Yeşil sentezlenen altın nanopartikülün akciğer kanseri hücre hattında boyut etkisinin incelenmesi
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Tarih
2023
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Yayıncı
Necmettin Erbakan Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Heterojen ve agresif fenotip gösteren küçük hücreli dışı akciğer kanseri (KHDAK), dünya çapında kanserden ölümlerin en sık nedenidir. Tüm erkek ve kadın popülasyonlarında en yaygın ve en ölümcül malignite olarak bilinmektedir. Akciğer kanseri vakalarının çoğuna geç evrelerde teşhis konulmaktadır ve genel sağkalım oranları düşüktür. Akciğer kanseri tedavisinde cerrahi hala kullanılan temel yaklaşımdır; ancak çok sayıda hasta nüks ve ilaç tedavisi başarısızlığı nedeniyle ölmektedir. Cerrahiyi takiben kullanılan çoğu kemoterapötik ilaç hem kanser hem de sağlıklı hücrelerde sistemik sitotoksisiteye ve ciddi yan etkilere neden olmaktadır. Bu nedenle, yeni terapötik hedeflerin daha fazla araştırılmasına ihtiyaç artmaktadır. Yeşil sentezlenen altın naopartiküllerin (AuNP) moleküler çözünürlükte hücrelerle etkileşime girebilmesi ve üstün biyouyumluluk sağlaması nedeniyle yakın zamanda insanlarda akciğer kanseri tedavisinde potansiyel terapötik ajanlar olarak hizmet edebileceği öngörülmektedir. Diğer nanopartiküllere göre daha kararlı ve biyouyumlu metal nanopartikül olan AuNP'ler antifungal, antibakteriyel, antiviral ve antikanser aktiviteleri sayesinde tıpta yaygın olarak kullanılma potansiyeline sahiptir. Bununla birlikte COX-2 seçici inhibitör olan Celecoxib, kanserin tedavisi ve önlenmesinde önemli bir rol oynamaktadır. COX-2 inhibitörleriyle birlikte çeşitli antineoplastik ilaçların kullanılması kanser kontrolü ve tedavisi için umut vaat etmektedir. AuNP ve Celecoxib ajanının kombine tedavisinin akciğer kanserinde tümör baskılayıcı aktivitelere aracılık ederek tümör ilerlemesini inhibe etmek için potansiyel yeni bir stratejiyi temsil edebileceği düşünülmektedir. Bu kapsamda yapılacak olan çalışmalar akciğer kanseri ilerlemesinin önlenmesi ve tedavisine büyük ölçüde katkı sağlayacaktır. Bu tez çalışmasında yeşil sentez ile antikanser etkisi bilinen Orchis Spitzelii (Salep) bitkisinin kök kısmının sulu ekstrakt konsantrasyonu değiştirilerek farklı boyutlarda AuNP sentezi gerçekleştirilmiştir. Sentezlenen AuNP'ler UV-Vis, XRD, FT-IR, DLS, Zeta potansiyel, TEM ve FESEM-EDX yöntemleri ile karakterize edilmiştir. Artan dozlarda yeşil sentezlenen AuNP'ler, Orchis Spitzelii (Salep) bitki ekstraktı ve Celecoxib ajanı ile NCI-H441 insan akciğer kanseri ve HUVEC hücre hattı 24 saat boyunca muamele edilmiştir. NCI-H441 insan akciğer kanseri ve HUVEC sağlıklı hücre hattı üzerindeki sitotoksik etkileri hücre canlılık testi ile belirlenmiştir. Belirlenen IC50 dozları ile PPARγ sinyal yolağı ve anjiyogenez, invazyon, metastaz, yağ asidi metabolizması ile ilgili hedef genler olan CD36, TIMP2, MMP2, PGE2, COX-2 ve VEGFR2 gen ekspresyonlarındaki değişiklikler qRT-PCR ile incelenmiştir. Ek olarak EMT sürecinde yer alan MMP2 protein düzeyi değişimi Western Blot yöntemiyle incelenmiştir. Yeşil sentezlenen AuNP'lerin hücre canlılığı testi sonucunda boyutları küçüldükçe sitotoksisitelerinin arttığı, qRT-PCR sonucunda Ekstrakt ve Celecoxib ajanından daha iyi seviyede anjiyogenez ve EMT süreci ile ilişkili olan PPARγ, CD36 ve TIMP2 gen ekspresyonunu istatistiksel olarak anlamlı bir şekilde upregüle ettiği ve MMP2, PGE2, COX-2 ve VEGFR2 genlerinin ekspresyonunu istatistiksel olarak anlamlı bir şekilde downregüle ettiği sonucuna ulaşılmıştır. Western Blot çalışması, gen ekspresyon çalışmalarıyla paralel olarak MMP2 protein düzeyinin istatistiksel olarak anlamlı bir şekilde baskılandığını göstermiştir. Çalışma kapsamında tüm veriler değerlendirildiğinde yeşil sentezlenen AuNP'lerin arzu edilen şekilde NCIH441 akciğer kanseri hücrelerine kıyasla sağlıklı HUVEC hücrelerine karşı daha yüksek biyouyumluluk sergilediği belirlenmiştir. Yeşil sentezlenen AuNP'lerin, akciğer kanseri hücrelerinde MMP-2'nin indüklediği EMT'yi ve VEGFR2'nin indüklediği anjiyogenezi inhibe ettiğini ve metastazı önlediğini gösterdik. PPARγ'nın AuNP'ler ile aktivasyonu, mezenkimal belirteç olan MMP2 ekspresyonunu önlemektedir. AuNP'lerin düşük toksisite profiliyle birlikte verilerimiz, bu nanopartiküllerin metastazı inhibe etmek için potansiyel terapötik ajanlar olarak hizmet edebileceğini göstermektedir.
