In vitro assessment of a computer-designed potential anticancer agent in cervical cancer cells

Abstract

BackgroundComputer-based technology is becoming increasingly essential in biological research where drug discovery programs start with the identification of suitable drug targets. 2-Methoxyestradiol (2ME2) is a 17β-estradiol metabolite that induces apoptosis in various cancer cell lines including cervical cancer, breast cancer and multiple myeloma. Owing to 2ME2’s poor in vivo bioavailability, our laboratory in silico-designed and subsequently synthesized a novel 2ME2 analogue, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol), using receptor- and ligand molecular modeling. In this study, the biological effects of ESE-15-ol (180 nM) and its parent molecule, 2ME2 (1 µM), were assessed on morphology and apoptosis induction in cervical cancer cells.ResultsTransmission electron microscopy, scanning electron microscopy and polarization-optical transmitted light differential interference contrast (PlasDIC) images demonstrated morphological hallmarks of apoptosis including apoptotic bodies, shrunken cells, vacuoles, reduced cell density and cell debris. Flow cytometry analysis showed apoptosis induction by means of annexin V-FITC staining. Cell cycle analysis showed that ESE-15-ol exposure resulted in a statistically significant increase in the G2M phase (72%) compared to 2ME2 (19%). Apoptosis induction was more pronounced when cells were exposed to ESE-15-ol compared to 2ME2. Spectrophotometric analysis of caspase 8 activity demonstrated that 2ME2 and ESE-15-ol both induced caspase 8 activation by 2- and 1.7-fold respectively indicating the induction of the apoptosis. However, ESE-15-ol exerted all of the above-mentioned effects at a much lower pharmacological concentration (180 nM) compared to 2ME2 (1 µM physiological concentration).ConclusionComputer-based technology is essential in drug discovery and together with in vitro studies for the evaluation of these in silico-designed compounds, drug development can be improved to be cost effective and time consuming. This study evaluated the anticancer potential of ESE-15-ol, an in silico-designed compound in vitro. Research demonstrated that ESE-15-ol exerts antiproliferative activity accompanied with apoptosis induction at a nanomolar concentration compared to the micromolar range required by 2ME2. This study is the first study to demonstrate the influence of ESE-15-ol on morphology, cell cycle progression and apoptosis induction in HeLa cells. In silico-design by means of receptor- and ligand molecular modeling is thus effective in improving compound bioavailability while preserving apoptotic activity in vitro.

A Combination of an Antimitotic and a Bromodomain 4 Inhibitor Synergistically Inhibits the Metastatic MDA-MB-231 Breast Cancer Cell Line

“…Furthermore, data from our cell cycle progression studies also revealed that the treatment of both MDA-MB-231 and MCF-7 breast cancer cells to ESE-15-ol alone leads to a significant increase in cells present in the G 2 /M phase. These results were consistent with previous studies from our laboratory which showed that ESE-15-ol caused a marked G 2 /M cell cycle arrest in cervical cancer (HeLa) and breast cancer (MCF-7, MDA-MB-231 and MCF-12A) cell lines [19, 20]. These results indicate that ESE-15-ol is able to induce a mitotic cell cycle arrest.…”

“…Previous studies in our laboratory have shown that ESE-15-ol is more potent than 2ME2 and that ESE-15-ol inhibits cell growth of the human tumorigenic breast epithelial cell line (MCF-7), human metastatic breast cell line (MDA-MB-231), human cervical adenocarcinoma cells (HeLa), and human nontumorigenic breast epithelial cell line (MCF-12A) [19, 20]. ESE-15-ol binds to the colchicine binding site on tubulin, thus triggering cells to undergo mitotic arrest which consequently leads to the induction of apoptosis [19, 20]. The MCF-12A cells were the least affected by 50 nM ESE-15-ol when compared to MDA-MB-231 and MCF-7 cells [19].…”

A bis-sulphamoylated estradiol derivative induces ROS-dependent cell cycle abnormalities and subsequent apoptosis

“…A stock solution of EMBS dissolved in dimethyl sulphoxide (DMSO) was prepared with a concentration of 10 mM and was stored at 4°C. Previous studies conducted in our laboratory demonstrated optimal antiproliferative activity after exposure to 0.4 μM EMBS for 24 h [ 15 ]. For this reason, for all subsequent experiments, cell lines were exposed to 0.4 μM EMBS.…”

“…We showed that EMBS exposure led to mitochondrial membrane depolarisation, caspase 6 and caspase 7 activation, while the extrinsic apoptotic pathway was also activated through caspase 8 [ 14 ]. Furthermore, 2ME2 exposure also resulted in increased reactive oxygen species (ROS) generation including hydrogen peroxide and superoxide [ 2 , 15 ]. 2ME2-dependent ROS induction was also responsible for endoreduplication in nasopharyngeal carcinoma cells via c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase family (MAPK) activation [ 15 ].…”

“…Furthermore, 2ME2 exposure also resulted in increased reactive oxygen species (ROS) generation including hydrogen peroxide and superoxide [ 2 , 15 ]. 2ME2-dependent ROS induction was also responsible for endoreduplication in nasopharyngeal carcinoma cells via c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase family (MAPK) activation [ 15 ].…”