Selim GENÇ, Suray PEHLİVANOGLU, Çiğdem AYDIN, Şükriye YEŞİLOT

VİTİS VİNİFERA ÇEKİRDEK ÖZÜTÜ KULLANILARAK ALTIN NANOPARTİKÜLLERİN YEŞİL SENTEZİ VE KOLON KANSERİ (HT-29) HÜCRELERİNDE ANTİKANSER ÖZELLİKLERİNİN DEĞERLENDİRİLMESİ

Son yıllarda metalik nanopartiküller antioksidan, antimikrobiyal ve antikanser etkinlikleri ile nanotıp alanında oldukça dikkat çekmektedir. Özellikle, kansertedavisinde farmakolojik etkinliği bilinen tıbbi bitkilerden yeşil sentez metodu ile üretilen metal nanopartiküller araştırmacıların ilgi odağı haline gelmiştir. Altınnanopartiküller; kendilerine özgü kimyasal, fiziksel ve nontoksik özellikleri ile ön plana çıkan metal nanopartiküllerden biridir. Çalışmamızda, üzüm (Vitis vinifera) çekirdeği sulu ekstraktı kullanılarak yeşil sentezi gerçekleştirilen altın nanopartiküllerin (Vv-AuNP) tek başına ve kemoterapötik bir ajan olan Gemsitabin ile kombine kullanımının HT-29 kolon kanseri hücre hattı üzerindeki sitotoksik, anti-proliferatif ve apoptotik etkilerinin değerlendirilmesi amaçlanmıştır.Altın nanopartiküllerin yeşil sentezi, Vitis vinifera sulu özütü kullanılarak gerçekleştirildi. Vv-AuNP’lerin UV-görünür spektrofotometre, taramalı elektronmikroskobu (SEM) ve Enerji Dağılımlı X-ışını Spektroskopisi (EDX) gibi çeşitli analitik tekniklerle karakterizasyonu yapıldı. Vv-AuNP'ler 0–80 μg/mL dozaralığında tek başına ve Gemsitabin (100 μg/mL veya 200 μg/mL sabit doz) ile kombine olacak şekilde HT29 hücreleri ile 72 saat süresince muamele edildi.Sitotoksik etkileri MTT testi ile değerlendirildi. Ayrıca, HT-29 hücreleri üzerine Vv-AuNP’lerin artan dozlarda (0-400μg/mL) tek başına ve Gemsitabin ile kombineantiproliferatif etkileri ise BrdU testi ile belirlendi. Altın nanopartiküllerin HT-29 hücreleri ile muamelesi sonrası Kaspaz-3 aktivasyon düzeyi Western-blot yöntemi ile analiz edildi.MTT sonuçları değerlendirildiğinde Vv-AuNP’lerin 80 μg/mL doza kadar çıkıldığında HT-29 hücrelerinde istenilen düzeyde sitotoksik etkiler göstermediği belirlendi. BrdU hücre proliferasyon testi sonuçlarına dayalı olarak, Vv-AuNP’lerin tek başına ve Gemsitabin ile birlikte uygulamalarında IC50 değerleri sırasıyla 147.9 ve 39.43 µg/mL olarak belirlendi. Vv-AuNP dozlarına bağlı kaspaz-3 aktivasyonunun kontrole kıyasla arttığı, Gemsitabin ile kombine kullanımının bu etkiyi güçlendirdiği gözlendi. Sonuç olarak, yeşil sentezlenmiş AuNP’ler in vitro koşullarda kolon kanseri hücreleri üzerinde anti-kanser özellikler sergilemiştir. Bu çalışmanın sonuçları, Vv-AuNP'lerin kolon kanseri tedavisi için potansiyel bir seçenek olarak düşünülebileceğini göstermektedir.

