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

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

1. Barta JA, Powell CA, Wisnivesky JP. Global epidemiology of lung cancer. Annals of global health 2019;85(1).
2. Singh T, Prasad R, Katiyar SK. Therapeutic intervention of silymarin on the migration of non-small cell lung cancer cells is associated with the axis of multiple molecular targets including class 1 HDACs, ZEB1 expression, and restoration of miR-203 and E-cadherin expression. Am J Cancer Res 2016;6(6):1287.
3. Sezer CV. An In Vitro Assessment of the Cytotoxic and Apoptotic Potency of Silymarin and Silymarin Loaded Solid Lipid Nanoparticles. Pakistan Journal of Zoology 2021;53(4).
4. D’Arcy MS. Cell death: a review of the major forms of apoptosis, necrosis and autophagy. Cell Biol Int 2019;43(6):582-92.
5. Kim S-H, Choo G-S, Yoo E-S, Woo J-S, Lee J-H, Han S-H, et al. Silymarin inhibits proliferation of human breast cancer cells via regulation of the MAPK signaling pathway and induction of apoptosis. Oncol Lett 2021;21(6):1-10.
6. Pfeffer CM, Singh AT. Apoptosis: a target for anticancer therapy. Int J Mol Sci 2018;19(2):448.
7. Bijak M. Silybin, a major bioactive component of milk thistle (Silybum marianum L. Gaernt.)—Chemistry, bioavailability, and metabolism. Molecules 2017;22(11):1942.
8. Hackett E, Twedt D, Gustafson D. Milk thistle and its derivative compounds: a review of opportunities for treatment of liver disease. J Vet Intern Med 2013;27(1):10-6.

9. Abenavoli L, Izzo AA, Milić N, Cicala C, Santini A, Capasso R. Milk thistle (Silybum marianum): A concise overview on its chemistry, pharmacological, and nutraceutical uses in liver diseases. Phytother Res 2018;32(11):2202-13.
10. Esmaeil N, Anaraki SB, Gharagozloo M, Moayedi B. Silymarin impacts on immune system as an immunomodulator: One key for many locks. Int Immunopharmacol 2017;50:194-201.
11. Hosseinabadi T, Lorigooini Z, Tabarzad M, Salehi B, Rodrigues CF, Martins N, et al. Silymarin antiproliferative and apoptotic effects: insights into its clinical impact in various types of cancer. Phytother Res 2019;33(11):2849-61.
12. Montgomery A, Adeyeni T, San K, Heuertz RM, Ezekiel UR. Curcumin sensitizes silymarin to exert synergistic anticancer activity in colon cancer cells. J Cancer 2016;7(10):1250.
13. Kalla PK, Chitti S, Aghamirzaei ST, Senthilkumar R, Arjunan S. Anti-cancer activity of silymarin on MCF-7 and NCIH-23 cell lines. Adv Biol Res 2014;8(2):57-61.
14. Kim SH, Choo GS, Yoo ES, Woo JS, Han SH, Lee JH, et al. Silymarin induces inhibition of growth and apoptosis through modulation of the MAPK signaling pathway in AGS human gastric cancer cells. Oncol Rep 2019;42(5):1904-14.
15. Fan L, Ma Y, Liu Y, Zheng D, Huang G. Silymarin induces cell cycle arrest and apoptosis in ovarian cancer cells. Eur J Pharmacol 2014;743:79-88.
16. Kacar S, Aykanat NEB, Sahinturk V. Silymarin inhibited DU145 cells by activating SLIT2 protein and suppressing expression of CXCR4. Med Oncol 2020;37(3):1-9.
17. Liakopoulou C, Kazazis C, Vallianou NG. Silimarin and cancer. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents) 2018;18(14):1970-4.
18. Jahanafrooz Z, Motamed N, Rinner B, Mokhtarzadeh A, Baradaran B. Silibinin to improve cancer therapeutic, as an apoptotic inducer, autophagy modulator, cell cycle inhibitor, and microRNAs regulator. Life Sci 2018;213:236-47.
19. Delmas D, Xiao J, Vejux A, Aires V. Silymarin and cancer: A dual strategy in both in chemoprevention and chemosensitivity. Molecules 2020;25(9):2009.
