Investigation of D-e-MAPP-derived Cytotoxicity on Human Prostate Cancer Cells

Öz

Sphingolipids play critical role in biological processes such cell death, survival and drug resistance in many cancers. Ceramide and dihydroceramide are proliferation and death associated sphingolipids. Recent cancer research are focused on clarifying cancer-sphingolipid metabolism. In last years, ceramide as a key molecule in sphingolipid metabolism relationship has been investigated for its anticancer activity via augmenting its intracellular level by ceramidase inhibitor application. Prostate cancer is amoung the most frequent human cancers and is reported as second most common cancer-related death cause. Prostate cancer is common at ages older than 65. The aim of this study was to investigate the potential cytotoxic activity of a ceramidease inhibitor (1S,2R)-D-erythro-2-(N-Myristoylamino)-1-phenyl-1-propanol (D-erythro-MAPP) on human prostate cancer DU-145 cells by using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay, flow cytometry, confocal and TEM microscopy. MTT findings showed that D-erythro-MAPP caused toxicity in low doses on DU-145 cells. Confocal and TEM microscopy findings indicated hole formation in cytoskeleton, chromatin condensation, horseshoe-shaped cell nuclei as morphological changes and blebbings on cell membrane, fragmentation of nuclei, chromatin condensation and loss of criastae of as ultrastructural changes, respectively. Flow cytometry findings showed that D-erythro-MAPP triggered apoptosis in short term application of 24 hours on DU-145 cells. According to results it can be conculded that D-erythro-MAPP decreased viability of DU-145 cells in dose-dependent manner.  Our findings stated the anti-cancer and cytotoxic potential of D-erythro-MAPP on DU-145 and this agent might be used in drug developing for cancer treatment after the further in vitro and in vivo studies.

Anahtar Kelimeler

• Sphingolipid,DU-145,Confocal microscopy

İnsan Prostat Kanseri Hücrelerinde D-e-MAPP kaynaklı Sitotoksisitenin Araştırılması

Öz

Sfingolipidler, birçok kanserde hücre ölümü, hayatta kalma ve ilaç direnci gibi biyolojik işlemlerde kritik rol oynamaktadır. Seramid ve dihidroseramid, proliferasyon ve ölüme bağlı olan sfingolipidlerdir. Son kanser araştırmaları, kanser-sfingolipid metabolizmasının netleştirilmesine odaklanmıştır. Son yıllarda, sfingolipid metabolizması ilişkisinde kilit bir molekül olarak seramid, antikanser aktivitesi için seramidaz inhibitör uygulaması ile hücre içi seviyesini arttırarak araştırılmıştır. Prostat kanseri, en sık görülen insan kanserleri arasındadır ve kansere bağlı en yaygın ikinci ölüm nedeni olarak rapor edilmektedir. Prostat kanseri, 65 yaşından büyüklerde yaygındır. Bu çalışmanın amacı, bir seramidaz inhibitörü olan  (1S,2R)-D-erythro-2-(N-Myristoylamino)-1-phenyl-1-propanol’un (D-eritro-MAPP) insan prostat kanseri DU-145 hücreleri üzerindeki potansiyel sitotoksik aktivitesini MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) testi, akış sitometrisi, konfokal ve TEM mikroskopisi kullanarak araştırmaktır. MTT bulguları D-eritro-MAPP'nin DU-145 hücrelerinde düşük dozlarda toksisiteye neden olduğunu göstermiştir. Konfokal ve TEM mikroskopi bulguları, hücre iskeletinde delik oluşumunu, kromatin yoğunlaşmasını, morfolojik değişiklikler olarak at nalı şeklindeki hücre çekirdeklerini ve hücre zarı üzerindeki tomurcuklanmaları, çekirdeklerin parçalanmasını, kromatin yoğunlaşmasını ve ultra yapısal değişiklikler olarak krista kaybını göstermiştir. Akış sitometrisi bulguları, D-eritro-MAPP'in DU-145 hücrelerine 24 saatlik kısa süreli uygulamasında apoptozu tetiklediğini göstermiştir. Elde edilen sonuçlara göre, D-eritro-MAPP'in DU-145 hücrelerinin canlılığını doza bağlı bir şekilde azalttığı söylenebilir. Bulgularımız, D-eritro-MAPP'in DU-145 hücreleri üzerindeki anti-kanser ve sitotoksik potansiyelini ortaya koymuştur ve bu ajanın, in vitro ve in vivo çalışmalardan sonra kanser tedavisi için ilaç geliştirmede kullanılabileceği belirtilmiştir.

Anahtar Kelimeler

• Sfingolipid,DU-145,konfokal mikroskopi

Kaynakça

1. [1] Ogretmen, B. (2006). Sphingolipids in cancer: Regulation of pathogenesis and therapy. FEBS Letters, 580, 5467–5476.
2. [2] Ogretmen, B., & Hannun, Y. A., (2004). Biologically active sphingolipids in cancer pathogenesis and treatment. Nat. Rev. Cancer 4, 604–616.
3. [3] Cowart, A. L., & Obeid L. M., (2007). Yeast sphingolipids: Recent developments in understanding biosynthesis, regulation, and function. Biochimica et Biophysica Acta, 1771, 421–431.
4. [4] Hannun, Y. A. & Obeid, L.M., (2018). Sphingolipids and their metabolism in physiology and disease. Nat Rev Mol Cell Biol. 19,(3), 175-191.
5. [5] Hannun, Y. A. & Obeid L.M., (2008). Principles of bioactive lipid signaling: lessons from sphingolipids. Molecular Cell Biology, 9,139-150.
6. [6] Hirabayashi, Y., Igarashi Y. & Merrill, A. H., (2006). Sphingolipid Biology. Springer, New York.
7. [7] Saieda E. M., & Arenza, C., (2016). Inhibitors of Ceramidases. Chemistry and Physics of Lipids, 197, 60–68.
8. [8] Voelkel-J. C., Norris J. S., & White, G.S., (2018). Interdiction of sphingolipid metabolism revisited: focus on prostate cancer. Adv Cancer Res, 140, 265-293.

