Nanoscale Investigation of DNA Demethylation in Leukemia Cells by Means of Ultrasensitive Vibrational Spectroscopy

DNA methylation is a crucial epigenetic hallmark of cancer development but the experimental methods able to prove nanoscale modifications are very scarce. Over time, Raman and its counterpart, surface-enhanced Raman scattering (SERS), became one of the most promising techniques capable to investigate nanoscale modifications of DNA bases. In our study, we employed Raman/SERS to highlight the differences between normal and leukemia DNA samples and to evaluate the effects of a 5-azacytidine treatment on leukemia cells. To obtain spectral information related to DNA base modifications, a DNA incubation step of 4 min at 94 °C, similar to the one performed in the case of RT-PCR experiments, was conducted prior to any measurements. In this way, reproducible Raman/SERS spectra were collected for all genomic DNA samples. Our Raman results allowed discrimination between normal and cancer DNAs based on their different aggregation behavior induced by the distinct methylation landscape present in the DNA samples. On the other hand, the SERS spectra collected on the same DNA samples show a very intense vibrational band located at 1008 cm−1 assigned to a rocking vibration of 5-methyl-cytosine. The intensity of this band strongly decreases in cancer DNA due to the modification of the methylation landscape occurring in cancers. We believe that under controlled experimental conditions, this vibrational band could be used as a powerful marker for demonstrating epigenetic reprogramming in cancer by means of SERS.

[1]  A. Jemal,et al.  Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries , 2021, CA: a cancer journal for clinicians.

[2]  J. Maciejewski,et al.  Decitabine- and 5-azacytidine resistance emerges from adaptive responses of the pyrimidine metabolism network , 2020, Leukemia.

[3]  R. Marginean,et al.  Solid Plasmonic Substrates for Breast Cancer Detection by Means of SERS Analysis of Blood Plasma , 2020, Nanomaterials.

[4]  Xiao-Liang Liu,et al.  Role of epigenetic in leukemia: From mechanism to therapy. , 2020, Chemico-biological interactions.

[5]  Kaige Wang,et al.  Label-Free Detecting of the Compaction and Decompaction of ctDNA Molecules Induced by Surfactants with SERS Based on a nanoPAA-ZnCl2-AuLs Solid Substrate , 2020, ACS omega.

[6]  A. Kudelski,et al.  Surface Enhanced Raman Spectroscopy for DNA Biosensors—How Far Are We? , 2019, Molecules.

[7]  N. Leopold,et al.  SERS assessment of the cancer-specific methylation pattern of genomic DNA: towards the detection of acute myeloid leukemia in patients undergoing hematopoietic stem cell transplantation , 2019, Analytical and Bioanalytical Chemistry.

[8]  Jeremy J. Baumberg,et al.  Present and Future of Surface-Enhanced Raman Scattering , 2019, ACS nano.

[9]  Bogdan Culic,et al.  The Effects of Low-Dose Irradiation on Human Saliva: A Surface-Enhanced Raman Spectroscopy Study , 2019, Diagnostics.

[10]  V. Lazar,et al.  SERS-based differential diagnosis between multiple solid malignancies: breast, colorectal, lung, ovarian and oral cancer , 2019, International journal of nanomedicine.

[11]  I. Ben-Sahra,et al.  Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides , 2019, Cancers.

[12]  Q. Zeng,et al.  Label-free rapid identification of tumor cells and blood cells with silver film SERS substrate. , 2018, Optics express.

[13]  H. Samaratunga,et al.  Epigenetically reprogrammed methylation landscape drives the DNA self-assembly and serves as a universal cancer biomarker , 2018, Nature Communications.

[14]  Shanshan Li,et al.  Reciprocal Regulation of Metabolic Reprogramming and Epigenetic Modifications in Cancer , 2018, Front. Genet..

[15]  Siva Umapathy,et al.  Challenges in application of Raman spectroscopy to biology and materials , 2018, RSC advances.

[16]  Luca Guerrini,et al.  Direct surface-enhanced Raman scattering (SERS) spectroscopy of nucleic acids: from fundamental studies to real-life applications. , 2018, Chemical Society reviews.

[17]  H. Hallen,et al.  DNA Methylation Detection Using Resonance and Nanobowtie-Antenna-Enhanced Raman Spectroscopy. , 2018, Biophysical journal.

[18]  A. Ardekani,et al.  Role of Epigenetics in Biology and Human Diseases , 2016, Iranian biomedical journal.

