CCL299, a Benzimidazole Derivative Induces G₁ Phase Arrest and Apoptosis in Cancer Cells

Abstract

Background/Aim: Benzimidazoles are considered potential anticancer candidates. We herein studied the anticancer activity of CCL299, benzonitrile. Materials and Methods: In this in vitro study, we used ATP assays, flow cytometry, western blotting, and caspase-3/7 assays to evaluate the effects of CCL299 on cell proliferation, cell-cycle progression and apoptosis. Results: ATP assays showed that CCL299 inhibited cell growth in the hepatoblastoma cell line HepG2 and the cervical cancer cell line HEp-2, without exhibiting cytotoxic effects on noncancer cells and TIG-1-20 fibroblasts. Flow cytometry, western blotting, and caspase-3/7 assays revealed that CCL299 induced G 1 -phase cell-cycle arrest followed by apoptosis that was associated with up-regulation of p-p53 (Ser15) and p21 expression and the down-regulation of p-CDK2 (Thr160) expression. Conclusion: CCL299 exhibits cytotoxic activity via apoptosis in a subset of cancer cells, and should be considered as a promising anticancer candidate agent.Cancer remains a significant cause of death worldwide (1, 2). Understanding the molecular mechanisms underlying cancers has enabled cancer diagnosis and improved patient survival. However, mortality in advanced cancer is partly attributable to tumour insensitivity towards anticancer drugs and due to acquired drug resistance (3-5). Identifying novel compounds with more significant cytotoxicity for cancer cells and fewer side-effects is of great importance.Benzimidazoles are well-known heterocyclic organic compounds containing benzene and imidazole groups. Benzimidazole derivatives are known for their antimicrobial and anti-parasitic activities, and they are potential candidates for anticancer therapy because they can alter tubulin-binding capacity (6-10). Mebendazole contains a benzimidazole skeleton and has been used worldwide as a vermifuge for echinococcosis and giardiasis in humans (11,12). Mebendazole's potential as an anticancer agent was investigated and was found to induce inhibition of cancer cell growth via M phase arrest (13-15). Similarly, albendazole, which is used to treat trichuriasis and hookworm infection (16), has anticancer activity that is mediated through M phase arrest in several human cancer cell lines (17)(18)(19)(20).Generally, mammalian cell-cycle progression is regulated by cyclin-dependent kinases (CDKs), along with cyclins and CDK inhibitory proteins. CDK2 binds to cyclin E, and the resultant cyclin E-CDK2 complex promotes G 1 phase progression (21). CDK2 activity is strongly controlled by the phosphorylated status of CDK2 at threonine (Thr) 160 ( 22). p53 and p21 are well known as key CDK inhibitors that are involved in several signalling pathways. Their protein expression levels increase in response to stress signalling and this induces cell-cycle arrest at the G 1 phase, followed by apoptosis (23). Therefore, both CDKs and CDK inhibitory proteins are highly validated as anticancer drug targets (24-26).Chiba Chemical Library is a molecule library that contains more than a thousand compounds,...