PANKREAS KANSERİ HÜCRELERİNDE 3-BROMOPÜRUVİK ASİDİN METABOLİK DEĞİŞİKLİKLER ÜZERİNDEKİ ANTİKANSER ETKİLERİNİN İN VİTRO OLARAK ARAŞTIRILMASI

Öz

Amaç: Pankreatik duktal adenokarsinom (PDAC), yüksek mortalite oranı ve kemoterapötiklere karşı direnç ile karakterize edilen en agresif kanser türlerinden biridir. TP53, SMAD4 ve CDKN2A genlerindeki sık mutasyonlar, metabolik yeniden programlamayı tetikleyerek tümör progresyonuna ve tedaviye karşı direnç gelişimine katkıda bulunur. Glikolizin kilit enzimlerinden biri olan Heksokinaz 2 (HK2), PDAC'da sıklıkla aşırı eksprese edilir ve umut verici bir terapötik hedef olarak öne çıkar. Bu çalışma, TP53, SMAD4 ve CDKN2A mutasyonlarını taşıyan BXPC-3 PDAC hücrelerinde 3-bromopürivik asidin (3-BrPA) metabolik yollar, oksidatif stres ve ferroptozis üzerindeki antikanser etkilerini araştırmayı amaçlamıştır. Gereç ve Yöntemler: BXPC-3 hücreleri farklı konsantrasyonlarda 3-BrPA ile muamele edilmiştir. Hücre canlılığı, IC₅₀ değerinin belirlenmesi amacıyla MTT testi ile değerlendirilmiştir. Morfolojik değişiklikler ışık mikroskobu ile incelenmiştir. HK2 ekspresyonu ile birlikte GST, G6PD, 6-PGD ve GR enzimlerinin aktiviteleri ölçülmüş; glutatyon redoks durumu ve hücre içi demir seviyeleri değerlendirilmiştir. Ayrıca, yağ asidi oksidasyonu yoluyla metabolik kompansasyonu değerlendirmek üzere CPT1C aktivitesi analiz edilmiştir. Bulgular: 3-BrPA uygulaması, doz bağımlı şekilde hücre canlılığını anlamlı düzeyde azaltmış ve HK2 ekspresyonunu baskılamıştır. Glikoliz ve pentoz fosfat yolunun enzimatik aktiviteleri inhibe edilmiş; antioksidan kapasite azalmış ve oksidatif stres belirteçleri artmıştır. Bu değişiklikler, hücre içi demir düzeylerinde artış ve GSH/GSSG oranında azalma ile birlikte gözlenmiş; ferroptozis indüksiyonunu işaret etmiştir. Ayrıca, CPT1C aktivitesinde artış saptanmıştır, bu da yağ asidi oksidasyonuna yönelik bir metabolik kaymaya işaret etmektedir. Sonuç: Elde edilen bulgular, 3-BrPA’nın HK2 inhibisyonu yoluyla glukoz metabolizmasını bozduğunu, oksidatif stresi artırarak ferroptozisi tetiklediğini ortaya koymaktadır. Bu çalışma, 3-BrPA’nın kanser metabolizması ve ferroptozisi hedefleyen terapötik potansiyeline dikkat çekmektedir.

Anahtar Kelimeler

• Pankreatik duktal adenokarsinom
• 3-bromopirüvik asit
• heksokinaz 2 inhibitörleri
• ferroptozis
• oksi datif stres
• pentoz fosfat yolu

