receptor extracellular domain bound to transforming growth factor α. Cell. 2002;110(6):763–73. https://doi.org/10.1016/s0092-8674(02)00940-6.CrossRefPubMed

10.

Seshacharyulu P, Ponnusamy MP, Haridas D, et al. Targeting the EGFR signaling pathway in cancer therapy. Expert Opin Ther Targets. 2012;16(1):15–31. https://doi.org/10.1517/14728222.2011.648617.CrossRefPubMedPubMedCentral

11.

Nestor JJ Jr, Newman SR, DeLustro B, et al. A synthetic fragment of rat transforming growth factor α with receptor binding and antigenic properties. Biochem Biophys Res Commun. 1985;129(1):226–32. https://doi.org/10.1016/0006-291x(85)91426-3.CrossRefPubMed

12.

Goleij Z, Hosseini HM, Sedighian H, et al. Breast cancer targeted/therapeutic with double and triple fusion immunotoxins. J Steroid Biochem Mol Biol. 2020. https://doi.org/10.1016/j.jsbmb.2020.105651.CrossRefPubMed

13.

Sawant SS, Patil SM, Gupta V, et al. Microbes as medicines: harnessing the power of bacteria in advancing cancer treatment. Int J Mol Sci. 2020;21(20):7575. https://doi.org/10.3390/ijms21207575.CrossRefPubMedCentral

14.

Amjadi G, Parivar K, Mousavi SF, et al. Anti-cancer effects of recombinant Arazyme from Serratia proteomaculans. J BUON Off J Balk Union Oncol. 2020;25(1):531–42.

15.

Bersanetti PA, Park H-Y, Bae KS, et al. Characterization of Arazyme, an exocellular metalloprotease isolated from Serratia proteamaculans culture medium. Enzyme Microb Technol. 2005;37(6):574–81. https://doi.org/10.1016/j.enzmictec.2005.01.041.CrossRef

16.

Pereira FV, Melo AC, de Melo FM, et al. TLR4-mediated immunomodulatory properties of the bacterial metalloprotease Arazyme in preclinical tumor models. Oncoimmunology. 2016;5(7): e1178420. https://doi.org/10.1080/2162402X.2016.1178420.CrossRefPubMedPubMedCentral

17.

Park J-K, Jeong D-H, Park H-Y, et al. Hepatoprotective effect of Arazyme on CCl₄-induced acute hepatic injury in SMP30 knock-out mice. Toxicology. 2008;246(2):132–42. https://doi.org/10.1016/j.tox.2008.01.006.CrossRefPubMed

18.

Rusnak DW, Alligood KJ, Mullin RJ, et al. Assessment of epidermal growth factor receptor (EGFR, ErbB1) and HER2 (ErbB2) protein expression levels and response to lapatinib (Tykerb, GW572016) in an expanded panel of human normal and tumour cell lines. Cell Prolif. 2007;40(4):580–94. https://doi.org/10.1111/j.1365-2184.2007.00455.x.CrossRefPubMedPubMedCentral

19.

Chen X, Zaro JL, Shen W-C. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013;65(10):1357–69. https://doi.org/10.1016/j.addr.2012.09.039.CrossRefPubMed

20.

Folin O, Ciocalteu V. On tyrosine and tryptophan determinations in proteins. J Biol Chem. 1927;73(2):627–50. https://doi.org/10.1016/S0021-9258(18)84277-6.CrossRef

21.

Yousefi F, Mousavi SF, Siadat SD, et al. Preparation and in vitro evaluation of antitumor activity of TGFαL3-SEB as a ligand-targeted superantigen. Technol Cancer Res Treat. 2016;15(2):215–26. https://doi.org/10.1177/1533034614568753.CrossRefPubMed

22.

Supino R. MTT assays. In: In vitro toxicity testing protocols. Springer; 1995. pp. 137–149. https://doi.org/10.1385/0-89603-282-5:137.

23.

Crowley LC, Marfell BJ, Scott AP, et al. Quantitation of apoptosis and necrosis by Annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harb Protoc. 2016. https://doi.org/10.1101/pdb.prot087288.CrossRefPubMed

24.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. Methods. 2001;25(4):402–8. https://doi.org/10.1006/meth.2001.1262.CrossRef

25.

Pereira FV, Ferreira-Guimarães CA, Paschoalin T, 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(4): e96141. https://doi.org/10.1371/journal.pone.0096141.CrossRefPubMedPubMedCentral

26.

Xu Q, Zhang X, Yue J, et al. Human TGFalpha-derived peptide TGFalphaL3 fused with superantigen for immunotherapy of EGFR-expressing tumours. BMC Biotechnol. 2010;10(1):1–10. https://doi.org/10.1186/1472-6750-10-91.CrossRef

27.

Maleki F, Sadeghifard N, Hosseini HM, et al. Growth-inhibitory effects of TGFαL3-SEB chimeric protein on colon cancer cell line. Biomed Pharmacother. 2019;110:190–6. https://doi.org/10.1016/j.biopha.2018.11.025.CrossRefPubMed

28.

Decker T, Lohmann-Matthes M-L. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods. 1988;115(1):61–9. https://doi.org/10.1016/0022-1759(88)90310-9.CrossRefPubMed

29.

