Investigation of the Anti-Cancer Effects of Kitosan Added Red Pine (Pinus Brutia Ten, Pinaceae) Cone Extracts

Öz

Cancer is a systemic disease that occurs with the accumulation of many mutations in the genome. There are many different treatment methods applied to cancer patients. During the treatment process, increasing toxicity and drug resistance occur over time and limit the success of the treatment. In order to overcome these limitations in cancer treatment, herbal products and synthetic derivatives can be preferred. At this point, chitosan, which is formed by deacetylating approximately half of the Korean pine (Pinus koraiensis) and long-chain polymer derivative chitin, which has been used in the treatment of many diseases in traditional medicine until today , stands out. In this study, the effects of red pine (Pinus brutia Ten) cone extract on MCF-7, VERO and HeLa cells after 24, 48 and 72 hours of incubation were investigated. The WST-1 cytotoxicity test was analyzed by applying the extracts to the cells at doses of 400, 200, 100, 50 and 25μg/mL. In the experimental results, while chitosan added green cone extract did not cause cytotoxic effects on cells after 24 hours of incubation, the IC50 value for the extract was calculated as 252.3 μg/mL at the end of 48 hours. The calculated IC50 value for the extract after 72 hours is 195.5μg/mL. IC50 values of chitosan added green pinecone extract in HeLa cells at 24, 48 and 72 hours incubation times were calculated as 200.5 μg/mL, 221.9 μg/mL and 352.3 μg/ml, respectively. IC50 values in HeLa cells in 24 and 48 hour incubations were calculated as 262.6 μg/mL and 161.9 μg/mL, respectively , in black pinecone extract with chitosan.

Anahtar Kelimeler

• MCF-7
• HeLa
• Cytotoxicity
• Chitosan
• Pinecone Extract

Kitosan Katkılı Kızılçam (Pinus Brutia Ten, Pinaceae) Kozalağı Ekstraktlarının Antikanser Etkilerinin Araştırılması

Öz

Kanser, genomdaki birçok mutasyonun birikimi ile ortaya çıkan sistemik bir hastalıktır. Kanser hastalarında uygulanan birçok farklı tedavi yöntemi bulunmaktadır. Tedavi süreci içerisinde zamanla artan toksisite ve ilaç direnci ortaya çıkmakta ve tedavi başarısını sınırlandırmaktadır. Kanser tedavisindeki bu sınırlandırmaları aşmak için bitkisel ürünler ve sentetik türevleri tercih edilebilmektedir. Bu noktada, geleneksel tıpta günümüze kadar birçok hastalığın tedavisinde kullanılan Kore çamı (Pinus koraiensis) ve uzun zincirli polimer türevi olan kitinin yaklaşık olarak yarısının deasetile edilmesiyle oluşturulan kitosan göze çarpmaktadır . Bu çalışmada kızılçam (Pinus brutia Ten) kozalağı ekstraktının MCF-7, VERO ve HeLa hücreleri üzerinde 24, 48 ve 72 saatlik inkübasyonlar sonundaki etkileri incelenmiştir. Ekstraktlar hücrelere 400, 200, 100, 50 ve 25μg/mL dozlarda uygulanarak maruziyet süreleri sonunda WST-1 sitotoksisite testi analiz edilmiştir. Deney sonuçlarında kitosan katkılı yeşil kozalak ekstraktı 24 saatlik inkübasyon sonunda hücrelerde sitotoksik etki yaratmazken, ekstrakt için 48 saat sonunda IC50 değeri 252.3 μg/mL olarak hesaplanmıştır. 72 saat sonrasında ekstrakt için hesaplanan IC50 değeri 195.5μg/mL ‘dir. Kitosan ilaveli yeşil çam kozalağı ekstraktının HeLa hücrelerinde 24, 48 ve 72 saatlik inkübasyon sürelerinde IC50 değerleri sırasıyla 200.5 μg/mL, 221.9 μg/mL ve 352.3 μg/ml olarak hesaplanmıştır. Kitosan ilaveli siyah çam kozalağı ekstraktında HeLa hücrelerinde 24 ve 48 saatlik inkübasyonlarda IC50 değerleri sırasıyla 262.6 μg/mL ve 161.9 μg/mL olarak hesaplanmıştır .

