HCT116 ve HT29 Kolon Kanseri Hücrelerinde 5-Florourasil Kaynaklı Hücre Ölümünün Fourier Dönüşümlü Kızılötesi Spektroskopisi ile İncelenmesi

Year 2023, Volume: 11 Issue: 1, 524 - 535, 31.01.2023

Dilşad Özerkan , Ferdane Danışman Kalındemirtaş Naci Tüzemen

https://doi.org/10.29130/dubited.993669

Abstract

Kolon kanseri, tüm dünyada yaygın olan ve yüksek oranda ölümcül bir kanser tipidir. Tüm kanserlerde olduğu gibi kolon kanserinin seyrinin izlenmesi kritik bir öneme sahiptir. 5-Florourasil (5-FU) kolon kanserlerinde sıklıkla kullanılan bir antikanser ilaçtır ve hücre ölümü esnasında hücrelerde bir takım biyokimyasal ve moleküler farklılıklara neden olmaktadır. Bu çalışmada HCT116 ve HT29 kolon kanseri hücreleri 5-FU kemoterapi ilacı ile inkübe edildikten sonra, 5FU’nun hücreler üzerinde oluşturduğu biyokimyasal değişikliğin tespiti için Fourier transform kızılötesi (FTIR) spektroskopisi kullanılmıştır. 5-FU'nun inhibisyon yüzdesinin 50'ye eşit olduğu konsantrasyonu (IC50), MTT yöntemi kullanılarak belirlendi. IC50 değeri saptandıktan sonra FTIR ölçümleri alındı. Buna göre, 5-FU IC50 değeri sırasıyla HCT116 hücreleri için 12,69 µg/ml, HT29 hücreleri için 10,10 µg/ml bulunmuştur. 5-FU’nun sebep olduğu hücre ölümünün göstergeleri olduğu söylenen lipit içeriğindeki artış ile nükleik asit oranı, total hücresel proteinlerin α-sarmal ve β-yaprak ikincil yapıları ve aminoasit kalıntılarının azalması, kollajen konformasyonundaki değişiklikler gibi IR biyobelirteçlerindeki değişiklikler FTIR spektroskopisi ile gösterilmiştir. Sonuç olarak hem ucuz ve hem de hızlı bir yöntem olan FTIR ile kanser hücre ölümünün incelenmesi alternatif bir yöntem olarak kullanılabilir.

Keywords

Kolon kanseri, 5-florourasil, FTIR, Colon cancer, 5-fluorouracil

Thanks

Çalışmada gerek laboratuvar imkanları gerek fikirleriyle bize yol gösteren sayın Prof. Dr. D. Serap KURUCA’ya teşekkürlerimizi iletiriz.

Investigation of 5-Fluorouracil-Induced Cell Death in HCT116 and HT29 Colon Cancer Cells by Fourier Transform Infrared Spectroscopy

Abstract

Colon cancer is a common and highly lethal type of cancer all over the world. As with all cancers, monitoring the course of colon cancer is critical. 5-Fluorouracil (5-FU) is an anticancer drug frequently used in colon cancers and causes some biochemical and molecular differences in cells during cell death. In this study, after incubation of HCT116 and HT29 colon cancer cells with 5-FU chemotherapy drug, Fourier transform infrared (FTIR) spectroscopy was used to detect the biochemical change caused by 5FU on cells. The concentration (IC50) of 5-FU at which percent inhibition equals 50 was determined using the MTT method. FTIR measurements were taken after the IC50 value was determined. Accordingly, the IC50 value of 5-FU was found to be 12.69 µg/ml for HCT116 cells and 10.10 µg/ml for HT29 cells, respectively. FTIR spectroscopy showed changes in IR biomarkers such as the increase in lipid content, which is said to be indicators of cell death caused by 5-FU, and the nucleic acid ratio, the α-helix and β-sheet secondary structures of total cellular proteins, and the reduction of amino acid residues, and changes in collagen conformation. As a result, examining cancer cell death with FTIR, which is both a cheap and fast method, can be used as an alternative method.

