Only a weak inhibition of telomere products was detected if the compound was added to the mixture between telomerase extension and PCR amplification. Therefore telomerase inhibition, rather than Taq inhibition, is responsible for the Andarine GTX-007 observed effect on TRAP. The in vitro potency of these triazine derivatives prompted us to investigate their effects in cultured cells. Ligand 115405 induced a dose dependent decrease in telomerase activity relative to untreated cells with an IC50 of 3 M, for a 24 h treatment. A lower inhibition of telomerase activity was induced by 12459, whereas no inhibition was detected for 5271, in agreement with their relative potency in vitro. The Gquadruplex stabilizing compounds were also active as antiproliferative agents on a panel of one human cancer cell lines and three immortalized human cell lines.
Ligand 115405 was active on the whole panel, including the ALT cell line GM847DM, the SV40 immortalized lung fibroblast cell line MRC5 V1, and the telomerase immortalized fibroblast hTERT BJ1 cell line. Ligand 12459 was active against only two cell lines, and 5271 and 5352, were devoid of significant antiproliferative activity. Antiproliferative properties were correlated with the in vitro stabilization of G quadruplexes, in contrast with other published G4 ligands. Ligand 115405 was found to induce major apoptosis within 24 h at micromolar concentrations in A549 cells. Recent reports indicate that short term and massive apoptosis may result from interference with telomere function when either hTERT or hTR are modified by mutations. The short term apoptosis observed here may either result from an effect on telomeres or a nonselective effect on other DNA targets at micromolar concentrations.
Further studies aimed at determining the causes of apoptosis have been undertaken through a microarray approach and will be presented elsewhere. We then examined whether G quadruplex ligands could interfere with telomere replication. Long term treatment at subcytotoxic doses of telomerase positive cultured cells was therefore undertaken, using cell lines of various initial telomere length. We hypothesized that if one part of the mechanism of action of these agents is linked to G quadruplex interaction at telomeres, we would observe a delayed impact on telomere length. To avoid short term apoptosis and other nonspecific events due to the narrow selectivity of these compounds that could render the detection of telomeric events difficult, subapoptotic concentrations of the G4 ligands were applied on A549 cells for long term exposure.
Population doubling was normal for up to a 45 day exposure for 12459 at 0.04 M, whereas treatment with 115405 induced a 25 30% decline in population doubling up to 40 days exposure, without affecting cell viability, suggesting an increase in cellcycle length. Prolonged exposure with either compound resulted in a population doubling plateau occurring at 40 45 days for both compounds, which ultimately led to cell division arrest at 70 and 53 days for 115405 and 12459, respectively and that corresponded to the failure of treated cells to replate. A 10 fold difference in drug concentration required to achieve growth arrest is observed between 12549 and 115405. This observation may be due to differences in drug permeation, because A549 express slight amounts of MRP 1.