Anahtar Kelimeler

• MCF-7
• HeLa
• Sitotoksisite
• Kitosan
• Çam Kozalağı Ekstraktı

Kaynakça

1. Akbayrak, N., et al. (1998). “İç Hastalıkları Hemşireliği El Kitabı“, 24-30.
2. Akyol, H. (2004). “Kemoterapinin temel ilkeleri, XIII.” TPOG Ulusal Pediatrik Kanser Kongresi, Hemşire Programı, 159-163.
3. Asmaz, H., (1993). Akdeniz peyzajında kızılçamın önemi. Uluslararası Kızılçam Sempozyumu, 18-23 Ekim, Marmaris-Muğla, Bildiriler Kitabı, s. 48-56.
4. Blagosklonny, M. V. (2005). “Molecular theory of cancer.” Cancer biology & therapy, 4:6: 621-627.
5. Chen, X., Zhang, Y., Wang, Z. ve Zu, Y. (2011). “In vivo antioxidant activity of Pinus koraiensis nut oil obtained by optimised supercritical carbon dioxide extraction. Natural product research”, 25(19): 1807-1816.
6. Choi, C., Nam, JP ve Nah, JW (2016). “Kitosan ve kitosan türevlerinin biyomalzeme olarak uygulanması. Endüstri ve Mühendislik Kimyası Dergisi”, 33: 1-10.
7. Croisier, F. ve Jérôme, C. (2013). “Chitosan-based biomaterials for tissue engineering. European Polymer Journal”, 49(4): 780-792.
8. Csaba N, Garcia-Fuentes M, Alonso MJ. (2006). “The performance of nanocarriers for transmucosal drug delivery. Expert Opin Drug Deliv.” 3:463- 478.

