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Formation and stabilization of the telomeric antiparallel G-quadruplex and inhibition of telomerase by novel benzothioxanthene derivatives with anti-tumor activity.

Zhang W, Chen M, Ling Wu Y, Tanaka Y, Juan Ji Y, Lin Zhang S, He Wei C, Xu Y - Sci Rep (2015)

Bottom Line: Results show that six compounds could differentially induce 26 nt G-rich oligonucleotides to form the G-quadruplex with high selectivity vs C-rich DNA, mutated DNA and double-stranded DNA, stabilize it with high affinity, promote apoptosis and inhibit mobility and telomerase activity of A549 cells and SGC7901 cells.Especially, S1, S3, S4 displayed stronger abilities, of which S3 was the most optimal with the maximum ΔTm value being up to 29.8 °C for G-quadruplex, the minimum IC50 value being 0.53 μM and the maximum cell inhibitory rate being up to 97.2%.This study suggests that this type of compounds that induce the formation of and stabilize the telomeric antiparallel G-quadruplex, and consequently inhibit telomerase activity, leading to cell apoptosis, can be screened for the discovery of novel antitumor therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Lab of Chemical Biology and Molecular Drug Design, College of Pharmaceutical Science, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, China.

ABSTRACT
G-quadruplexes formed in telomeric DNA sequences at human chromosome ends can be a novel target for the development of therapeutics for the treatment of cancer patients. Herein, we examined the ability of six novel benzothioxanthene derivatives S1-S6 to induce the formation of and stabilize an antiparallel G-quadruplex by EMSA, UV-melting and CD techniques and the influence of S1-S6 on A549 and SGC7901 cells through real-time cell analysis, wound healing, trap assay methods. Results show that six compounds could differentially induce 26 nt G-rich oligonucleotides to form the G-quadruplex with high selectivity vs C-rich DNA, mutated DNA and double-stranded DNA, stabilize it with high affinity, promote apoptosis and inhibit mobility and telomerase activity of A549 cells and SGC7901 cells. Especially, S1, S3, S4 displayed stronger abilities, of which S3 was the most optimal with the maximum ΔTm value being up to 29.8 °C for G-quadruplex, the minimum IC50 value being 0.53 μM and the maximum cell inhibitory rate being up to 97.2%. This study suggests that this type of compounds that induce the formation of and stabilize the telomeric antiparallel G-quadruplex, and consequently inhibit telomerase activity, leading to cell apoptosis, can be screened for the discovery of novel antitumor therapeutics.

No MeSH data available.


Related in: MedlinePlus

CD spectra for the interaction between benzothioxanthene derivatives S1, S3, S4 and 26 nt telomeric G-rich DNA sequences of human chromosome in 10 mM K+ by titration of S1, S3 and S4 into DNA.All experiments were performed in a buffer (pH 7.4) containing 10 mM Tris–HCl, 10 mM KCl, 0.1 mM EDTA, and samples of 4 μM G-rich DNA (in single strand) and 2 μM double-stranded DNA (in double strand) dissolved in the buffer were incubated for 24 h after annealing at 95 °C. (a) The CD spectrum for G-rich-DNA in the presence/absence of S1 at the ratio of S1 to DNA (r), 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, of which the inset is the change of CD with the r ([compounnd]/[DNA]) at 292.5 nm. (b) The CD spectrum for G-rich-DNA in the presence/absence of S3 at the ratio of S3 to DNA (r), 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 10, of which the inset is the change of CD with the r at 292.5 nm. (c) The CD spectrum for G-rich-DNA in the presence/absence of S4 at the ratio of S4 to DNA (r), 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, of which the inset is the change of CD with the r at 292.5 nm. (d) The CD spectrum for ds-DNA in the presence/absence of S3 at the ratio of S3 to DNA (r), 0.0, 1.0, 2.0, 4.0, 8.0, 16, of which the inset is the enlarged view of ICD in the range from 355 to 455 nm. Arrows denote the increase of the compound concentration. Dashed line: DNA alone; dotted line: compound alone.
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f3: CD spectra for the interaction between benzothioxanthene derivatives S1, S3, S4 and 26 nt telomeric G-rich DNA sequences of human chromosome in 10 mM K+ by titration of S1, S3 and S4 into DNA.All experiments were performed in a buffer (pH 7.4) containing 10 mM Tris–HCl, 10 mM KCl, 0.1 mM EDTA, and samples of 4 μM G-rich DNA (in single strand) and 2 μM double-stranded DNA (in double strand) dissolved in the buffer were incubated for 24 h after annealing at 95 °C. (a) The CD spectrum for G-rich-DNA in the presence/absence of S1 at the ratio of S1 to DNA (r), 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, of which the inset is the change of CD with the r ([compounnd]/[DNA]) at 292.5 nm. (b) The CD spectrum for G-rich-DNA in the presence/absence of S3 at the ratio of S3 to DNA (r), 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 10, of which the inset is the change of CD with the r at 292.5 nm. (c) The CD spectrum for G-rich-DNA in the presence/absence of S4 at the ratio of S4 to DNA (r), 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, of which the inset is the change of CD with the r at 292.5 nm. (d) The CD spectrum for ds-DNA in the presence/absence of S3 at the ratio of S3 to DNA (r), 0.0, 1.0, 2.0, 4.0, 8.0, 16, of which the inset is the enlarged view of ICD in the range from 355 to 455 nm. Arrows denote the increase of the compound concentration. Dashed line: DNA alone; dotted line: compound alone.

