İnsan HTR1B İfadesinin İnsan Hücre Hatlarında ve Ortoloğu Ser-4’ün Caenorhabditis elegans’da Hipoksik Koşullarda Cevabının Deneysel ve In silico İncelenmesi

Abstract

Serotonin ve serotonin reseptörleri ile kanser arasındaki ilişki son yıllarda özellikle araştırılmıştır. Bazı çalışmalar, serotonin reseptörlerinin kanser hücrelerinin büyümesini, yayılmasını ve metastazını destekleyebileceğini ileri sürmektedir. Serotonin ayrıca C. elegans'ta önemli bir rol oynar. Bu organizmada serotonin, sinir sistemi ve diğer dokulardaki çeşitli fizyolojik süreçlerin düzenlenmesinde kullanılan bir nörotransmitter olarak işlev görür. C. elegans, serotonin sisteminin evrimsel olarak oldukça korunduğunu ve insanlarda benzer biyolojik işlevlere sahip olduğunu göstermektedir. Hücrelerdeki oksijen seviyelerinde azalmaya hipoksi denir ve bazı klinik çalışmalar hipoksinin tümör büyümesini desteklediğini ve tedavi direnciyle ilişkili olduğunu göstermiştir. HIF, oksijen seviyelerindeki değişikliklere yanıt olarak gen ekspresyonunu düzenler ve hipoksi koşulları altında hücrelerin yanıt verdiği DNA'nın belirli bölgelerine bağlanarak gen ekspresyonunu etkiler. Hipoksik çalışmalarda kanserle ilişkili genlerin araştırılmasında yeni bir model olarak C. elegans'ın kullanılabilirliği önemlidir. Bu amaçla iki farklı modelde (insan hücre hatları (HUVEC ve PC-3) ve C. elegans) hipoksik koşullar oluşturuldu ve son yıllarda kanserle ilişkili olduğu belirlenen serotonin reseptörleri HTR1B ve onun ortologu Ser-4'ün ekspresyon değişiklikleri incelendi. Yapılan biyoinformatik analizlerde bu iki genin %87 benzer olduğu ve benzer hücresel sinyal yollarını etkilediği görülmüştür. HTR1B'nin hipoksik koşullarda 48 ve 72. saatlerde HUVEC hücre hattında ekspresyonunun normal koşullara kıyasla arttığını bulduk. PC-3 hücre hattında ise 48. saatte hipoksik yanıt gözlendi. HTR1B C. elegans ortolog geni olan Ser-4'ün ekspresyonu da 1. saatte hipokside artmıştır. HTR1B geninin çeşitli hücre hatları üzerindeki etkileri, serotoninerjik sistemin karmaşık dinamiklerini anlamada kritik bir rol oynamaktadır. Sonuç olarak, HTR1B geninin çeşitli hücre hatları üzerindeki etkileri, bu genin kanserdeki işlevselliğini ve potansiyel terapötik kullanımlarını anlamada önemli bir adım oluşturmaktadır.

Keywords

• Serotonin
• HTR1B
• Ser-4
• HUVEC
• PC-3
• C.elegans
• Kanser
• Hipoksi

Experimental and In silico Analysis of the Hypoxic Response of Human HTR1B Expression in Human Cell Lines and Its Ortholog Ser-4 Expression in Caenorhabditis elegans

