Silymarin Inhibited A549 Cells by Activating SLIT2 Protein and Suppressing Expression of CXCR4

Yıl 2021, , 327 - 334, 01.12.2021

Sedat Kaçar , Nuriye Ezgi Bektur Aykanat

https://doi.org/10.32708/uutfd.971366

Öz

Lung cancer is one of the leading causes of cancer death in both men and women worldwide. SLIT2/ROBO1 signaling is a crucial pathway that has been reported to inhibit various types of cancer. CXCR4 is a chemokine receptor involved in cancer progression. Silymarin is a phytochemical that has been suggested to have anti-carcinogenic activity in various cancers, especially liver diseases and lung cancer. How-ever, there is no study examining the effect of silymarin on the SLIT2–ROBO1–CXCR4 axis in lung cancer. Here, our aim is to investigate the cytotoxic and morphological effects of silymarin on A549 cells and to reveal its role in the SLIT2-ROBO1-CXCR4 pathway. Firstly, 24, 48 and 72 hour long cytotoxicity tests were performed for silymarin dose analysis, followed by H-E staining for morphological evaluation with varying doses of silymarin. According to MTT analysis, IC50 concentrations of silymarin against A549 cells were determined as 930.1, 432.1 and 99.8 μM for 24, 48 and 72 hours administration, respectively. When stained with H-E, cytoplasmic vacuoles, shrunken hetero-chromatin nuclei and cells with basophilic cytoplasm were observed. Then, western blot and immunocytochemistry analyzes were performed for SLIT2, ROBO1 and CXCR4 proteins with 750 μM silymarin. 750 μM silymarin increased SLIT2 and ROBO1 expressions while de-creased CXCR4 compared to control group. In conclusion, silymarin dose-dependently inhibited A549 cells by activating SLIT2 and ROBO1 protein expression and inhibiting CXCR4 expression. The effects of silymarin on lung cancer have been reported in the literature. This is the first study to examine the interaction between SLIT2– ROBO1 –CXCR4 proteins and silymarin in A549 cells. We believe that our study will open new horizons for future researches.

Anahtar Kelimeler

Lung cancer, A549, Silymarin, Slit 2, Robo 1, CXCR4

Silimarin SLIT2 Proteinini Aktive Ederek ve CXCR4 Ekspresyonunu Baskılayarak A549 Hücrelerini İnhibe Etti

Öz

Akciğer kanseri, dünya çapında hem erkeklerde hem de kadınlarda kansere bağlı önde gelen ölüm nedenlerindendir. SLIT2/ROBO1 sinyali, çeşitli kanser tiplerini inhibe ettiği bildirilen çok önemli bir yolaktır. CXCR4, kanser ilerlemesinde rol oynayan bir kemokin reseptörüdür. Silimarin, başta karaciğer hastalıkları olmak üzere akciğer kanseri de dahil çeşitli kanserlerde anti-kanserojen aktivitesi öne sürülen bir fitokimyasaldır. Ancak silimarinin akciğer kanserinde SLIT2–ROBO1–CXCR4 ekseni üzerindeki etkisini inceleyen çalışma bulunmamaktadır. Burada amacımız silimarinin A549 hücreleri üzerindeki sitotoksik ve morfolojik etkilerini araştırmak ve SLIT2-ROBO1-CXCR4 yolağındaki rolünü ortaya çıkarmaktır. İlk olarak, silimarinin doz analizi için 24, 48 ve 72 saat uzunluğunda sitotoksisite testleri yapıldı. Ardından değişen dozlarda silimarin ile morfolojik değerlendirme için hücreler H-E ile boyandı. Daha sonra SLIT2, ROBO1 ve CXCR4 proteinleri için western blot ve immünositokimya analizleri yapıldı. MTT analizine göre, A549 hücrelerine karşı silimarinin IC50 konsantrasyonları 24, 48 ve 72 saatlik uygulamaları için sırasıyla 930.1, 432.1 ve 99.8 μM olarak saptandı. H-E boyama yapılarak morfolojik olarak incelendiğinde sitoplazmik vakuoller, küçülmüş heterokromatin çekirdek ve bazofilik sitoplazmalı hücreler gözlendi. 750 μM silimarin ile SLIT2, ROBO1 ve CXCR4 proteinleri için Western blot ve immünositokimya analizleri yapıldı. 750 μM silimarin, kontrol grubuna kıyasla SLIT2 ve ROBO1 ekspresyonlarını arttırırken CXCR4'ü azalttı. Sonuç olarak silimarin, SLIT2 ve ROBO1 protein ekspresyonunu aktive ederek ve CXCR4 ekspresyonunu inhibe ederek A549 hücrelerini doza bağlı olarak inhibe etmiştir. Silimarinin akciğer kanseri üzerindeki etkileri literatürde belirtilmiştir. Ancak bu çalışma, A549 hücrelerinde SLIT2–ROBO1–CXCR4 proteinleri ile silimarin arasındaki etkileşimi inceleyen ilk çalışmadır. Çalışmamızın bundan sonraki araştırmalara yeni ufuklar açacağına inanıyoruz.

Anahtar Kelimeler

Akciğer kanseri, A549, Silimarin, Slit 2, Robo 1, CXCR4

Kaynakça

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