ALTERATIONS IN THE APOPTOSIS-ASSOCIATED GENE EXPRESSIONS IN LUNG CANCER CELLS IN THE PRESENCE OF IXAZOMIB AND GIVINOSTAT

Öz

Objective: Proteasome inhibitors and histone deacetylase (HDAC) inhibitors represent promising therapeutic agents in lung cancer, particularly by enhancing cancer cell sensitivity to treatment. Among these, the combination of ixazomib, a proteasome inhibitor, and givinostat, an HDAC inhibitor, has demonstrated synergistic cytotoxicity in non-small cell lung cancer (NSCLC) cell lines. This strategy holds potential for overcoming therapeutic resistance. Furthermore, macrophage co-culture systems have been shown to modulate gene expression patterns in lung cancer cells. In this context, the present study aimed to investigate the effects of ixazomib and givinostat, individually and in combination, along with differentiated M1 macrophages, on the expression of apoptosis-related genes in NSCLC cells. Material and Method: The IC50 values of ixazomib and givinostat in A549 lung cancer cells were determined using the MTT assay after 24 hours of treatment with a range of concentrations (400, 100, 10, and 1 µM). Based on the determined IC50 values, A549 cells were treated with ixazomib alone or in combination with givinostat for 24 hours. In parallel, A549 cells treated with the ixazomib–givinostat combination were co-cultured with THP-1 monocyte–derived M1 macrophages. Total RNA was then isolated, and RT-PCR analysis was performed to evaluate alterations in apoptosis-related gene expression. Result and Discussion: Based on the results of the anti-proliferative effect analysis, the IC50 values for ixazomib and givinostat were determined to be 2.42 µM and 7.32 µM, respectively. According to the RT-PCR results, variations of up to 48.99-fold were observed in the NFKB2, NFKBIA, NFKBIB, RELB, IL-6, IL-8, TP53, PIK3CA, and BCR genes. Significant alterations were particularly observed in genes involved in apoptosis-related pathways, especially in the presence of M1 macrophages. Additional investigation is warranted to thoroughly explore the heightened inflammatory response observed in lung cancer cells in the presence of these drugs.

Anahtar Kelimeler

• Apoptosis
• givinostat
• ixazomib
• lung cancer
• M1 macrophage

İXAZOMİB VE GİVİNOSTAT VARLIĞINDA AKCİĞER KANSERİ HÜCRELERİNDE APOPTOZLA İLİŞKİLİ GEN EKSPRESYON DÜZEYLERİNDEKİ DEĞİŞİKLİKLER

Öz

Amaç: Proteazom inhibitörleri ve histon deasetilaz (HDAC) inhibitörleri, özellikle kanser hücrelerinin tedaviye duyarlılığını artırmaları açısından akciğer kanserinde umut vadeden tedavi ajanları olarak değerlendirilmektedir. Bu ajanlar arasında, bir proteazom inhibitörü olan ixazomib ile bir HDAC inhibitörü olan givinostat’ın kombinasyonu, küçük hücreli olmayan akciğer kanseri (KHOAK) hücre hatlarında sinerjistik sitotoksisite göstermiştir. Bu tedavi yaklaşımı, terapötik dirençlerin aşılması açısından potansiyel taşımaktadır. Ayrıca, makrofaj ile ortak kültür sistemlerinin, akciğer kanseri hücrelerinde gen ekspresyon paternlerini değiştirdiği gösterilmiştir. Bu bağlamda, bu çalışma, ixazomib ve givinostat’ın ayrı ayrı ve birlikte uygulanmasının yanı sıra farklılaşmış M1 makrofajların etkisinin, KHOAK hücrelerinde apoptozla ilişkili genlerin ekspresyonu üzerindeki etkilerini araştırmayı amaçlamıştır. Gereç ve Yöntem: İxazomib ve givinostat’ın A549 akciğer kanseri hücrelerindeki IC₅₀ değerleri, farklı konsantrasyonlarda (400, 100, 10 ve 1 µM) 24 saatlik uygulamayı takiben MTT testi kullanılarak belirlenmiştir. Belirlenen IC₅₀ değerlerine göre A549 hücreleri 24 saat boyunca yalnızca ixazomib veya ixazomib+givinostat kombinasyonu ile muamele edilmiştir. Paralel olarak, ixazomib–givinostat kombinasyonu ile muamele edilen A549 hücreleri, THP-1 monositlerinden türetilmiş M1 makrofajlar ile ko-kültüre edilmiştir. Ardından total RNA izole edilmiş ve apoptozla ilişkili gen ekspresyonundaki değişimleri değerlendirmek amacıyla RT-PCR analizleri gerçekleştirilmiştir. Sonuç ve Tartışma: Anti-proliferatif etki analizine dayalı olarak, ixazomib ve givinostat için IC₅₀ değerleri sırasıyla 2.42 µM ve 7.32 µM olarak belirlenmiştir. RT-PCR sonuçlarına göre, NFKB2, NFKBIA, NFKBIB, RELB, IL-6, IL-8, TP53, PIK3CA ve BCR genlerinde 48.99 kata varan değişiklikler gözlemlenmiştir. Özellikle M1 makrofajların varlığında, apoptozla ilişkili yollaklarda yer alan genlerde anlamlı düzeyde değişiklikler saptanmıştır. Bu ilaçların varlığında akciğer kanseri hücrelerinde gözlenen artmış inflamatuar yanıtın detaylı olarak incelenmesi için ek çalışmalara ihtiyaç vardır.

Anahtar Kelimeler

• Apoptoz
• akciğer kanseri
• givinostat
• ixazomib
• M1 makrofaj

Kaynakça

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