High Keratin Secretion of T47D Cell under Hypoxic Condition
Abstract
BACKGROUND: Hypoxia adaptation in cancer cells is mediated by hypoxia inducible factor (HIF)-1 that induces the expression of various proteins. Up to date, the analysis of T47D breast cancer cells-secreted protein is still limited. The aim of this study was to compare the protein profile secreted by T47D breast cancer cells under hypoxic and to normoxic conditions and identify the proteins as candidate for hypoxia marker proteins in T47D breast cancer cells.
METHODS: T47D breast cancer cells were cultured under standard conditions. Cells were subcultured in normoxic and hypoxic conditions. The normoxic group was incubated with 20% oxygen and the hypoxic groups were incubated in a hypoxic chamber with 0.5% and 5% oxygen for 6, 24, and 48 hours in serum free medium. Proteins in the culture media were isolated and precipitated. Protein concentrations released by the cells were then measured by bicinchoninic assay (BCA). Protein bands were visualized by the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method. The bands that had differences between the hypoxic and normoxic groups were further analyzed by liquid chromatography-mass spectrophotometer (LC-MS).
RESULTS: Hypoxic groups showed higher secretory protein than normoxic group. Protein bands were found in the 0.5% hypoxic group with a size of 50-75 kDa. Secretory proteins identified by LC-MS were keratin 1, 2, 9, and 10.
CONCLUSION: The T47D cell line under 0.5% hypoxic treatment showed higher secretory proteins that identified as keratin 1, 2, 9, and 10.
KEYWORDS: hypoxia, secretory protein, liquid chromatography-mass spectrophotometer
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Barrak NH, Khajah MA, Luqmani YA. Hypoxic environment may enhance migration/penetration of endocrine resistant MCF7- derived breast cancer cells through monolayers of other non-invasive cancer cells in vitro. Sci Rep. 2020; 10(1): 1127, CrossRef.
Gilkes D, Semenza G. Role of hypoxia-inducible factors in breast cancer metastasis. Futur Oncol. 2015; 9(11): 1623-36, CrossRef.
Rahman MN, Wijaya CR, Novalentina M. Survivin clinical features in cervical cancer. Mol Cell Biomed Sci. 2017; 1(1): 6-16, CrossRef.
Caroline W, Sven P, Bexell, Daniel. Therapeutic targeting of hypoxia and hypoxia-inducible factors in cancer. Pharmacol Ther. 2016; 164: 152-69, CrossRef.
Iwona B, Latosinska A, Metzger J, Marx D, Vlahou A, Mischak H, et al. Proteomics biomarkers for solid tumors: Current status and future prospects. Mass Spec Rev. 2017; 38(1): 49-78, CrossRef.
Kozakiewicz AMB, Budzik MP, Przybylsky J. Hypoxia in breast cancer. Pol J Pathol. 2015; 66(4): 337-46, CrossRef.
Sidharta VM, Herningtyas EH, Lagonda CA, Fauza D, Kusnadi Y, Susilowati R, et al. High VEGF level is produced by human umbilical cord mesenchymal stem cells (hUC-MSCs) in amino acid-rich medium and under hypoxia condition. Indones Biomed J. 2018; 10(3): 222-30, CrossRef.
Meiliana A, Dewi NM, Wijaya A. Cancer stem cell hypothesis: Implication for cancer prevention and treatment. Indones Biomed J. 2016; 8(1): 21-36, CrossRef.
Li H, DeSouza LV, Ghanny S, Li W, Romaschin AD, Colgan TJ, et al. Identification of candidate biomarker proteins released by human endometrial and cervical cancer cells using two-dimensional liquid chromatography/tandem mass spectrometry. J Proteome Res. 2007; 6(7): 2615-22, CrossRef.
Lilis L, Siregar NC, Andrijono A. The association of parametrial invasion with the expression of vascular endothelial growth factor-C and other factors in squamous cell cervical carcinoma stage IB and IIA. Indonesian Biomedical Journal. 2019; 11(3): 279-85, CrossRef.
Huang LJ, Chen SX, Luo WJ, Jiang HH, Zhang PF, Yi H. Proteomic analysis of secreted proteins of non-small cell lung cancer. Ai Zheng. 2006; 25(11): 1361-7, PMID.
Ralhan R, Desouza LV, Matta A, Tripathi SC, Ghanny S, Datta Gupta S, et al. Discovery and verification of head-and-neck cancer biomarkers by differential protein expression analysis using iTRAQ labeling, multidimensional liquid chromatography, and tandem mass spectrometry. Mol Cell Proteomics. 2008; 7(6): 1162-73, CrossRef.