20. Shen X, Li L, He Y, Lv X, Ma J. Raddeanin A inhibits proliferation, invasion, migration and promotes apoptosis of cervical cancer cells via regulating miR-224-3p/Slit2/Robo1 signaling pathway. Aging (Albany NY) 2021;13(5):7166.
21. Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res 2010;16(11):2927-31.
22. Lombardi L, Tavano F, Morelli F, Latiano TP, Di Sebastiano P, Maiello E. Chemokine receptor CXCR4: role in gastrointestinal cancer. Crit Rev Oncol Hematol 2013;88(3):696-705.
23. Verdura S, Cuyàs E, Ruiz-Torres V, Micol V, Joven J, Bosch-Barrera J, et al. Lung Cancer Management with Silibinin: A Historical and Translational Perspective. Pharmaceuticals 2021;14(6):559.
24. Mateen S, Raina K, Agarwal R. Chemopreventive and anti-cancer efficacy of silibinin against growth and progression of lung cancer. Nutr Cancer 2013;65(sup1):3-11.
25. Khojaste E, Ahmadizadeh C. Catechin Metabolites along with Curcumin Inhibit Proliferation and Induce Apoptosis in Cervical Cancer Cells by Regulating VEGF Expression In-Vitro. Nutr Cancer 2021:1-10.
26. Aykanat NEB, Kacar S, Karakaya S, Sahinturk V. Silymarin suppresses HepG2 hepatocarcinoma cell progression through downregulation of Slit-2/Robo-1 pathway. Pharmacol Rep 2020;72(1):199-207.
27. Gao T, Zhou X-L, Liu S, Rao C-X, Shi W, Liu J-C. In vitro effects of nicotine on the non-small-cell lung cancer line A549. J Pak Med Assoc 2016;66(4):368-72.
28. Tang Y, Fu Q, He W, Sun Y, Wang Y, Wang X. Non-apoptotic programmed cell death induced by extract of Spatholobus suberctus in human lung cancer A549 cells. Zhongguo Zhong yao za zhi= Zhongguo zhongyao zazhi= China journal of Chinese materia medica 2008;33(16):2040-4.
29. Shanmugam MK, Manu KA, Ong TH, Ramachandran L, Surana R, Bist P, et al. Inhibition of CXCR4/CXCL12 signaling axis by ursolic acid leads to suppression of metastasis in transgenic adenocarcinoma of mouse prostate model. Int J Cancer 2011;129(7):1552-63.
30. Shanmugam MK, Ahn KS, Hsu A, Woo CC, Yuan Y, Tan KHB, et al. Thymoquinone inhibits bone metastasis of breast cancer cells through abrogation of the CXCR4 signaling axis. Front Pharmacol 2018;9:1294.
31. Zhu WB, Zhao ZF, Zhou X. AMD3100 inhibits epithelial–mesenchymal transition, cell invasion, and metastasis in the liver and the lung through blocking the SDF‐1α/CXCR4 signaling pathway in prostate cancer. J Cell Physiol 2019;234(7):11746-59.
32. Liu Y, Ren C-C, Yang L, Xu Y-M, Chen Y-N. Role of CXCL12-CXCR4 axis in ovarian cancer metastasis and CXCL12-CXCR4 blockade with AMD3100 suppresses tumor cell migration and invasion in vitro. J Cell Physiol 2018;234(4):3897-909.
33. Shah M, Jan H, Drouet S, Tungmunnithum D, Shirazi JH, Hano C, et al. Chitosan Elicitation Impacts Flavonolignan Biosynthesis in Silybum marianum (L.) Gaertn Cell Suspension and Enhances Antioxidant and Anti-Inflammatory Activities of Cell Extracts. Molecules 2021;26(4):791.
34. Lama S, Vanacore D, Diano N, Nicolucci C, Errico S, Dallio M, et al. Ameliorative effect of Silybin on bisphenol A induced oxidative stress, cell proliferation and steroid hormones oxidation in HepG2 cell cultures. Sci Rep 2019;9(1):1-10.
