THIOSTREPTON, MDA-MB-231 HÜCRELERİNDE TLR4 EKSPRESYONUNU DÜZENLER VE APOPTOZU İNDÜKLER: IN VITRO VE IN SILICO ANALİZ

Year 2024, Volume: 25 Issue: 3, 209 - 221, 30.09.2024

Funda Demırtaş Korkmaz , Zekeriya Düzgün , Asuman Deveci Özkan

https://doi.org/10.69601/meandrosmdj.1540223

Abstract

Amaç: Toll-benzeri reseptörler (TLR'ler), tümör oluşumu, apoptoz ve metastazda kilit rol oynayan desen tanıma reseptörleridir. Üçlü negatif meme kanseri (TNBC), kötü bir prognoza sahip, son derece agresif bir malignitedir. TLR'lerin meme kanserindeki rolü yeterince araştırılmamış olmasına rağmen, son çalışmalar TNBC'de TLR'leri hedeflemenin faydalı olabileceğini öne sürmektedir. Bu çalışmada, sedef hastalığı inflamasyonunda TLR7-9'un yeni bir inhibitörü olarak tanımlanan antibiyotik Thiostrepton, TNBC hücrelerinde (MDA-MB-231) TLR3, TLR4 ve TLR9 ekspresyonu üzerindeki etkileri açısından incelenmiştir.
Materyal ve Metotlar : Thiostrepton’un sitotoksisitesi MTT testi kullanılarak değerlendirildi. TLR3, TLR4, TLR9, Bax, Bcl-2, Nf-κB ve E-cadherin gen ekspresyon seviyeleri RT-PCR ile ölçüldü. Hücre morfolojisi değişiklikleri Acridine Orange/Ethidium Bromide (AO/EtBr) boyaması ile analiz edildi. Thiostreptonun TLR4-MD-2 kompleksi ile etkileşimleri moleküler kenetlenme ve dinamik simülasyonlar ile gösterildi.
Bulgular: Thiostrepton, konsantrasyon ve zamana bağlı olarak hücre canlılığında azalmaya yol açtı. TLR4 ve Bcl-2 gen ekspresyonunu önemli ölçüde azaldığını ve TLR3, Bax ve Nf-κB seviyelerinin ise arttığı gözlenmiştir. Bax ve Bcl-2 gen ekspresyonundaki değişiklikler ve hücre morfolojisindeki değişiklikler, thiostreptonun MDA-MB-231 hücrelerinde apoptozu teşvik ettiğini göstermiştir. TLR9 ekspresyonundaki azalma anlamlı olmasa da, thiostrepton TLR3 ekspresyonunu önemli ölçüde artırmış ve TLR4 ekspresyonunu azaltmıştır. Üç bağımsız moleküler dinamik simülasyonu, thiostreptonun TLR4-MD2 domainine yüksek bağlanma affinitesi göstererek kararlı bir şekilde bağlandığını gösterdi.
Sonuç: Thiostrepton, TNBC hücrelerinde apoptozu etkin bir şekilde indüklemekte ve hücre canlılığını azaltmaktadır. In silico analizler, thiostreptonun TLR4'ü modüle edebileceğini öne sürerek, daha fazla araştırma ve terapötik geliştirme için aday olma potansiyelini vurgulamaktadır.

