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The spectrum was limited to approximately 1. The bandpass filter was a commercially available 1. Therefore, if the degree of global methylation is reduced, the intensity of the resonance peaks observed using THz-TDS will be decreased after exposure to high-power THz radiation Fig. Global demethylation using high-power THz radiation of approximately 1. The 1. The radiation is expected to influence only the methyl-DNA bond because the THz energy is not sufficient to cause ionization. The bandwidth-limited THz radiation was applied on a genome-wide scale.
If global demethylation is caused by exposure to resonant high-power THz radiation, the amplitude of the resonance peak measured by THz-TDS could decrease after exposure at the resonant frequency. The sample holder in the setup was designed to irradiate THz radiation vertically.
Targeting DNA Methylation and Chromatin for Cancer Therapy
The radiation average power density is 2. The centre of resonance peaks for methylated DNA in blood cancer is approximately 1. Before the experiment of the blood cancer cell lines, we attempted to reduce the degree of DNA methylation in artificially methylated DNA samples.
The values from the two measurement techniques were compared by normalization. This result indicates that the resonant high-power THz radiation demethylated MT DNA and that THz demethylation is a physicochemical reaction induced by a resonance phenomenon. Because some physicochemical reactions are dependent on reaction time, the efficiency of demethylation may depend on the exposure time of THz radiation. To check this relationship, we changed the exposure time of THz radiation by minute increments. As shown in Fig. As we predicted based on Fig.
Because it was demonstrated that high-power THz radiation has the ability to demethylate artificially methylated DNA, we applied this technique to assess the demethylation effect on blood cancer DNA. These results may be a strong evidence that the demethylation of the blood cancer DNA occurs in the specific resonance frequency.
The reason for the difference in the reduction ratio was not fully elucidated, but we hypothesize that it is attributed to the difference in collective vibrations according to the arrangement or distribution of DNA methylation. In summary, resonant high-power THz radiation significantly induces global demethylation in genomic blood cancer DNA, and the rate of reduction varies according to cell line.
The types of blood cancer cell lines were the same as those used in the experiment associated with Fig. The degree of methylation is mostly in good agreement between the THz red bar and ELISA blue bar results, which was normalized to compare the results of both techniques.
The THz-TDS results were obtained by the average of three independent waveforms taking a mean value of three measurements at a point. Each measurement was performed twice and averaged.
DNA methylation and cancer therapy.
The impact of DNA methylation in molecular cancer therapy has been emphasized in recent years 26 , Some researchers have shown that demethylation has a potential in cancer therapeutic applications. DNA methylation inhibitors reduce the risk of cancer progression by preventing the hypermethylation of tumor suppressors or tumor metastasis 28 , Several chemical inhibitors and enzymes for DNA demethylation, which remove methyl group indirectly, already have been developed 30 , 31 , However, there is no current approach using an optical technique for directly manipulating the DNA methylation.
Demethylation using THz radiation might have a potential for use in clinical applications as an active demethylation technique; this method is a non-invasive, non-ionizing and non-labelling technique applied to DNA samples. Previous researches have reported that high-power THz radiation could occur the genomic instability 33 , cell division 34 , and chromosome activity 35 , Especially, Titova et al.
In this study, we suppose that the impact of other DNA structure may be minimal because the filtered THz radiation around the specific resonance frequency has lower power. However, this issue should be checked by additional experiments. We observed the resonance of the methylated DNA in various blood cancer cell lines at a frequency of 1.
We further developed this discovery into achieving demethylation by breaking the methyl-DNA bond using the resonant high-power THz radiation. Although we demonstrated the demethylation of cancer DNA from blood cancer cell lines, the molecular dynamics have not been clearly elucidated. No statistical methods were used to predetermine the sample size. No samples were excluded from the data analysis. All experiments were approved and performed in accordance with relevant guidelines and regulations by University of Seoul, and Seoul National University Boramae Medical Centre and Bondang Hospital.
To operate the THz-TDS system, the laser beam was separated into two beam lines by a polarizing beam splitter, and each beam was sent toward the emission or detection part of the system. The THz radiation was collected and transformed into a parallel beam by a parabolic mirror in front of the crystal. The sample holder was customized to freeze aqueous solution stably The liquid samples were dropped on a z-cut quartz window and covered with an upper window that was composed of hydrophobic material Teflon and used to flatten the surface of samples.
The loss of THz radiation was minimized because the refractive index is similar to that of ice. The upper window was removed after the sample was completely frozen.
The laser beam from the oscillator was amplified to 0. The tilted-pulse-front beam was incident on a LiNbO 3 crystal doped with 0. The residual laser beam was blocked by a wide silicon window, through which THz radiation could penetrate. The high-power THz radiation was gathered by a parabolic mirror and focused vertically to the centre position of the samples by another parabolic mirror. The bandwidth of THz radiation was limited by a cross-shaped bandpass filter at a 1.
Epigenetic Therapy: Histone Acetylation, DNA Methylation and Anti-Cancer Drug Discovery
The bandpass filter passed only the THz waves and blocked other infrared waves, including the nm wave that was used to generate the high-power THz radiation. A microcentrifuge tube, which contained the DNA aqueous solution samples, was held vertically by the sample holder Supplementary Fig.
The temporal shape of the THz beam was obtained using an electro-optical sampling technique with a ZnTe direction crystal with a thickness of 0. The temporal THz pulse was obtained by finding the mean among three measured data at each sampling point. We then averaged three independent measurements of the temporal THz pulse to deduce the final data. The temporal data were modified based on the presence of an electric field in the frequency domain by the fast Fourier transform FFT algorithm. A numerical optimization algorithm was applied to solve this relationship using a method proposed by Duvillaret et al.
The culture medium was replaced every 2 days to preserve cell condition. The genomic DNAs were dissolved in distilled water after purification to produce an isolated environment and to remove the noise from other residue. Before the baseline correction, the absorption coefficients consist of both the DNA methylation signal and the baseline signal originating from ice, extra DNA structures, and residual materials used to produce the DNA samples. We applied the baseline correction algorithm to separate the DNA methylation signal 17 , We set a Gaussian function as the baseline because ice has the absorption coefficient of a Gaussian form in the range of 0.
The baseline was subtracted from the measurement data. Then, we obtained the resonance peak of DNA methylation. We divided the sample into three equal portions and measured the OD data two times. The final data was obtained by averaging six data sets. We defined the comparison value as. We normalized the degree of DNA methylation obtained from both methods to the highest value as the reference for direct comparison. To assess the dependence of exposure time, the MT DNA sample was divided into five portions and irradiated with high-power THz radiation for different times at minute increments.
The samples were also divided into two portions, one of which served as the control. Egger, G. Epigenetics in human disease and prospects for epigenetic therapy. Nature , — Robertson, K. DNA methylation and human disease.
Portela, A. Epigenetic modifications and human disease. Kelly, T. Epigenetic modifications as therapeutic targets. Duffy, M.
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