Tumor Florescence Imaging Method Using 5-ALA in Ht-29 Cells

Öz

Objective: 5-ALA, also known as 5-aminolevulinic acid, is a naturally occurring amino acid that plays a crucial role in the biosynthesis of heme, a vital component of hemoglobin and various enzymes in the body. Specifically, it is used in technique called photodynamic diagnosis (PDD) and photodynamic therapy (PDT) as a photosensitizer. When exposed to a specific wavelength of light, the accumulated 5-ALA causes fluorescence in these target areas, allowing for enhanced visualization and detection during surgical procedures, such as in neurosurgery or urology. The purpose of this study was to evaluate the conditions for optimal photodynamic diagnosis using 5-ALA in colon cancer. Methods: HT-29 cell line which was administered different 5-ALA (100, 200, 300, 500, 1000, 1500 μM) concentrations for 3 hours incubation time, were performed on cell viability, fluorescence intensity, apoptosis analysis. Results: While there was no difference in cell viability between the control group and low 5-ALA doses (100,200 and 300 μM), a significant difference was found at higher 5-ALA doses (1000 and 1500 μM) (p<,0001). 5-ALA doses increased in parallel with the fluorescence intensity, and the highest fluorescence intensity was at 1500 μM 5-ALA (p<,0001). The apoptosis/dead ratio was significantly showed to be highest at 1000 μM and 1500 μM 5-ALA which had the highest fluorescence intensity (p<0.05). Conclusion: the optimum dose of 5-ALA concentration was determined to be 500 μM in HT-29 cells. High concentrations of 5-ALA have been shown to cause apoptosis in HT29 cells.

Anahtar Kelimeler

• 5-aminolevulinic acid (ALA)
• Florescence Imaging
• Tumor cells
• Apoptosis
• Photodynamic Diagnosis

Destekleyen Kurum

Aydın Adnan Menderes University Scientific Research Projects Unit

Proje Numarası

TPF-17006

Ht-29 Hücrelerinde 5-ALA Kullanılarak Tümör Floresan Görüntüleme Yöntemi

Öz

Amaç: 5-aminolevulinik asit olarak da bilinen 5-ALA, hemoglobinin önemli bir bileşeni olan heme ve vücuttaki çeşitli enzimlerin biyosentezinde çok önemli bir rol oynayan doğal olarak oluşan bir amino asittir. Mitokondride glisin ve süksinil-CoA'dan sentezlenir. Spesifik olarak, fotodinamik tanı (PDD) ve fotodinamik terapi (PDT) adı verilen teknikte bir ışığa duyarlılaştırıcı olarak kullanılır. Fotodinamik tanıda (PDD), 5-ALA tümör hücreleri veya kanser öncesi lezyonlar gibi belirli doku veya hücrelerde seçici olarak birikir. Spesifik bir dalga boyundaki ışığa maruz kaldığında, biriken 5-ALA, bu hedef alanlarda floresana neden olarak beyin cerrahisi veya üroloji gibi cerrahi prosedürler sırasında gelişmiş görselleştirme ve algılamaya olanak tanır. Bu çalışmanın amacı, kolon kanserinde 5-ALA kullanılarak optimal fotodinamik tanı koşullarını belirlemektir. Yöntem: HT-29 hücre hattına 3 saat farklı 5-ALA (100, 200, 300, 500, 1000, 1500 μM) konsantrasyonları uygulanmıştır. Uygulanan hücrelerde hücre canlılığı, floresans şiddeti, apoptoz analizleri yapıldı. Bulgular: Kontrol grubu ile düşük 5-ALA dozları (100,200 ve 300 μM) arasında hücre canlılığı açısından fark yokken, yüksek 5-ALA dozlarında (1000 ve 1500 μM) anlamlı fark bulundu (p<,0001) 5-ALA dozları floresan şiddeti ile paralel olarak artmış ve en yüksek floresans şiddeti 1500 μM 5-ALA'da olmuştur (p<,0001). Apoptoz/ölü oranı, floresans yoğunluğunun en yüksek olduğu 1000 μM ve 1500 μM 5-ALA'da anlamlı olarak en yüksek olduğu gösterildi (p<0.05). Sonuç: HT-29 hücrelerinde 5-ALA konsantrasyonunun optimum dozu 500 μM olduğu belirlendi. 5-ALA'nın yüksek konsantrasyonlarının HT29 hücrelerinde apoptoza neden olduğu gösterilmiştir.

Anahtar Kelimeler

• 5-aminolevulinik asit (ALA)
• Floresans görüntüleme
• Tümör hücreleri
• Apoptoz
• Fotodinamik Tanı

