Cell fate following radiation of MDA-MB-231 and MCF-7 breast cancer cells pre-exposed to a tetrahydroisoquinoline analogue

Abstract

Chemotherapy and radiation, together with surgery, remain the mainstays of cancer therapeutics. There remains a need to identify agents which circumvent multidrug resistance, radioresistance and minimise the side effect profile of the treatment modalities. To increase the efficacy of 2-Methoxyestradiol (2ME2) as an anticancer agent, a sulfamoylated analogue was designed to retain the functional groups of the parent compound presented on a non-steroidal scaffold. A tetrahydroisoquinoline (THIQ) core was used to mimic the A- and B rings of 2ME2. In this study, 2-(3-Bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline (STX3451) was used to pre-treat MDA-MB-231- and MCF-7 breast cancer cells before radiation in order to investigate the cell fate as a result of this combination treatment. The concentration at which growth cell proliferation was inhibited by 50% (GI50) was determined via spectrophotometric analysis of crystal violet (CV) staining. Thereafter, cells were exposed to 0.07 μM STX3451 for 24-hours prior to a single dose of 6 Gray (Gy) radiation. In order to determine the temporal intracellular events in response to the combination parallel to the individual treatment controls, experiments were terminated 2-, 24-, or 48-hours after radiation. Cell cycle progression and induction of apoptosis were investigated via flow cytometric analysis. Morphological changes were investigated using light microscopy. The extent of radiation-induced deoxyribonucleic acid (DNA) damage was assessed via micronuclei (Mn) quantification. Early in vitro response to the combination therapy included quantification of reactive oxygen species (ROS) generation using flow cytometric analysis of hydroethidine (HE) staining. DNA-damage repair signalling as a response to the treatments was investigated by semi-quantitative analysis of ataxia-telangiectasia mutated (ATM) expression using Western blots. Long-term cellular survival was assessed via clonogenic studies. Cytotoxicity studies on both cell lines revealed half maximum growth inhibitory concentration (GI50) values in the micromolar range. MCF-7 and MDA-MB-231 cells displayed a metaphase block, with a concurrent decrease in viability and an increase in apoptosis in response to the iv combination treatment, a phenomenon that was attributed to the STX3451 pre-treatment and the radiation. On microscopy, an increased number of rounded cells were observed in response to combination treatment. Both cell lines displayed significantly more Mn formation in response to the combination treatment when compared to the experimental controls. MCF-7 cells exposed to the combination treatment resulted in significantly more ROS generation 48-hours after radiation, in alignment with the drug control. ATM expression was suppressed in response to the combination-and compound-only treated MDA-MB-231- and MCF-7 cells, which indicates possible suppression of the DNA damage response (DDR). Both MCF-7 and MDA-MB-231 cells displayed a significant decrease in long-term cell survival in response to combination treatment when compared to experimental controls. In conclusion, STX3451 demonstrated potential radiosensitizing effects in breast cancer cells. By increasing DNA damage, inhibiting expression of the essential DNA damage repair protein ATM, and decreasing the long-term viability of breast cancer cells, this combination treatment shows promise in reducing the amount of radiation required to treat breast cancer. Future studies should involve senescence-associated β-galactosidase detection to determine whether these cells undergo senescence in response to STX3451 and radiation combination treatment.