Abstract

The relationship between serotonin receptors and cancer has been particularly investigated in recent years. Some studies suggest that serotonin receptors may promote the growth, spread, and metastasis of cancer cells. Serotonin also plays an important role in C. elegans. The simple nervous system of C. elegans provides an ideal system to study to understand the functions of neurons and neuromodulators. The Serotonin system is highly conserved evolutionarily in humans and C. elegans. A decrease in oxygen levels in cells is called hypoxia and hypoxia promotes tumor growth and is associated with treatment resistance. The usability of C. elegans as a new model in the investigation of cancer-related genes in hypoxic studies is important. For this purpose, hypoxic conditions were created in two different models (human cell lines (HUVEC and PC-3) and C. elegans) and the expression changes of serotonin receptors HTR1B and its ortholog Ser-4 were examined. Bioinformatic analyses showed that these two genes were 87% similar and affected similar cellular signaling pathways. The expression of HTR1B was increased in the HUVEC cell line at 48 and 72 hours under hypoxic conditions. A hypoxic response was observed in the PC-3 cell line at 48 hours. The expression of Ser-4, the HTR1B C. elegans ortholog gene, was also increased in hypoxia at 1 hour. The effects of the HTR1B gene on various cell lines play a critical role in understanding the complex dynamics of the serotonergic system. In conclusion, the effects of the HTR1B gene on various cell lines constitute an important step in understanding the functionality of this gene in cancer and its potential therapeutic uses.

Keywords

• Serotonin
• HTR1B
• Ser-4
• HUVEC
• PC-3
• C.elegans
• Cancer
• Hypoxia

References

1. Anderson, A., Laurenson-Schafer, H., Partridge, F.A., Hodgkin, J., & McMullan, R. (2013). Serotonergic chemosensory neurons modify the C. elegans immune response by regulating G-protein signaling in epithelial cells. PLoS pathogens, 9(12), e1003787. https://doi.org/10.1371/journal.ppat.1003787
2. Balakrishna, P., George, S., Hatoum, H., & Mukherjee, S. (2021). Serotonin Pathway in Cancer. International Journal of Molecular Sciences, 22(3), 1268. https://doi.org/10.3390/ijms22031268
3. Curran, K.P., & Chalasani, S.H. (2012). Serotonin circuits and anxiety: what can invertebrates teach us? Invertebrate Neuroscience, 12, 81-92. https://doi.org/10.1007/s10158-012-0140-y
4. Dag, U., Nwabudike, I., Kang, D., Gomes, M.A., Kim, J., Atanas, A.A., ... & Flavell, S.W. (2023). Dissecting the functional organization of the C. elegans serotonergic system at whole-brain scale. Cell, 186(12), 2574-2592. https://doi.org/10.1016/j.cell.2023.04.023
5. David, D.J., & Gardier, A.M. (2016). Les bases de pharmacologie fondamentale du système sérotoninergique: application à la réponse antidépressive. L'encéphale, 42(3), 255-263. https://doi.org/10.1016/j.encep.2016.03.012
6. Dizeyi, N., Bjartell, A., Nilsson, E., Hansson, J., Gadaleanu, V., Cross, N., & Abrahamsson, P.A. (2004). Expression of serotonin receptors and role of serotonin in human prostate cancer tissue and cell lines. Prostate, 59(3), 328-36. https://doi.org/10.1002/pros.10374
7. Gürel, G., Gustafson, M.A., Pepper, J.S., Horvitz, H.R., & Koelle, M.R. (2012). Receptors and other signaling proteins required for serotonin control of locomotion in Caenorhabditis elegans. Genetics, 192(4), 1359-1371. https://doi.org/10.1534/genetics.112.142125
8. Karmakar, S., & Lal, G. (2021). Role of serotonin receptor signaling in cancer cells and anti-tumor immunity. Theranostics, 11(11), 5296. https://doi.org/10.7150/thno.55986

