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Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents.

Huang WC, Tseng TY, Chen YT, Chang CC, Wang ZF, Wang CL, Hsu TN, Li PT, Chen CT, Lin JJ, Lou PJ, Chang TC - Nucleic Acids Res. (2015)

Bottom Line: In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells.In this study, we use fluorescence lifetime imaging microscopy to verify the existence of mtDNA G4s in live cells.Bioactivity studies indicate that interactions between these anti-cancer agents and mtDNA G4 can suppress mitochondrial gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan.

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Related in: MedlinePlus

(A) Confocal images of HeLa and MRC-5 cells incubated with 5 μM BMVC-12C-P for 24 h (left) and then stained by 20 nM Mitotracker red (middle) together with their merges (right). The images were taken to visualize intracellular localization of BMVC-12C-P under optimized condition. Scale bar is 25 μm. (B) Cell viability assays of various cancer cell lines (solid line) and normal cell lines (dash line) after incubation with BMVC-12C-P from 0 μM to 20 μM for 72 h measured by MTT assay. (C) Time-dependent fluorescence intensity of 1 μM BMVC-12C-P treated HeLa and MRC-5 cells measured by flow cytometry. (D) Delayed anti-proliferation activity of BMVC-12C-P. CL1–0 cancer and MRC-5 normal cell lines were treated with 0.5 μM BMVC-12C-P. The numbers of cells were counted during the passages and the population doubling was determined. (E) BMVC-12C-P suppresses tumor growth. Nude mice (n = 4) were each injected with 1 × 106 H1299 cells. BMVC-12C-P at 3 mg/kg was injected every 3 d. The tumor size and body weight (inset) were measured every 3 d.
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Figure 2: (A) Confocal images of HeLa and MRC-5 cells incubated with 5 μM BMVC-12C-P for 24 h (left) and then stained by 20 nM Mitotracker red (middle) together with their merges (right). The images were taken to visualize intracellular localization of BMVC-12C-P under optimized condition. Scale bar is 25 μm. (B) Cell viability assays of various cancer cell lines (solid line) and normal cell lines (dash line) after incubation with BMVC-12C-P from 0 μM to 20 μM for 72 h measured by MTT assay. (C) Time-dependent fluorescence intensity of 1 μM BMVC-12C-P treated HeLa and MRC-5 cells measured by flow cytometry. (D) Delayed anti-proliferation activity of BMVC-12C-P. CL1–0 cancer and MRC-5 normal cell lines were treated with 0.5 μM BMVC-12C-P. The numbers of cells were counted during the passages and the population doubling was determined. (E) BMVC-12C-P suppresses tumor growth. Nude mice (n = 4) were each injected with 1 × 106 H1299 cells. BMVC-12C-P at 3 mg/kg was injected every 3 d. The tumor size and body weight (inset) were measured every 3 d.

Mentions: o-BMVC is a G4 ligand with no appreciable cytotoxic effects on cancer cells. However, the BMVC derivative, BMVC-12C-P, is a fluorescent anti-cancer agent. The strong fluorescence of BMVC-12C-P accumulates primarily in the mitochondria of HeLa cancer cells, whereas weak fluorescence is distributed in the cytoplasm of MRC-5 normal fibroblasts (Figure 2A). Variations in the intensity of BMVC-12C-P fluorescence between cancer and normal cells were further confirmed using flow cytometry (Figure 2B). These variations can be attributed to a significant increase in the fluorescence of BMVC-12C-P upon interacting with DNA (Supplementary Figure S1) and also to a greater accumulation of BMVC-12C-P in the mitochondria of cancer cells than in normal cells.


Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents.

Huang WC, Tseng TY, Chen YT, Chang CC, Wang ZF, Wang CL, Hsu TN, Li PT, Chen CT, Lin JJ, Lou PJ, Chang TC - Nucleic Acids Res. (2015)

(A) Confocal images of HeLa and MRC-5 cells incubated with 5 μM BMVC-12C-P for 24 h (left) and then stained by 20 nM Mitotracker red (middle) together with their merges (right). The images were taken to visualize intracellular localization of BMVC-12C-P under optimized condition. Scale bar is 25 μm. (B) Cell viability assays of various cancer cell lines (solid line) and normal cell lines (dash line) after incubation with BMVC-12C-P from 0 μM to 20 μM for 72 h measured by MTT assay. (C) Time-dependent fluorescence intensity of 1 μM BMVC-12C-P treated HeLa and MRC-5 cells measured by flow cytometry. (D) Delayed anti-proliferation activity of BMVC-12C-P. CL1–0 cancer and MRC-5 normal cell lines were treated with 0.5 μM BMVC-12C-P. The numbers of cells were counted during the passages and the population doubling was determined. (E) BMVC-12C-P suppresses tumor growth. Nude mice (n = 4) were each injected with 1 × 106 H1299 cells. BMVC-12C-P at 3 mg/kg was injected every 3 d. The tumor size and body weight (inset) were measured every 3 d.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4666356&req=5

Figure 2: (A) Confocal images of HeLa and MRC-5 cells incubated with 5 μM BMVC-12C-P for 24 h (left) and then stained by 20 nM Mitotracker red (middle) together with their merges (right). The images were taken to visualize intracellular localization of BMVC-12C-P under optimized condition. Scale bar is 25 μm. (B) Cell viability assays of various cancer cell lines (solid line) and normal cell lines (dash line) after incubation with BMVC-12C-P from 0 μM to 20 μM for 72 h measured by MTT assay. (C) Time-dependent fluorescence intensity of 1 μM BMVC-12C-P treated HeLa and MRC-5 cells measured by flow cytometry. (D) Delayed anti-proliferation activity of BMVC-12C-P. CL1–0 cancer and MRC-5 normal cell lines were treated with 0.5 μM BMVC-12C-P. The numbers of cells were counted during the passages and the population doubling was determined. (E) BMVC-12C-P suppresses tumor growth. Nude mice (n = 4) were each injected with 1 × 106 H1299 cells. BMVC-12C-P at 3 mg/kg was injected every 3 d. The tumor size and body weight (inset) were measured every 3 d.
Mentions: o-BMVC is a G4 ligand with no appreciable cytotoxic effects on cancer cells. However, the BMVC derivative, BMVC-12C-P, is a fluorescent anti-cancer agent. The strong fluorescence of BMVC-12C-P accumulates primarily in the mitochondria of HeLa cancer cells, whereas weak fluorescence is distributed in the cytoplasm of MRC-5 normal fibroblasts (Figure 2A). Variations in the intensity of BMVC-12C-P fluorescence between cancer and normal cells were further confirmed using flow cytometry (Figure 2B). These variations can be attributed to a significant increase in the fluorescence of BMVC-12C-P upon interacting with DNA (Supplementary Figure S1) and also to a greater accumulation of BMVC-12C-P in the mitochondria of cancer cells than in normal cells.

Bottom Line: In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells.In this study, we use fluorescence lifetime imaging microscopy to verify the existence of mtDNA G4s in live cells.Bioactivity studies indicate that interactions between these anti-cancer agents and mtDNA G4 can suppress mitochondrial gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan.

Show MeSH
Related in: MedlinePlus