Non-small cell lung cancer (NSCLC), which exhibits a heterogeneous and aggressive phenotype, is the most common cause of cancer deaths worldwide. It is known as the most common and deadliest malignancy in all male and female populations. Most cases of lung cancer are diagnosed in the late stages and overall survival rates are low. Surgery is still the main approach used in the treatment of lung cancer; however, many patients die due to relapse and drug treatment failure. Most chemotherapeutic drugs used following surgery cause systemic cytotoxicity and serious side effects in both cancer and healthy cells. Therefore, there is an increasing need for further investigation of new therapeutic targets. It is predicted that green synthesized gold nanoparticles (AuNP) may recently serve as potential therapeutic agents in treating lung cancer in humans, as they can interact with cells at molecular resolution and provide superior biocompatibility. AuNPs, which are metal nanoparticles that are more stable and biocompatible than other nanoparticles, have the potential to be widely used in medicine thanks to their antifungal, antibacterial, antiviral, and anticancer activities. However, Celecoxib, a COX-2 selective inhibitor, plays an important role in the treatment and prevention of cancer. The use of various antineoplastic drugs in combination with COX-2 inhibitors holds promise for cancer control and treatment. It is thought that the combined treatment of AuNP and Celecoxib agent may represent a potential new strategy to inhibit tumor progression by mediating tumor suppressor activities in lung cancer. Studies to be carried out in this context will greatly contribute to preventing and treating lung cancer progression. In this thesis study, AuNPs of different sizes were synthesized by changing the aqueous extract concentration of the root part of the Orchis Spitzelii (Salep) plant, which is known to have an anticancer effect, with green synthesis. Synthesized AuNPs were characterized by UV-Vis, XRD, FT-IR, DLS, Zeta potential, TEM, and FESEM-EDX methods. NCI-H441 human lung cancer and HUVEC cell line were treated with increasing doses of green synthesized AuNPs, Orchis Spitzelii (Salep) plant extract, and Celecoxib agent for 24 hours. The cytotoxic effects of NCI-H441 on human lung cancer and the HUVEC healthy cell line were determined by cell viability testing. With the determined IC50 doses, changes in the gene expressions of CD36, TIMP2, MMP2, PGE2, COX-2, and VEGFR2, which are target genes related to the PPARγ signaling pathway and angiogenesis, invasion, metastasis, and fatty acid metabolism, were examined by qRT-PCR. In addition, the MMP2 protein level change involved in the EMT process was examined by the Western Blot method. As a result of the cell viability test, green synthesized AuNPs showed that their cytotoxicity increased as their size decreased, and as a result of qRT-PCR, they statistically significantly upregulated PPARγ, CD36, and TIMP2 gene expression, which are associated with angiogenesis and EMT process, at a better level than Extract and Celecoxib agent, and MMP2, PGE2. It was concluded that it downregulated the expression of COX-2 and VEGFR2 genes in a statistically significant way. Western Blot study showed statistically significant suppression of MMP2 protein level in parallel with gene expression studies. When all data were evaluated within the scope of the study, it was determined that green synthesized AuNPs exhibited higher biocompatibility against healthy HUVEC cells compared to NCIH441 lung cancer cells, as desired. We showed that green synthesized AuNPs inhibited MMP-2-induced EMT and VEGFR2-induced angiogenesis in lung cancer cells and prevented metastasis. Activation of PPARγ with AuNPs prevents the expression of MMP2, a mesenchymal marker. Together with the low toxicity profile of AuNPs, our data suggest that these nanoparticles may serve as potential therapeutic agents to inhibit metastasis.