GREEN SYNTHESIS OF GOLD NANOPARTICLES USING VITIS VINIFERA SEED EXTRACT AND EVALUATION OF ANTI-CANCER PROPERTIES IN COLON CANCER (HT-29) CELLS

In recent years, metallic nanoparticles have attracted the attention in the field of nanomedicine with their antioxidant, antimicrobial and anticancer activities. Especially, metal nanoparticles produced by green synthesis method from medicinal plants with known pharmacological efficacy in cancer treatment have been spotlighted by researchers.. Gold nanoparticles; become prominentamong metal nanoparticles with their unique chemical, physical and non-toxic properties. In this study, it was aimed to perform green synthesis of gold nanoparticles using Vitis vinifera (grape seed) aqueous extract and to evaluate cytotoxic, anti-proliferative and apoptotic effects of gold nanoparticles (Vv-AuNP) alone and with Gemcitabine co-administration on HT-29 human colon cancer cell line. Green synthesis of gold nanoparticles was carried out using the aqueous extract of Vitis vinifera. Vv-AuNPs were characterized by various analytical techniques such as UV-visible spectrophotometer, scanning electron microscope (SEM) and Energy Dispersive X-ray Spectroscopy (EDX). Vv-AuNPs were treated with HT-29 cells at a dose range of 0–80 μg / mL for 72 hours, alone and in combination with Gemcitabine (100 μg / mL or 200 μg / mL fixed dose). Cytotoxic effects were evaluated by MTT assay. In addition, the antiproliferative effects of Vv-AuNPs at increasing doses (0-400 μg / mL) alone and combined with Gemcitabine were determined by the BrdU test. After treatment of gold nanoparticles with HT-29 cells, the level of Caspase-3 activation was analyzed by Western-blot method. According to MTT results, when the dose of VvAuNPs was increased to 80 μg / mL, it was determined that HT-29 cells did not show the expected level of cytotoxic effect. According to the results of the BrdU cell proliferation test, IC50 values of Vv-AuNPs alone and combined with Gemcitabine were determined as 147.9 and 39.43 µg / mL, respectively. It was observed that the activation of caspase-3 due to VvAuNP doses increased compared to control, and its combined use with Gemcitabine reinforced this effect. As a result, green synthesized AuNPs exhibited anti-cancer properties on colon cancer cells in vitro. The results of this study show that Vv-AuNPs can be considered as a potential option for colon cancer treatment.

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1. Merlo LM, Pepper JW, Reid BJ, Maley CC. Cancer as an evolutionary and ecological process. Nat Rev Cancer 2006;6(12):924-935.
2. Mármol I, Sánchez-de-Diego C, Pradilla Dieste A, Cerrada E, Rodriguez Yoldi M. Colorectal Carcinoma. A General Overview and Future Perspectives in Colorectal Cancer. Int J Mol Sci 2017;18(1):197.
3. Ahmed M. Colon cancer: a clinician's perspective in 2019. Gastroenterology Res 2020;13(1):1–10.
4. Baykara O. Kanser tedavisinde güncel yaklaşımlar. Balikesir Saglik Bil Derg 2016;5(3):154-165.
5. Aydın Acar Ç, Pehlivanoğlu S. Biosynthesis of Silver Nanoparticles Using Rosa canina Extract and Its Anti-cancer and Antimetastatic Activity on Human Colon Adenocarcinoma Cell Line HT29. MAKU J. Health Sci. Inst. 2019;7(2):124-131.
6. Osuwa JC, Anusionwu PC. Some advances and prospects in nanotechnology: a review. Asian J Inf Technol 2011;10:96-100.
7. Rana A, Yadav K, Jagadevan S. A comprehensive review on green synthesis of nature-inspired metal nanoparticles: Mechanism, application and toxicity. Journal of Cleaner Production 2020;272:122880.
8. Mohanpuria P, Rana NK, Yadav SK. Biosynthesis of nanoparticles: technological concepts and future applications. J Nanopart Res 2008;10:507–517.
9. Kumar V, Yadav SK. Plant‐mediated synthesis of silver and gold nanoparticles and their applications. J Chem Technol Biotechnol 2009;84:151–157.
10. Kamran U, Bhatti HN, Iqbal M, Nazir A. Green synthesis of metal nanoparticles and their applications in different fields: a review. Zeitschrift für Physikalische Chemie 2019;233(9):1325-1349.
11. Yesilot S, Aydin CA. Silver nanoparticles; a new hope in cancer therapy?. Eastern Journal of Medicine 2019;24(1):111-116.
12. Kwatra B. A review on potential properties and therapeutic applications of grape seed extract. World J. Pharm. Res 2020;9:2519-2540.
