Sains Malaysiana 50(9)(2021): 2701-2711

http://doi.org/10.17576/jsm-2021-5009-17

Ranibizumab Inhibits Human Tenon’s Fibroblast Proliferation via p21 Dependent p53 Mechanisms

(Ranibizumab Merencat Percambahan Fibroblas Tenon Manusia melalui Mekanisme p21 Bersandar p53)

 SITI MUNIRAH MD NOH¹*, SITI HAMIMAH SHEIKH ABDUL KADIR^2,3 & SUSHIL VASUDEVAN²

¹University of Malaya Centre of Innovation and Commercialization (UMCIC), University of Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

²Faculty of Medicine, Universiti Teknologi MARA, Cawangan Sungai Buloh, 47000 Sungai Buloh, Selangor Darul Ehsan, Malaysia

³Institute for Pathology, Laboratory and Forensic Medicine (I-PPerForM), Universiti Teknologi MARA, Cawangan Sungai Buloh, 47000 Sungai Buloh, Selangor Darul Ehsan, Malaysia

Received: 11 August 2020/Accepted: 19 January 2021

ABSTRACT

Trabeculectomy is the gold standard procedure performed in glaucoma when topical medication and laser intervention failed. In a trabeculectomy, number of clinical trials have shown the efficacy of ranibizumab in minimizing extracellular matrix accumulation at the filtering site. Ranibizumab (Lucentis^TM) is a drug that targets vascular endothelial growth factor (VEGF). However, to date the actual mechanisms of this anti-VEGF in trabeculectomy is still not well understood. Hence, in here we aimed to elucidate the effects of ranibizumab on human Tenon’s fibroblast (HTF) isolated from patients undergoing trabeculectomy. In our previous study, we had reported that ranibizumab reduces the level of spermidine metabolite whereby spermidine is an important polyamine for cell proliferation. For this current study, cultured HTFs were divided into untreated, control IgG, ranibizumab only, difluoromethylornithine (DFMO; inhibitor of spermidine) only and ranibizumab with DFMO. All cells were extracted for PCR array (expression of CDKN1A, CDK2, and CDK4) and protein expression of p53, p21, CDK2, and CDK4 by Western Blot. In here, our result demonstrated that cells treated with ranibizumab or DFMO and cells treated with ranibizumab-DFMO have similar effects as both show increased in p53 and p21. Meanwhile, no significant differences in expression of CDKN1A, CDK2 and CDK4 were observed in all groups. In essence, our findings suggest that ranibizumab action is mediated by p21 and p53.

Keywords: Human Tenon’s fibroblast; ranibizumab; vascular endothelial growth factor

ABSTRAK

Trabekulektomi adalah prosedur utama yang dijalankan bagi penyakit glaukoma apabila ubatan topikal dan intervensi laser telah gagal. Dalam trabekulektomi, beberapa ujian klinikal telah menunjukkan keberkesanan ranibizumab dalam menurunkan pengumpulan metriks ekstrasel pada tapak penurasan. Ranibizumab (Lucentis^TM) adalah sejenis dadah yang mensasarkan faktor pertumbuhan endotelium vaskular (VEGF). Namun, sehingga kini masih belum difahami sepenuhnya bagaimana anti-VEGF bekerja dalam mengurangkan kesan sampingan bagi trabekulekomi. Oleh kerana itu, kami mensasarkan untuk mengkaji kesan ranibizumab ke atas fibroblas Tenon manusia (HTF) yang telah diambil daripada pesakit yang melalui prosedur trabekulektomi. Dalam kajian lepas, kami telah melaporkan bahawa ranibizumab menurunkan kadar metabolit spermidin, iaitu sejenis poliamina penting untuk pertumbuhan sel. Dalam kajian semasa ini, kultur HTFs dibahagikan kepada tanpa rawatan, IgG kawalan, ranibizumab, difluorometilornitin (DFMO; perencat spermidin) dan ranibizumab bersama DFMO. Semua sel diekstrak untuk tatasusunan PCR (ekspresi CDKN1A, CDK2 dan CDK4) dan ekspresi protein p53, p21, CDK2 dan CDK4 secara pemblotan western. Keputusan menunjukkan sel yang dirawat ranibizumab atau DFMO serta ranibizumab dengan DFMO menyebabkan peningkatan ekspresi p53 dan p21. Sementara, tiada perubahan signifikan dilihat bagiCDKN1A, CDK2 dan CDK4 dalam semua sel. Secara ringkasnya, keputusan kami mencadangkan bahawa ranibizumab mungkin bertindak melalui protein p21 dan p53.

Kata kunci: Faktor pertumbuhan endotelium vaskular; fibroblas Tenon manusia; ranibizumab

REFERENCES

Abukhdeir, A.M. & Park, B.H. 2008. p21 and p27: Roles in carcinogenesis and drug resistance. Expert Reviews in Molecular Medicine 10: e19. https://doi.org/10.1017/S1462399408000744.

Agarwal, M.L., Agarwal, A., Taylor, W.R. & Stark, G.R. 1995. P53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts. Proceedings of the National Academy of Sciences of the United States of America 92(18): 8493-8497. https://doi.org/10.1073/pnas.92.18.8493.

Akiyode, O. & Tran, C. 2016. Overview of ocular anti-vascular endothelial growth factor therapy in the management of diabetic eye complications. Diabetes Spectrum 29(1): 44-49. https://doi.org/10.2337/diaspect.29.1.44.