Keywords

Thiostrepton, toll like reseptörler, TLR4 reseptör, üçlü negatif meme kanseri

THIOSTREPTON MODULATES TLR4 EXPRESSION AND INDUCES APOPTOSIS IN MDA MB 231 CELLS: AN IN VITRO AND IN SILICO ANALYSIS

Abstract

Objective: Toll-like receptors (TLRs) are key pattern recognition receptors involved in tumorigenesis, apoptosis, and metastasis. Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with a poor prognosis. Although the role of TLRs in breast cancer remains underexplored, recent studies suggest targeting TLRs in TNBC could be beneficial. In this study Thiostrepton, an antibiotic and novel inhibitor of TLR7-9 in psoriatic inflammation, was investigated for its effects on TLR3, TLR4, and TLR9 expression in TNBC cells (MDA-MB-231).
Materials and Methods: The cytotoxicity of thiostrepton was assessed using the MTT assay. RT-PCR was used to measure gene expression levels of TLR3, TLR4, TLR9, Bax, Bcl-2, Nf-κB, and E-cadherin. Cell morphology changes were analyzed with Acridine Orange/Ethidium Bromide (AO/EtBr) staining. Molecular docking and dynamics simulations examined interactions between thiostrepton and the TLR4-MD-2 complex.
Results: Thiostrepton led to a concentration- and time-dependent decrease in cell viability. It significantly inhibited TLR4, Bcl-2 gene expression and increased TLR3, Bax, and Nf-κB levels. The changes in Bax and Bcl-2 gene expression, along with alterations in cell morphology, demonstrated that thiostrepton promoted apoptosis in MDA-MB-231 cells. While TLR9 expression reduction was not significant, thiostrepton notably increased TLR3 expression and decreased TLR4 expression. The three independent molecular dynamics simulations demonstrated that thiostrepton binds stably to the TLR4-MD2 domain, exhibiting a high binding affinity as indicated by the binding free energy calculations.
Conclusion: Thiostrepton effectively induces apoptosis and reduces cell viability in TNBC cells. In silico analysis suggest thiostrepton could modulate TLR4, highlighting its potential as a candidate for further research and therapeutic development.

