Triple Negatif Meme Kanseri Hücrelerinde Hidroksiklorokin ve Timokinon Kombinasyon Uygulaması Sonrası Otofajik Aktivitenin Azalması

Öz

Amaç: Otofaji, triple negatif meme kanseri alt tipi dahil olmak üzere meme kanserinde tümör oluşumunda etkili bir mekanizmadır. Araştırmalar, hidroksiklorokin ve timokinon’un otofajiyi düzenleyerek aktivitesini potansiyel olarak baskıladığını göstermektedir. Ancak, bu maddelerin kombine uygulanmasının triple negatif meme kanserinde otofaji üzerindeki etkileri henüz aydınlatılmamıştır. Bu çalışma, hidroksiklorokin ve timokinon kombinasyonunun triple negatif meme kanseri hücrelerinde hücrelerinde antikanser ve otofajik etkilerini in vitro olarak incelemeyi hedeflemektedir. Gereç ve Yöntem: Hidroksiklorokin (10-210 µM) ve timokinon (5-45 µM)’nun 24 ve 48 saat boyunca MDA-MB-231 hücrelerine uygulanması sonucu hücrelerin canlılığı WST-1 testi ile değerlendirildi. Kombinasyonlarının etkileri, CompuSyn (v.10) ile kombinasyon indeksi ve doz azaltma indeksi hesaplanarak Chou-Talalay yöntemiyle analiz edildi. Hücre canlılığının azalmasındaki otofaji etkisini gözlemlemek adına, otofajik veziküllerin tanımlanması ve görüntülenmesi için “Autophagy Detection Kit” ve floresan mikroskobu kullanıldı. İstatistiksel analizler, GraphPad Prism (v.8.3.0) kullanılarak gerçekleştirildi (p<0,0001). Bulgular: Hidroksiklorokin ve timokinon’un 24 ve 48 saatlik uygulamaları sonucu hücre canlılığında anlamlı bir azalma gözlemlendi (p<0,0001). Kombinasyon uygulamaları sonucu sinerjistik etkileri 24 saatte belirgin olarak görülürken, 48 saat uygulanmaları sonucu ise antagonistik etkiler elde edildi (kombinasyon indeksi>1). Doz azaltmada olumlu etkiler 24 saatte belirgin iken (doz azaltma indeksi >1), 48 saatte tam tersi etkiler elde edildi (doz azaltma indeksi<1). Sonuç olarak, kombinasyonun 24 saatlik uygulamasıyla karşılaşılan sinerjistik etkilerin otofajik vezikülleri azalttığı gösterildi (p<0,0001). Sonuç: Bu çalışma, hidroksiklorokin ve timokinon tarafından indüklenen otofaji mekanizması aracılığıyla triple negatif meme kanseri hücrelerinde zamanla bağımlı canlılık azalmasını ilk kez ortaya koymuştur. Bu kombinasyon ileriki çalışmalarla birlikte triple negatif meme kanseri tedavisi ve otofaji modülasyonunda etkin rol oynama potansiyeli taşımaktadır.

Anahtar Kelimeler

• Hidroksiklorokin
• ilaç kombinasyonu
• meme kanseri
• otofaji
• timokinon

Etik Beyan

Etik kurul iznine gerek yoktur.

Decreased Autophagic Activity in Triple-Negative Breast Cancer Cells upon Hydroxychloroquine and Thymoquinone Combination Treatment

Öz

Objective: Autophagy plays a significant role in breast cancer tumorigenesis, including triple-negative breast cancer. Research indicates that hydroxychloroquine and thymoquinone modulate autophagy, potentially suppressing its activity. However, their combined effects on autophagy in triple-negative breast cancer remain unexplored. In this study, we investigated the potential anti-cancer and autophagy-modulating effects of hydroxychloroquine-thymoquinone combination on triple-negative breast cancer cells in vitro. Material and Method: The viability of MDA-MB-231 cells was evaluated after treatment with hydroxychloroquine (10-210 µM) and thymoquinone (5-45 µM) for 24 and 48 hours using the WST-1 assay. Combination effects were analyzed using the Chou-Talalay method with CompuSyn (v.10). Autophagic vesicles were visualized using an Autophagy Detection Kit and fluorescence microscopy to investigate their role in the decrease in cell viability. Statistical analysis was performed with GraphPad Prism (v.8.3.0). Results: At both 24- and 48-hour intervals post-treatment, a significant decrease in viability was observed for both hydroxychloroquine and thymoquinone treatments individually (p<0.0001). The combination of these drugs revealed pronounced synergistic effects at 24 hours, whereas antagonistic effects were noted at 48 hours (combination index>1). At 24 hours, favorable dose reduction effects were evident (dose reduction index >1), while the 48-hour results showed an unfavorable reduction (dose reduction index<1). Consequently, the 24-hour synergistic effects resulted in a reduction in autophagic vesicles (p<0.0001).(p<0.0001). Conclusion: This study revealed, for the first time, a time-dependent decrease in triple-negative breast cancer cell viability via the autophagy mechanism induced by hydroxychloroquine and thymoquinone, highlighting their novel implications for triple-negative breast cancer treatment and autophagy modulation.

Anahtar Kelimeler

• Autophagy
• breast cancer
• drug-combination
• hydroxychloroquine
• thymoquinone

Etik Beyan

Not applicable.

Kaynakça

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