Leukemia (2007) 21, 1300–1346 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu
LETTERS TO THE EDITOR Inhibition of myc promoter and telomerase activity and induction of delayed apoptosis by SYUIQ-5, a novel G-quadruplex interactive agent in leukemia cells
Leukemia (2007) 21, 1300–1302. doi:10.1038/sj.leu.2404652; published online 29 March 2007
G-quadruplex structures are involved in many cellular events, including control of gene expression and inhibition of telomerase activity.1,2 Besides telomeric single-stranded overhang, G-quadruplexes were also present in the promoter or regulatory regions of important oncogene such as c-myc, bcl2, c-Fos and cABL.2,3 C-myc is an important proto-oncogene, which is linked to potentiation of cellular proliferation and to inhibition of differentiation and acts as both a transcriptional activator and repressor. In addition, c-myc plays a function in the apoptotic response.4,5 Studies demonstrated that the nuclease hypersensitive element (NHEIII) of the c-myc gene promoter was conducive to the formation of G-quadruplex structures.5,6 Owing to the important function of c-myc, the strategy to target c-myc, especially G-quadruplex structure has emerged as another attractive anti-cancer therapy. We have tested the formation and stabilization of G-quadruplex of DNA promoted by quindoline derivatives, especially SYUIQ-5 (N0 -(10H-indolo (3,2-b) quinolin-11-yl)-N,N-dimethyl-propane-1,
3-diamine) via UV and circular dichroism spectroscopy. Our previous study has proved that SYUIQ-5 could induce the formation of G-quadruplex in telomere structures and induce senescence and telomere shortening in cancer cells.7 C-myc is an important oncogene and is overexpressed in many human malignancies including leukemia. We report here that SYUIQ-5 can also interact with G-quadruplex in the c-myc gene promoter and inhibit its expression. Meanwhile, it can potently induce delayed apoptosis of leukemia cells. We performed a specific PCR stop assay to explore the effects of quindoline derivatives on c-myc gene promoter activity in a cell-free system. In this case, quindoline derivative SYUIQ-5 obviously inhibited the formation of the final double-stranded PCR product in a dose-dependent manner under SYUIQ-5 concentrations from 0.6 to 10 mg/ml whereas Pu22myc oligomer was used as substrates (Figure 1a). It indicated that in the presence of SYUIQ-5, the Pu22myc oligomer was induced to form G-quadruplex that prevented hybridization with a complementary strand overlapping the last G repeat. To demonstrate further that the inhibition induced by SYUIQ-5 was not owing to the inhibition of Taq polymerase, a parallel experiment with a mutant test oligomer that contains two mutations from G to A and is unable to form G quadruplex was performed (Pu22mu).
Figure 1 (a) Effect of SYUIQ-5 on the formation of the PCR-stop assay with G-quadruplex forming Pu22myc oligomer or with control mutated Pu22mu oligomer. SYUIQ-5 were added to G-quadruplex forming Pu22myc oligomer or to mutated Pu22mu oligomer. Effects of SYUIQ-5 on c-myc expression in HL-60 (b) and K562 cells (c) treated by SYUIQ-5 for 72 h. Effects of SYUIQ-5 on E2F1, hTERT (d) and TRF1, TRF2 (e) expression in HL-60 cells treated by SYUIQ-5 for 72 h.
