Cyclophosphamide Exerts an Anti-Metastatic Effect by Reducing the Expression of MMP-2 and -9 in Saos-2 Osteosarcoma Cells

Year 2023, , 69 - 74, 16.04.2023

Gülistan Sanem Sarıbaş

https://doi.org/10.46332/aemj.1140022

Abstract

Purpose: Although osteosarcoma is the most common primary malignant bone tumor among adolescents and young adults, the molecular mechanisms underlying the development of cancer are not fully elucidated. Cyclophosphamide (CYC) is an alkylating antineoplastic agent widely used in the treatment regimens of various cancers and chronic inflammatory diseases. The aim of this study is to reveal the effect of CYC on Saos-2 cells at the molecular level by determining the expression levels of some matrix metalloprotease proteins (MMP-2 and MMP-9) of cancer progression-related genes.

Materials and Methods: Cytotoxicity analyzes were performed by applying CYC to the developed human osteosarcoma (Saos-2) cell cultures. The determined dose of CYC was applied to the 2D cell lines for 12, 24 and 48 hours. As a result of agent application, the expression levels of MMPs, which are metastatic markers in these cultures, were determined by the immunocytochemical method.

Results: While MMP-2 and MMP-9 protein expression levels increased in control (PBS) groups in correlation with the extent of duration in the cell cultures created, decreased with CYC administration.

Conclusion: It was determined that the expression levels of genes that play a role in metastasis, such as MMP-2 and MMP-9, were decreased by CYC application to Saos-2 cells. These results shed light on molecular studies for the treatment of osteosarcoma cancer.

Keywords

cancer, cyclophosphamide, MMP, osteosarcoma, Saos-2

Supporting Institution

Scientific Researches Project Unit at Kirsehir Ahi Evran University

Project Number

TIP.A4.20.002

Siklofosfamid, Saos-2 Osteosarkom Hücrelerinde MMP-2 ve -9'un Ekspresyonunu Azaltarak Anti-Metastatik Etki Gösterir

Abstract

Amaç: Osteosarkoma, ergenler ve genç yetişkinler arasında en sık rastlanan primer malign kemik tümörü olmakla birlikte, kanser gelişiminin altında yatan moleküler mekanizmalar tam anlamıyla aydınlatılamamıştır. Siklofosfamid (CYC), çeşitli kanserlerin ve kronik inflamatuvar hastalıkların tedavi rejimlerinde oldukça yaygın kullanılan alkilleyici antineoplastik bir ajandır. Bu çalışmanın amacı, CYC’nin Saos-2 hücreleri üzerine olan etkisini kanser progresyonu ilişkili genlere ait bazı matriks metalloproteaz (MMP-2 ve MMP-9) proteinlerinin ekspresyon seviyelerini belirleyerek moleküler düzeyde ortaya koymaktır.

Araçlar ve Yöntem: Geliştirilen insan osteosarkoma (Saos-2) hücre kültürlerinde CYC uygulanarak sitotoksisite analizleri yapıldı. Belirlenen dozda CYC, iki boyutlu hücre hatlarına 12, 24 ve 48 saatlik sürelerde uygulandı. Ajan uygulaması sonucunda bu kültürlerdeki metastatik belirteçler olan MMP’lerin ekspresyon seviyeleri immünositokimyasal yöntem ile belirlendi.

Bulgular: MMP2 ve MMP-9 protein ekspresyon seviyeleri oluşturulan hücre kültürlerinde artan süre ile korele olarak kontrol (PBS) gruplarında artarken; CYC uygulaması ile birlikte azalmıştır.

Sonuç: Saos-2 hücrelerine CYC uygulaması ile MMP-2 ve MMP-9 gibi metastazda rol oynayan genlerin ekspresyon düzeylerini azaltıldığı belirlendi. Bu sonuçlar osteosarkoma kanseri tedavisi için moleküler çalışmalara ışık tutacak niteliktedir.

