Abstract
Background2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) is a unique, in silico-designed compound with possible anticancer properties, which were identified in our laboratory. This compound is capable of interfering with microtubule dynamics and is believed to have potential carbonic anhydrase IX inhibiting activity.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 0.2 μM with an exposure time of 24 hours.ResultsQualitative results were obtained via 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, thus confirming metaphase block.Quantitative results were obtained via flow cytometry and spectrophotometry. Cell death via apoptosis in ESE-16-treated cells was quantitatively confirmed by the Annexin V-FITC apoptosis detection assay. Flow cytometry and spectrophotometry revealed dissipation of mitochondrial membrane potential and an increase in superoxide levels in the ESE-16-treated cells when compared to the relevant controls. Both initiator caspase 9 and effector caspase 3 activities were increased, which demonstrates that ESE-16 causes cell death in a caspase-dependent manner.ConclusionsThis 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.
Highlights
Microtubule-interfering drugs (MIDs) are one of the most promising classes of cancer chemotherapeutic drugs available [1,2,3]
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
Summary
Microtubule-interfering drugs (MIDs) are one of the most promising classes of cancer chemotherapeutic drugs available [1,2,3]. MIDs target the cell cycle by binding to and interfering with the microtubule machinery, thereby inhibiting the normal function of the mitotic spindle and preventing hyperproliferation of cancer cells [1,4,5,6]. The compound influences the spindle assembly checkpoint (SAC) by interacting with the colchicine binding site situated between the α- and β-dimers of the tubulin protein [8,9,14,17,18] This interaction causes abnormal spindle formation and activation of the spindle checkpoint, which leads to metaphase arrest, inhibition of further cell proliferation and the induction of cell death [6,10,11,19]. ESE-16 (Figure 1B) was developed when potential carbonic anhydrase IX (CAIX) inhibitors, capable of interfering with microtubule dynamics were identified in our laboratory with the use of bioinformatics software [8]
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