Letters to the Editor
1301 No inhibition was observed even at the highest concentration evaluated (Figure 1a). It indicated that the inhibition was specific and G-quadruplex formation played an important role in this process. SYUIQ-5 could inhibit c-myc gene promoter activity in a cellfree system, which prompted us to investigate whether SYUIQ-5 could inhibit its expression in cultured cells. HL-60 cells and K562 cells were cultured in the presence of SYUIQ-5 at indicated concentrations for 72 h. The expression of c-myc was markedly inhibited in mRNA and protein levels of HL-60 (Figure 1b) and K562 cells (Figure 1c). Oncoprotein c-MYC can induce E2F1 and hTERT expression in carcinogenesis. E2F1 is the downstream gene of c-myc; hTERT, the catalytic subunit of telomerase is under transcriptional control in part by the c-myc transcription factor.6 The telomere-binding proteins TRF1 and TRF2 are major regulators of telomere stability. These proteins directly interact with TTAGGG repeats and a number of other proteins to ensure proper telomere maintenance. Having shown an inhibitory effect of SYUIQ-5 on c-myc expression, thereby it is of interest to knowing whether it would affect the expression of E2F1, hTERT, TRF1 and TRF2. The results indicated that both E2F1 and hTERT were obviously inhibited in HL-60 cells at a dose-dependent manner (Figure 1d). The levels of TRF2 protein decreased, but the levels of TRF1 protein only have a slight decrease following SYUIQ-5 treatment (Figure 1e). It could be that the dissociation of TRF2 from telomeres was much more drastic than that of TRF1, and TRF2 is easily degraded while dissociation from telomere.8 Furthermore, we investigated the effect of SYUIQ-5 on telomerase activity in HL-60 cells. TRAP-ELISA assay was employed. HL-60 cells were cultured in the presence of SYUIQ-5. After 3 days, cells were lysed, and telomerase activity was measured in HL-60 cells. Treatment with SYUIQ-5 resulted in inhibition of telomerase activity by 22.6, 41.5, 52.8 and 64.2% respectively contrary to untreated HL-60 cells. To explore the long-term effect of SYUIQ-5 on HL-60 cells, the concentrations of SYUIQ-5 were determined in which telomerase activity could be inhibited without extensive inhibition of cell proliferation firstly. Short-term cell viability were determined in a 3-day cytotoxic assay using various concentrations of SYUIQ-5. The IC50 value of SYUIQ-5 for HL-60 cells was 1.6 mg/ml (Figure 2a). Therefore, the concentrations of 0.1, 0.2, 0.3, 0.4 mg/ml of SYUIQ-5, which had no obvious effect on short-term cell viability or proliferation, were employed in the following long-term cultivation experiments respectively. We characterized the growth properties of SYUIQ-5-treated cells. Treatment of HL-60 cells with SYUIQ-5 at 0.4 mg/ml induced a plateau at day 9 on growth curves. In contrast, the cells treated
with SYUIQ-5 at 0.1, 0.2, 0.3 mg/ml were able to grow continuously after 12 days, but the population doubling time obviously increased (Figure 2b). Cyclin-dependent kinase inhibitors P16, P21 and P27 are regarded as key effectors of cellular senescence. To identify whether P16, P21 and P27 were involved in SYUIQ-5-induced growth inhibition, the expression of these proteins were determined using immunoblot analysis. As shown in Figure 3a, P16, P21 and P27 were upregulated by SYUIQ-5 in HL-60 cells, which suggested that growth arrest in HL-60 cells induced by SYUIQ-5 could be because of upregulation of the expression of P16, P21 and P27. DNA fragmentation assay and flow cytometry are utilized to assess apoptosis. As shown in Figure 3b, caspase-3 and PARP were cleaved. Pro-caspase-3 was cleaved to yield a 17 kDa fragmentation and PARP was cleaved to 89 kDa fragmentation after treatment for 9 days. SubG1 peak obviously appeared after HL-60 cells with 0.4 mg/ml of SYUIQ-5 treatment for 9 days (Figure 3c). It indicated that treatment of HL-60 cells with 0.4 mg/ml of SYUIQ-5 resulted in induction of delayed apoptosis. Our previous studies have shown that SYUIQ-5 could stabilize intramolecular G-quadruplexes, potentially induce telomere shortening and cell senescence in K562 and SW620 cells.7 The NHEIII upstream of the P1 promoter of c-myc controls 85–90% of the transcriptional activation of this gene. It has been demonstrated that the purine-rich strand of the DNA in this region can form intramolecular G-quadruplex.2 Therefore, in the present study, we examined effect of SYUIQ-5 on c-myc gene promoter activity and c-myc expression. Using a specific PCR-stop assay and immunoblot