[19]  S. Flis,et al.  DNA methyltransferase inhibitors and their emerging role in epigenetic therapy of cancer. , 2013, Anticancer research.

[20]  V. Chiș,et al.  Adsorption geometry of propranolol enantiomers on silver nanoparticles , 2013 .

[21]  G. Fan,et al.  DNA Methylation and Its Basic Function , 2013, Neuropsychopharmacology.

[22]  R. Momparler.  A Perspective on the Comparative Antileukemic Activity of 5-Aza-2′-deoxycytidine (Decitabine) and 5-Azacytidine (Vidaza) , 2012, Pharmaceuticals.

[23]  Helen Brady,et al.  A Comparison of Azacitidine and Decitabine Activities in Acute Myeloid Leukemia Cell Lines , 2010, PloS one.

[24]  Peter A. Jones,et al.  Epigenetics in cancer. , 2010, Carcinogenesis.

[25]  K. Faulds,et al.  Surface-enhanced Raman scattering as a detection technique for molecular diagnostics , 2009, Expert review of molecular diagnostics.

[26]  David Erickson,et al.  Surface enhanced Raman spectroscopy and its application to molecular and cellular analysis , 2009 .

[27]  F. Lyko,et al.  Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine , 2008, International journal of cancer.

[28]  N. Halas,et al.  Surface-enhanced Raman spectroscopy of DNA. , 2008, Journal of the American Chemical Society.

[29]  Keith D. Robertson,et al.  DNA Methylation Inhibitor 5-Aza-2′-Deoxycytidine Induces Reversible Genome-Wide DNA Damage That Is Distinctly Influenced by DNA Methyltransferases 1 and 3B , 2007, Molecular and Cellular Biology.

[30]  P. Vandenabeele,et al.  Reference database of Raman spectra of biological molecules , 2007 .

[31]  F. Šorm,et al.  5-Azacytidine, a new, highly effective cancerostatic , 1964, Experientia.

[32]  Peter A. Jones,et al.  Role of the DNA methyltransferase variant DNMT3b3 in DNA methylation. , 2004, Molecular cancer research : MCR.

[33]  B. Dutrillaux,et al.  Segmentation of human chromosomes induced by 5-ACR (5-azacytidine) , 1976, Human Genetics.

[34]  Bernhard Lendl,et al.  A New Method for Fast Preparation of Highly Surface-Enhanced Raman Scattering (SERS) Active Silver Colloids at Room Temperature by Reduction of Silver Nitrate with Hydroxylamine Hydrochloride , 2003 .

[35]  J. Christman,et al.  5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy , 2002, Oncogene.

[36]  V. Bloomfield,et al.  Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence. , 1999, Biopolymers.

[37]  R. Aroca,et al.  Surface enhanced vibrational spectra of thymine , 1999 .

[38]  S. Sánchez‐Cortés,et al.  SERS of cytosine and its methylated derivatives on metal colloids , 1992 .

[39]  G. Prendergast,et al.  Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region. , 1991, Science.

[40]  Jan Greve,et al.  Surface-enhanced Raman spectroscopy of DNA bases , 1986 .

[41]  Donald Macleod,et al.  A fraction of the mouse genome that is derived from islands of nonmethylated, CpG-rich DNA , 1985, Cell.

[42]  G. Thomas,et al.  Characterization of DNA structures by laser Raman spectroscopy , 1984, Biopolymers.

[43]  M. Moskovits.  Surface selection rules , 1982 .

[44]  P. Jones,et al.  Mutagenicity of 5-azacytidine and related nucleosides in C3H/10T 1/2 clone 8 and V79 cells. , 1982, Cancer research.

[45]  A. Bird.  DNA methylation and the frequency of CpG in animal DNA. , 1980, Nucleic acids research.

[46]  A. Čihák.  Biological effects of 5-azacytidine in eukaryotes. , 1974, Oncology.

[47]  W. Benedict,et al.  Chromatid Breakage: Differential Effect of Inhibitors of DNA Synthesis during G2 Phase , 1972, Science.

[48]  L H Li,et al.  Cytotoxicity and mode of action of 5-azacytidine on L1210 leukemia. , 1970, Cancer research.

[49]  S. Halle.  5-Azacytidine as a mutagen for arboviruses , 1968, Journal of virology.

[50]  F. Šorm,et al.  Nucleic acids components and their analogues. LI. Synthesis of 1-glycosyl derivatives of 5-azauracil and 5-azacytosine , 1964 .