13. Unusan N. Proanthocyanidins in grape seeds: An updated review of their health benefits and potential uses in the food industry. Journal of Functional Foods 2020;67:103861.
14. Nassiri‐Asl M, Hosseinzadeh H. Review of the Pharmacological Effects of Vitisvinifera (Grape) and its Bioactive Constituents:An Update. Phytother. Res. 2016;30:1392–1403.
15. Akhtar M, Panwar J, Yun YS. Biogenic synthesis of metallic nanoparticles by plant extracts. ACS Sustain Chem Eng 2013;1:591-602.
16. Conde J, Doria G, Baptista P. Noble metal nanoparticles applications in cancer. J Drug Deliv 2012;2012:751075.
17. Singh P, Kim YJ, Wang C, Mathiyalagan R, El-Agamy Farh M, Yang DC. Biogenic silver and gold nanoparticles synthesized using red ginseng root extract, and their applications. Artif Cells Nanomed Biotechnol 2016;44(3):811-6.
18. Tan G, Onur MA, Sağlam N. Utilization of gold nanostructures in biomedical applications. Turk J Biol 2012; 36:607- 621.
19. Biao L, Tan S, Meng Q, Gao J, Zhang X, Liu Z, Fu Y. Green synthesis, characterization and application of proanthocyanidins-functionalized gold nanoparticles. Nanomaterials 2018;8(1):53.
20. Hamelian M, Hemmati S, Varmira K, Veisi H. Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using Pistacia Atlantica extract. Journal of the Taiwan Institute of Chemical Engineers 2018;93:21-30.
21. Usman AI, Aziz AA, Noqta OA. Application of green synthesis of gold nanoparticles: A review. Jurnal Teknologi 2019;81(1):171-182.
22. Noah N. Green synthesis: Characterization and application of silver and gold nanoparticles. Micro and Nano Technologies 2019;111-135.
23. Noble S, Goa KL. Gemcitabine. Drugs 1997;54(3):447-472.
24. Guo X, Xu B, Pandey S, Goessl E, Brown J, Armesilla AL, Wang W. Disulfiram/copper complex inhibiting NFκB activity and potentiating cytotoxic effect of gemcitabine on colon and breast cancer cell lines. Cancer letters 2010;290(1):104-113.
25. Ramsden J. Essentials of nanotechnology. Nanotechnology © 2009 Jeremy Ramsden & Ventus Publishing ApS, ISBN 978-87-7681-418-2; 2009.
26. Cai W, Gao T, Hong H, Sun J. Applications of gold nanoparticles in cancer nanotechnology. Nanotechnol Sci Appl 2008;1(1):17-32.
27. Nejati K, Dadashpour M, Gharibi T, Mellatyar H, Akbarzadeh A. Biomedical Applications of Functionalized Gold Nanoparticles: A Review. Journal of Cluster Science 2021.https://doi.org/10.1007/s10876-020-01955-9
28. Datkhile KD, Patil SR, Durgawale PP. et al. Biogenic synthesis of gold nanoparticles using Argemone mexicana L. and their cytotoxic and genotoxic effects on human colon cancer cell line (HCT-15). J Genet Eng Biotechnol 2021;19:9.
29. Zhang Y, Elechalawar CK, Hossen MN, Francek ER, Dey A, Wilhelm S, et al. Gold nanoparticles inhibit activation of cancer-associated fibroblasts by disrupting communication from tumor and microenvironmental cells. Bioactive materials 2021;6(2):326-332.
30. Kuppusamy P, Yusoff MM, Maniam GP, Govindan N. Biosynthesis of metallic nanoparticles using plant derivatives and their new avenues in pharmacological applications – An updated report. Saudi Pharm J 2016;24(4):473-84.
31. Baran MF, Saydut A. Altın nanomalzeme sentezi ve karekterizasyonu. DÜMF Müh Der. 2019;10(3):1033-1040.
32. Umamaheswari C, Lakshmanan A, Nagarajan NS. Green synthesis, characterization and catalytic degradation studies of gold nanoparticles against congo red and methyl orange. J Photoch Photobio B 2018;178:33-39.
33. Zhang P, Wang P, Yan L, Liu L. Synthesis of gold nanoparticles with Solanum xanthocarpum extract and their in vitro anticancer potential on nasopharyngeal carcinoma cells. In j nanomed 2018;13:7047.