INVESTIGATING THE ANTICANCER EFFECTS OF 3-BROMOPYRUVIC ACID ON METABOLIC ALTERATIONS IN PANCREATIC CANCER CELLS IN VITRO

Öz

Objective: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, characterised by a high mortality rate and resistance to chemotherapeutics. Frequent mutations in TP53, SMAD4, and CDKN2A drive metabolic reprogramming and contribute to tumour progression and treatment resistance. Hexokinase 2 (HK2), a key glycolytic enzyme, is commonly overexpressed in PDAC and represents a promising therapeutic target. This study aimed to investigate the anticancer effects of 3-bromopyruvic acid (3-BrPA) on metabolic pathways, oxidative stress, and ferroptosis in BXPC-3 PDAC cells harbouring TP53, SMAD4, and CDKN2A mutations. Material and Methods: BXPC-3 cells were administered with various concentrations of 3-BrPA, and cell viability was assessed using the MTT assay to determine the IC₅₀. The morphological changes were evaluated via light microscopy. HK2 expression and the enzymatic activities of GST, G6PD, 6-PGD, and GR were measured, along with the glutathione redox status and intracellular iron levels. CPT1C activity was additionally assessed to evaluate the metabolic alterations associated with the fatty acid oxidation pathways. Results: 3-BrPA administration significantly decreased cell viability in a dose-dependent manner and downregulated HK2 expression. It inhibited glycolysis and the pentose phosphate pathway enzymes, reduced the antioxidant capacity, and elevated oxidative stress markers. These changes were associated with increased intracellular iron levels and decreased GSH/GSSG ratios, indicating the induction of ferroptosis. Furthermore, CPT1C activity was upregulated, indicating increased fatty acid oxidation. Conclusion: Our findings demonstrate that 3-BrPA disrupts glucose metabolism, induces oxidative stress, and triggers ferroptosis in PDAC cells through HK2 inhibition. This study highlights 3-BrPA’s therapeutic potential in targeting cancer metabolism and ferroptosis.

Anahtar Kelimeler

• Pancreatic ductal adenocarcinoma
• 3-bromopyruvic acid
• hexokinase 2 inhibitors
• ferroptosis
• oxidative stress
• pentose phosphate pathway

Kaynakça

1. Sun Y, Ren D, Zhou Y, Shen J, Wu H, Jin X. Histone acetyltransferase 1 promotes gemcitabine resistance by regulating the PVT1/EZH2 complex in pancreatic cancer. Cell Death Dis 2021;12(10):878. google scholar
2. Khaira R, Sharma J, Saini V. Development and characterization of nanoparticles for the delivery of gemcitabine hydrochloride. ScientificWorldJournal 2014;2014:560962. google scholar
3. Ju HQ, Gocho T, Aguilar M, Wu M, Zhuang ZN, Fu J, et al. Mechanisms of overcoming intrinsic resistance to gemcitabine in pancreatic ductal adenocarcinoma through the redox modulation. Mol Cancer Ther 2015;14(3):788-98. google scholar
4. Orth M, Metzger, P, Gerum S, Mayerle J, Schneider G, Belka C et al. Pancreatic ductal adenocarcinoma: biological hallmarks, current status, and future perspectives of combined modality treatment approaches. Radiation Oncology 2019;14(1):141. google scholar
5. Liu J, Zhang C, Hu W, Feng Z. Tumor suppressor p53 and metabolism. J Mol Cell Biol 2019;11(4):284-92. google scholar
6. Liu P, Wang Y, Li X. Targeting the untargetable KRAS in cancer therapy. Acta Pharm Sin B 2019;9(5):871-9. google scholar
7. Ghanavat M, Shahrouzian M, Deris Zayeri Z, Banihashemi S, Kazemi SM, Saki N. Digging deeper through glucose metabolism and its regulators in cancer and metastasis. Life Sci 2021;264:118603. google scholar
8. Liu M, Liu W, Qin Y, Xu X, Yu X, Zhuo Q, et al. Regulation of metabolic reprogramming by tumor suppressor genes in pancreatic cancer. Exp Hematol Oncol 2020;9(1):23. google scholar

