PİRAZOL-AKRİDİN TÜREVİ SENTEZİNİN SKBR-3 İNSAN MEME KANSERİ HÜCRE HATTINDA POTANSİYEL ANTİKANSER AKTİVİTESİNİN DEĞERLENDİRİLMESİ

Öz

Amaç: Antikanser aktiviteye sahip heterosiklik bileşikler arasında yer alan, içerdikleri iki nitrojen atomu ile pirazol ve bir nitrojen atomu ile akridin türevleri, kanser, nörolojik bozukluklar ve bulaşıcı hastalıkların tedavisinde umut verici sonuçlar elde edilmiştir. Bu çalışmanın temel hedefi, sentezlenen pirazol ve akridin bileşiğinin, özellikle insan meme kanseri (SKBR-3) hücreleri üzerindeki antikanser aktivitesini araştırılmasıdır. Gereç ve Yöntemler: Bu çalışma kapsamında, 2- hidrazinobenzotiyazol ve 4-kloroasetofenon kullanılarak sentezlenen hidrazona dayalı olarak pirazol-4-karbaldehit elde edilmiştir. Pirazol-4-karbaldehit, 5,5-dimetilsikloheksan-1,3- dion ve 4-nitroanilin kullanılarak halkalandırma yöntemiyle yeni bir pirazol-akridin türevine (3-ACH) dönüştürülmüştür. 3- ACH, Fourier Dönüşümlü Kızılötesi Spektroskopisi, Nükleer Manyetik Rezonans, Kütle Spektrometrisi ve elemental analiz kullanılarak karakterize edilmiştir. Bu çalışmada, SKBR-3 hücrelerinde 3-ACH'nin sitotoksik etkilerini değerlendirmek amacıyla farklı dozlarda (50, 100 ve 150 μg/mL) ve farklı sürelerde (12 ve 24 saat) hücre canlılığı testi ile analiz edilmiştir. Ayrıca, 3-ACH uygulamasının ardından BAX, Kaspaz-3, Kaspaz-8 ve Kaspaz-9 apopitoz yollarını immün boyama yöntemiyle incelemiştir. Bulgular: 3-ACH'nin insan meme kanseri hücrelerinde sitotoksik etkiler gösterdiğini, bu etkilerin dozaj ve süre ile ilişkili olduğunu gösterdik. Apopitozun içsel yolaklardan sorumlu olan Kaspaz-9 ve BAX, dışsal yolaklardan sorumlu olan Kaspaz-8 sentezi immün boyama yöntemi ile artışı gösterilmiştir. Ayrıca hem içsel hem de dışsal yollardan sorumlu olan Kaspaz-3’ün protein sentezi de belirgin bir şekilde artmıştır. Sonuç: Bu bulgular, 3-ACH’nin hem içsel hem de dışsal apopitoz yollarını aktive ederek sitotoksik etkilerine katkıda bulunabilir. Araştırmamızın bulguları, 3-ACH'ın kanser tedavisi için umut verici bir ajan olarak değerlendirilmesine yönelik kanıtlar sunmaktadır. 3-ACH tedavisinin apopitotik yanıtındaki rolü daha detaylı şekilde anlamak için ek araştırmalara ihtiyaç vardır.

Anahtar Kelimeler

• SKBR-3
• akridin
• pirazol
• sitotoksisite
• apoptoz

Evaluation of the Potential Anticancer Activity of Pyrazole-Acridine Derivative Synthesis on SKBR-3 Human Breast Cancer Cell Line

Öz

Objective: Among heterocyclic compounds with anticancer activity, derivatives of pyrazole containing two nitrogen atoms and acridine containing one nitrogen atom have shown promising results in the treatment of cancer, neurological disorders, and infectious diseases. The main objective of this study is to investigate the anticancer activity of the synthesized pyrazole and acridine compound, particularly on human breast cancer (SKBR-3) cells. Material and Methods: In this study, pyrazole-4-carbaldehyde was obtained based on hydrazone (HT) synthesized using 2- hydrazinobenzothiazole and 4-chloroacetophenone. Pyrazole-4- carbaldehyde was converted to a new pyrazole-acridine derivative (3-ACH) by cyclization using 5,5- dimethylcyclohexane-1,3-dione and 4-nitroaniline. 3-ACH was characterized using Fourier Transform Infrared Spectroscopy, Nuclear Magnetic Resonance, Mass Spectrometry, and elemental analysis. The cytotoxic effects of 3-ACH on SKBR-3 cells were evaluated using the cell viability test at different doses (50, 100, and 150 μg/mL) and durations (12 and 24 hours). Additionally, the synthesis of BAX, Caspase-3, Caspase- 8, and Caspase-9 apoptotic pathways was examined through immunostaining after 3-ACH application. Results: We demonstrated that 3-ACH exhibits cytotoxic effects on human breast cancer cells, and these effects are dose and duration-dependent. The synthesis increase of Caspase-9 and BAX responsible for intrinsic pathways and Caspase-8 responsible for extrinsic pathways was shown through immunostaining. Moreover, the protein synthesis of Caspase-3, responsible for both intrinsic and extrinsic pathways, significantly increased. Conclusion: These findings suggest that 3-ACH may contribute to its cytotoxic effects by activating both intrinsic and extrinsic apoptotic pathways. The results of our study provide strong evidence for considering 3-ACH as a promising agent for cancer treatment. Further research is needed to understand the role of 3-ACH in the apoptotic response in more detail.