Keywords

kanser, siklofosfamid, MMP, osteosarkoma, Saos-2

Project Number

TIP.A4.20.002

References

• 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018;68(6):394-424.
• 2. Koutsogiannouli E, Papavassiliou AG, Papanikolaou NA. Complexity in cancer biology: is systems biology the answer? Cancer Med. 2013;2(2):164-177.
• 3. Yokota J. Tumor progression and metastasis. Carcinogenesis. 2000;21(3):497-503.
• 4. Ram Kumar RM, Boro A, Fuchs B. Involvement and clinical aspects of microRNA in osteosarcoma. Int. J. Mol. Sci. 2016;17(6):877.
• 5. Pautke C, Schieker M, Tischer T, et al. Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts. Anticancer Res. 2004;24(6):3743-3748.
• 6. Coggins PR, Ravdin RG, Eisman SH. Clinical evaluation of a new alkylating agent: Cytoxan (cyclophosphamide). Cancer. 1960;13(6):1254-1260.
• 7. Koyama H, Wada T, Nishizawa Y, et al. Cyclophosphamide‐induced ovarian failure and its therapeutic significance in patients with breast cancer. Cancer. 1977;39(4):1403-1409.
• 8. Messerschmitt PJ, Garcia RM, Abdul-Karim FW, Greenfield EM, Getty PJ. Osteosarcoma. JAAOS- J Am Acad Orthop Surg. 2009;17(8):515-527.
• 9. Lindsey BA, Markel JE, Kleinerman ES. Osteosarcoma overview. Rheumatol Ther. 2017;4(1): 25-43.
• 10. Lopez SG, Luderer U. Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis. Free Radic. Biol. Med. 2004;36(11):1366-1377.
• 11. Kawabata TT, Chapman MY, Dong-Hyun K, Stevens WD, Holsapple MP. Mechanisms of in vitro immunosuppression by hepatocyte-generated cyclophosphamide metabolites and 4-hydroperoxy cyclophosphamide. Biochem. Pharmacol. 1990;40(5): 927-935.
• 12. Singh N, Nigam M, Ranjan V, et al. Resveratrol as an adjunct therapy in cyclophosphamide‐treated MCF‐7 cells and breast tumor explants. Cancer Sci. 2011;102(5):1059-1067.
• 13. Sliwowska I, Kopczyński Z. Zymography--method for quantitation of activity on gelatinase A (pro-MMP-2, 72 kDa) and gelatinase B (pro-MMP-9, 92 kDa) in serum of patients with breast cancer. Wiad. Lek. 2007;60(5-6):241-247.
• 14. Wu ZS, Wu Q, Yang JH, et al. Prognostic significance of MMP‐9 and TIMP‐1 serum and tissue expression in breast cancer. Int. J. Cancer Res. 2008;122(9):2050-2056.
• 15. Provatopoulou X, Gounaris A, Kalogera E, et al. Circulating levels of matrix metalloproteinase-9 (MMP-9), neutrophil gelatinase-associated lipocalin (NGAL) and their complex MMP-9/NGAL in breast cancer disease. BMC cancer. 2009;9(1):1-7.
• 16. Sun Z, Yzu T, Chen J, et al. Effects of ulinastatin and cyclophosphamide on the growth of xenograft breast cancer and expression of CXC chemokine receptor 4 and matrix metalloproteinase-9 in cancers. Int. J. Med. Res. 2010;38(3):967-976.
• 17. Guo H, Ge Y, Li X, et al. Targeting the CXCR4/CXCL12 axis with the peptide antagonist E5 to inhibit breast tumor progression. Signal Transduct. Target. Ther. 2017;2(1):1-8.
• 18. Qin Y, Zhang Q, Lee S, et al. Doxycycline reverses epithelial-to-mesenchymal transition and suppresses the proliferation and metastasis of lung cancer cells. Oncotarget. 2015;6(38):40667.
• 19. Paulus P, Stanley ER, Schäfer R, Abraham D, Aharinejad S. Colony-stimulating factor-1 antibody reverses chemoresistance in human MCF-7 breast cancer xenografts. Cancer Res. 2006;66(8):4349-4356.
• 20. Man S, Zhang Y, Gao W, Yan L, Ma C. Cyclophosphamide promotes pulmonary metastasis on mouse lung adenocarcinoma. Clin. Exp. Metastasis. 2008;25(8):855-864.
• 21. Hung CM, Hsu YC, Chen TY, Chang CC, Lee MJ. Cyclophosphamide promotes breast cancer cell migration through CXCR4 and matrix metalloproteinases. Cell Biol. Int. 2017;41(3):345-352.
• 22. Zhang Y, Wu G, Hu X, Zhang J. Effect of Cyclophosphamide on Expression of MMP-9 and TGF-β1 in Renal Tissue of Rats with Diabetes Mellitus. Cell Biochem. Biophys. 2015;72(2):399-403.
• 23. Izdebska M, Zielińska W, Krajewski A, et al. Downregulation of MMP-9 enhances the anti-migratory effect of cyclophosphamide in MDA-MB-231 and MCF-7 breast cancer cell lines. Int. J. Mol. Sci. 2021;22(23):12783.