Sains Malaysiana 53(6)(2024): 1333-1341

http://doi.org/10.17576/jsm-2024-5306-09

Dimethyloxalylglycine (DMOG)-Induced Hypoxia Promotes Migratory and Invasive Properties of HCT116 Colon Cancer Cell Line

(Hipoksia Aruhan Dimetiloksalilglisin (DMOG) Menggalakkan Sifat Migrasi dan Invasif Titisan Sel Kanser Kolon HCT116)

NOR EZLEEN QISTINA AHMAD^1,2, AMIRAH ALHUSNA MOHD YUSOFF¹, NUR FARIESHA MD HASHIM¹, NURUL AKMARYANTI ABDULLAH¹, NORAINA MUHAMAD ZAKUAN^1,*

¹Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

²Institute of Medical Science Technology, Universiti Kuala Lumpur, 43000 Kajang,

Selangor, Malaysia

Diserahkan: 1 November 2023/Diterima: 20 Mei 2024

Abstract

Hypoxia, a condition characterised by low oxygen levels, leads to increased production of a protein called hypoxia-inducible factor-1 alpha (HIF-1α) in cancer cells. This protein is involved in driving processes such as vascularization, cytoskeletal reorganisation, and epithelial-to-mesenchymal transformation (EMT), which contribute to metastasis. Previous studies used hypoxic workstations, chambers, and incubators to evaluate the effects of hypoxia on colon cancer cell lines. In a cell culture model, hypoxic conditions can also be induced using dimethyloxalylglycine (DMOG) as the hypoxia-mimicking agent. This study aims to investigate the effects of DMOG-induced hypoxia on colon cancer metastasis, focusing on cell migration and invasion. HCT116 cells were subjected to hypoxic conditions by treating them with DMOG, and the expression of HIF-1α proteins was measured at various time points, followed by wound healing and invasion assays. It was found that HIF-1α protein expression increases after 6 h of DMOG induction and persists for 24 h. At 6 and 24 h, a significantly higher percentage of hypoxic cells migrated compared to normoxic cells. The invasion assay demonstrated that hypoxic cells were more invasive than normoxic cells within 24 h. Thus, the increase in migration and invasion of cells is comparable to the increase in HIF-1α expression at 6 and 24 h. These findings suggest that DMOG induces HIF-1α expression in colon cancer cells, leading to enhanced cell migration and invasiveness. The established model can be further utilised in gene knockdown or drug treatment studies to evaluate the effects of hypoxia on cancer cells.

Keywords: Colorectal cancer; DMOG; HIF-1α; hypoxia; metastasis

Abstrak

Hipoksia, merupakan keadaan yang dicirikan oleh paras oksigen yang rendah, membawa kepada peningkatan pengeluaran protein yang dipanggil hypoxia-inducible factor-1 alpha (HIF-1α) dalam sel kanser. Protein ini terlibat dalam memacu beberapa proses seperti vaskularisasi, penyusunan semula sitoskeleton dan transformasi epitelium-ke-mesenkima (EMT) yang menyumbang kepada metastasis. Kajian terdahulu menggunakan stesen kerja, kebuk dan inkubator hipoksik untuk menilai kesan hipoksia pada titisan sel kanser kolon. Dalam model kultur sel, keadaan hipoksik juga boleh diaruh menggunakan dimetiloksalilglisin (DMOG) sebagai agen memimik hipoksia. Kajian ini bertujuan untuk mengkaji kesan hipoksi yang diaruh DMOG pada metastasis kanser kolon dengan memberi tumpuan kepada migrasi dan invasi sel. Sel HCT116 menjadi hipoksik dengan merawatnya menggunakan DMOG, kemudian ekspresi protein HIF-1α diukur pada pelbagai titik masa, diikuti dengan ujian migrasi dan ujian invasi. Didapati bahawa pengekspresan protein HIF-1α meningkat selepas 6 jam aruhan DMOG dan berterusan selama 24 jam. Pada 6 dan 24 jam, peratusan migrasi sel hipoksik meningkat dengan signifikan berbanding sel normoksik. Ujian pencerobohan menunjukkan bahawa sel hipoksik lebih invasif daripada sel normoksik dalam masa 24 jam. Oleh itu, peningkatan dalam migrasi dan pencerobohan sel adalah selari dengan peningkatan ekspresi HIF-1α pada 6 dan 24 jam. Penemuan ini menunjukkan bahawa DMOG mengaruh pengekspresan HIF-1α dalam sel kanser kolon yang membawa kepada peningkatan kadar migrasi dan pencerobohan sel. Model ini boleh digunakan selanjutnya dalam kajian berkaitan penindasan gen atau rawatan ubat untuk menilai kesan hipoksia pada sel kanser.