34. Asariha M, Chahardoli A, Karimi N, Gholamhosseinpour M, Khoshroo A, Nemati H, et al. Green synthesis and structural characterization of gold nanoparticles from Achillea wilhelmsii leaf infusion and in vitro evaluation. Bull Mater Sci 2020;43:57.
35. Islam NU, Jalil K, Shahid M, Muhammad N, Rauf A. Pistacia integerrima gall extract mediated green synthesis of gold nanoparticles and their biological activities. Arab J Chem 2019;12(8):2310-2319.
36. Zhang X, Fan L, Cui Y, Cui T, Chen S, Ma G, et al. Green synthesis of gold nanoparticles using longan polysaccharide and their reduction of 4-nitrophenol and biological applications. Nano 2020;15(1):2050002.
37. Kumar PV, Kala SMJ, Prakash KS. Green synthesis of gold nanoparticles using Croton Caudatus Geisel leaf extract and their biological studies. Mater Lett 2019;236:19-22.
38. Izadiyan Z, Shameli K, Hara H, Taib SHM. Cytotoxicity assay of biosynthesis gold nanoparticles mediated by walnut (Juglans regia) green husk extract. Journal of Molecular Structure 2018;1151:97-105.
39. Abu-Tahon MA, Ghareib M, Abdallah WE. Environmentally benign rapid biosynthesis of extracellular gold nanoparticles using Aspergillus flavus and their cytotoxic and catalytic activities. Process Biochemistry 2020;95:1-11.
40. Divakaran D, Lakkakula JR, Thakur M, Kumawat MK, Srivastava R. Dragon fruit extract capped gold nanoparticles: Synthesis and their differential cytotoxicity effect on breast cancer cells. Materials Letters 2019;236:498-502.
41. Shahriari M, Hemmati S, Zangeneh A, Zangeneh MM. Biosynthesis of gold nanoparticles using Allium noeanum Reut. ex Regel leaves aqueous extract; characterization and analysis of their cytotoxicity, antioxidant, and antibacterial properties. Applied organometallic chemistry 2019;33(11):e5189.
42. Dey A, Yogamoorthy A, SM S. Green synthesis of gold nanoparticles and evaluation of its cytotoxic property against colon cancer cell line. Research Journal of Life Sciences, Bioinformatics, Pharmaceutical and Chemical Sciences 2018;4:1-17.
43. Carmichael J. The role of gemcitabine in the treatment of other tumours. Br J Cancer 1998;78:21-25.
44. Sarvepalli D, Rashid MU, Rahman AU, Ullah W, Hussain I, Hasan B, et al. Gemcitabine: a review of chemoresistance in pancreatic cancer. Critical Reviews™ in Oncogenesis 2019;24(2).
45. Wang F, Wang Y, Ma Q, Cao Y, Yu B. Development and characterization of folic acid-conjugated chitosan nanoparticles for targeted and controlled delivery of gemcitabinein lung cancer therapeutics. Artif Cells Nanomed B 2017;45(8):1530-1538.
46. Yuan YG, Peng QL, Gurunathan S. Silver nanoparticles enhance the apoptotic potential of gemcitabine in human ovarian cancer cells: combination therapy for effective cancer treatment. Int J Nanomedicine 2017;12:6487–6502.
47. Devi L, Gupta R, Jain SK, Singh S, Kesharwani P. Synthesis, characterization and in vitro assessment of colloidal gold nanoparticles of Gemcitabine with natural polysaccharides for treatment of breast cancer. J Drug Deliv Sci Tec 2020;56:101565.
48. Baharara J, Ramezani T, Divsalar A, Mousavi M, Seyedarabi A. Induction of Apoptosis by Green Synthesized Gold Nanoparticles Through Activation of Caspase-3 and 9 in Human Cervical Cancer Cells. Avicenna J Med Biotechnol. 2016;8(2):75-83.
49. Mata R, Nakkala JR, Sadras SR. Polyphenol stabilized colloidal gold nanoparticles from Abutilon indicum leaf extract induce apoptosis in HT-29 colon cancer cells, Colloids and Surfaces B: Biointerfaces, 2016;143:499-510.