Keywords

Thiostrepton, toll like receptors, TLR4 receptor, triple-negative breast cancer

References

• 1. Foulkes WD, Smith IE, Reis-Filho JS. Triple-Negative Breast Cancer. N Engl J Med. 2010;363(20):1938–48.
• 2. Javaid N, Choi S. Toll-like Receptors from the Perspective of Cancer Treatment. Cancers 2020;12(2):297.
• 3. Rakoff-Nahoum S, Medzhitov R. Toll-like receptors and cancer. Nat Rev Cancer. 2009;9(1):57–63.
• 4. Ren T, Wen ZK, Liu ZM, Liang YJ, Guo ZL, Xu L. Functional expression of TLR9 is associated to the metastatic potential of human lung cancer cell. Cancer Biol Ther. 2007;6(11):1704-1709.
• 5. Butkowsky C, Aldor N, Poynter SJ. Toll-like receptor 3 ligands for breast cancer therapies (Review). Mol Clin Oncol. 2023;19(2):1-8.
• 6. Shi S, Xu C, Fang X, Zhang Y, Li H, Wen W, et al. Expression profile of Toll-like receptors in human breast cancer. Mol Med Rep. 2020; 21(2):786-794.
• 7. Fan L, Sui XY, Jin X, Zhang WJ, Zhou P, Shao ZM. High expression of TLR3 in triple-negative breast cancer predicts better prognosis—data from the Fudan University Shanghai Cancer Center cohort and tissue microarrays. BMC Cancer. 2023;23(1):298.
• 8. Tuomela J, Sandholm J, Karihtala P, Ilvesaro J, Vuopala KS, Kauppila JH, et al. Low TLR9 expression defines an aggressive subtype of triple-negative breast cancer. Breast Cancer Res Treat. 2012;135(2):481-493.
• 9. Ahmed M, Maldonado AM, Durrant JD. From byte to bench to bedside: molecular dynamics simulations and drug discovery. BMC Biol. 2023;21(1):299.
• 10. Salo-Ahen OMH, Alanko I, Bhadane R, Bonvin AMJJ, Honorato RV, Hossain S, et al. Molecular dynamics simulations in drug discovery and pharmaceutical development. Processes. 2020;9(1):71.
• 11. Muhammed MT, Aki-Yalcin E. Molecular docking: principles, advances, and its applications in drug discovery. Lett Drug Des Discov. 2024;21(3):480–95.
• 12. Nascimento IJ dos S, de Aquino TM, da Silva-Júnior EF. The new era of drug discovery: The power of computer-aided drug design (CADD). Lett Drug Des Discov. 2022;19(11):951–5.
• 13. Ain Q ul, Batool M, Choi S. TLR4-targeting therapeutics: structural basis and computer-aided drug discovery approaches. Molecules. 2020;25(3):627.
• 14. Bailly C. The bacterial thiopeptide thiostrepton. An update of its mode of action, pharmacological properties and applications. Eur J Pharmacol. 2022;914:174661.
• 15. Bhat UG, Halasi M, Gartel AL. Thiazole antibiotics target FoxM1 and induce apoptosis in human cancer cells. PLoS One. 2009;4(5):e5592.
• 16. Lai C-Y, Yeh D-W, Lu C-H, Liu Y-L, Huang L-R, Kao C-Y, et al. Identification of Thiostrepton as a Novel Inhibitor for Psoriasis-like Inflammation Induced by TLR7–9. J Immunol. 2015;195(8).
• 17. Kwok JMM, Myatt SS, Marson CM, Coombes RC, Constantinidou D, Lam EWF. Thiostrepton selectively targets breast cancer cells through inhibition of forkhead box M1 expression. Mol Cancer Ther. 2008;7(7):2022–32.
• 18. Cai X, Xiao W, Shen J, Lian H, Lu Y, Liu X, et al. Thiostrepton and miR‑216b synergistically promote osteosarcoma cell cytotoxicity and apoptosis by targeting FoxM1. Oncol Lett [Internet]. 2020;20(6):1–1.
• 19. Demirtas Korkmaz F, Dogan Turacli I, Esendagli G, Ekmekci A. Effects of thiostrepton alone or in combination with selumetinib on triple-negative breast cancer metastasis. Mol Biol Rep [Internet]. 2022;49(11):10387–97.
• 20. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162(1):156-159.
• 21. Park BS, Song DH, Kim HM, Choi B-S, Lee H, Lee J-O. The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex. Nature. 2009;458(7242):1191–5.
• 22. Kim S, Thiessen PA, Bolton EE, Chen J, Fu G, Gindulyte A, et al. PubChem Substance and Compound databases. Nucleic Acids Res. 2016;44(D1):D1202–13.
• 23. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010;31(2):455–61.
• 24. Dallakyan S, Olson AJ. Small-Molecule Library Screening by Docking with PyRx. In 2015 p. 243–50.
• 25. Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1–2:19–25.