Shin J, Kim HJ, Kim G, Song M, Woo SJ, Lee ST, et al. Discovery of melanotransferrin as a serological marker of colorectal cancer by secretome analysis and quantitative proteomics. J Proteome Res. 2014; 13(11): 4919-31, CrossRef.
Wanandi SI, Lestari DR, Hilbertina N, Siregar NC, Jusman SW, Abdullah M. Secretomes of primary cancer-associated fibroblasts upregulate the expression of stemness markers in HT-29 human colorectal carcinoma cells. Indones Biomed J. 2020; 12(4): 333-9, CrossRef.
Widowati W, Jasaputra DK, Sumitro SB, Widodo MA, Afifah E, Rizal R, et al. Direct and indirect effect of TNFα and IFNγ toward apoptosis in breast cancer cells. Mol Cell Biomed Sci. 2018; 2(2): 60-9, CrossRef.
Han ZB, Ren H, Zhao H, Chi Y, Chen K, Zhou B, et al. Hypoxia-inducible factor (HIF)-1α directly enhances the transcriptional activity of stem cell factor (SCF) in response to hypoxia and epidermal growth factor (EGF). Carcinogenesis. 2008; 29(10): 1853-61, CrossRef.
Mbeunkui F, Metge BJ, Shevde LA, Pannell LK. Identification of differentially secreted biomarkers using LC-MS/MS in isogenic cell lines representing a progression of breast cancer. J Proteome Res. 2007; 6(8): 2993-3002, CrossRef.
Qutub AA, Popel AS. A computational model of intracellular oxygen sensing by hypoxia-inducible factor HIF1 alpha. J Cell Sci. 2006; 119(Pt 16): 3467-80, CrossRef.
Lundgren K, Nordenskjöld B, Landberg G. Hypoxia, Snail and incomplete epithelial-mesenchymal transition in breast cancer. Br J Cancer. 2009; 101(10): 1769-81, CrossRef.
Jafari R, Rahbarghazi R, Ahmadi M, Hassanpour M, Rezaie J. Hypoxic exosomes orchestrate tumorigenesis: molecular mechanisms and therapeutic implications. J Transl Med. 2020; 18(1): 474, CrossRef.
Welsh JA, Jenkins LM, Kepley J, Lyons GC, Moore DM, Traynor T, et al. High sensitivity protein gel electrophoresis label compatible with mass-spectrometry. Biosensors (Basel). 2020; 10(11): 160, CrossRef.
Jacob JT, Coulombe PA, Kwan R, Omary MB. Types I and II keratin intermediate filament. Cold Spring Harb Perspect Biol. 2018; 10: a018275, CrossRef.
Alix-Panabières C, Vendrell JP, Slijper M, Pellé O, Barbotte E, Mercier G, et al. Full-length cytokeratin-19 is released by human tumor cells: a potential role in metastatic progression of breast cancer. Breast Cancer Res. 2009; 11(3): R39, CrossRef.
Palazzolo G, Albanese NN, DI Cara G, Gygax D, Vittorelli ML, Pucci-Minafra I. Proteomic analysis of exosome-like vesicles derived from breast cancer cells. Anticancer Res. 2012; 32(3): 847-60, PMID.
Green TM, Alpaugh ML, Barsky SH, Rappa G, Lorico A. Breast Cancer-Derived Extracellular Vesicles: Characterization and Contribution to the Metastatic Phenotype. Biomed Res Int. 2015; 2015: 634865, CrossRef.
Lee JW, Bae SH, Jeong JW, Kim SH, Kim KW. Hypoxia-inducible factor (HIF-1)alpha: its protein stability and biological functions. Exp Mol Med. 2004; 36(1): 1-12, CrossRef.
Hu X, Wu R, Shehadeh LA, Zhou Q, Jiang C, Huang X, et al. Severe hypoxia exerts parallel and cell-specific regulation of gene expression and alternative splicing in human mesenchymal stem cells. BMC Genomics. 2014; 15: 303, CrossRef.
Ni N, Navdeep S, Juan L, Karen M. Mitochondria; reactive oxygen species are require for hypoxia-induced degradation of keratin intermediate filaments. FASEB J. 2010; 24(3): 799-809, CrossRef.
Lubec G, Afjehi-Sadat L. Limitations and pitfalls in protein identification by mass spectrometry. Chem Rev. 2007; 107(8): 3568-84, CrossRef.
DOI: https://doi.org/10.18585/inabj.v14i4.1999
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