9. Kitson, S.L. (2007). 5-hydroxytryptamine (5-HT) receptor ligands. Current pharmaceutical design, 13(25), 2621-2637. https://doi.org/10.2174/138161207781663000
10. Leiser, S.F., Fletcher, M., Begun, A., & Kaeberlein, M. (2013). Life-span extension from hypoxia in Caenorhabditis elegans requires both HIF-1 and DAF-16 and is antagonized by SKN-1. Journals of Gerontology Series A: Biomedical Sciences and Medical Sciences, 68(10), 1135-1144. https://doi.org/10.1093/gerona/glt016
11. Miller, D.L., & Roth, M.B. (2009). C. elegans are protected from lethal hypoxia by an embryonic diapause. Current Biology, 19(14), 1233-1237. https://doi.org/10.1016/j.cub.2009.05.066
12. Nystul, T.G., Goldmark, J.P., Padilla, P.A., & Roth, M.B. (2003). Suspended animation in C. elegans requires the spindle checkpoint. Science, 302(5647), 1038-1041. https://doi.org/10.1126/science.1089705
13. Poyrazlı, F., Okuyan, D., Köçkar, F., & Türkoğlu, S.A. (2024). Hypoxic Regulation of the KLK4 Gene in two Different Prostate Cancer Cells Treated with TGF-β. Cell Biochemistry and Biophysics, 82(3), 2797-2812. https://doi.org/10.1007/s12013-024-01396-5
14. Rascón, B., & Harrison, J.F. (2010). Lifespan and oxidative stress show a non-linear response to atmospheric oxygen in Drosophila. Journal of Experimental Biology, 213(20), 3441-3448. https://doi.org/10.1242/jeb.044867
15. Riccio, C. (2019). Extracting total RNA from Caenorhabditis elegans using phase-lock gel separation tubes. https://doi.org/10.17504/protocols.io.5sug6ew
16. Riddle, D.L., Blumenthal, T., Meyer, J.B., & Priess, J.R. (1997). C. elegans II, 2nd edition’’. Cold Spring Harbor Mongraph Series 1222pp.
17. Sarrouilhe, D., Clarhaut, J., Defamie, N., & Mesnil, M. (2015). Serotonin and cancer: what is the link?. Current molecular medicine, 15(1), 62-77. https://doi.org/10.2174/1566524015666150114113411
18. Siddiqui, E.J., Shabbir, M., Mikhailidis, D.P., Thompson, C.S., & Mumtaz, F.H. (2006). The role of serotonin (5-hydroxytryptamine1A and 1B) receptors in prostate cancer cell proliferation. J Urol., 176(4 Pt 1), 1648-53. https://doi.org/10.1016/j.juro.2006.06.087
19. Savaş, N., Öğüt, S., & Olgun, A. (2018). Toksikolojik Araştirmalarda Alternatif Bir Organizma: Caenorhabditis elegans (C. elegans). Adnan Menderes Üniversitesi Sağlık Bilimleri Fakültesi Dergisi, 2(2), 99-106.
20. Smith, C., Smith, M., Cunningham, R., & Davis, S. (2020). Recent advances in Antiemetics: New formulations of 5-HT: 3: Receptor antagonists in adults. Cancer Nursing, 43(4), E217-E228. https://doi.org/10.1097/NCC.0000000000000694
21. Turkoglu, S.A., & Kockar, F. (2016). SP1 and USF differentially regulate ADAMTS1 gene expression under normoxic and hypoxic conditions in hepatoma cells. Gene, 575(1), 48-57. https://doi.org/10.1016/j.gene.2015.08.035
22. Türkoğlu, S.A., Dayi, G., & Köçkar, F. (2020). Upregulation of PSMD4 gene by hypoxia in prostate cancer cells. Turkish Journal of Biology, 44(5), 275-283. https://doi.org/10.3906/biy-2002-71
23. Türkoğlu, S.A., Poyrazlı, F., Babacan, D., & Köçkar, F. (2021). Hipoksi ve kanser. Journal of Advanced Research in Natural and Applied Sciences, 7(3), 450-463. https://doi.org/10.28979/jarnas.930938
24. Veenstra-VanderWeele, J., Anderson, G.M., & Cook Jr, E.H. (2000). Pharmacogenetics and the serotonin system: initial studies and future directions. European journal of pharmacology, 410(2-3), 165-181 https://doi.org/10.1016/S0014-2999(00)00814-1
25. Walther, D.J., Peter, J.U., Bashammakh, S., Hörtnagl, H., Voits, M., Fink, H., & Bader, M. (2003). Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science, 299(5603), 76. https://doi.org/10.1126/science.1078197
26. White, J.G., Southgate, E., Thomson, J.N., & Brenner, S. (1986). The structure of the nervous system of the nematode Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci, 314(1165), 1-340. https://doi.org/10.1098/rstb.1986.0056