Keywords

Colon cancer, 5-fluorouracil

References

• [1]T. Reya, S.J. Morrison, M.F. Clarke, and I.L. Weissman, “Stem cells, cancer, and cancer stem cells,” Nature, vol. 414, pp. 105–111, 2001.
• [2]L.H. Kushi, T. Byers, C. Doyle, C. L. Rock, W. Demark‐Wahnefried, E. V. Bandera, et al. “American cancer society guidelines on nutrition and physical activity for cancer prevention: reducing the risk of cancer with healthy food choices and physical activity,” CA Cancer J Clin, vol. 56, pp. 254‑281, 2006.
• [3]F. Bray, J. Ferlay, I Soerjomataram, R.L. Siegel, L.A. Torre, and A. Jemal, “Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries,” CA Cancer J Clin, vol. 68, pp. 394-424, 2018.
• [4]K. Sasaki, N.H. Tsuno, E. Sunami, G. Tsurita, K. Kawai, Y. Okaji, et al. “Chloroquine potentiates the anti cancer effect of 5 fluorouracil on colon cancer cells,” BMC Cancer, vol. 10, pp. 370, 2010.
• [5]G. Folprecht and C.H. Kohne, “The role of new agents in the treatment of colorectal cancer,” Oncology, vol. 66, pp. 1‑17, 2004.
• [6]M.D. Wyatt and D.M. Wilson, “Participation of DNA repair in the response to 5‑fluorouracil,” Cell Mol Life Sci, vol. 66, no.3, pp. 788‑799, 2009.
• [7]A.L. Stelling, D. Toher, O. Uckermann, J. Tavkin, E. Leipnitz, J. Schweizer, et al. “Infrared spectroscopic studies of cells and tissues: triple helix proteins as a potential biomarker for tumors,” PLoS One, vol. 8, pp. e58332, 2013.
• [8]M.J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, et al. (2014). “Using Fourier transform IR spectroscopy to analyze biological materials,” Nature protocols, vol.9(8), pp. 1771-1791, 2014.
• [9]A. Dazzi, C. B. Prater, “AFM-IR: Technology and applications in nanoscale infrared spectroscopy and chemical imaging.” Chemical reviews, vol. 117, no.7, pp. 5146-5173, 2017.
• [10]G. Bellisola and C. Sorio, “Infrared spectroscopy and microscopy in cancer research and diagnosis,” Am J Cancer Res, vol. 2, pp. 121, 2012.
• [11]G. Güler, E. Acikgoz, N.Ü.K. Yavasoglu, B. Bakan, E. Goormaghtigh ve H. Aktug, “Deciphering the biochemical similarities and differences among mouse embryonic stem cells, somatic and cancer cells using ATR-FTIR spectroscopy,” Analyst, vol. 143, no. 7, pp. 1624-1634, 2018.
• [12]A. Sala, D.J. Anderson, P.M. Brennan, H.J. Butler, J.M. Cameron, M.D. Jenkinson, et al.“Biofluid diagnostics by FTIR spectroscopy: A platform technology for cancer detection,” Cancer letters, vol. 477, pp. 122-130, 2020.
• [13]P. Giamougiannis, C.L. Morais, B. Rodriguez, N.J. Wood, P. L. Martin-Hirsch, and F. L. Martin, “Detection of ovarian cancer (±neo-adjuvant chemotherapy effects) via ATR-FTIR spectroscopy: comparative analysis of blood and urine biofluids in a large patient cohort,”.Analytical and bioanalytical chemistry, pp. 1-13, 2021.
• [14]P. Thirukumaran, A. Shakila, S. Muthusamy, “Synthesis and characterization of novel bio-based benzoxazines from eugenol,” Rsc Advances, vol. 4, no. 16, pp. 7959-7966, 2014.
• [15]C. Junhom, N. Weerapreeyakul, W. Tanthanuch, K. Thumanu, “FTIR microspectroscopy defines early drug resistant human hepatocellular carcinoma (HepG2) cells,” Exp. cell Res, vol. 340, no. 1, pp. 71–80, 2016.
• [16]P. G. Andrus and R.D. Strickland, “Cancer grading by Fourier transform infrared spectroscopy,” Biospectros. vol. 4, no. 1, pp. 37–46, 1998.
• [17]X. Li, J. Lin, J. Ding, S. Wang, Q. Liu and S. Qing, “Raman spectroscopy and fluorescence for the detection of liver cancer and abnormal liver tissue,” In Engineering in Medicine and Biology Society, IEMBS’04, 26th Annual International Conference of the IEEE, San Francisco, CA, 2004, pp. 212–215.