Mentions: As shown in Fig. 3a–c, CD spectra of the G-rich DNA alone (dashed line) presented a strong positive band at 292.5 nm, a shoulder band around 275 nm and a relatively shallow negative band at 243 nm, which is characteristic of a hybrid type of G-quadruplex, also named a 3 + 1 G-quadruplex structure33, possibly including a small amount of antiparallel one which is in good agreement with EMSA showing in Fig 1; whereas three compounds alone all are non optically active there existing not any CD bands (dotted line). More importantly, CD spectrum of the G-rich DNA exhibited great difference in peak intensity, the feature of induced CD (ICD) and stoichiometry of compound-DNA interaction in the presence of S1, S3 and S4, respectively. In Fig. 3a, with gradual titration of S1 into the G-rich DNA at the ratios of compound/DNA, 0.25:1, 0.5:1, 1:1, 1.5:1, 2.0:1, 2.5:1, 3.0:1 and 4.0:1, the 292.5 nm positive peak intensity of the G-rich DNA CD spectrum progressively increased up to saturation at 2:1, and gradually increasing and shifting to 260 nm negative bands and 240 nm positive bands, characteristic of an antiparallel G-quadruplex structure36, appeared, with induced CD (ICD) spectra ranging from 560 nm to 425 nm and from 410 nm to 335 nm. Furthermore, The CD profiles have two isoelliptic points at 275 nm and 244 nm, indicating the two-state nature of the structural transition between the quadruplexes upon S1 binding. In addition, in the inset of Fig. 3a, that the positive CD signals at 292.5 nm were plotted against compound–DNA ratio indicates stoichiometry of a 2:1 S1-DNA complex, presumably their interaction mode being by stacking of S1 on two terminal G-tetrads of the G-quadruplex37. These results suggest that the G-quadruplex structures formed by the G-rich DNA alone gradually convert to ones dominatingly including an antiparallel one in the presence of S1, consistent with gel electrophoresis results shown in Fig. 1, and there exists stronger binding of S1 to the antiparallel G-quadruplex at the ratio of 2:1 than to the 3 + 1 one. Likewise, the CD spectra of G-rich DNA in the presence of S4 were obtained (Fig. 3c), showing that there was almost the same characteristics of the CD spectra as ones observed in the presence of S1, with exception of slightly strong ICD signals ranging from 555 nm to 425 nm and from 410 nm to 335 nm. However, very striking, as shown in Fig. 3b, were several quite differences in binding affinity, ICD, binding ratio of S3 to the G-rich DNA in comparison with S1 and S4. With increasing concentration of S3, the positive peak intensity at 292.5 nm much increase up to saturation at the ratio of S3 to DNA 4:1 and a 240 nm distinct band and stronger ICD spectrum bands ranging from 500 nm to 435 nm appeared; the inset in Fig. 3B of the CD signal intensity at 292.5 nm as a function of S3/DNA (r = 0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 10) indicated that there were two binding modes of S3 to the antiparallel G-quadruplex formed through inducing the 3 + 1 structure at the ratios of 2:1 and 4:1, respectively. In contrast, S1, S3 and S4 have no influence on C-rich DNA as a reference, a complementary strand of 26 nt telomeric G-rich DNA, at tested compound concentrations of 4, 20, 40 μM, which can be assigned to a single strand DNA structure with a positive maximum at 271 nm and a negative minimum at 248 nm (Fig. S4A, B and C)3839.