Alexiou, G.A., Tsamis, K.I., Vartholomatos, E., Peponi, E., Tzima, E., Tasiou, I., Lykoudis, E., Tsekeris, P. & Kyritsis, A.P. 2015. Combination treatment of TRAIL, DFMO and radiation for malignant glioma cells. Journal of Neuro-Oncology 123(2): 217-224. https://doi.org/10.1007/s11060-015-1799-9.

Alhonen, L., Parkkinen, J.J., Keinänen, T., Sinervirta, R., Herzig, K.H. & Jänne, J. 2000. Activation of polyamine catabolism in transgenic rats induces acute pancreatitis. Proceedings of the National Academy of Sciences of the United States of America 97(15): 8290-8295. https://doi.org/10.1073/pnas.140122097.

Brugarolas, J., Moberg, K., Boyd, S.D., Taya, Y., Jacks, T. & Lees, J.A. 1999. Inhibition of cyclin-dependent kinase 2 by P21 is necessary for retinoblastoma protein-mediated G1 arrest after γ-irradiation. Proceedings of the National Academy of Sciences of the United States of America 96(3): 1002-1007. https://doi.org/10.1073/pnas.96.3.1002.

Dulić, V., Kaufmann, W.K., Wilson, S.J., Tisty, T.D., Lees, E., Harper, J.W., Elledge, S.J. & Reed, S.I. 1994. P53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell 76(6): 1013-1023. https://doi.org/10.1016/0092-8674(94)90379-4.

Elmekawey, H. & Khafagy, A. 2014. Intracameral ranibizumab and subsequent mitomycin C augmented trabeculectomy in neovascular glaucoma. Journal of Glaucoma 23(7): 437-440. https://doi.org/10.1097/IJG.0b013e3182946398.

Herbst, R.S., Johnson, D.H., Mininberg, E., Carbone, D.P., Henderson, T., Kim, E.S., Blumenschein Jr., G., Lee, J.J., Liu, D.D., Truong, M.T., Hong, W.K., Tran, H., Tsao, A., Xie, D., Ramies, D.A.,  Mass, R., Seshagiri, S., Eberhard, D.A., Kelley, S.K. & Sandler, A. 2005. Phase I/II trial evaluating the anti-vascular endothelial growth factor monoclonal antibody bevacizumab in combination with the HER-1/epidermal growth factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent non-small-cell lung cancer. Journal of Clinical Oncology 23(11): 2544-2555.

Huang, Y., Pledgie, A., Rubin, E., Marton, L.J., Woster, P.M., Saraswati Sukumar, Casero, R.A. & Davidson, N.E. 2005. Role of P53/P21Waf1/Cip1 in the regulation of polyamine analogue-induced growth inhibition and cell death in human breast cancer cells. Cancer Biology and Therapy 4(9): 1006-1013. https://doi.org/10.4161/cbt.4.9.1970.

Liang, M., Ekblad, E., Hellstrand, P. & Nilsson, B.O. 2004. Polyamine synthesis inhibition attenuates vascular smooth muscle cell migration. Journal of Vascular Research 41(2): 141-147. https://doi.org/10.1159/000077133.

Li, L., Li, J., Rao, J.N., Li, M., Bass, B.L. & Wang, J.Y. 1999. Inhibition of polyamine synthesis induces P53 gene expression but not apoptosis. American Journal of Physiology - Cell Physiology 276(4): C946-C954. https://doi.org/10.1152/ajpcell.1999.276.4.c946.

Napoleone, F. 2010. Pathways mediating VEGF-independent tumor angiogenesis. Cytokine and Growth Factor Reviews 21(1): 21-26. https://doi.org/10.1016/j.cytogfr.2009.11.003.

Napoleone, F. 2004. Vascular endothelial growth factor: Basic science and clinical progress. Endocrine Reviews 25(4): 581-611. https://doi.org/10.1210/er.2003-0027.

Nissen, N.N., Polverini, P.J., Koch, A.E., Volin, M.V., Gamelli, R.L. & DiPietro, L.A. 1998. Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing. American Journal of Pathology 152(6): 1445-1452.

Patocka, J. & Kuehn, G.D. 2000. Natural polyamines and their biological consequence in mammals. Acta Medica (Hradec Králové)/Universitas Carolina, Facultas Medica Hradec Králové 43(4): 119-124. https://doi.org/10.14712/18059694.2019.124.

Siti Munirah Md. Noh, Siti Hamimah Sheikh Abdul Kadir & Sushil Vasudevan. 2019. Important metabolites in maintaining folate cycle, homocysteine, and polyamine metabolism associated with ranibizumab treatment in cultured human Tenon’s fibroblasts. Biomolecules 9(6): 1-12. https://doi.org/10.3390/biom9060243.

Siti Munirah Md Noh, Siti H. Sheikh Abdul Kadir, Zakaria M. Bannur, Gabriele Anisah Froemming, Narimah Abdul Hamid Hasani, Hapizah Mohd Nawawi, Jonathan G. Crowston & Sushil Vasudevan. 2014. Effects of ranibizumab on the extracellular matrix production by human tenon’s fibroblast. Experimental Eye Research 127: 236-242. https://doi.org/10.1016/j.exer.2014.08.005.

Van Bergen, T., Van de Velde, S., Vandewalle, E., Moons, L. & Stalmans, I. 2014. Improving patient outcomes following glaucoma surgery: State of the art and future perspectives. Clinical Ophthalmology 8: 857-867. https://doi.org/10.2147/OPTH.S48745.

*Corresponding author; email: sitimunirah.umcic@um.edu.my