analysis, we found that quindoline derivative SYUIQ-5 could inhibit the c-myc promoter activity and decrease c-myc expression at both the mRNA and protein levels. We have also found in this study that SYUIQ5 could inhibit E2F1 and hTERT expression and induce a delayed apoptosis of HL-60 cells. The G-quadruplex, which is very different from classical double-strand, provides a good structural basis for selective recognition. We have demonstrated that SYUIQ-5 could interact with G-quadruplexes in human telomere sequence.7 In this study, we observed that SYUIQ-5 could stabilize G-quadruplex structure of c-myc gene promoter. It suggests that SYUIQ-5 is not only able to stabilize telomeric but also c-myc G-quadruplex structures. We also detected the effect of SYUIQ-5 on telomerase activity. SYUIQ-5 could obviously inhibit telomerase activity in HL-60 cells. When exposure of cancer cells to non-cytotoxic concentrations of SYUIQ-5, SYUIQ-5 did not immediately cause a growth arrest. Instead, in the beginning cancer cells continued
Figure 2 (a) Effects of SYUIQ-5 on HL-60 cell viability after 72 h treatment. Cells were treated with different concentrations of SYUIQ-5 for 72 h. Cell viability was determined by trypan blue staining. (b) Growth arrest induced by SYUIQ-5 on HL-60 cells. Long-term exposure with nonacute cytotoxic concentrations. Cells were exposed to SYUIQ-5. Every 3 days, the cells in control and drug-exposed flasks were counted using a hematocytometer and flasks reseeded with the same number cells. Then, the growth curve was drawn. Each experiment was performed three times at each point. Leukemia
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Figure 3 SYUIQ-5 induced a delayed apoptosis in HL-60 cells. (a) The levels of P16, P21 and P27 protein in HL-60 cells after 0.4 mg/ml of SYUIQ-5 treatment for 6, 9, 12 days. (b) Cleavage of caspase-3 and PARP in HL-60 cells after 0.4 mg/ml of SYUIQ-5 treatment for 6, 9, 12 days. (c) HL-60 cells were treated with or without 0.4 mg/ml of SYUIQ-5 for 3, 6, 9, 12 days. The cells were collected, stained with propidium iodide. Then the cells were analyzed with flow cytometry.
to proliferate, but the population doubling time increased, finally the cells underwent delayed apoptosis. Also, the growth inhibition of HL-60 cells induced by SYUIQ-5 was associated with upregulation of cyclin-dependent kinase inhibitors P16, P21 and P27. It indicates that P16, P21 and P27 could mediate the cessation of growth. Growth inhibition of HL-60 cells was accompanied by delayed apoptosis, which was not detected in the first 6 days. After 9 days, apoptosis were observed in HL-60 cells treated with 0.4 mg/ml of SYUIQ-5. The induction of delayed apoptosis by SYUIQ-5 was characterized by caspase-3 and PARP cleavage. Apoptosis induction may be related to severe telomere disruption. The dissociation of TRF2 from telomeres results in loss of 30 overhang of telomeres and exposed telomere are no longer able to protect chromosomal integrity.8 And the apoptosis associated with DNA damage signaling may be mediated by the ATM-dependent pathway. However, the mechanism of delayed apoptosis will be elucidated by our future studies. In conclusion, c-myc is an important proto-oncogene and plays a vital role in the process of cellular growth and differentiation, leading to its association with a number of human and animal malignancies, including carcinomas of the breast, colon and myelodid leukemias.5 The G-quadruplex structure formed in the c-myc promoter region provides a new molecular target for cancer treatment. Therefore, the highly specific and novel potent G-quadruplex-interactive agents, quindoline derivatives especially SYUIQ-5 can inhibit c-myc gene promoter activity and telomerase activity, induced cell growth arrest and delayed apoptosis by interacting with G-quadruplex and therefore is a worthwhile agent for anti-leukemia treatment.
Acknowledgements This work was supported by grants from the National Nature Science Foundation of China (30400556, 20472117) and the Fund of Southern China National Research Center for Integrated Leukemia
Biosciences and the specialized Research Fund for the Doctoral program of Higher Education (20060558038).
J-N Liu1, R Deng1, J-F Guo1, J-M Zhou1, G-K Feng1, Z-S Huang2, L-Q Gu2, Y-X Zeng1 and X-F Zhu1 1 Department of Signal Transduction and Anti-Cancer Drug, State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou, China; 2 Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China E-mail:
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