Anticancer Potential of Lyophilised Medicinal Leech (Hirudo verbana) of Saliva Extract Against Pancreatic Cancer (MIA PaCa-2) Cell Lines

Yıl 2025, Cilt: 10 Sayı: 2, 167 - 173

Serkan Özdemir , Hüseyin Ayhan

https://doi.org/10.35229/jaes.1619391

Öz

Cancer is the second leading cause of death worldwide, after cardiovascular disease. It can affect any part of the body and spread to other organs. Pancreatic cancer is a tough disease to diagnose and treat. It is the fourteenth most common and the seventh deadliest cancer worldwide. New technology and innovative methods, combined with a range of therapeutic agents, have led to promising new anticancer treatments. The presence of various bioactive components in the secretions of medicinal leeches has prompted a re-evaluation of these organisms as a popular method in traditional medicine. In this study, the effect of the anticancer potential of Lyophilised medicinal leech secretion on pancreatic cancer cell line (MIA PaCa-2) was investigated using XTT assay. A cell viability test was conducted to ascertain the degree of cytotoxicity following the administration of varying concentrations of medicinal leech secretion to cell lines over a period of 24, 48, and 72 hours. The percentage viability of cancer cells was determined at each concentration. The doses were adjusted using dilution procedures with ratios of 75 µg/ml, 150 µg/ml, 300 µg/ml, 600 µg/ml, and 1200 µg/ml. IC50 value was determined at 24th hour: 484.48 µg/ml; at 48th hour: 330.92 µg/ml; at 72nd hour: 542,75 µg/ml. It was observed that the effect of Hirudo verbana leech secretion on pancreatic cancer (MIA PaCa-2) cell viability was not linear. It was determined that 600 µg/ml and 1200 µg/ml extracts had cytotoxic and anti-proliferative effects. These results indicate that the leech saliva extract has anti proliferative and cytotoxic effects and may have a promising role in developing new anticancer drugs.

Anahtar Kelimeler

Anti-cancer, Hirudo verbana, Pancreatic Cancer, MIA PaCa-2, XTT

Etik Beyan

Ethics committee authorisation is not required since the secretion was obtained from a live medical leech (Hirudo verbana) which is an invertebrate animal. In addition, no living creature was harmed.

Teşekkür

The authors would like to thank Kübranur ÜNAL and Mustafa GÜNGÖRMÜŞ for their help in laboratory studies and support of the study. We also thank Fatma ÇORUK for checking the English of the article. We thank AYBÜ MERLAB for their support in all techniques.

Liyofilize Tıbbi Sülük (Hirudo verbana) Tükürük Ekstraktının Pankreas Kanseri (MIA PaCa-2) Hücre Hatlarına Karşı Antikanser Potansiyeli

Öz

Kanser, kardiyovasküler hastalıklardan sonra dünya çapında ikinci önde gelen ölüm nedenidir. Vücudun herhangi bir bölümünü etkileyebilir ve diğer organlara yayılabilir. Pankreas kanseri teşhis ve tedavisi zor bir hastalıktır. Dünya çapında en yaygın on dördüncü ve en ölümcül yedinci kanserdir. Yeni teknoloji ve yenilikçi yöntemler, bir dizi terapötik ajanla birleşerek umut verici yeni antikanser tedavilerine yol açmıştır. Tıbbi sülüklerin salgılarında çeşitli biyoaktif bileşenlerin bulunması, bu organizmaların geleneksel tıpta popüler bir yöntem olarak yeniden değerlendirilmesine neden olmuştur. Bu çalışma, liyofilize tıbbi sülük salgısının pankreas kanseri hücre hattı (MIA PaCa-2) üzerindeki antikanser potansiyelinin etkisi XTT testi kullanılarak incelenmiştir. Hücre hatlarına 24, 48 ve 72 saatlik bir süre boyunca değişen konsantrasyonlarda tıbbi sülük salgısının uygulanmasının ardından sitotoksisite derecesini tespit etmek için bir hücre canlılık testi yapılmıştır. Kanser hücrelerinin canlılık yüzdesi her bir konsantrasyonda belirlenmiştir. Dozlar 75 µg/ml, 150 µg/ml, 300 µg/ml, 600 µg/ml ve 1200 µg/ml oranlarında seyreltme prosedürleri kullanılarak ayarlanmıştır. IC50 değeri 24. saatte: 484,48 µg/ml; 48. saatte: 330,92 µg/ml; 72. saatte 542,75 µg/ml olarak belirlenmiştir. Hirudo verbana sülük salgısının pankreas kanseri (MIA PaCa-2) hücre canlılığı üzerindeki etkisinin doğrusal olmadığı gözlenmiştir. 600 µg/ml ve 1200 µg/ml ekstraktların sitotoksik ve anti-proliferatif etkileri olduğu belirlenmiştir. Bu sonuçlar, sülük tükürüğü ekstraktının anti proliferatif ve sitotoksik etkilere sahip olduğunu ve yeni antikanser ilaçların geliştirilmesinde umut verici bir role sahip olabileceğini göstermektedir.