Formation and stabilization of the telomeric antiparallel G-quadruplex and inhibition of telomerase by novel benzothioxanthene derivatives with anti-tumor activity.

Zhang W, Chen M, Ling Wu Y, Tanaka Y, Juan Ji Y, Lin Zhang S, He Wei C, Xu Y - Sci Rep (2015)

CD spectra for the interaction between benzothioxanthene derivatives S1, S3, S4 and 26 nt telomeric G-rich DNA sequences of human chromosome in 10 mM K+ by titration of S1, S3 and S4 into DNA.All experiments were performed in a buffer (pH 7.4) containing 10 mM Tris–HCl, 10 mM KCl, 0.1 mM EDTA, and samples of 4 μM G-rich DNA (in single strand) and 2 μM double-stranded DNA (in double strand) dissolved in the buffer were incubated for 24 h after annealing at 95 °C. (a) The CD spectrum for G-rich-DNA in the presence/absence of S1 at the ratio of S1 to DNA (r), 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, of which the inset is the change of CD with the r ([compounnd]/[DNA]) at 292.5 nm. (b) The CD spectrum for G-rich-DNA in the presence/absence of S3 at the ratio of S3 to DNA (r), 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 10, of which the inset is the change of CD with the r at 292.5 nm. (c) The CD spectrum for G-rich-DNA in the presence/absence of S4 at the ratio of S4 to DNA (r), 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, of which the inset is the change of CD with the r at 292.5 nm. (d) The CD spectrum for ds-DNA in the presence/absence of S3 at the ratio of S3 to DNA (r), 0.0, 1.0, 2.0, 4.0, 8.0, 16, of which the inset is the enlarged view of ICD in the range from 355 to 455 nm. Arrows denote the increase of the compound concentration. Dashed line: DNA alone; dotted line: compound alone.
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Show All Figures
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f3: CD spectra for the interaction between benzothioxanthene derivatives S1, S3, S4 and 26 nt telomeric G-rich DNA sequences of human chromosome in 10 mM K+ by titration of S1, S3 and S4 into DNA.All experiments were performed in a buffer (pH 7.4) containing 10 mM Tris–HCl, 10 mM KCl, 0.1 mM EDTA, and samples of 4 μM G-rich DNA (in single strand) and 2 μM double-stranded DNA (in double strand) dissolved in the buffer were incubated for 24 h after annealing at 95 °C. (a) The CD spectrum for G-rich-DNA in the presence/absence of S1 at the ratio of S1 to DNA (r), 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, of which the inset is the change of CD with the r ([compounnd]/[DNA]) at 292.5 nm. (b) The CD spectrum for G-rich-DNA in the presence/absence of S3 at the ratio of S3 to DNA (r), 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 10, of which the inset is the change of CD with the r at 292.5 nm. (c) The CD spectrum for G-rich-DNA in the presence/absence of S4 at the ratio of S4 to DNA (r), 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, of which the inset is the change of CD with the r at 292.5 nm. (d) The CD spectrum for ds-DNA in the presence/absence of S3 at the ratio of S3 to DNA (r), 0.0, 1.0, 2.0, 4.0, 8.0, 16, of which the inset is the enlarged view of ICD in the range from 355 to 455 nm. Arrows denote the increase of the compound concentration. Dashed line: DNA alone; dotted line: compound alone.
Mentions: As shown in Fig. 3a–c, CD spectra of the G-rich DNA alone (dashed line) presented a strong positive band at 292.5 nm, a shoulder band around 275 nm and a relatively shallow negative band at 243 nm, which is characteristic of a hybrid type of G-quadruplex, also named a 3 + 1 G-quadruplex structure33, possibly including a small amount of antiparallel one which is in good agreement with EMSA showing in Fig 1; whereas three compounds alone all are non optically active there existing not any CD bands (dotted line). More importantly, CD spectrum of the G-rich DNA exhibited great difference in peak intensity, the feature of induced CD (ICD) and stoichiometry of compound-DNA interaction in the presence of S1, S3 and S4, respectively. In Fig. 3a, with gradual titration of S1 into the G-rich DNA at the ratios of compound/DNA, 0.25:1, 0.5:1, 1:1, 1.5:1, 2.0:1, 2.5:1, 3.0:1 and 4.0:1, the 292.5 nm positive peak intensity of the G-rich DNA CD spectrum progressively increased up to saturation at 2:1, and gradually increasing and shifting to 260 nm negative bands and 240 nm positive bands, characteristic of an antiparallel G-quadruplex structure36, appeared, with induced CD (ICD) spectra ranging from 560 nm to 425 nm and from 410 nm to 335 nm. Furthermore, The CD profiles have two isoelliptic points at 275 nm and 244 nm, indicating the two-state nature of the structural transition between the quadruplexes upon S1 binding. In addition, in the inset of Fig. 3a, that the positive CD signals at 292.5 nm were plotted against compound–DNA ratio indicates stoichiometry of a 2:1 S1-DNA complex, presumably their interaction mode being by stacking of S1 on two terminal G-tetrads of the G-quadruplex37. These results suggest that the G-quadruplex structures formed by the G-rich DNA alone gradually convert to ones dominatingly including an antiparallel one in the presence of S1, consistent with gel electrophoresis results shown in Fig. 1, and there exists stronger binding of S1 to the antiparallel G-quadruplex at the ratio of 2:1 than to the 3 + 1 one. Likewise, the CD spectra of G-rich DNA in the presence of S4 were obtained (Fig. 3c), showing that there was almost the same characteristics of the CD spectra as ones observed in the presence of S1, with exception of slightly strong ICD signals ranging from 555 nm to 425 nm and from 410 nm to 335 nm. However, very striking, as shown in Fig. 3b, were several quite differences in binding affinity, ICD, binding ratio of S3 to the G-rich DNA in comparison with S1 and S4. With increasing concentration of S3, the positive peak intensity at 292.5 nm much increase up to saturation at the ratio of S3 to DNA 4:1 and a 240 nm distinct band and stronger ICD spectrum bands ranging from 500 nm to 435 nm appeared; the inset in Fig. 3B of the CD signal intensity at 292.5 nm as a function of S3/DNA (r = 0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 10) indicated that there were two binding modes of S3 to the antiparallel G-quadruplex formed through inducing the 3 + 1 structure at the ratios of 2:1 and 4:1, respectively. In contrast, S1, S3 and S4 have no influence on C-rich DNA as a reference, a complementary strand of 26 nt telomeric G-rich DNA, at tested compound concentrations of 4, 20, 40 μM, which can be assigned to a single strand DNA structure with a positive maximum at 271 nm and a negative minimum at 248 nm (Fig. S4A, B and C)3839.