9. De la Fuente M, Csaba N, Garcia-Fuentes M, Alonso MJ. (2008). “Nanoparticles as protein and gene carriers to mucosal surfaces. Nanomedicine (Lond).” 3: 845- 857.
10. Gibot, L., Chabaud, S., Bouhout, S., Bolduc, S., Auger, F. A., Moulin, V. J. (2015). “Anticancer properties of chitosan on human melanoma are cell line dependent. International journal of biological macromolecules”, 72: 370-379.
11. Gürboy, B. (2007). “Kuzey Kıbrıs’ta doğal olarak yetişen kızılçam (pinus brutia Ten.)’ın lif morfolojisi. Türkiye Ormancılık Dergisi”, 8(2): 119-127.
12. Hasegawa, M., Yagi, K., Iwakawa, S. ve Hirai, M. (2001). “Chitosan induces apoptosis via caspase‐3 activation in bladder tumor cells. Japanese journal of cancer research”, 92(4): 459-466.
13. Kantarcı, M.D., (1991). “Akdeniz Bölgesi’nin yetişme ortamı bölgesel sınıflandırması.” T.C. Tarım Orman ve Köyişleri Bakanlığı Orman Genel Müdürlüğü Yayını, Sıra No: 668, Seri No: 64, Ankara.
14. Karatepe, Y., Özçelik, R., Gürlevik, N., Yavuz, H., Kırış, R. (2014). “Ecological evaluation of vegetation structure in Turkish red pine forests (Pinus brutia Ten.) in different sites of western Mediterranean region of Turkey.” Türkiye Ormancılık Dergisi, 15(1): 1-8.
15. Kızılcı, S. (1999). “Kemoterapi alan kanserli hastalar ve yakınlarının yaşam kalitesini etkileyen faktörler.” Cumhuriyet Üniversitesi Hemşirelik Yüksekokulu Dergisi, 3(2): 18-26.
16. Kuruoğlu, A. (2017). “Karboplatinin etoposide dirençli A549 hücre hattında sitotoksik ve anti-metastatik özelliklerinin araştırılması”. Yüksek lisans tezi, Akdeniz üniversitesi, Antalya- Türkiye, 72.
17. Kvols, L. K. (2005). “Radiation sensitizers: a selective review of molecules targeting DNA and non-DNA targets.” Journal of Nuclear Medicine, 46(1): 187S.
18. Larrayoz, I. M., Huang, J. D., Lee, J. W., Pascual, I., Rodríguez, I. R. (2010). “7-Ketocholesterol–induced inflammation: involvement of multiple kinase signaling pathways via NFκB but independently of reactive oxygen species formation.” Investigative ophthalmology & visual science, 51(10): 4942-4955.
19. Lee, J. H., Yang, H. Y., Lee, H. S., Hong, S. K. (2008). “Chemical composition and antimicrobial activity of essential oil from cones of Pinus koraiensis.” Microbiol Biotechnol, 18(3): 497-502.
20. Lee, T. K., Park, J. Y., Yu, J. S., Jang, T. S., Oh, S. T., Pang, C, Kim, K. H. (2018). 7α, “15-Dihydroxydehydroabietic acid from Pinus koraiensis inhibits the promotion of angiogenesis through downregulation of VEGF, p-Akt and p-ERK in HUVECs. Bioorganic & medicinal chemistry letters”, 28(6): 1084-1089.
21. Lee, T. K., Roh, H. S., Yu, J. S., Baek, J., Lee, S., Ra, M., Kim, K. H. (2017). “Pinecone of Pinus koraiensis Inducing Apoptosis in Human Lung Cancer Cells by Activating Caspase‐3 and its Chemical Constituents. Chemistry & biodiversity”, 14(4): e1600412.
22. Li, K., Li, Q., Li, J., Zhang, T., Han, Z., Gao, D., Zheng, F. (2007). “Antitumor activity of the procyanidins from Pinus koraiensis bark on mice bearing U14 cervical cancer.” Yakugaku Zasshi, 127(7): 1145-1151.
23. Liu, J., Bai, J., Jiang, G., Li, X., Wang, J., Wu, D., Li, W. (2015). “Anti-tumor effect of Pinus massoniana bark proanthocyanidins on ovarian cancer through induction of cell apoptosis and inhibition of cell migration. PloS one”, 10(11): e0142157.
24. Loeb, L. A., Loeb, K. R., Anderson, J. P. (2003). “Multiple mutations and cancer.” Proceedings of the National Academy of Sciences, 100(3): 776-781.
25. Ma, H., Lai, F., Xie, H., Wang, J., Wang, H. (2008). “Involvement of the Bcl‐2 family members in Pinus massoniana bark extract induced apoptosis in HeLa cells. Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives”, 22(11): 1472-1476.
26. Ma, H., Liu, B., Feng, D., Xie, H., Li, R., Yuchi, Y., Wang, J. (2010). “Pinus massoniana bark extract selectively induces apoptosis in human hepatoma cells, possibly through caspase-dependent pathways. International journal of molecular medicine”, 25(5): 751-759.
