Pharmacodynamics of the G-Quadruplex-Stabilizing Telomerase Inhibitor 3,11-Difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) in Vitro: Activity in Human Tumor Cells Correlates with Telomere Length and Can Be Enhanced, or Antagonized, with Cytotoxic Agents

Abstract

Telomeric integrity is required to maintain the replicative ability of cancer cells and is a target for the G-quadruplex-stabilizing drug 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4). We report a senescent-like growth arrest in MCF-7 breast cancer cells, within 14 to 17 days, and a reduction in telomere length (from 5.2 kilobases (kb) to 4.7 and 4.3 kb after 17 days of treatment at 0.5 and 1 M, respectively). These effects occurred at noncytotoxic drug concentrations (doses Ͻ 1 M over a 14-day exposure) compatible with long-term drug dosing. The telomere length of cancer cells influences their sensitivity to growth inhibition by RHPS4: mutant (mt) human telomerase reverse transcriptase (hTERT)-expressing MCF-7 cells [short telomere restriction fragment (TRF) length, 1.9 kb; IC 50 , 0.2 M] were 10 times more sensitive to RHPS4 compared with wild-type (wt) hTERT-expressing, vector-transfected control cells (longer TRF-length 5.2 kb; IC 50 2 M) in the 5 day SRB assay. This relationship was corroborated in a panel of 36 human tumor xenografts grown in vitro showing a positive correlation between telomere length and growth inhibitory potency of RHPS4 (15-day clonogenic assay, r ϭ 0.75). These observations are consistent with loss of the protective capping status of telomeres mediated by RHPS4 G-quadruplex-stabilization, thus leading to greater susceptibility of cells with shorter telomeres. In combination studies, paclitaxel (Taxol), doxorubicin (Adriamycin), and the experimental therapeutic agent 17-(allylamino)-17-demethoxygeldanamycin, which inhibits the 90-kDa heat shock protein, conferred enhanced sensitivity in RHPS4 treated MCF-7 cells, whereas the DNA-interactive temozolomide and cisplatin antagonized the action of RHPS4. Our results support the combined use of certain classes of cytotoxic anticancer agents with RHPS4 to enhance potential clinical benefit.

Telomere uncapping by the G-quadruplex ligand RHPS4 inhibits clonogenic tumour cell growth in vitro and in vivo consistent with a cancer stem cell targeting mechanism

“…First, mutant (dominant negative) hTERT-expressing cancer cells show reduced telomerase activity (inhibition), reduction in telomere length, chromosome fusions, slowing and eventual arrest of cell growth related to initial telomere length, and reduced tumourigenicity in immunodeficient nude mice (Hahn et al, 1999). Our own mutant-hTERT MCF-7 breast cancer cell line model also demonstrated markedly reduced clonogenicity and tumourigenicity (Cookson et al, 2005; data not shown). Second, GRN163L, a modified antisense oligonucleotide directed against the telomerase RNA component and the first telomerase inhibitor to enter clinical trials, produces in vitro telomerase inhibition, progressive telomere shortening, reduced clonogenicity and tumourigenicity of breast cancer cell lines, and suppression of tumour growth and lung metastases in animal models in vivo (Dikmen et al, 2005;Kelland, 2005;Hochreiter et al, 2006;Burger, 2007).…”

“…Although, the in vitro data demonstrated that RHPS4 can act synergistically with Taxol in the UXF1138L cell line (Figure 5), as well as in MCF-7 cells, DNA-cross linking and alkylating agents such as cisplatin and temozolomide were antagonistic, possibly because these agents preferentially react at G-rich DNA sequences. Synergism between RHPS4 and other drugs were observed if there was an overlap between the molecular mechanism of the combination partners (Cookson et al, 2005).…”

“…Southern blotting was performed with the Telo-TAGGG-telomere length kit from Roche (Penzberg, Germany) and analysed as described before Cookson et al, 2005).…”

“…RHPS4, however, exerts clear tumour growth inhibitory effects in longer term growth assays in vitro in various experimental models (Gowan et al, 2001;Leonetti et al, 2004;Cookson et al, 2005); phenotypic changes are consistent with a G-quadruplex-stabilizing mechanism of action at telomeres, with the consequent inhibition of telomerase. Sensitivity to growth inhibition by RHPS4 appears correlated to telomere length as shown in a panel of human tumour lines that were grown in the clonogenic assay, also known as the human tumour stem cell assay (HTCA) (Hamburger and Salmon, 1977;Cookson et al, 2005). The most sensitive tumour line was the uterus carcinoma UXF1138L, which possesses short telomeres (mean TRF 2.7 kb).…”

“…A characteristic of RHPS4 is a low overall growth-inhibitory activity in short-term cytotoxicity assays such as the 48 h sulforhodamine B assay used by the NCI 60 cell line screen (mean IC 50 13.18 mM), but potent inhibition of telomerase enzyme activity (IC 50 0.33 mM) (Heald et al, 2002). RHPS4, however, exerts clear tumour growth inhibitory effects in longer term growth assays in vitro in various experimental models (Gowan et al, 2001;Leonetti et al, 2004;Cookson et al, 2005); phenotypic changes are consistent with a G-quadruplex-stabilizing mechanism of action at telomeres, with the consequent inhibition of telomerase. Sensitivity to growth inhibition by RHPS4 appears correlated to telomere length as shown in a panel of human tumour lines that were grown in the clonogenic assay, also known as the human tumour stem cell assay (HTCA) (Hamburger and Salmon, 1977;Cookson et al, 2005).…”