9. [9] Realini, N., Solorzano, C., Pagliuca, C., Pizzirani, D., Armirotti, A., Luciani, R., Costi, M.P., Bandiera, T., & Piomelli, D., (2013). Discovery of highly potent acid ceramidase inhibitors with in vitro tumor chemosensitizing activity. Sci. Reports, 3, 1-7.
10. [10] Shaw, J., Costa, P. P., Patterson, L., Drews, K., Spiegel, S., & Kester, M., (2018). Novel sphingolipid-based cancer therapeutics in the personalized medicine era. Adv. Cancer Res., 140, 327-366.
11. [11] Gangoiti, P., Camacho, L., Arana, L., Ouro, A., Granado, M. H., Brizuela, L., Casas, J., Fabrias, G., Abad, J. L., Delgado, A., & Gomez, M. A., (2010). Control of metabolism and signaling of simple bioactive sphingolipids: Implications in disease. Prog. Lipid Res., 49, 316− 334.
12. [12] Dimanche-Boitrel, M. & T., Dimanche-Boitrel, A., (2013). Sphingolipids and response to chemotherapy. Handb. Exp. Pharmacol., 216, 73−91.
13. [13] Salvemini, D., Doyle, T., Kress, M., & Nicol, G., (2013). Therapeutic targeting of the ceramide-to-sphingosine 1-phosphate pathway in pain. Trends Pharmacol. Sci., 34, 110−118.
14. [14] Patti, G.J., Yanes, O., Shriver, L.P., Courade, J.P., Tautenhahn, R., Manchester, M., & Siuzdak, G., (2012). Metabolomics implicates altered sphingolipids in chronic pain of neuropathic origin. Nat Chem Biol. Jan 22;8, (3), 232-4.
15. [15] Mao, C., & Obeid, L. M., (2008). Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids, 1781, 424−434.
16. [16 ] Spiegel, S., & Milstien, S., (2003). Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat. Rev. Mol. Cell Biol., 4, 397−407.
17. [17] Takabe , K., & Spiegel, S., (2014). Export of sphingosine-1-phosphate and cancer progression. J. Lipid Res., 55, 1839−1846.
18. [18] Huang, W. C., Chen, C. L., Lin, Y. S., & Lin, C. F., (2011). Apoptotic sphingolipid ceramide in cancer therapy. J. Lipids, 565316.
19. [19] Bielawska, A., Linardic, C. M., & Hannun, Y. A., (1992). Ceramide-mediated biology. Determination of structural and stereospecific requirements through the use of N-acylphenylaminoalcohol analogs. J. Biol. Chem., 267, 18493−18497.
20. [20] Maceyka, M. (2014). Spiegel, S. Sphingolipid metabolites in inflammatory disease. Nature, 510, 58−67.
21. [21] Pettus, B. J., Chalfant, C. E., & Hannun, Y. A., (2002). Ceramide in apoptosis: an overview and current perspectives. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids, 1585, 114−125.
22. [23] Nussbaumer, P. (2008). Medicinal chemistry aspects of drug targets in sphingolipid metabolism. ChemMedChem, 3, 543−551.
23. [24] Adan-Gokbulut, A., Kartal-Yandim, M., Iskender, G., & Baran, Y., (2013). Novel agents targeting bioactive sphingolipids for the treatment of cancer. Curr. Med. Chem., 20, 108−122.
24. [25] Wymann, M. P., & Schneiter, R., (2008). Lipid signalling in disease. Nat. Rev. Mol. Cell Biol., 9, 162−176.
25. [26] Morad, S. A., & Cabot, M. C., (2013). Ceramide-orchestrated signalling in cancer cells. Nat. Rev. Cancer, 13, 51−65.
26. [27] Kolesnick, R. (2002). The therapeutic potential of modulating the ceramide/ sphingomyelin pathway. J. Clin. Invest., 110 3–8.
27. [28] Strelow, K., Bernardo, S., Adam-Klages, T., Linke, K., Sandhoff, M., & Kronke, D. A., (2000). Overexpression of acid ceramidase protects from tumor necrosis factor-induced cell death. J. Exp. Med., 192 601–612.
28. [29] Bielawska, A., Greenberg, M. S., Perry, D., Jayade, S., Shayman, J. A, McKay, C., & Hannun Y. A., (1996). (1S,2R)-D-erythro-2-(N-Myristoylamino)-1-phenyl-1-propanol as an Inhibitor of Ceramidase. The Journal Of Biologıcal Chemistry, (271), 21,12646–12654.
29. [30] Choia, M. S., Mary A. A., Zhongjian, Z., Drazen B. Z., Nicolae, P., & Anil, M., (2003). Neutral ceramidase gene: role in regulating ceramide-induced apoptosis. Gene, 315, 113–122.
30. [31] Zdzislaw M. S., Nalini, M., AiPing, B., Bielawskia, J,. Xiang, L., James, S. N., Yusuf A. H., & Alicja B., (2008). Novel Analogs of D-e-MAPP and B13. Part 1. Synthesis and Evaluation as Potential Anticancer Agent. Bioorg Med Chem., 15, 16(2), 1015–1031.