• [18]T. Mosmann, “Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays,” J Immunol Methods, vol. 65, pp. 55–63,1983.
• [19]S. Chakrabarty, “Regulation of human colon-carcinoma cell adhesion to extracellular matrix by transforming growth factor beta 1,” Int J Cancer, vol. 50, no. 6, pp. 968–73,1992.
• [20]S.R. Choi, M. Cho, H.R. Kim, D.H. Ahn, M.H. Sleisenger, Y.S. Kim, “Biological properties and expression of mucins in 5-fluorouracil resistant HT29 human colon cancer cells,” Int J Oncol, vol.17(1), pp. 141–147, 2000.
• [21]M.J. Demers, S. Thibodeau, D. Noël, N. Fujita, T. Tsuruo et al. “Gauthier R, Arguin M, Vachon PH. Intestinal epithelial cancer cell anoikis resistance: EGFR-mediated ­sustained activation of Src overrides Fak-dependent signaling to MEK/Erk and/or PI3-K/Akt-1,” J Cell Biochem, vol. 107, no. 4, pp. 639–654, 2009.
• [22]F. Lokiec, F. Ghiringhelli, A. Bernard, A. “Isolated Lung Perfusion as an Adjuvant Treatment of Colorectal Cancer Lung Metastases: A Preclinical Study in a Pig Model,” PLoS One, vol. 8, no. 3, p. e59485, 2013.
• [23]G. B. Park, Y. H. Chung, D. Kim, “2-Deoxy-D-glucose suppresses the migration and reverses the drug resistance of colon cancer cells through ADAM expression regulation,” Anti-Cancer Drugs, vol. 28, no. 4, pp. 410-420, 2017.
• [24]M. Khanmohammadi, and A. B. Garmarudi, “Infrared spectroscopy provides a green analytical chemistry tool for direct diagnosis of cancer,” TrAC Trends Analytical Chem, vol. 30, no. 6, pp. 864– 874, 2011.
• [25]G. E. Menzies, H. R. Fox, C. Marnane, L. Pope, V. Prabhu, S. Winter, P. D. Lewis, “Fourier transform infrared for noninvasive optical diagnosis of oral, oropharyngeal, and laryngeal cancer,” Translational Res, vol. 163, no. 1, pp. 19–26, 2014.
• [26]L. Chiriboga, P. Xie, H. Yee, V. Vigorita, D. Zarou, D. Zakim and M. Diem, “Infrared spectroscopy of human tissue. I. Differentiation and maturation of epithelial cells in the human cervix,” Biospectrosc, vol. 4, no. 1, pp. 47–53,1998.
• [27]N. Fujioka, Y. Morimoto, T. Arai, M. Kikuchi, “Discrimination between normal and malignant human gastric tissues by Fourier transform infrared spectroscopy,” Cancer Detect. Prev, vol. 28, no. 1, pp. 32–36, 2004.
• [28]J. Liu, Q.H. Zhang, F. Ma, S.F. Zhang, Q. Zhou and A.M. Huang, “Three-step identification of infrared spectra of similar tree species to Pterocarpus santalinus covered with beeswax,” Journal of Molecular Structure, vol. 1218, p. 128484, 2020.
• [29]G.I. Dovbeshko, N.Y. Gridina, E.B. Kruglova and O.P. Pashchuk, “FTIR spectroscopy studies of nucleic acid damage,” Talanta, vol. 53, no. 1, pp. 233–246, 2000.
• [30]S. Prabhakar, N. Jain and R.A. Singh, “Infrared spectra in monitoring biochemical parameters of human blood,” In Journal of Physics: Conference Series, IOP Publishing, 2012, vol. 365, no. 1, p. 012059.
• [31]Y. Liu, Y. Xu, Y. Zhang, D. Wang, D. Xiu, Z. Xu and X. Ling, “Detection of cervical metastatic lymph nodes in papillary thyroid carcinoma by Fourier transform infrared spectroscopy,” Br. J. Surg, vol. 98, no. 3, pp. 380–384, 2011.
• [32]I. Dreissig, S. Machill, R. Salzer and C. Krafft, “Quantification of brain lipids by FTIR spectroscopy and partial least squares regression,” Spectrochim. Acta Part A: Mol. Biomol. Spectros, vol. 71, no. 5, pp. 2069–2075, 2009.
• [33]N. Q. Trung, P.T. Phuong Nam, N.T. Phuong Chi and N. Van Tuyen, “[Fe (III)(MeO‐salen) Cl] complexes and their in vitro cytotoxicity against KB and HepG2 human cancer cells,” Vietnam Journal of Chemistry, vol. 56, no. 6, pp. 689-694, 2018.