Anahtar Kelimeler

• SKBR-3
• acridine
• pyrazole
• cytotoxicity
• apoptosis

Kaynakça

1. Sung H, Ferlay J, Siegel RL, Laversanne M, ve ark. Global cancer statistics 2020: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209-249.
2. Kabir E, Uzzaman M. A review on biological and medicinal impact of heterocyclic compounds. Results in Chemistry. 2022;4:100606.
3. Varakumar P, Rajagopal K, Aparna B, et al. Acridine as an anti-tumour agent: A critical review. Molecules. 2023;28(1):193.
4. Mishra BB, Kumar D, Singh AS, Tripathi RP, Tiwari VK. Ionic liquids-prompted synthesis of biologically relevant five- and six-membered heterocyclic skeletons: An update. In: Brahmachari G, ed. Green Synthetic Approaches for Biologically Relevant Heterocycles. 1. basım. Boston. Elsevier, 2015:437-493.
5. Das A, Banik BK. Microwave-assisted synthesis of N- heterocycles. In: Das A, Banik B, eds. Microwaves in Chemistry Applications. 1. basım. Amsterdam. Elsevier, 2021:143-198.
6. Khandelwal S, Tailor YK, Rushell E, Kumar M. 9 - Use of sustainable organic transformations in the construction of heterocyclic scaffolds. In: Banik BK, ed. Green Approaches in Medicinal Chemistry for Sustainable Drug Design. 1. basım. Amsterdam. Elsevier, 2020:245-352.
7. Duarte SS, Silva DKF, Lisboa TMH, ve ark. Anticancer effect of a spiro-acridine compound involves immunomodulatory and anti-angiogenic actions. Anticancer Res. 2020;40(9):5049-5057.
8. Naim MJ, Alam O, Nawaz F, Alam MJ, Alam P. Current status of pyrazole and its biological activities. J Pharm Bioallied Sci. 2016;8(1):2-17.

9. Karrouchi K, Radi S, Ramli Y, ve ark. Synthesis and pharmacological activities of pyrazole derivatives: A review. Molecules. 2018;23(1):134.
10. Ashourpour M, Mostafavi Hosseini F, Amini M, ve ark. Pyrazole derivatives induce apoptosis via ROS generation in the triple negative breast cancer cells, MDA-MB-468. Asian Pac J Cancer Prev. 2021;22(7):2079-2087.
11. Deweshri N, Kishor D, Vijayshri R, Ruchi S, Ujwala M. Pyrazole scaffold: Strategies toward the synthesis and their applications. In: Premlata K, Amit BP, eds. Strategies for the Synthesis of Heterocycles and Their Applications. 1. Edition. Rijeka. IntechOpen, 2022:254.
12. Elmusa M, Elmusa S, Mert S, ve ark. One-pot three- component synthesis of novel pyrazolo-acridine derivatives and assessment of their acetylcholinesterase inhibitory properties: An in vitro and in silico study. J Mol Struct. 2023;1274:134553.
13. Sachin S. Wazalwar ARB, Franc Perdih. Aqueous phase synthesis, crystal structure and biological study of isoxazole extensions of pyrazole-4-carbaldehyde derivatives. J Mol Struct. 2017;1150:258-267.
14. Hongjin Z, Zhang S, Ampomah-Wireko M, ve ark.. Pyrazole: An important core in many marketed and clinical drugs. Russ J Bioorg Chem. 2022;48(6):1175-1189.
15. G Mohan, Sridhar G, Laxminarayana E, Chary MT. Synthesis and biological evaluation of 1,2,4-oxadiazole incorporated 1,2,3-triazole-pyrazole derivatives as anticancer agents. Chem Data Coll. 2021;34:100735.
16. Kang M, Pandit N, Kim AY, et al. PCW-1001, a novel pyrazole derivative, exerts antitumor and radio-sensitizing activities in breast cancer. Front Oncol. 2022;12:835833.
17. Ramaswamy B, Mrozek E, Kuebler JP, Bekaii-Saab T, Kraut EH. Phase II trial of pyrazoloacridine (NSC#366140) in patients with metastatic breast cancer. Invest New Drugs. 2011;29(2):347-351.
18. Sun YW, Niu TK, Yang JM, Kwon CH, Chen KY, Chen KM. Potentiation of the growth inhibition activity of 2- ({4-[4-(acridin-9-ylamino)phenylthio]phenyl}(2- hydroxyethyl)amino)ethan-1-ol (CK0402) by Herceptin in SKBR-3 human breast cancer cells. Exp Ther Med. 2010;1(3):513-518.
19. Sun YW, Chen KY, Kwon CH, Chen KM. CK0403, a 9‑aminoacridine, is a potent anti‑cancer agent in human breast cancer cells. Mol Med Rep. 2016;13(1):933-938.
20. Pérez SA, de Haro C, Vicente C, Donaire A, Zamora A, Zajac J, ve ark. New acridine thiourea gold(I) anticancer agents: Targeting the nucleus and inhibiting vasculogenic mimicry. ACS Chem Biol. 2017;12(6):1524-1537.
21. Kumar NP, Sharma P, Reddy TS, Shankaraiah N, Bhargava SK, Kamal A. Microwave-assisted one-pot synthesis of new phenanthrene fused-tetrahydrodibenzo- acridinones as potential cytotoxic and apoptosis inducing agents. Eur J Med Chem. 2018;151:173-185.