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

Year 2024, , 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

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