RHPS4 G-quadruplex ligand induces anti-proliferative effects in brain tumor cells

Abstract

Background: Telomeric 3' overhangs can fold into a four-stranded DNA structure termed G-quadruplex (G4), a formation which inhibits telomerase. As telomerase activation is crucial for telomere maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure.

Methods: We exposed brain tumor cells to the G4 ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) and investigated proliferation, cell cycle dynamics, telomere length, telomerase activity and activated c-Myc levels.

Results: Although all cell lines tested were sensitive to RHPS4, PFSK-1 central nervous system primitive neuroectodermal cells, DAOY medulloblastoma cells and U87 glioblastoma cells exhibited up to 30-fold increased sensitivity compared to KNS42 glioblastoma, C6 glioma and Res196 ependymoma cells. An increased proportion of S-phase cells were observed in medulloblastoma and high grade glioma cells whilst CNS PNET cells showed an increased proportion of G1-phase cells. RHPS4-induced phenotypes were concomitant with telomerase inhibition, manifested in a telomere length-independent manner and not associated with activated c-Myc levels. However, anti-proliferative effects were also observed in normal neural/endothelial cells in vitro and ex vivo.

Conclusion: This study warrants in vivo validation of RHPS4 and alternative G4 ligands as potential anti-cancer agents for brain tumors but highlights the consideration of dose-limiting tissue toxicities.

Figure 1. Acute RHPS4 exposure inhibits proliferation of high grade brain tumor cells in vitro.

Proliferation of tumor cells was impaired in malignant brain tumor cells after acute 72 hours exposure to RHPS4. (A–C) PFSK-1, DAOY, U87 and (E) Res196 cells exhibited IC₅₀ values of 2.7, 2.2, 1.1, and 1.6 µM respectively when 0.5–5.0 µM RHPS4 was used, representing a significant inhibition of cell proliferation (p≤0.05 for each drug concentration versus untreated). (D, F–G) Within this concentration range, KNS42, C6 and GB-1 cells were resistant to RHPS4. (H–I) At higher concentrations of RHPS4 exposure C6 and GB-1 cells exhibited IC₅₀ values of 26 µM and 32 µM respectively, representing a significant inhibition of cell proliferation (p≤0.05 for each drug concentration versus untreated). Error bars indicate standard error from three independent experiments. (J–M) Light microscopy of PFSK-1, DAOY, C6 and GB-1 cells showing a marked reduction in cellular density after RHPS4 exposure. Magnifications, x20; Scale bar = 25 µm.

Figure 2. Acute RHPS4 exposure alters cell cycle dynamics of brain tumor cells in vitro.

PFSK-1 cells exhibited a dose-dependent increase in the proportion of cells in G1-phase. In contrast DAOY, C6 and GB-1 cells exhibited a dose-dependent increase in the proportion of cells in S-phase. PFSK-1 further shows a moderate accompanying increase of sub-G0/1 cells at the higher RHPS4 concentration (5 µM). Percentages are the mean from three independent experiments. Asterisk denotes a significant difference relative to untreated cells.

Figure 3. Telomere length measurement and RHPS4-mediated inhibition of Taq polymerase in vitro.

(A) Mean TRF lengths for PFSK-1/DAOY cells (3.8 kb/7.8 kb) and C6/GB-1 (7.5 kb/3.9 kb) cells were determined prior to RHPS4 exposure. (B) Quantitative TRAP assay showing telomerase-mediated telomere extension after 30 minutes (standard TRAP assay) or 5 minutes extension time in non-drug exposed cells. (C) PCR gel showing telomere extension products after 5 minutes extension time in non-drug exposed cells. 1, no lysate control; 2–5, PFSK-1, DAOY, C6 and GB-1. (D) PFSK-1 and DAOY showed low levels of telomerase activity at low RHPS4 concentrations and complete absence of activity at high RHPS4 concentrations, when RHPS4 was added pre-telomere extension. C6 and GB-1 showed complete absence of telomerase activity at both RHPS4 concentrations analyzed. (E) Telomerase activity was absent in all cell lines and at both RHPS4 concentrations when RHPS4 was added post-telomere extension. CHAPS, CHAPS buffer only no lysate control; IC, internal control 61-bp telomere substrate oligonucleotide.

Figure 4. Acute RHPS4 exposure is associated with telomerase inhibition in brain tumor cells in vitro.

(A) TRAP assay using ethanol-precipitated telomere extended DNA products after 30 minutes extension in non-drug treated brain tumor cells. High levels of telomerase activity are observed in each cell line. (B–D) TRAP assays in RHPS4-treated brain tumor lysates reveals complete telomerase inhibition in all cell lines at each drug concentration. 0.1 µg of total protein lysate was loaded per well in each TRAP assay. CHAPS, CHAPS buffer only no lysate control; TS, telomere substrate internal control 61-bp oligonucleotide.

Figure 5. c-Myc activation is not associated with degree of RHPS4 sensitivity.

(A) c-Myc transcription factor assay. Jurkat cell nuclear extracts show activation and specificity of c-Myc proportional to concentration of extract analyzed and in the presence of wild-type or mutant competitor. (B–C) No significant difference in c-Myc activation was observed between untreated PFSK-1 or C6 cells and RHPS4-treated cells. Asterisk denotes significant reduction in c-Myc levels when either PFSK-1 or C6 untreated cells were exposed to a wild-type oligonucleotide competitor (p≤0.05). (D–E) c-Myc quantitative reverse transcriptase PCR. No difference in PFSK-1 or C6 c-Myc gene expression was observed between representative RHPS4-treated cells and untreated cells.

Figure 6. RHPS4 sensitivity in normal neural and endothelial cells in vitro and ex vivo.

(A) C17.2 cerebellar progenitor cells and (B) HBMEC endothelial cells are sensitive to RHPS4 with an IC₅₀ of 15 µM and 5 µM respectively. (C) Primary rat ependymal ex vivo cultures exhibited functional impairment of ependymal CBF after 3 µM or 30 µM RHPS4 exposure (p≤0.01). (D) A significant reduction in cilia tip distance was observed after either 3 µM or 30 µM RHPS4 exposure (p≤0.01). Error bars represent standard error of the mean from four separate rat brains/experiments.