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Bis-indole derivatives with antitumor activity turn out to be specific ligands of human telomeric G-quadruplex.

Amato J, Iaccarino N, Pagano B, Morigi R, Locatelli A, Leoni A, Rambaldi M, Zizza P, Biroccio A, Novellino E, Randazzo A - Front Chem (2014)

Bottom Line: Bis-indolinone derivatives having either 2,6-disubstituted pyridine core (1a and 1b) or 1,10-disubstituted phenanthroline core (2a and 2b), already known to have antitumor activity, have been tested as potential G-quadruplex binders.Compounds 2a and 2b are able to selectively stabilize G-quadruplex over duplex DNA, and also to discriminate among different G-quadruplex structures, having a particular affinity for the parallel form of the human telomeric G-quadruplex.Both compounds are also able to induce telomeric DNA damage that may explain the activity of these compounds.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Naples "Federico II" Naples, Italy.

ABSTRACT
Bis-indolinone derivatives having either 2,6-disubstituted pyridine core (1a and 1b) or 1,10-disubstituted phenanthroline core (2a and 2b), already known to have antitumor activity, have been tested as potential G-quadruplex binders. Compounds 2a and 2b are able to selectively stabilize G-quadruplex over duplex DNA, and also to discriminate among different G-quadruplex structures, having a particular affinity for the parallel form of the human telomeric G-quadruplex. Both compounds are also able to induce telomeric DNA damage that may explain the activity of these compounds.

No MeSH data available.


Related in: MedlinePlus

Analysis of telomere damage by bis-indole derivatives 2a and 2b. BJ-EHLT fibroblasts were grown in absence (−) or in presence of the bis-indole derivatives 2a and 2b at the reported concentration. Upon 24 h, each sample was processed for IF analysis by using antibodies against γH2AX (green) and TRF1 (red) to mark DNA damage and telomeres, respectively. DAPI staining was used to mark nuclei. (A) Representative IF images acquired by using a Leica Deconvolution microscope (magnification 63×). Enlarged views of TIFs are reported on the right of each picture. (B,C) Quantification of TIF-positive cells (B) and mean of TIFs per nucleus (C) from IF experiments reported in (A). Data are means ± SD of three independent experiments. p-values were calculated using the student t-test (**p < 0.005; ***p < 0.001).
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Figure 6: Analysis of telomere damage by bis-indole derivatives 2a and 2b. BJ-EHLT fibroblasts were grown in absence (−) or in presence of the bis-indole derivatives 2a and 2b at the reported concentration. Upon 24 h, each sample was processed for IF analysis by using antibodies against γH2AX (green) and TRF1 (red) to mark DNA damage and telomeres, respectively. DAPI staining was used to mark nuclei. (A) Representative IF images acquired by using a Leica Deconvolution microscope (magnification 63×). Enlarged views of TIFs are reported on the right of each picture. (B,C) Quantification of TIF-positive cells (B) and mean of TIFs per nucleus (C) from IF experiments reported in (A). Data are means ± SD of three independent experiments. p-values were calculated using the student t-test (**p < 0.005; ***p < 0.001).

Mentions: To evaluate whether γH2AX was phosphorylated in response to dysfunctional telomeres, the most effective drug concentrations of both compounds were tested by double immunofluorescence (IF). The analysis performed by deconvolution microscopy revealed that both compounds induced γH2AX foci that colocalized with TRF1, an effective marker for telomeres, generating the so-called telomere-dysfunction induced foci (TIFs) (Takai et al., 2003) (Figure 6), clearly indicating that the tested compounds caused telomere localized damage. Consistent with these data, results from quantitative analysis revealed that both 2a and 2b significantly increased the percentage of cells with more than four γH2AX/TRF1 colocalizations (Pearson's correlation coefficient ≥0.45), with a mean of about 6 TIFs per nucleus (Figure 6).


Bis-indole derivatives with antitumor activity turn out to be specific ligands of human telomeric G-quadruplex.

Amato J, Iaccarino N, Pagano B, Morigi R, Locatelli A, Leoni A, Rambaldi M, Zizza P, Biroccio A, Novellino E, Randazzo A - Front Chem (2014)

Analysis of telomere damage by bis-indole derivatives 2a and 2b. BJ-EHLT fibroblasts were grown in absence (−) or in presence of the bis-indole derivatives 2a and 2b at the reported concentration. Upon 24 h, each sample was processed for IF analysis by using antibodies against γH2AX (green) and TRF1 (red) to mark DNA damage and telomeres, respectively. DAPI staining was used to mark nuclei. (A) Representative IF images acquired by using a Leica Deconvolution microscope (magnification 63×). Enlarged views of TIFs are reported on the right of each picture. (B,C) Quantification of TIF-positive cells (B) and mean of TIFs per nucleus (C) from IF experiments reported in (A). Data are means ± SD of three independent experiments. p-values were calculated using the student t-test (**p < 0.005; ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Analysis of telomere damage by bis-indole derivatives 2a and 2b. BJ-EHLT fibroblasts were grown in absence (−) or in presence of the bis-indole derivatives 2a and 2b at the reported concentration. Upon 24 h, each sample was processed for IF analysis by using antibodies against γH2AX (green) and TRF1 (red) to mark DNA damage and telomeres, respectively. DAPI staining was used to mark nuclei. (A) Representative IF images acquired by using a Leica Deconvolution microscope (magnification 63×). Enlarged views of TIFs are reported on the right of each picture. (B,C) Quantification of TIF-positive cells (B) and mean of TIFs per nucleus (C) from IF experiments reported in (A). Data are means ± SD of three independent experiments. p-values were calculated using the student t-test (**p < 0.005; ***p < 0.001).
Mentions: To evaluate whether γH2AX was phosphorylated in response to dysfunctional telomeres, the most effective drug concentrations of both compounds were tested by double immunofluorescence (IF). The analysis performed by deconvolution microscopy revealed that both compounds induced γH2AX foci that colocalized with TRF1, an effective marker for telomeres, generating the so-called telomere-dysfunction induced foci (TIFs) (Takai et al., 2003) (Figure 6), clearly indicating that the tested compounds caused telomere localized damage. Consistent with these data, results from quantitative analysis revealed that both 2a and 2b significantly increased the percentage of cells with more than four γH2AX/TRF1 colocalizations (Pearson's correlation coefficient ≥0.45), with a mean of about 6 TIFs per nucleus (Figure 6).

Bottom Line: Bis-indolinone derivatives having either 2,6-disubstituted pyridine core (1a and 1b) or 1,10-disubstituted phenanthroline core (2a and 2b), already known to have antitumor activity, have been tested as potential G-quadruplex binders.Compounds 2a and 2b are able to selectively stabilize G-quadruplex over duplex DNA, and also to discriminate among different G-quadruplex structures, having a particular affinity for the parallel form of the human telomeric G-quadruplex.Both compounds are also able to induce telomeric DNA damage that may explain the activity of these compounds.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Naples "Federico II" Naples, Italy.

ABSTRACT
Bis-indolinone derivatives having either 2,6-disubstituted pyridine core (1a and 1b) or 1,10-disubstituted phenanthroline core (2a and 2b), already known to have antitumor activity, have been tested as potential G-quadruplex binders. Compounds 2a and 2b are able to selectively stabilize G-quadruplex over duplex DNA, and also to discriminate among different G-quadruplex structures, having a particular affinity for the parallel form of the human telomeric G-quadruplex. Both compounds are also able to induce telomeric DNA damage that may explain the activity of these compounds.

No MeSH data available.


Related in: MedlinePlus