Anahtar Kelimeler

Anti-kanser, Hirudo verbana, MIA PaCa-2, Pankreas Kanseri, XTT

Etik Beyan

Salgı, omurgasız bir hayvan olan canlı bir tıbbi sülükten (Hirudo verbana) elde edildiği için etik kurul onayı gerekmemektedir. Ayrıca hiçbir canlıya zarar verilmemiştir.

Teşekkür

The authors would like to thank Kübranur ÜNAL and Mustafa GÜNGÖRMÜŞ for their help in laboratory studies and support of the study. We also thank Fatma ÇORUK for checking the English of the article. We thank AYBÜ MERLAB for their support in all techniques.

Kaynakça

• Abdualkader, A.M., Merzouk, A., Ghawi, A.M. & Alaama, M. (2011). Some biological activities of Malaysian leech saliva extract. IIUM Engineering Journal, 12(4). DOI: 10.31436/iiumej.v12i4.156
• Abdualkader, A.M., Ghawi, A.M., Alaama, M., Awang, M. & Merzouk, A. (2013). Leech therapeutic applications. Indian journal of pharmaceutical sciences, 75(2), 127-137.
• Alaama, M., Kucuk, O., Bilir, B., Merzouk, A., Ghawi, A. M., Yerer, M. B., Ahmado, M. A., Abdualkader, A. M. & Helaluddin, A. B. M. (2024). Development of Leech extract as a therapeutic agent: A chronological review. Pharmacological Research-Modern Chinese Medicine, 10, 100355. DOI: 10.1016/j.prmcm.2023.100355
• Ammar, A.E., Hassona, M.H., Meckling, G.R., Chan, L.G., Chin, M.Y., Abdualkader, A., Alaama, M., Merzouk, A., Helaluddin, A., Ghawi, A., Kucuk, O. & Guns, E.S. (2015). Assessment of the antitumor activity of leech (huridinaria manillensis) saliva extract in prostate cancer. Cancer Research, 75(15_Supplement), 5130- 5130. DOI:10.1158/1538-7445.AM2015-5130
• Ammar, A., Guns, E., Kucuk, O., Abdualkader, A., Alaama, M., Uddin, A.H., ... & Hassona, M. (2017). Mechanism of anticancer activity of BPS- 001 (lyophilized leech saliva extract). Cancer Research, 77 (13_Supplement), 107-107. DOI: 10.1158/1538-7445.AM2017-107
• Ayhan, H. & Mollahaliloğlu, S. (2018). Tıbbi sülük tedavisi: Hirudoterapi. Ankara Medical Journal, 18(1), 141-148. DOI: 10.17098/amj.409057
• Ayhan, H., Özyurt Koçakoğlu, N. & Candan, S. (2021). Functional morphology of the suckers and teeth of the medicinal leech Hirudo verbana Carena, 1820 (Annelida; Clitellata; Hirudinida): A scanning electron microscope study. Microscopy Research and Technique, 84(12), 2930-2935. DOI: 10.1002/jemt.23851
• Ayhan, H., Sevin, S., Karaaslan, S. & Ayaz, F. (2025). Immunomodulatory effects of medicinal leech saliva extract on in vitro activated macrophages. Immunologic Research, 73(1), 1-8. DOI: 10.1007/s12026-024-09575-5
• Aysin, F., Özek, N.Ş., Acet, N. & Koc, K. (2024). The Anti-Proliferative Effects of Ficus carica Latex on Cancer and Normal Cells. Journal of Anatolian Environmental and Animal Sciences, 9(2), 145- 151. DOI: 10.35229/jaes.1412816
• Baskova, I.P., Kostrjukova, E.S., Vlasova, M.A., Kharitonova, O.V., Levitskiy, S.A., Zavalova, L.L., Moshkovskii, S.A. & Lazarev, V.N. (2008). Proteins and peptides of the salivary gland secretion of medicinal leeches Hirudo verbana, H. medicinalis, and H. orientalis. Biochemistry (Moscow), 73, 315-320. DOI: 10.1134/S0006297908030127