9. Liu M, Liu W, Qin Y, Xu X, Yu X, Zhuo Q, et al. Regulation of metabolic reprogramming by tumor suppressor genes in pancreatic cancer. Exp Hematol Oncol 2020;9(1):23. google scholar
10. Yang Y, Zhang ZJ, Wen Y, Xiong L, Huang YP, et al. Novel perspective in pancreatic cancer therapy: Targeting ferroptosis pathway. World J Gastrointest Oncol 2021;13(11):1668-79. google scholar
11. Fan T, Sun G, Sun X, Zhao L, Zhong R, Peng Y. Tumor energy metabolism and potential of 3-bromopyruvate as an ınhibitor of aerobic glycolysis: Implications in tumor treatment. Cancers (Basel) 2019;11(3):317. google scholar
12. Akella NM, Ciraku L, Reginato MJ. Fueling the fire: emerging role of the hexosamine biosynthetic pathway in cancer. BMC Biol 2019;17(1):52. google scholar
13. Otahal A, Aydemir D, Tomasich E, Minichsdorfer C. Delineation of cell death mechanisms induced by synergistic effects of statins and erlotinib in non small cell lung cancer cell (NSCLC) lines. Sci Rep 2020;10(1):959. google scholar
14. Aydemir D, Hashemkhani M, Acar HY, Ulusu NN. In vitro interaction of glutathione S‐transferase‐pi enzyme with glutathione‐coated silver sulfide quantum dots: A novel method for biodetection of glutathione S‐transferase enzyme. Chem Biol Drug Des 2019;94(6):2094-102. google scholar
15. Aydemir D, Hashemkhani M, Acar HY, Ulusu NN. Evaluation of the biocompatibility of the GSH-coated Ag2S quantum dots in vitro: a perfect example for the non-toxic optical probes. Mol Biol Rep 2020;47(6):4117-29. google scholar
16. Fan K, Fan Z, Cheng H, Huang Q, Yang C, Jin K, et al. Hexokinase 2 dimerization and interaction with voltage‐dependent anion channel promoted resistance to cell apoptosis induced by gemcitabine in pancreatic cancer. Cancer Med 2019;8(13):5903-15. google scholar
17. Aydemir D, Öztürk K, Arslan FB, Çalis S, Ulusu NN. Gemcitabine-loaded chitosan nanoparticles enhanced apoptotic and ferroptotic response of gemcitabine treatment alone in the pancreatic cancer cells in vitro. Naunyn Schmiedebergs Arch Pharmacol 2024;397:9051-66. google scholar
18. Zheng P, Pan HH, Zhou XH, Qiu YY, Hu J, Qin ZS, et al. Glucose 6 phosphatase dehydrogenase (G6PD): a novel diagnosis marker related to gastrointestinal cancers. Am J Transl Res 2023;15(4):2304-28. google scholar
19. Anapali M, Kaya-Dagistanli F, Akdemir AS, Aydemir D, Ulusu NN, Ulutin T, et al. Combined resveratrol and vitamin D treatment ameliorate inflammation related liver fibrosis, ER stress, and apoptosis in a high-fructose diet/ streptozotocin-induced T2DM model. Histochem Cell Biol 2022;158(3):279-96. google scholar
20. Li L, Yu X, Gao L, Cheng L, Sun B, Wang G. Diabetic ferroptosis and pancreatic cancer: Foe or friend? Antioxid Redox Signal 2022;37(16-18):1206-21. google scholar
21. Aydemir D, Malik AN, Kulac I, Basak AN, Lazoglu I, Ulusu NN. Impact of the amyotrophic lateral sclerosis disease on the biomechanical properties and oxidative stress metabolism of the lung tissue correlated with the human mutant SOD1G93A protein accumulation. Front Bioeng Biotechnol 2022;10:810243. google scholar
22. Cao JY, Dixon SJ. Mechanisms of ferroptosis. Cell Mol Life Sci 2016;73(11-12):2195-209. google scholar
23. Huang Z, Ma Y, Sun Z, Cheng L, Wang G. Ferroptosis: potential targets and emerging roles in pancreatic diseases. Arch Toxicol 2024;98(1):75-94. google scholar
24. Zhou Y, Chen Y, Zhao P, Xian T, Gao Y, Fan S, et al. The YY1-CPT1C signaling axis modulates the proliferation and metabolism of pancreatic tumor cells under hypoxia. Biochem Pharmacol 2024;227:116422. google scholar
25. Fadó R, Zagmutt S, Herrero L, Muley H, Rodríguez-Rodríguez R, Bi H, et al. To be or not to be a fat burner, that is the question for cpt1c in cancer cells. Cell Death Dis 2023;14(1):57. google scholar