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

Yıl 2025, Cilt: 49 Sayı: 4-14th International Symposium on Pharmaceutical Sciences (ISOPS-14), 1140 - 1150, 28.12.2025

Elif Kaya Tilki , Selin Engür Öztürk

https://doi.org/10.33483/jfpau.1747620

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

• 1. Ma, M., Tan, H., Yan, H., Zheng, K. (2024). The impact of family cancer history on tumor metabolism and prognosis in patients with non-small cell lung cancer. Scientific Reports, 14(1), 22632. [CrossRef]
• 2. Xiang, Y., Liu, X., Wang, Y., Zheng, D., Meng, Q., Jiang, L., Yang, S., Zhang, S., Zhang, X., Liu, Y. (2024). Mechanisms of resistance to targeted therapy and immunotherapy in non-small cell lung cancer: Promising strategies to overcoming challenges. Frontiers in Immunology, 15, 1366260. [CrossRef]
• 3. Sosa Iglesias, V., Giuranno, L., Dubois, L.J., Theys, J., Vooijs, M. (2018). Drug resistance in non-small cell lung cancer: A potential for NOTCH targeting? Frontiers in Oncology, 8, 267. [CrossRef]
• 4. Soave, C.L., Guerin, T., Liu, J., Dou, Q.P. (2017). Targeting the ubiquitin-proteasome system for cancer treatment: Discovering novel inhibitors from nature and drug repurposing. Cancer and Metastasis Reviews, 36, 717-736. [CrossRef]
• 5. Engür, S., Dikmen, M., Öztürk, Y. (2016). Comparison of antiproliferative and apoptotic effects of a novel proteasome inhibitor MLN2238 with bortezomib on K562 chronic myeloid leukemia cells. Immunopharmacology and Immunotoxicology, 38(2), 87-97. [CrossRef]
• 6. Engür, S., Dikmen, M. (2017). The evaluation of the anti-cancer activity of ixazomib on Caco2 colon solid tumor cells, comparison with bortezomib. Acta Clinica Belgica, 72(6), 391-398. [CrossRef]
• 7. Cinek, T., Karalı, N. (2021). Histone deacetylase inhibitors providing an epigenetic treatment in cancer. Istanbul Journal of Pharmacy, 51(2), 283-290. [CrossRef]
• 8. Contreras-Sanzón, E., Prado-Garcia, H., Romero-Garcia, S., Nuñez-Corona, D., Ortiz-Quintero, B., Luna-Rivero, C., Martínez-Cruz, V., Carlos-Reyes, Á. (2022). Histone deacetylases modulate resistance to the therapy in lung cancer. Frontiers in Genetics, 13, 960263. [CrossRef]
• 9. Chifotides, H.T., Bose, P., Verstovsek, S. (2020). Givinostat: An emerging treatment for polycythemia vera. Expert Opinion on Investigational Drugs, 29(6), 525-536. [CrossRef]
• 10. Oikonomou, A., Watrin, T., Valsecchi, L., Scharov, K., Savino, A.M., Schliehe-Diecks, J., Bardini, M., Fazio, G., Bresolin, S., Biondi, A. (2024). Synergistic drug interactions of the histone deacetylase inhibitor givinostat (ITF2357) in CRLF2-rearranged pediatric B-cell precursor acute lymphoblastic leukemia identified by high-throughput drug screening. Heliyon, 10(13). [CrossRef]
• 11. Lamb, Y.N. (2024). Givinostat: First approval. Drugs, 84(7), 849-856. [CrossRef]
• 12. Engür-Öztürk, S., Dikmen, M. (2022). Proteasome inhibitor immunotherapy for the epithelial to mesenchymal transition: Assessing the A549 lung cancer cell microenvironment and the role of M1, M2a and M2c ‘hydrocortisone-polarised’macrophages. Molecular Biology Reports, 49(6), 4777-4793. [CrossRef]
• 13. Pan, Y., Yu, Y., Wang, X., Zhang, T. (2020). Tumor-associated macrophages in tumor immunity. Frontiers in Immunology, 11, 583084. [CrossRef]
• 14. Zhou, W., Yang, F., Zhang, X. (2024). Roles of M1 Macrophages and Their Extracellular Vesicles in Cancer Therapy. Cells, 13(17), 1428. [CrossRef]
• 15. Haffani, Y.Z., Louati, K., Tilki, E.K., Mami, N. Ben, Mbarek, S., Halim, N. Ben, Boudhrioua, N., Ozturk, Y., Chekir, R.B.C., Dikmen, M. (2023). Anti-tumoral activity of Allium roseum compounds on breast cancer cells MCF7 and MDA-MB231. Advances in Traditional Medicine, 1-11. [CrossRef]
• 16. Baghirova, L., Kaya Tilki, E., Ozturk, A.A. (2023). Evaluation of cell proliferation and wound healing effects of vitamin A palmitate-loaded PLGA/Chitosan-Coated PLGA Nanoparticles: Preparation, characterization, release, and release Kinetics. ACS Omega, 8(2), 2658-2668. [CrossRef]
• 17. Kaya-Tilki, E., Dikmen, M. (2021). Neuroprotective effects of some epigenetic modifying drugs’ on Chlamydia pneumoniae-induced neuroinflammation: A novel model. Plos One, 16(11), e0260633. [CrossRef]
• 18. Tilki, E.K., Öztürk, S.E. (2024). The proteasome inhibitor ixazomib targets epigenetic chromatin modification enzymes upregulated by m2c macrophage polarisation in lung cancer. İstanbul Journal of Pharmacy, 54(2), 215-222. [CrossRef]
