In Vitro Evaluation of Anti-cancer Effect of Various Ecto-5' - Nucleotidase Inhibitors

Abstract

In vitro Evaluation of Anti-cancer Effect of Various Ecto-5′- Nucleotidase Inhibitors Ecto-nucleotidases play an important role in the transmission of purinergic signaling by hydrolyzing extracellular nucleotides into their respective nucleoside and free phosphate group. Among the different types of ecto-nucleotidases, ecto-5'- nucleotidase involves the dephosphorylation reaction i.e., conversion of extracellular AMP into adenosine and phosphate group. Adenosine either activates transmembrane adenosine receptors or can be internalized though dipyridamole-sensitive carriers. Adenosine acts as barrier on different cell types (e.g., endothelia, epithelia) and results in various physiological functions such as regulation of endothelial permeability, stimulation of epithelial chloride secretion and attenuation of neutrophil adhesion. The nucleoside, adenosine is involved in various processes that leads to adaptation to hypoxic condition such as circulatory and respiratory response. In addition to enzymatic hydrolysis, e5′NT/CD73 behaves as an adhesive molecule and interacts with extracellular matrix glycoprotein, such as fibronectin and laminin. Irrespective of its enzymatic role, e5′NT also function as co-signaling molecule on T lymphocytes and showed expression on many cancerous tissue and tumor cell lines. Its overexpression is linked with invasive cancer phenotype, tumor-promoting functions and drug resistance. In such conditions, potent and selective inhibitors of e5'NT might be helpful candidates for the prevention or treatment of these pathological conditions. In this present work, various derivatives of ionic liquid and pyridine group were selected and studied for their potential behaviour to inhibit the membrane bounded e5'NT. The data obtained from different experiments exhibited that 2p (pyrido coumarin), 10a (4-aminopyridine derivatives), 4a (thiazole), 5c (quinoline) and 3d (pyrazolo-pyridine) were found highly potent and selective inhibitors of e5′NT. Moreover, ionic liquid based derivatives were also evaluated for their inhibitory activity against e5′NT. The imidazole based derivatives F-4 (imidazole based) and A 6 (amine based) derivative showed potent inhibition. Among all potent derivatives of different series, 10a exhibited the highest inhibitory potential (IC50 value of 0.25 ± 0.05 µM) on h-e5′NT. Molecular docking study exhibited the binding interactions of these selective inhibitors within the active site of enzyme, also supported the enzyme inhibition results. The cell line based in vitro assays (malachite green assay, cytotoxic Dimethyl–2–thiazolyl–2,5–diphenyl–2H–tetrazolium bromide (MTT) assay and flow cytometric assay) were performed on the most potent and selective inhibitors of e5′NT. In malachite green assay, the HeLa cell lysate was employed and the production of free phosphate was significantly inhibited by these derivatives. Cytotoxic results (MTT assay) from both normal baby hamster kidney cells (BHK-21) and cervical cancer cell line (HeLa, Henrietta Lacks) showed that these inhibitors selectively toxic to cancer cell line with no effect on normal cells. Apoptosis analysis on HeLa cells was carried out using flow cytometer and the apoptotic results were further confirmed by visualization of fragmented nuclei under fluorescence microscope using staining dyes i.e.,DAPI. It was observed that the compounds which exhibited maximum anticancer potential also induced maximum apoptosis in HeLa cell lines. Furthermore, the inhibition mechanism of the potent derivatives at molecular level i.e., effect on mRNA (transcriptional level) level was also evaluated by using quantitative polymerase chain reaction (qPCR) technique. All potent inhibitors show reduction in up-regulation of e5′NT enzyme at mRNA level. The assay was carried out at different time interval and it was found as the time of incubation of HeLa cells with inhibitor increased, there is reduction in expression of e5′NT enzyme at molecular level. Finally, the protein level in treated HeLa cells were evaluated that confirmed the qPCR results as e5′NT protein was decreased at different time interval with maximum reduction at longer incubation of these inhibitors. The obtained results revealed that among the selected deivatives, pyridine derivatives were found as potent and selective inhibitors of e5′NT with strong anticancer potential. The ionic liquid derivatives also displayed potent anticancer potential, but less than pyridine derivatives. Hence, the identified inhibitors can be used in future as potential leads to synthesize more derivatives that can be employed for the treatment of cervical cancer linked with the abberent expression of enzyme. Additionally, these inhibitors can be explored further though in vivo studies to determine the anticancer mechanisms in future.