Hashimi SM, Grant B, Alqurashi N, et al. EGF ligand fused to truncated Pseudomonas aeruginosa exotoxin A specifically targets and inhibits EGFR-positive cancer cells. Oncol Rep. 2018;40(5):2690–7. https://doi.org/10.3892/or.2018.6685.CrossRefPubMed

30.

Yang Y, Tian Z, Ding Y, et al. EGFR-targeted immunotoxin exerts antitumor effects on esophageal cancers by increasing ROS accumulation and inducing apoptosis via inhibition of the Nrf2-Keap1 pathway. J Immunol Res. 2018;2018:1090287. https://doi.org/10.1155/2018/1090287.CrossRefPubMedPubMedCentral

31.

Kim IS, Kim MJ, Shin DH, et al. Arazyme inhibits cytokine expression and upregulates skin barrier protein expression. Mol Med Rep. 2013;8(2):551–6. https://doi.org/10.3892/mmr.2013.1520.CrossRefPubMed

32.

Attie AD, Raines RT. Analysis of receptor–ligand interactions. J Chem Educ. 1995;72(2):119–24. https://doi.org/10.1021/ed072p119.CrossRefPubMedPubMedCentral

33.

Nagata S. Apoptosis and clearance of apoptotic cells. Annu Rev Immunol. 2018;36(1):489–517. https://doi.org/10.1146/annurev-immunol-042617-053010.CrossRefPubMed

34.

Szondy Z, Sarang Z, Kiss B, et al. Anti-inflammatory mechanisms triggered by apoptotic cells during their clearance. Front Immunol. 2017. https://doi.org/10.3389/fimmu.2017.00909.CrossRefPubMedPubMedCentral

35.

Morse DL, Gray H, Payne CM, et al. Docetaxel induces cell death through mitotic catastrophe in human breast cancer cells. Mol Cancer Ther. 2005;4(10):1495–504. https://doi.org/10.1158/1535-7163.MCT-05-0130.CrossRefPubMed

36.

Hernández-Vargas H, Palacios J, Moreno-Bueno G. Molecular profiling of docetaxel cytotoxicity in breast cancer cells: uncoupling of aberrant mitosis and apoptosis. Oncogene. 2007;26(20):2902–13. https://doi.org/10.1038/sj.onc.1210102.CrossRefPubMed

37.

Radha G, Raghavan SC. BCL2: a promising cancer therapeutic target. Biochim Biophys Acta Rev Cancer. 2017;1868(1):309–14. https://doi.org/10.1016/j.bbcan.2017.06.004.CrossRefPubMed

38.

Ghorbani-Abdi-Saedabad A, Hanafi-Bojd MY, Parsamanesh N, et al. Anticancer and apoptotic activities of parthenolide in combination with epirubicin in MDA-MB-468 breast cancer cells. Mol Biol Rep. 2020;47(8):5807–15. https://doi.org/10.1007/s11033-020-05649-3.CrossRefPubMed

39.

McIlwain DR, Berger T, Mak TW. Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol. 2015. https://doi.org/10.1101/cshperspect.a008656.CrossRefPubMedPubMedCentral

40.

Hyatt DC, Ceresa BP. Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells. Exp Cell Res. 2008;314(18):3415–25. https://doi.org/10.1016/j.yexcr.2008.08.020.CrossRefPubMedPubMedCentral

41.

Nowsheen S, Cooper T, Stanley JA, et al. Synthetic lethal interactions between EGFR and PARP inhibition in human triple negative breast cancer cells. PLoS ONE. 2012;7(10): e46614. https://doi.org/10.1371/journal.pone.0046614.CrossRefPubMedPubMedCentral

42.

Bauvois B. New facets of matrix metalloproteinases MMP-2 and MMP-9 as cell surface transducers: outside-in signaling and relationship to tumor progression. Biochim Biophys Acta Rev Cancer. 2012;1825(1):29–36. https://doi.org/10.1016/j.bbcan.2011.10.001.CrossRef

43.

Amjadi G, Parivar K, Mousavi SF, et al. Effect of metalloprotease Arazyme on the expression of MMP2 and MMP9 genes in metastasis of colon and ovarian cancer cell lines. Thrita. 2019. https://doi.org/10.5812/thrita.100004.CrossRef

44.

Rahimi N. VEGFR-1 and VEGFR-2: two non-identical twins with a unique physiognomy. Front Biosci J Virtual Libr. 2006;11:818. https://doi.org/10.2741/1839.CrossRef

45.

Safari E, Zavaran Hosseini A, Hassan Z, et al. Cytotoxic effect of immunotoxin containing the truncated form of pseudomonas exotoxin A and anti-VEGFR2 on HUVEC and MCF-7 cell lines. Cell J. 2014;16(2):203–10.PubMedPubMedCentral

Titel: ARA-linker-TGFαL3: a novel chimera protein to target breast cancer cells
verfasst von: Abdolamir Ghadaksaz
Abbas Ali Imani Fooladi
Hamideh Mahmoodzadeh Hosseini
Taher Nejad Satari
Mohsen Amin
Publikationsdatum: 01.08.2021
Verlag: Springer US
Erschienen in: Medical Oncology / Ausgabe 8/2021
Print ISSN: 1357-0560
Elektronische ISSN: 1559-131X
DOI: https://doi.org/10.1007/s12032-021-01546-2

Springer Medizin

ARA-linker-TGFαL3: a novel chimera protein to target breast cancer cells

Abstract

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Springer Medizin

Abstract

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