27. Narazaki, M., Segarra, M., Hou, X., Tanaka, T., Li, X., Tosato, G. (2010). “Oligoguanosine nucleotide induces neuropilin-1 internalization in endothelial cells and inhibits angiogenesis.” Blood, 116(16): 3099-3107.
28. Ong, S. Y., Wu, J., Moochhala, S. M., Tan, M. H., Lu, J. (2008). “Development of a chitosan-based wound dressing with improved hemostatic and antimicrobial properties.” Biomaterials, 29(32): 4323-4332.
29. Öner, ö. ü. e. k. (2020). “Odun dışı bitkisel ürünler. Research ın medıcınal and aromatıc plants”, 95.
30. Özkan, S. A., Dedeoğlu, A., Karadaş Bakırhan, N., Özkan, Y. (2019). “Nanocarriers Used Most in Drug Delivery and Drug Release: Nanohydrogel, Chitosan, Graphene, and Solid Lipid. Turkish Journal of Pharmaceutical Sciences”, 16(4).
31. Piao, Z. J., Tang, L. N., Swihart, R. K., Wang, S. X. (2011). “Human-wildlife competition for Korean pine seeds: vertebrate responses and implications for mixed forests on Changbai Mountain, China. Annals of Forest Science”, 68: 911–919.
32. Proboningrat, A., Fadholly, A., Iskandar, R. P. D., Achmad, A. B., Rantam, F. A., Sudjarwo, S. A. (2019). “The potency of chitosan-based Pinus merkusii bark extract nanoparticles as anti-cancer on HeLa cell lines. Veterinary World”, 12(10): 1616.
33. Qi, L., Xu, Z., Chen, M. (2007). “In vitro and in vivo suppression of hepatocellular carcinoma growth by chitosan nanoparticles. European journal of cancer”, 43(1): 184-193.
34. Sakka, L., Delétage, N., Chalus, M., Aissouni, Y., Sylvain-Vidal, V., Gobron, S., Coll, G. (2017). “Assessment of citalopram and escitalopram on neuroblastoma cell lines: Cell toxicity and gene modulation.” Oncotarget, 8(26): 42789.
35. Şefik, Y. (1964). Kızılçam (Pinus brutia Ten.) “kozalak ve tohumu üzerine araştırmalar.” İÜ Orman Fakültesi Dergisi, Seri A, 14(2): 35-70.
36. Takimoto, H., Hasegawa, M., Yagi, K., Nakamura, T., Sakaeda, T., Hirai, M. (2004). “Proapoptotic effect of a dietary supplement: water soluble chitosan activates caspase-8 and modulating death receptor expression.” Drug metabolism and Pharmacokinetics, 19(1): 76-82.
37. Ullah S, Zainol I, Idrus RH. (2017). “Incorporation of zinc oxide nanoparticles into chitosan-collagen 3D porous scaffolds: effect on morphology, mechanical properties andcytocompatibility of 3D porous scaffolds.” Int J Biol Macromol;104: 1020–1029.
38. Wahba, M. I. (2019). “Enhancement of the mechanical properties of chitosan. Journal of Biomaterials Science, Polymer Edition”, 1-26.
39. Yesil‐Celiktas, O., Ganzera, M., Akgun, I., Sevimli, C., Korkmaz, K. S., Bedir, E. (2009). “Determination of polyphenolic constituents and biological activities of bark extracts from different Pinus species. Journal of the Science of Food and Agriculture”, 89(8): 1339-1345.
40. Yi, J., Wang, Z., Bai, H., Yu, X., Jing, J., Zuo, L. (2015). “Optimization of purification, identification and evaluation of the in vitro antitumor activity of polyphenols from pinus koraiensis pinecones. Molecules”, 20(6): 10450-10467.
41. Zhang X, Lin Y, Gillies RJ. (2010). “Tumor pH and its measurement.” J Nucl Med. 51: 1167-1170.
42. Zhang, H., Wu, F., Li, Y., Yang, X., Huang, J., Lv, T., Liu, G. (2016). “Chitosanbased nanoparticles for improved anticancer efficacy and bioavailability of mifepristone. Beilstein journal of nanotechnology”, 7(1): 1861-1870.
43. Zhang, H., Zhao, H., Yao, L., Yang, X., Shen, S., Wang, J., Geng, L. (2017). “Isolation, physicochemical properties, and in vitro antioxidant activity of polysaccharides extracted from different parts of Pinus koraiensis.” Journal of wood chemistry and technology, 37(3): 225-240.
44. Zhang, J. H., Feng, D. R., Ma, H. L., Liu, B., Wang, H. B., Xie, H., Wang, J. F. (2012). “Antitumor effects of Pinus massoniana bark extract in murine sarcoma S180 both in vitro and in vivo. The American journal of Chinese medicine, 40(04): 861-875.