Non-small cell lung cancer (NSCLC), which exhibits a heterogeneous and aggressive phenotype, is the most common cause of cancer deaths worldwide. It is known as the most common and deadliest malignancy in all male and female populations. Most cases of lung cancer are diagnosed in the late stages and overall survival rates are low. Surgery is still the main approach used in the treatment of lung cancer; however, many patients die due to relapse and drug treatment failure. Most chemotherapeutic drugs used following surgery cause systemic cytotoxicity and serious side effects in both cancer and healthy cells. Therefore, there is an increasing need for further investigation of new therapeutic targets. It is predicted that green synthesized gold nanoparticles (AuNP) may recently serve as potential therapeutic agents in treating lung cancer in humans, as they can interact with cells at molecular resolution and provide superior biocompatibility. AuNPs, which are metal nanoparticles that are more stable and biocompatible than other nanoparticles, have the potential to be widely used in medicine thanks to their antifungal, antibacterial, antiviral, and anticancer activities. However, Celecoxib, a COX-2 selective inhibitor, plays an important role in the treatment and prevention of cancer. The use of various antineoplastic drugs in combination with COX-2 inhibitors holds promise for cancer control and treatment. It is thought that the combined treatment of AuNP and Celecoxib agent may represent a potential new strategy to inhibit tumor progression by mediating tumor suppressor activities in lung cancer. Studies to be carried out in this context will greatly contribute to preventing and treating lung cancer progression. In this thesis study, AuNPs of different sizes were synthesized by changing the aqueous extract concentration of the root part of the Orchis Spitzelii (Salep) plant, which is known to have an anticancer effect, with green synthesis. Synthesized AuNPs were characterized by UV-Vis, XRD, FT-IR, DLS, Zeta potential, TEM, and FESEM-EDX methods. NCI-H441 human lung cancer and HUVEC cell line were treated with increasing doses of green synthesized AuNPs, Orchis Spitzelii (Salep) plant extract, and Celecoxib agent for 24 hours. The cytotoxic effects of NCI-H441 on human lung cancer and the HUVEC healthy cell line were determined by cell viability testing. With the determined IC50 doses, changes in the gene expressions of CD36, TIMP2, MMP2, PGE2, COX-2, and VEGFR2, which are target genes related to the PPARγ signaling pathway and angiogenesis, invasion, metastasis, and fatty acid metabolism, were examined by qRT-PCR. In addition, the MMP2 protein level change involved in the EMT process was examined by the Western Blot method. As a result of the cell viability test, green synthesized AuNPs showed that their cytotoxicity increased as their size decreased, and as a result of qRT-PCR, they statistically significantly upregulated PPARγ, CD36, and TIMP2 gene expression, which are associated with angiogenesis and EMT process, at a better level than Extract and Celecoxib agent, and MMP2, PGE2. It was concluded that it downregulated the expression of COX-2 and VEGFR2 genes in a statistically significant way. Western Blot study showed statistically significant suppression of MMP2 protein level in parallel with gene expression studies. When all data were evaluated within the scope of the study, it was determined that green synthesized AuNPs exhibited higher biocompatibility against healthy HUVEC cells compared to NCIH441 lung cancer cells, as desired. We showed that green synthesized AuNPs inhibited MMP-2-induced EMT and VEGFR2-induced angiogenesis in lung cancer cells and prevented metastasis. Activation of PPARγ with AuNPs prevents the expression of MMP2, a mesenchymal marker. Together with the low toxicity profile of AuNPs, our data suggest that these nanoparticles may serve as potential therapeutic agents to inhibit metastasis.
Açıklama
Yüksek Lisans Tezi
Anahtar Kelimeler
Altın Nanopartikül, Boyut Etkisi, Celecoxib, Küçük Hücreli Dışı Akciğer Kanseri, Matriks Metalloproteinaz 2, Orchis Spitzelii (Salep), Yeşil Sentez, Gold Nanoparticle, Size Effect, Non-small Cell Lung Cancer, Matrix Metalloproteinase 2, Green Synthesis
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Künye
Doğan, P. E. (2023). Yeşil sentezlenen altın nanopartikülün akciğer kanseri hücre hattında boyut etkisinin incelenmesi. (Yayımlanmamış yüksek lisans tezi). Necmettin Erbakan Üniversitesi, Fen Bilimleri Enstitüsü Moleküler Biyoloji ve Genetik Anabilim Dalı, Konya.