• 26. Lindorff-Larsen K, Piana S, Palmo K, Maragakis P, Klepeis JL, Dror RO, et al. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins. 2010;78(8):1950–8.
• 27. Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG. A smooth particle mesh Ewald method. J Chem Phys. 1995;103(19):8577.
• 28. Hess B, Bekker H, Berendsen HJC, Fraaije JGEM. LINCS: A Linear Constraint Solver for Molecular Simulations. J Comput Chem. 1997;18:1463–72.
• 29. Lemak AS, Balabaev NK. On the Berendsen thermostat. Mol Simul. 1994;13(3):177–87.
• 30. Martoňák R, Laio A, Parrinello M. Predicting Crystal Structures: The Parrinello-Rahman Method Revisited. Phys Rev Lett. 2003;90(7): 075503.
• 31. Wang E, Sun H, Wang J, Wang Z, Liu H, Zhang JZH, et al. End-Point Binding Free Energy Calculation with MM/PBSA and MM/GBSA: Strategies and Applications in Drug Design [Internet]. Vol. 119, Chemical Reviews. American Chemical Society.2019 p. 9478–508.
• 32. Kumari R, Kumar R, Lynn A, Lynn A. g_mmpbsa—A GROMACS Tool for High-Throughput MM-PBSA Calculations. J Chem Inf Model. 2014;54(7):1951–62.
• 33. Gartel AL. FoxM1 inhibitors as potential anticancer drugs. Expert Opin Ther Targets. 2008;12(6):663–5.
• 34. Wongkhieo S, Numdee K, Lam EWF, Choowongkomon K, Kongsema M, Khongkow M. Liposomal Thiostrepton Formulation and Its Effect on Breast Cancer Growth Inhibition. J Pharm Sci. 2021;110(6):2508–16.
• 35. Kongsema M, Wongkhieo S, Khongkow M, Lam E, Boonnoy P, Vongsangnak W, et al. Molecular mechanism of Forkhead box M1 inhibition by thiostrepton in breast cancer cells. Oncol Rep . 2019;42(3):953–62.
• 36. Lai CY, Su YW, Lin KI, Hsu LC, Chuang TH. Natural Modulators of Endosomal Toll-Like Receptor-Mediated Psoriatic Skin Inflammation. Journal of Immunology Research. 2017;2017:1-15.
• 37. Jiang L, Wang P, Chen L, Chen H. Down-regulation of FoxM1 by thiostrepton or small interfering RNA inhibits proliferation, transformation ability and angiogenesis, and induces apoptosis of nasopharyngeal carcinoma cells. Int J Clin Exp Pathol. 2014;7(9):5450–60.
• 38. Liu SX, Zhou Y, Zhao L, Zhou LS, Sun J, Liu GJ, et al. Thiostrepton confers protection against reactive oxygen species-related apoptosis by restraining FOXM1-triggerred development of gastric cancer. Free Radic Biol Med. 2022 20;193:385–404.
• 39. Kuttikrishnan S, Prabhu KS, Khan AQ, Alali FQ, Ahmad A, Uddin S. Thiostrepton inhibits growth and induces apoptosis by targeting FoxM1/SKP2/MTH1 axis in B-precursor acute lymphoblastic leukemia cells. Leuk Lymphoma. 2021;62(13):3170–80.
• 40. Peng W, Hong Z, Chen X, Gao H, Dai Z, Zhao J, et al. Thiostrepton reactivates latent HIV-1 through the p-TEFb and NF-κB pathways mediated by heat shock response. Antimicrob Agents Chemother. 2020;64(5):10-1128.
• 41. Lai C-Y, Yeh D-W, Lu C-H, Liu Y-L, Huang L-R, Kao C-Y, et al. Thiostrepton inhibits psoriasis-like inflammation induced by TLR7, TLR8, and TLR9. J Immunol. 2016 1;196(1_Supplement):124.41.
• 42. Hsu Y Bin, Lan MC, Kuo YL, Huang CYF, Lan MY. A preclinical evaluation of thiostrepton, a natural antibiotic, in nasopharyngeal carcinoma. Invest New Drugs. 2020;38(2): 264-273.
• 43. Salaun B, Coste I, Rissoan M-C, Lebecque SJ, Renno T. TLR3 Can Directly Trigger Apoptosis in Human Cancer Cells. J Immunol. 2006;176(8):4894–901.
• 44. Butkowsky C, Aldor N, Poynter S. Toll‑like receptor 3 ligands for breast cancer therapies (Review). Mol Clin Oncol. 2023;19(2):1-8.
• 45. Esparza K, Oliveira SD, Castellon M, Minshall RD, Onyuksel H. Thiostrepton-Nanomedicine, a TLR9 Inhibitor, Attenuates Sepsis-Induced Inflammation in Mice. Yokota S, editor. Mediators Inflamm. 2023;2023:1–11.
• 46. Niu X, Yu Y, Guo H, Yang Y, Wang G, Sun L, et al. Molecular modeling reveals the inhibition mechanism and binding mode of ursolic acid to TLR4-MD2. Comput Theor Chem. 2018;1123:73–8.
• 47. Sun M, Yao L, Yu Q, Duan Y, Huang J, Lyu T, et al. Screening of Poria cocos polysaccharide with immunomodulatory activity and its activation effects on TLR4/MD2/NF-κB pathway. Int J Biol Macromol. 2024;273:132931.