Inhibition of Cancer Cell Migration and Glycolysis by Terahertz Wave Modulation via Altered Chromatin Accessibility

Abstract

Metastasis and metabolic disorders contribute to most cancer deaths and are potential drug targets in cancer treatment. However, corresponding drugs inevitably induce myeloid suppression and gastrointestinal toxicity. Here, we report a nonpharmaceutical and noninvasive electromagnetic intervention technique that exhibited long-term inhibition of cancer cells. Firstly, we revealed that optical radiation at the specific wavelength of 3.6 μm (i.e., 83 THz) significantly increased binding affinity between DNA and histone via molecular dynamics simulations, providing a theoretical possibility for THz modulation- (THM-) based cancer cell intervention. Subsequent cell functional assays demonstrated that low-power 3.6 μm THz wave could successfully inhibit cancer cell migration by 50% and reduce glycolysis by 60%. Then, mRNA sequencing and assays for transposase-accessible chromatin using sequencing (ATAC-seq) indicated that low-power THM at 3.6 μm suppressed the genes associated with glycolysis and migration by reducing the chromatin accessibility of certain gene loci. Furthermore, THM at 3.6 μm on HCT-116 cancer cells reduced the liver metastasis by 60% in a metastatic xenograft mouse model by splenic injection, successfully validated the inhibition of cancer cell migration by THM in vivo. Together, this work provides a new paradigm for electromagnetic irradiation-induced epigenetic changes and represents a theoretical basis for possible innovative therapeutic applications of THM as the future of cancer treatments.

Scheme 1

Schematic of the suppression effects of THM on cancer cells. THM at a specific wavelength (3.6  μm) significantly inhibited the migration and glycolysis of cancer cells by altering the chromatin accessibility of genes.

Figure 1

The binding free energy between histones and DNA before and after being irritated by THz wave. (a) Schematic of the simulated nucleosome core particle with histones in the middle wrapped by DNA helices and immersed in NaCl solution. (b) Four decomposed energetic terms of the total binding free energy between protein and DNA in the baseline case. (c) Two typical salt bridges (blue dashed lines) formed between PO4- of adenine (A)/guanine (G) bases and the positively charged side chains of arginine and lysine residues. (d) Lysine and arginine moieties of the histones located within 7 Å of the DNA. (e) Variations of the four energetic terms under 3.64  μm THM.

Figure 2

THM (3.6  μm) profoundly inhibited cancer cell migration and glycolysis at a power of 0.3 mW. (a) The red line represents the relationship between the theoretically calculated phase matching angle and the output wavelength, while the green line represents the measured values. (b) Different phase matching angles correspond to the average power of infrared light generated at different wavelengths. (c) Temperature changes after THM for 300 s ( $P=5/0.3$  mW; $\lambda =3.6$ μm). (d and e) MTS results showing the effect of different THM power ( $P=5/0.3$  mW) on SW480 cell proliferation. (f and g) EdU results showing the effects of different THM power ( $P=5/0.3$  mW) on SW480 cell growth. Scale bar, 100  μm (left). (h and i) Transwell migration assay results showing the effects of different THM power ( $P=5/0.3$  mW) on SW480 cell migration. Scale bar, 100  μm (left). (j) Effects of THM ( $P=0.3$  mW) on extracellular acidification rate. (k) THM downregulated maximum glycolysis, glycolytic metabolic capacity, and glycolytic reserve ability of SW480 cells. (l) Effects of THM ( $P=0.3$  mW) on oxygen consumption rate. (m) THM did not influence basal respiratory capacity, maximum respiratory capacity, or respiratory reserve capacity of SW480 cells. All data are shown as $\text{means}\pm \text{SEM}$ , ^# $P<0.05$ , and ^## $P<0.01$ . NS: not significant.