Kata kunci: DMOG; HIF-1α; hipoksia; kanser kolorektal; metastasis

RUJUKAN

Biller, H.L. & Schrag, D. 2021. Diagnosis and treatment of metastatic colorectal cancer. JAMA 325(7): 669.

Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R.L., Torre, L.A. & Jemal, A. 2018. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians 68(6): 394-424.

Bray, F., Laversanne, M., Weiderpass, E. & Soerjomataram, I. 2021. The ever‐increasing importance of cancer as a leading cause of premature death worldwide. Cancer 127(16): 3029-3030.

Chaffer, C.L. & Weinberg, R.A. 2011. A perspective on cancer cell metastasis. Science 331(6024): 1559-1564.

Chai, X., Wu, X., Ren, J., Du, K., Wu, X., Feng, F. & Zheng, J. 2022. Expression of HIF-1α, ANXA3, CD133 and their associations with clinicopathological parameters in human colon carcinoma. Translational Cancer Research 11(6): 1644-1651.

Chan, M.C., Ilott, N.E., Schödel, J., Sims, D., Tumber, A., Lippl, K., Mole, D.R., Pugh, C.W., Ratcliffe, P.J., Ponting, C.P. & Schofield, C.J. 2016. Tuning the transcriptional response to hypoxia by inhibiting hypoxia-inducible factor (HIF) prolyl and asparaginyl hydroxylases. Journal of Biological Chemistry 291(39): 20661-20673.

Davis, C.K., Jain, S.A., Bae, O-N., Majid, A. & Rajanikant, G.K. 2019. Hypoxia mimetic agents for ischemic stroke. Frontiers in Cell and Developmental Biology 6: 175.

Hamad, H.A., Enezei, H.H., Alrawas, A., Zakuan, N.M., Abdullah, N.A., Cheah, Y.K. & Hashim, N.F.M. 2020. Identification of potential chemical substrates as fuel for hypoxic tumors that may be linked to invadopodium formation in hypoxia-induced MDA-MB-231 breast-cancer cell line. Molecules 25(17): 3876.

Hanna, S.C., Krishnan, B., Bailey, S.T., Moschos, S.J., Kuan, P-F., Shimamura, T., Osborne, L.D., Siegel, M.B., Duncan, L.M., O’Brien, E.T., Superfine, R., Miller, C.R., Simon, M.C., Wong, K-K. & Kim, W.Y. 2013. HIF1α and HIF2α independently activate SRC to promote melanoma metastases. The Journal of Clinical Investigation 123(5): 2078-2093.

Imran Khan, M. 2022. Exploration of metabolic responses towards hypoxia mimetic DMOG in cancer cells by using untargeted metabolomics. Saudi Journal of Biological Sciences 29(10): 103426.

Jaakkola, P., Mole, D.R., Tian, Y-M., Wilson, M.I., Gielbert, J., Gaskell, S.J., von Kriegsheim, A., Hebestreit, H.F., Mukherji, M., Schofield, C.J., Maxwell, P.H., Pugh, C.W. & Ratcliffe, P.J. 2001. Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O₂-regulated prolyl hydroxylation. Science 292(5516): 468-472.

Majmundar, A.J., Wong, W.J. & Simon, M.C. 2010. Hypoxia-inducible factors and the response to hypoxic stress. Molecular Cell 40(2): 294-309.

Ministry of Health Malaysia. 2019. Malaysia National Cancer Registry Report 2012-2016.

National Cancer Patient Registry-Colorectal Cancer and Clinical Research Centre (CRC). 2014. The Second Report of the National Cancer Patient Registry-Colorectal Cancer (NCPR-CRC), 2008-2013. Putrajaya: Ministry of Health.

Neophytou, C.M., Panagi, M., Stylianopoulos, T. & Papageorgis, P. 2021. The role of tumour microenvironment in cancer metastasis: Molecular mechanisms and therapeutic opportunities. Cancers 13(9): 2053.