• 19. Chen, X., Wu, X., Li, L., Zhu, X. (2024). Development of proteasome inhibitors for cancer therapy. International Journal of Drug Discovery and Pharmacology, 100004. [CrossRef]
• 20. Testa, U. (2009). Proteasome inhibitors in cancer therapy. Current Drug Targets, 10(10), 968-981. [CrossRef]
• 21. Iveland, T.S., Hagen, L., de Sousa, M.M.L., Liabakk, N.B., Aas, P.A., Sharma, A., Kavli, B., Slupphaug, G. (2025). Cytotoxic mechanisms of pemetrexed and HDAC inhibition in non-small cell lung cancer cells involving ribonucleotides in DNA. Scientific Reports, 15(1), 2082. [CrossRef]
• 22. Del Bufalo, D., Desideri, M., De Luca, T., Di Martile, M., Gabellini, C., Monica, V., Busso, S., Eramo, A., De Maria, R., Milella, M. (2014). Histone deacetylase inhibition synergistically enhances pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer. Molecular Cancer, 13, 1-16. [CrossRef]
• 23. Yunna, C., Mengru, H., Lei, W., Weidong, C. (2020). Macrophage M1/M2 polarization. European Journal of Pharmacology, 877, 173090. [CrossRef]
• 24. Cui, K., Ardell, C.L., Podolnikova, N.P., Yakubenko, V.P. (2018). Distinct migratory properties of M1, M2, and resident macrophages are regulated by αDβ2 and αMβ2 integrin-mediated adhesion. Frontiers in Immunology, 9, 2650. [CrossRef]
• 25. Rostam, H.M., Reynolds, P.M., Alexander, M.R., Gadegaard, N., Ghaemmaghami, A.M. (2017). Image based Machine Learning for identification of macrophage subsets. Scientific Reports, 7(1), 3521. [CrossRef]
• 26. Heinrich, F., Lehmbecker, A., Raddatz, B. B., Kegler, K., Tipold, A., Stein, V.M., Kalkuhl, A., Deschl, U., Baumgärtner, W., Ulrich, R. (2017). Morphologic, phenotypic, and transcriptomic characterization of classically and alternatively activated canine blood-derived macrophages in vitro. PloS One, 12(8), e0183572. [CrossRef]
• 27. Yao, P.-L., Lin, Y.-C., Wang, C.-H., Huang, Y.-C., Liao, W.-Y., Wang, S.-S., Chen, J.J.W., Yang, P.-C. (2005). Autocrine and paracrine regulation of interleukin-8 expression in lung cancer cells. American Journal of Respiratory Cell and Molecular Biology, 32(6), 540-547. [CrossRef]
• 28. Liu, H., Zhao, Q., Tan, L., Wu, X., Huang, R., Zuo, Y., Chen, L., Yang, J., Zhang, Z.-X., Ruan, W. (2023). Neutralizing IL-8 potentiates immune checkpoint blockade efficacy for glioma. Cancer Cell, 41(4), 693-710. [CrossRef]
• 29. Bos, T., Ratti, J.A., Harada, H. (2021). Targeting stress-response pathways and therapeutic resistance in head and neck cancer. Frontiers in Oral Health, 2, 676643. [CrossRef]
• 30. Yerlikaya, A., Okur, E., Ulukaya, E. (2012). The p53-independent induction of apoptosis in breast cancer cells in response to proteasome inhibitor bortezomib. Tumor Biology, 33(5), 1385-1392. [CrossRef]
• 31. Uo, T., Veenstra, T.D., Morrison, R.S. (2009). Histone deacetylase inhibitors prevent p53-dependent and p53-independent Bax-mediated neuronal apoptosis through two distinct mechanisms. Journal of Neuroscience, 29(9), 2824-2832. [CrossRef]
• 32. Vilborg, A., Wilhelm, M.T., Wiman, K.G. (2010). Regulation of tumor suppressor p53 at the RNA level. Journal of Molecular Medicine, 88(7), 645-652. [CrossRef]
• 33. Rahmani, M., Aust, M.M., Benson, E.C., Wallace, L., Friedberg, J., Grant, S. (2014). PI3K/mTOR inhibition markedly potentiates HDAC inhibitor activity in NHL cells through BIM-and MCL-1-dependent mechanisms in vitro and in vivo. Clinical Cancer Research, 20(18), 4849-4860. [CrossRef]
• 34. Mondello, P., Derenzini, E., Asgari, Z., Philip, J., Brea, E. J., Seshan, V., Hendrickson, R.C., de Stanchina, E., Scheinberg, D.A., Younes, A. (2017). Dual inhibition of histone deacetylases and phosphoinositide 3-kinase enhances therapeutic activity against B cell lymphoma. Oncotarget, 8(8), 14017. [CrossRef]
• 35. Ozturk, S., Kaya Tilki, E., Dikmen, M., Cantürk, Z. (2021). Comparison of the apoptotic effects of bortezomib using 2D and 3D co-culture models of THP-1 derived macrophage and A549 lung cancer. Journal Of Research in Pharmacy, 25(4). [CrossRef]
• 36. Engür‐Öztürk, S., Kaya‐Tilki, E., Cantürk, Z., Dİkmen, M. (2024). Enhanced angiogenesis of human umbilical vein endothelial cells via THP‐1‐derived M2c‐like macrophages and treatment with proteasome inhibitors ‘bortezomib and ixazomib.’ Apmis, 132(8), 594-607. [CrossRef]