Proje Numarası

TPF-17006

Kaynakça

1. 1. Knowlton, C. A., Mackay, M. K., Speer, T. W., Vera, R. B., Arthur, D. W., Wazer, D. E.et al. (2022). Colon Cancer. Encyclopedia of Radiation Oncology, 77–77.
2. 2. Kitada, M., Ohsaki, Y., Matsuda, Y., Hayashi, S., & Ishibashi, K. (2015). Photodynamic diagnosis of pleural malignant lesions with a combination of 5-aminolevulinic acid and intrinsic fluorescence observation systems. BMC Cancer, 15(1).
3. 3. Kausch, I., Sommerauer, M., Montorsi, F., Stenzl, A., Jacqmin, D., Jichlinski, P., et al. (2010). Photodynamic diagnosis in non-muscle-invasive bladder cancer: a systematic review and cumulative analysis of prospective studies. European Urology, 57(4), 595–606.
4. 4. Kobuchi, H., Moriya, K., Ogino, T., Fujita, H., Inoue, K., Shuin, T., et al. (2012). Mitochondrial localization of ABC transporter ABCG2 and its function in 5-aminolevulinic acid-mediated protoporphyrin IX accumulation. PloS One, 7(11).
5. 5. Kenan, S., Liang, H., Goodman, H. J., Jacobs, A. J., Chan, A., Grande, D. A., et al. (2020). 5-Aminolevulinic acid tumor paint and photodynamic therapy for myxofibrosarcoma: an in vitro study. Journal of Orthopaedic Surgery and Research, 15(1).
6. 6. Koizumi, N., Harada, Y., Minamikawa, T., Tanaka, H., Otsuji, E., Takamatsu, T. (2016). Recent advances in photodynamic diagnosis of gastric cancer using 5-aminolevulinic acid. World Journal of Gastroenterology, 22(3), 1289–1296.
7. 7. Inoue, Y., Tanaka, R., Komeda, K., Hirokawa, F., Hayashi, M., Uchiyama, K. (2014). Fluorescence detection of malignant liver tumors using 5-aminolevulinic acid-mediated photodynamic diagnosis: principles, technique, and clinical experience. World Journal of Surgery, 38(7), 1786–1794.
8. 8. Nishimura, M., Murayama, Y., Harada, K., Kamada, Y., Morimura, R., Ikoma, H., et al. (2016). Photodynamic Diagnosis of Hepatocellular Carcinoma Using 5-Aminolevulinic Acid. Anticancer Research, 36(9), 4569– 4574.

9. 9. Nokes, B., Apel, M., Jones, C., Brown, G., Lang, J. E. (2013). Aminolevulinic acid (ALA): photodynamic detection and potential therapeutic applications. The Journal of Surgical Research, 181(2), 262–271.
10. 10. Fukuhara, H., Yamamoto, S., Karashima, T., Inoue, K. (2021). Photodynamic diagnosis and therapy for urothelial carcinoma and prostate cancer: new imaging technology and therapy. International Journal of Clinical Oncology, 26(1), 18–25.
11. 11. Brunner, H., Hausmann, F., Knuechel, R. (2003). New 5-aminolevulinic acid esters--efficient protoporphyrin precursors for photodetection and photodynamic therapy. Photochemistry and Photobiology, 78(5), 481.
12. 12. Casas, A. (2020). Clinical uses of 5-aminolaevulinic acid in photodynamic treatment and photodetection of cancer: A review. Cancer Letters, 490, 165–173.
13. 13. Ramonaite, R., Petrolis, R., Unay, S., Kiudelis, G., Skieceviciene, J., Kupcinskas, L., et al. (2019). Mathematical morphology-based imaging of gastrointestinal cancer cell motility and 5-aminolevulinic acid-induced fluorescence. Biomedizinische Technik. Biomedical Engineering, 64(6).
14. 14. Kenan, S., Liang, H., Goodman, H. J., Jacobs, A. J., Chan, A., Grande, D. A., et al. (2020). 5-Aminolevulinic acid tumor paint and photodynamic therapy for myxofibrosarcoma: an in vitro study. Journal Of Orthopaedic Surgery and Research, 15(1), 94.
15. 15. Srikoon, P., Chaemsawang, W., Phimsen, S., Vaeteewoottacharn, K., Khongkaew, P., Chamsawang, W. (2021). Deoxycholic acid-formulated curcumin enhances caspase 3/7-dependent apoptotic induction in cholangiocarcinoma cell lines. Journal of Basic and Applied Pharmacology, 1(1).1-9.
16. 16. Hirano, T., Hagiya, Y., Fukuhara, H., Inoue, K., Shuin, T., Matsumoto, K., et al. (2013). Improvement of aminolevulinic acid (ALA)-mediated photodynamic diagnosis using n-propyl gallate. Photodiagnosis and Photodynamic Therapy, 10(1), 28–32.
17. 17. Nakanishi, T., Ogawa, T., Yanagihara, C., Tamai, I. (2015). Kinetic Evaluation of Determinant Factors for Cellular Accumulation of Protoporphyrin IX Induced by External 5-Aminolevulinic Acid for Photodynamic Cancer Therapy. Journal of Pharmaceutical Sciences, 104(9), 3092–3100.
18. 18. Kitajima, Y., Ishii, T., Kohda, T., Ishizuka, M., Yamazaki, K., Nishimura, Y., et al. (2019). Mechanistic study of PpIX accumulation using the JFCR39 cell panel revealed a role for dynamin 2-mediated exocytosis. Scientific Reports, 9(1).
19. 19. Kawczyk-Krupka, A., Latos, W., Latos, M., Czuba, Z. P., Sieroń, A. (2016). ALA-induced photodynamic effect on viability, apoptosis, and secretion of S100 protein, secreted by colon cancer cells in vitro. Photodiagnosis and Photodynamic Therapy, 15, 218–227.
20. 20. Jones, P. S., Yekula, A., Lansbury, E., Small, J. L., Ayinon, C., Mordecai, S., et al. (2019). Characterization of plasma-derived protoporphyrin-IX-positive extracellular vesicles following 5-ALA use in patients with malignant glioma. EBioMedicine, 48, 23–35.
21. 21. Kushibiki, T., Noji, T., Ebihara, Y., Hontani, K., Ono, M., Kuwabara, S., et al. (2017). 5-Aminolevulinic-acid- mediated Photodynamic Diagnosis Enhances the Detection of Peritoneal Metastases in Biliary Tract Cancer in Mice. In Vivo (Athens, Greece), 31(5), 905–908.
22. 22. 2Ito, H., Kurokawa, H., Suzuki, H., Indo, H. P., Majima, H. J., Matsui, H. (2019). 5-Aminolevulinic acid induced apoptosis via oxidative stress in normal gastric epithelial cells. Journal of Clinical Biochemistry and Nutrition, 65(2), 83–90.