Figure 3

RNA-seq revealed differentially expressed genes under 3.6  μm THM. (a) Identification of differentially expressed genes by RNA-seq induced by THM (genes with a log10-fold $\text{change}>0.176$ and a $P$ value < 0.01 are colored red, while those with a log10-fold $\text{change}<-0.176$ and a $P$ value less than 0.01 are colored blue; others are colored green). (b) Heat map of significantly differentially expressed genes between the control and 3.6  μm THM groups (with $P<0.01$ ), with red indicating upregulation and blue indicating downregulation. Hierarchical clustering of transcriptional profiles in the control group and 3.6  μm THM group. The color key represents FPKM normalized log2 transformed counts, and each row represents a gene. (c) GO analysis of differentially expressed genes. Each row represents a BP term; the size indicates the number of genes enriched; the color intensity indicates the -log ( $P$ value). (d) KEGG pathway analysis of differentially expressed genes (DEGs). Each row represents a KEGG term; the size indicates the number of genes enriched; the color indicates the -log ( $P$ value). (e) Top five GSEA pathways. The color intensity indicates the fold change, and the size indicates the number of genes enriched. (f) GSEA analysis revealed that glycolysis- and hypoxia-associated genes were inhibited.

Figure 4

Genomic chromatin accessibility altered by THM (3.6  μm and 0.3 mW). (a) Identification of differentially accessible genes by ATAC-seq induced by THM. The horizontal axis indicates the normalized and log2-transformed average degree of the opening of each peak, the vertical axis indicates the difference on the normalized and log2-transformed average degree of the opening of each peak resulted by THM (specific peaks only detected in the THM group are colored red; specific peaks only detected in the control group are colored blue; common peaks detected in both groups are colored grey). (b) Density distribution of reads in chromosomes (the horizontal axis represents the normalized gene location, and the vertical axis represents the read density). TSS: transcription start site; TES: transcription stop site. (c) GO analysis of differential peaks. Each row represents a BP term; the size indicates the number of genes enriched; the color indicates the -log ( $P$ value). (d) KEGG pathway analysis of differential peaks. Each row represents a KEGG term; the size indicates the number of genes enriched; the color intensity indicates the -log ( $P$ value). (e) Cytoscape pathway analysis of differentially opened peaks. (f) Cytoscape pathway analysis of differentially closed peaks. (g and h) TFs binding to the regions with differentially opened and closed peaks.

Figure 5

Altered genomic chromatin accessibility resulted in expression changes of genes governing the migration and glycolysis of cancer cells. (a) GO analysis of differential peaks (the genes exhibited >1.5-fold change in mRNA levels with changes in chromatin accessibility peaks). Each row represents a BP term; the size indicates the number of genes enriched; the color indicates the -log ( $P$ value). (b) Circos plot displaying interconnectivity among genes altered by THM ( $\lambda =3.6$ μm; $P=0.3$  mW). (c) RT-qPCR confirmed the increased levels of CPQ, ABHD5, AB13BP, RBMS3, and CGNL1 under THM. (d) RT-qPCR confirmed the decreased levels of ITGA5, TMOD2, RTN4R, and CCBE1 under THM. Results are expressed as $\text{means}\pm \text{SEM}$ ( ^## $P<0.01$ ). NS: not significant.

Figure 6

THM at 3.6  μm decreased the liver metastasis of cancer cells in vivo. (a) Body weight of the mice after splenic injection of HCT-116 cancer cells. (b) The bioluminescence images of all mice 4 weeks after splenic injection. The upper panel showed the mice of control group, while the lower panel displayed the THM group. The bioluminescence signals are presented in color: blue for the lowest and red for the highest intensity. (c) The images of the livers with and without metastasis harvested 4 weeks after splenic injection. The blue arrows indicated the metastasis foci. (d) The comparison on the number of hepatic nodules between the THM group and the control group. (e) The comparison on the bioluminescence signals between the two groups. (f) The comparison on the weight of liver between the two groups ( $n=7$ for each group, ^# $\text{P}<0.05$ ).