• Baykara, O. (2016). Kanser Tedavisinde Güncel Yaklaşimlar. Balıkesir Sağlık Bilimleri Dergisi, 5(3), 154-165.
• Bray, F., Ren, J.S., Masuyer, E. & Ferlay, J. (2013). Global estimates of cancer prevalence for 27 sites in the adult population in 2008. International journal of cancer, 132(5), 1133-1145. DOI: 10.1002/ijc.27711
• Daniel D. Von Hoff. (2018). Pancreatic Cancer: Harrison’s Principles of Internal Medicine 20th ed., Mc Graw Hill Education.
• Davies, R.W. (1991). Annelida: Leeches, Polychaetes and Acanthobdellids. In: Ecology and Classification of North American Freshwater Invertabrates. In J. H., Thorp, A. P., Covich, (Eds.) Academic Press, New York, 437-79.
• Deng, S., Shanmugam, M.K., Kumar, A.P., Yap, C.T., Sethi, G. & Bishayee, A. (2019). Targeting autophagy using natural compounds for cancer prevention and therapy. Cancer, 125(8), 1228- 1246. DOI: 10.1002/cncr.31978
• Dong, P., Rakesh, K.P., Manukumar, H.M., Mohammed, Y.H.E., Karthik, C.S., Sumathi, S., ... & Qin, H.L. (2019). Innovative nano- carriers in anticancer drug delivery-a comprehensive review. Bioorganic chemistry, 85, 325-336. DOI:10.1016/j.bioorg.2019.01.019
• Ferlay, J., Colombet, M., Soerjomataram, I., Mathers, C., Parkin, D.M., Piñeros, M., Znaor, A. & Bray, F. (2019). Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. International journal of cancer, 144(8), 1941-1953. DOI: 10.1002/ijc.31937
• Fesinmeyer, M.D., Austin, M.A., Li, C.I., De Roos, A.J. & Bowen, D.J. (2005). Differences in survival by histologic type of pancreatic cancer. Cancer Epidemiology Biomarkers & Prevention, 14(7), 1766-1773. DOI: 10.1158/1055-9965.EPI-05- 0120
• Fitzmaurice, C., Dicker, D., Pain, A., Hamavid, H., Moradi-Lakeh, M., MacIntyre, M.F. & Naghavi, M. (2015). The global burden of cancer 2013. Global Burden of Disease Cancer Collaboration. JAMA oncology, 1(4), 505-527. DOI: 10.1001/jamaoncol.2015.0735
• Gödekmerdan, A., Arusan, S., Bayar, B. & Sağlam, N. (2011). Tıbbi sülükler ve hirudoterapi. Turkish Journal of Parasitology, 35(4), 234-239.
• Guo, Y., Tian, X. & Xiao, Z. (2006). Study on inhibition effects of freeze-thawing leech extract on HepG2 cells. Chinese Journal of Information on Traditional Chinese Medicine, (12).
• Ilic, M. & Ilic, I. (2016). Epidemiology of pancreatic cancer. World journal of gastroenterology, 22(44), 9694. DOI: 10.3748/wjg.v22.i44.9694
• Kern, S.E., Hruban, R.H., Hidalgo, M. & Yeo, C.J. (2002). An introduction to pancreatic adenocarcinoma genetics, pathology and therapy. Cancer Biology & Therapy, 1(6), 607-613. DOI: 10.4161/cbt.307
• McGuigan, A., Kelly, P., Turkington, R.C., Jones, C., Coleman, H.G. & McCain, R.S. (2018). Pancreatic cancer: A review of clinical diagnosis, epidemiology, treatment and outcomes. World Journal of Gastroenterology, 24(43), 4846. DOI: 10.3748/wjg.v24.i43.4846
• Modi B. & Shires G.T. (2020). Pancreatic cancer, cystic pancreatic neoplasms, and other nonendocrine pancreatic tumor. In: Feldman M, Friedman L, Brandt L; (Eds.) Sleisenger and Fordtran’s Gastrointestinal and Liver Disease, 11th edition. Philadelphia: Elsevier, 947-965.