• [34]D.G. Conceição, B.H.R. Gonçalves, F.F.D. Hora, A.S. Faleiro, L.S. Santos and S.P. Ferrão, “Use of FTIR-ATR spectroscopy combined with multivariate analysis as a screening tool to identify adulterants in raw milk,” Journal of the Brazilian Chemical Society, vol. 30, pp. 780-785, 2019.
• [35]K.M. Ostrowska, A. Garcia, A. D. Meade, A. Malkin, I. Okewumi, J.J. O’Leary, and F.M. Lyng, “Correlation of p16 INK4A expression and HPV copy number with cellular FTIR spectroscopic signatures of cervical cancer cells,” Analyst, vol. 136, no. 7, pp. 1365–1373, 2011.
• [36]A.R. Garifzyanov, N.V. Davletshina, L.I. Akhmadullina, I.T. Safiullin and R.A. Cherkasov, “Synthesis of New Aminophosphabetaines,” Russian Journal of General Chemistry, vol. 88, no. 11, pp. 2445-2448. 2018.
• [37]S.W. Fogarty, I.I. Patel, J. Trevisan, T. Nakamura, C. J. Hirschmugl, N. J., Fullwood, and F. L. Martin, “Sub-cellular spectrochemical imaging of isolated human corneal cells employing synchrotron radiation-based Fourier-transform infrared microspectroscopy,” Analyst, vol. 138, pp. 240-248, 2013.
• [38]T. Li, Y. Qian, H. Li, J. Deng, “Combination of serum lipids and cancer antigens as a novel marker for colon cancer diagnosis,” Lipids Health Dis, vol. 17, p. 261, 2018.
• [39]F. Probert, V. Ruiz-Rodado, D. Te Vruchte, E.R. Nicoli, T.D.W. Claridge, et al. “NMR analysis reveals significant differences in the plasma metabolic profiles of Niemann Pick C1 patients, heterozygous carriers, and healthy controls,” Sci. Rep, vol. 7, p. 6320, 2017.
• [40]E. Batard, F. Jamme, D. Boutoille, C. Jacqueline, J. Caillon, G. Potel, and P. Dumas, “Fourier transform infrared microspectroscopy of endocarditis vegetation,” Appl Spectrosc, vol. 64, pp. 901-906, 2010.
• [41]G.W. Abbott, B. Ramesh and S.K. Srai, “Interaction between soluble and membrane‑embedded potassium channel peptides monitored by fourier transform infrared spectroscopy,” PLoS One, vol. 7, p. e49070, 2012.
• [42] H.H. Mantschn and R.A. Shaw, “Vibrational spectroscopy and medicine: an alliance in the making,” Vib Spectrosc, vol. 30, pp. 31‑41, 2002.
• [43]A. Bonifacio and V. Serg, “Effects of sample orientation in Raman microspectroscopy of collagen fibers and their impact on the interpretation of the amide III band,” Vibrational Spectroscopy, vol. 53, no. 2, pp. 314-317, 2010.
• [44]Y. Liu and W.F. Bodmer, “Analysis of P53 mutations and their expression in 56 colorectal cancer cell lines,” Proc Natl Acad Sci USA, vol. 103, pp. 976–981, 2006.
• [45]S.L. Volchenboum, C. Li, S. Li, E. F. Attiyeh, C. P. Reynolds, J. M. Maris, et al, “Comparison of primary neuroblastoma tumors and derivative early-passage cell lines using genome-wide single nucleotide polymorphism array analysis,” Cancer Res, vol. 69, pp. 4143–4149, 2009.
• [46]E.J. Douglas, H. Fiegler, A. Rowan, S. Halford, D. C. Bicknell, W. Bodmer, et al. “Array comparative genomic hybridization analysis of colorectal cancer cell lines and primary carcinomas,” Cancer Res, vol. 64, pp. 4817–4825, 2004.
• [47]J.K Willson, G.N. Bittner, T.D. Oberley, L.F. Meisner, and J.L. Weese, “Cell culture of human colon adenomas and carcinomas,” Cancer Res, vol. 47, pp. 2704–2713, 1987.
• [48]T. M. Yeung, S. C. Gandhi, J. L. Wilding, R. Muschel, and W.F. Bodmer, “Cancer stem cells from colorectal cancer-derived cell lines,” Proceedings of the National Academy of Sciences, vol. 107, no. 8, 3722-3727, 2010.
• [49]J.L. Arrondo, A. Muga, J. Castresana, F.M. Goni, “Quantitative studies of the structure of proteins in solution by Fourier-transform infrared spectroscopy,” Prog Biophys Mol Biol. vol. 59, no.1, pp.23-56, 1993.
• [50]J.L. Arrondo, F.M. Goni, “Structure and dynamics of membrane proteins as studied by infrared spectroscopy,” Prog Biophys Mol Biol, vol.72, no. 4, pp.367-405, 1999.