Ogle, M.E., Gu, X., Espinera, A.R. & Wei, L. 2012. Inhibition of prolyl hydroxylases by dimethyloxaloylglycine after stroke reduces ischemic brain injury and requires hypoxia inducible factor-1α. Neurobiology of Disease 45(2): 733-742.

Paul, C.D., Mistriotis, P. & Konstantopoulos, K. 2017. Cancer cell motility: Lessons from migration in confined spaces. Nature Reviews Cancer 17(2): 131-140.

Pavlacky, J. & Polak, J. 2020. Technical feasibility and physiological relevance of hypoxic cell culture models. Frontiers in Endocrinology 11: 57.

Rana, N.K., Singh, P. & Koch, B. 2019. CoCl₂ simulated hypoxia induce cell proliferation and alter the expression pattern of hypoxia associated genes involved in angiogenesis and apoptosis. Biological Research 52: 12.

Rankin, E.B., Nam, J-M. & Giaccia, A.J. 2016. Hypoxia: Signaling the metastatic cascade. Trends in Cancer 2(6): 295-304.

Rinderknecht, H., Ehnert, S., Braun, B., Histing, T., Nussler, A.K. & Linnemann, C. 2021. The art of inducing hypoxia. Oxygen 1(1): 46-61.

Sebestyén, A., Kopper, L., Dankó, T. & Tímár, J. 2021. Hypoxia signaling in cancer: From basics to clinical practice. Pathology and Oncology Research 27: 1609802.

Semenza, G.L. 2010. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 29(5): 625-634.

Semenza, G.L., Jiang, B-H., Leung, S.W., Passantino, R., Concordet, J-P., Maire, P. & Giallongo, A. 1996. Hypoxia response elements in the aldolase a, enolase 1, and lactate dehydrogenase a gene promoters contain essential binding sites for hypoxia-inducible factor 1. Journal of Biological Chemistry 271(51): 32529-32537.

Semenza, G.L. 2012. Hypoxia-inducible factors in physiology and medicine. Cell 148(3): 399-408.

Seyfried, T.N. & Huysentruyt, L.C. 2013. On the origin of cancer metastasis. Critical Reviews in Oncogenesis 18(1-2): 43-73.

Singh, A., Wilson, J.W., Schofield, C.J. & Chen, R. 2020. Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors induce autophagy and have a protective effect in an in-vitro ischaemia model. Scientific Reports 10(1): 1597.

Singh, E., Joffe, M., Cubasch, H., Ruff, P., Norris, S.A. & Pisa, P.T. 2017. Breast cancer trends differ by ethnicity: A report from the South African National Cancer Registry (1994-2009). European Journal of Public Health 27(1): 173-178.

Sung, H., Ferlay, J., Siegel, R.L., Laversanne, M., Soerjomataram, I., Jemal, A. & Bray, F. 2021. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians 71(3): 209-249.

Takeda, K., Cowan, A. & Fong, G-H. 2007. Essential role for prolyl hydroxylase domain protein 2 in oxygen homeostasis of the adult vascular system. Circulation 116(7): 774-781.

Uchida, T., Rossignol, F., Matthay, M.A., Mounier, R., Couette, S., Clottes, E. & Clerici, C. 2004. Prolonged hypoxia differentially regulates hypoxia-inducible factor (HIF)-1α and HIF-2α expression in lung epithelial cells. Journal of Biological Chemistry 279(15): 14871-14878.

Wang, G. & Semenza, G. 1993. Desferrioxamine induces erythropoietin gene expression and hypoxia- inducible factor 1 DNA-binding activity: Implications for models of hypoxia signal transduction. Blood 82(12): 3610-3615.

Wicks, E.E. & Semenza, G.L. 2022. Hypoxia-inducible factors: cancer progression and clinical translation. The Journal of Clinical Investigation 132(11): e159839.

Wong, C.C.L., Gilkes, D.M., Zhang, H., Chen, J., Wei, H., Chaturvedi, P., Fraley, S.I., Wong, C-M., Khoo, U-S., Ng, I.O.L., Wirtz, D. & Semenza, G.L. 2011. Hypoxia-inducible factor 1 is a master regulator of breast cancer metastatic niche formation. Proceedings of the National Academy of Sciences 108(39): 16369-16374.

Zeisberg, M. & Neilson, E.G. 2009. Biomarkers for epithelial-mesenchymal transitions. Journal of Clinical Investigation 119(6): 1429-1437.

*Pengarang untuk surat-menyurat; email: noraina@upm.edu.my