Bottom Line: Results show that six compounds could differentially induce 26 nt G-rich oligonucleotides to form the G-quadruplex with high selectivity vs C-rich DNA, mutated DNA and double-stranded DNA, stabilize it with high affinity, promote apoptosis and inhibit mobility and telomerase activity of A549 cells and SGC7901 cells.Especially, S1, S3, S4 displayed stronger abilities, of which S3 was the most optimal with the maximum ΔTm value being up to 29.8 °C for G-quadruplex, the minimum IC50 value being 0.53 μM and the maximum cell inhibitory rate being up to 97.2%.This study suggests that this type of compounds that induce the formation of and stabilize the telomeric antiparallel G-quadruplex, and consequently inhibit telomerase activity, leading to cell apoptosis, can be screened for the discovery of novel antitumor therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Lab of Chemical Biology and Molecular Drug Design, College of Pharmaceutical Science, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, China.

ABSTRACT
G-quadruplexes formed in telomeric DNA sequences at human chromosome ends can be a novel target for the development of therapeutics for the treatment of cancer patients. Herein, we examined the ability of six novel benzothioxanthene derivatives S1-S6 to induce the formation of and stabilize an antiparallel G-quadruplex by EMSA, UV-melting and CD techniques and the influence of S1-S6 on A549 and SGC7901 cells through real-time cell analysis, wound healing, trap assay methods. Results show that six compounds could differentially induce 26 nt G-rich oligonucleotides to form the G-quadruplex with high selectivity vs C-rich DNA, mutated DNA and double-stranded DNA, stabilize it with high affinity, promote apoptosis and inhibit mobility and telomerase activity of A549 cells and SGC7901 cells. Especially, S1, S3, S4 displayed stronger abilities, of which S3 was the most optimal with the maximum ΔTm value being up to 29.8 °C for G-quadruplex, the minimum IC50 value being 0.53 μM and the maximum cell inhibitory rate being up to 97.2%. This study suggests that this type of compounds that induce the formation of and stabilize the telomeric antiparallel G-quadruplex, and consequently inhibit telomerase activity, leading to cell apoptosis, can be screened for the discovery of novel antitumor therapeutics.

No MeSH data available.


Related in: MedlinePlus