Abstract:
Unique, in silico-designed compounds with possible anticancer properties were identified in our laboratory. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16), with potential carbonic anhydrase IX inhibiting activity, is capable of interfering with microtubule dynamics.
In this study, it was investigated whether ESE-16 is capable of inducing apoptosis in vitro in the esophageal carcinoma SNO cell line via the intrinsic pathway at a concentration of 0.2μM with an exposure time of 24 hours.
Qualitative results were obtained via polarization-optical transmitted light differential interference contrast microscopy, light microscopy, transmission electron microscopy and confocal microscopy. Results showed hallmarks of apoptosis in the ESE-16-treated cells. In addition, data revealed an increase in the number of ESE-16-treated cells blocked in metaphase. Cell death via apoptosis in the ESE-16-treated cells was confirmed by studying the internal ultrastructure of the cells via transmission electron microscopy, while confocal microscopy revealed abnormal spindle formation and condensed chromatin in ESE-16-treated cells, confirming metaphase block.
Quantitative results were obtained via flow cytometry and spectrophotometry. Cell death via apoptosis in ESE-16-treated cells were quantitatively confirmed by cell cycle progression analysis and the Annexin V-FITC apoptosis detection assay. Metaphase block due to ESE-16 exposure was confirmed by demonstrating an increase in cyclin B levels in the ESE-16-treated cells. In addition, flow cytometry and spectrophotometry revealed dissipation of mitochondrial membrane potential, an increase in superoxide levels, changes in the redox status and an increase in cytochrome c levels in the cytosol of the ESE-16-treated cells. Both initiator caspase 9 and effector caspase 3 activities were increased, which demonstrates that ESE-16 causes cell death in a caspase-dependent manner.
This was the first in vitro study conducted to investigate the action mechanism of ESE-16 on an esophageal carcinoma cell line. The results provided valuable information on the action mechanism of this potential anticancer agent. It can be concluded that the novel in silico-designed compound exerts an anti-proliferative effect on the esophageal carcinoma SNO cell line by disrupting microtubule function resulting in metaphase block. This culminates in apoptotic cell death via the intrinsic apoptotic pathway. This research provided cellular targets warranting in vivo assessment of ESE-16’s potential as an anticancer agent.