• Muhammad, N., Usmani, D., Tarique, M., Naz, H., Ashraf, M., Raliya, R., Tabrez S., Zughaibi, T. A., Alsaieedi A., Hakeem, I.J. & Suhail, M. (2022). The role of natural products and their multitargeted approach to treat solid cancer. Cells, 11(14), 2209. DOI: 10.3390/cells11142209
• Neubert, E. & Nesemann H. (1999). Annelida, Clitellata: Branchiobdellida, Acanthobdellea, Hirudinea. Süβwasserfauna von Mitteleuropa 6/2. Spektrum Akademischer Verlag, 176.
• Pavlopoulou, A., Spandidos, D.A. & Michalopoulos, I. (2015). Human cancer databases. Oncology Reports, 33(1), 3-18. DOI: 10.3892/or.2014.3579
• Sağlam, N. (2004). Tatlı Su ve Deniz Sülükleri Tanı Anahtarı. Fırat Üniversitesi Basım Evi.
• Sawyer, R.T. (1986). Leech Biology and Behaviour. Clarendon Press, Oxford.
• Shakouri, A. & Wollina, U. (2021). Time to change theory; medical leech from a molecular medicine perspective leech salivary proteins playing a potential role in medicine. Advanced Pharmaceutical Bulletin, 11(2), 261. DOI: 10.34172/apb.2021.038
• Shakouri, A., Kahroba, H., Hamishekar, H. & Abdolalizadeh, J. (2021). Nanoencapsulation of Hirudo medicinalis proteins in liposomes as a nanocarrier for inhibiting angiogenesis through targeting VEGFA in the Breast cancer cell line (MCF-7). BioImpacts: BI, 12(2), 115. DOI: 10.34172/bi.2021.39
• Singh, S.K. & Rajoria, K. (2020). Medical leech therapy in Ayurveda and biomedicine–A review. Journal of Ayurveda and Integrative Medicine, 11(4), 554- 564. DOI: 10.1016/j.jaim.2018.09.003
• Surh, Y.J. (2003). Cancer chemoprevention with dietary phytochemicals. Nature Reviews Cancer, 3(10), 768-780.
• Taherian, M., Wang, H. & Wang, H. (2022). Pancreatic ductal adenocarcinoma: molecular pathology and predictive biomarkers. Cells, 11(19), 3068. DOI: 10.3390/cells11193068
• Tang, L., Duan, Q.H. & Fan, P.S. (2012). Clinical study on leech in treating hypercoagulable state of malignant tumors. Clinical Journal of Traditional Chinese Medicine, 24, 871-872.
• Tural, Ö. & Ayhan, H. (2024). The Anti-Proliferative Activity of Lyophilised Medicinal Leech (Hirudo Verbana) Saliva Extract on Breast Cancer Cell Line (MCF-7). Journal of Anatolian Environmental and Animal Sciences, 9(4), 508- 513. DOI: 10.35229/jaes.1541179
• Ünal, K., Tırık, N., Erol, M., İbrahimkhanlı, L., Elçi, M. & Ayhan, H. (2023). The Investigation of Effects of Medicinal Leech Saliva Extract on the Breast Fibroblast Cell Line In Vitro: An Experimental Study. Geleneksel ve Tamamlayıcı Tıp Dergisi, 6(2). DOI: 10.5336/jtracom.2022- 92875
• Wolfgang, C.L., Herman, J.M., Laheru, D.A., Klein, A.P., Erdek, M.A., Fishman, E.K. & Hruban, R.H. (2013). Recent progress in pancreatic cancer. CA: A Cancer Journal for Clinicians, 63(5), 318-348. DOI: 10.3322/caac.21190
• World Health Organization (WHO). (2023). Noncommunicable disease. https://www.who.int/news-room/fact- sheets/detail/noncommunicable-diseases Access date: 28.07.2024