Abstract 3476: Effect of long term treatment with BRACO 19 on HeLa proliferation and senescence

Abstract

Telomerase, the enzyme responsible for the length maintenance of telomeres is expressed in about 85% of cancer cells, being a key to tumorigenesis. Telomerase inhibitor drugs have been studied as a promising cancer therapy due to its specificity, with less side effects than conventional approach. However, the effects expected from these therapy require many cell divisions, and the consequences of these long-term therapies are poorly understood. This work evaluated the effects of a long term treatment in cancer cells (HeLa) using BRACO-19, a G-quadruplex-interactive molecule, evaluating the consequences of its use in cell viability and its potential to induce senescence. Telomerase positive HeLa cells were grown in DMEM medium supplemented with 10% fetal bovine serum at pattern conditions. Culture medium was changed every 2 days. For preliminary citotoxicity assay, cells were seeded in 96 wells plate at 3.2x104 cells/cm2, incubated for 24 hours and treated for 24 hours with increasing concentrarions of BRACO-19 and vehicle (0.1% H2O). Cell viability was measured by MTT assay. For the long-term treatment, a subtoxic concentration was chosen. The cells (5.5x102 cells/cm2) were grown under continuous presence of the drug or 0.02% H2O for 11 weeks. During the long-term exposure to BRACO-19, the cells were splitted every 7 - 20 days, depending on its proliferation rate, viable and unviable cells were counted at these points through trypan blue dye exclusion assay. β-Galactosidase (β-gal) activity was used as cell senescence marker to evaluate the senescence after 24 hours treatment and after a long exposure to the telomerase inhibitor. Changes in cell morphology were accessed by phase contrast microscopy. The short-term (24h) treatment with BRACO-19 showed a dose-dependent effect on cell viability. The IC50% for HeLa was 5.25 µM. The subtoxic concentration of 1µM was used for long-term treatment, showing effect on cell proliferation in a way that HeLa cells exposed to the drug showed their proliferative profile decreased when compared to control. Furthemore, no senescent cells were found in control group, even after many passages; on the other hand, 24 hours and long-term exposure to BRACO-19 led to senescence. Long-term treatment was also associated with visual changes on cell morphology. The telomerase inhibitor BRACO-19 showed an expected dose-dependent effect on HeLa cells at short-term exposition, since cytotoxic effect of this compound has been demonstrated in several cell lines, but the impact of the long-term treatment on cell proliferation pattern points BRACO-19 as a potential prototype of a new telomere related drug for cancer therapy. Also, it was demonstrated the senescence inducing action of this drug, perhaps due to erosion of HeLa telomeres. These results show that HeLa is sensitive do BRACO-19 even under subtoxic concentrations. Its effectiveness against other cell lines and tumors is under investigation. Citation Format: Ana Flavia Reis Guimaraes, Diêgo M. de Oliveira. Effect of long term treatment with BRACO 19 on HeLa proliferation and senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3476. doi:10.1158/1538-7445.AM2017-3476