35. Rugamba A, Kang DY, Sp N, Jo ES, Lee J-M, Bae SW, et al. Silibinin Regulates Tumor Progression and Tumorsphere Formation by Suppressing PD-L1 Expression in Non-Small Cell Lung Cancer (NSCLC) Cells. Cells 2021;10(7):1632.
36. Singh RP, Deep G, Chittezhath M, Kaur M, Dwyer-Nield LD, Malkinson AM, et al. Effect of silibinin on the growth and progression of primary lung tumors in mice. J Natl Cancer Inst 2006;98(12):846-55.
37. Bosch-Barrera J, Queralt B, Menendez JA. Targeting STAT3 with silibinin to improve cancer therapeutics. Cancer Treat Rev 2017;58:61-9.
38. Tyagi A, Singh RP, Ramasamy K, Raina K, Redente EF, Dwyer-Nield LD, et al. Growth inhibition and regression of lung tumors by silibinin: modulation of angiogenesis by macrophage-associated cytokines and nuclear factor-κB and signal transducers and activators of transcription 3. Cancer Prevention Research 2009;2(1):74-83.
39. Mateen S, Raina K, Jain AK, Agarwal C, Chan D, Agarwal R. Epigenetic modifications and p21-cyclin B1 nexus in anticancer effect of histone deacetylase inhibitors in combination with silibinin on non-small cell lung cancer cells. Epigenetics 2012;7(10):1161-72.
40. Chu SC, Chiou HL, Chen PN, Yang SF, Hsieh YS. Silibinin inhibits the invasion of human lung cancer cells via decreased productions of urokinase‐plasminogen activator and matrix metalloproteinase‐2. Molecular Carcinogenesis: Published in cooperation with the University of Texas MD Anderson Cancer Center 2004;40(3):143-9.
41. Jiang Z, Liang G, Xiao Y, Qin T, Chen X, Wu E, et al. Targeting the SLIT/ROBO pathway in tumor progression: molecular mechanisms and therapeutic perspectives. Ther Adv Med Oncol 2019;11:1758835919855238.
42. Qin F, Zhang H, Ma L, Liu X, Dai K, Li W, et al. Low expression of Slit2 and Robo1 is associated with poor prognosis and brain-specific metastasis of breast cancer patients. Sci Rep 2015;5(1):1-11.
43. Werbowetski-Ogilvie T, Sadr MS, Jabado N, Angers-Loustau A, Agar N, Wu J, et al. Inhibition of medulloblastoma cell invasion by Slit. Oncogene 2006;25(37):5103-12.
44. Tseng R-C, Lee S-H, Hsu H-S, Chen B-H, Tsai W-C, Tzao C, et al. SLIT2 attenuation during lung cancer progression deregulates β-catenin and E-cadherin and associates with poor prognosis. Cancer Res 2010;70(2):543-51.
45. Wang LJ, Zhao Y, Han B, Ma YG, Zhang J, Yang DM, et al. Targeting Slit–Roundabout signaling inhibits tumor angiogenesis in chemical‐induced squamous cell carcinogenesis. Cancer Sci 2008;99(3):510-7.
46. Mertsch S, Schmitz N, Jeibmann A, Geng J-G, Paulus W, Senner V. Slit2 involvement in glioma cell migration is mediated by Robo1 receptor. J Neurooncol 2008;87(1):1-7.
47. Wang Y, Zhang S, Bao H, Mu S, Zhang B, Ma H, et al. MicroRNA-365 promotes lung carcinogenesis by downregulating the USP33/SLIT2/ROBO1 signalling pathway. Cancer Cell Int 2018;18(1):1-14.
48. Narayan G, Goparaju C, Arias-Pulido H, Kaufmann AM, Schneider A, Dürst M, et al. Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression. Mol Cancer 2006;5(1):1-10.
49. Kim HK, Zhang H, Li H, Wu T-T, Swisher S, He D, et al. Slit2 inhibits growth and metastasis of fibrosarcoma and squamous cell carcinoma. Neoplasia 2008;10(12):1411-20.
50. Chatterjee S, Azad BB, Nimmagadda S. The intricate role of CXCR4 in cancer. Adv Cancer Res 2014;124:31-82.