Ethyl 2-[N-m-chlorobenzyl-(2′-methyl)]anilino-4-oxo-4,5-dihydrofuran-3-carboxylate (JOT01006) Induces Apoptosis in Human Cervical Cancer HeLa Cells

Human cervical cancer is potentially lethal, and therefore the development of effective and tolerable therapeutic options is vital. In the present study, the in vitro effect of the synthetized compound JOT01006 (C21H20C1NO4) on human cervical epithelioid carcinoma cell line (HeLa) was examined. The results demonstrated that JOT01006 induced morphological changes and cytotoxicity (decreased the percentage of viable cells) in a dose-dependent manner. JOT01006 induced apoptosis which was analyzed by flow cytometric methods and confirmed by DAPI staining and DNA fragmentation analyzed by DNA gel electrophoresis. JOT01006 also induced reactive oxygen species (ROS) overproduction before causing endoplasmic reticulum (ER) stress which was also confirmed by the increased levels of Grp78 and Gadd153. Western blotting was selected to demonstrate that JOT01006 promoted p53, Bak, PARP, caspase-3 levels and decreased the levels of Bcl-2 and Bcl-xL. Our results also showed that JOT01006 also promoted caspase-12 production followed by apoptosis. The results also showed that JOT01006 inhibited the migration of HeLa cells potentially through inhibition of MMP-2 and -9. Induction of apoptosis is one of the mechanisms of action of anticancer agents. It has been demonstrated that apoptosis can be induced through mitochondria-dependent and -independent pathways (1). It is well-known that many chemotherapeutic agents can trigger the mitochondrial apoptotic pathway by means of various stress signals (1). These signals can induce mitochondrial membrane permeabilization leading to cytochrome C release, caspase-3 activation and then apoptosis. Endoplasmic reticulum (ER) stress also causes apoptosis. Many agents have been shown to induce apoptosis via the overproduction of reactive oxygen species (ROS) before leading to the mitochondria- dependent pathway for apoptosis (2, 3). ROS have also been shown to cause ER stress (4). ER stress results in damage to most of the principal biological macromolecules giving the potential for apoptotic cell death (5). Our previous studies have shown that JOT01006 (formula: C21H20C1NO4) induced cytotoxicity in mice leukemia WEHI-3 cells (6). In this study, human cervical epithelioid carcinoma HeLa cells were treated with JOT01006 and analyzed for the correlation between ROS and apoptosis. The effects of JOT01006 on the cell cycle and the signal pathway for apoptosis associated with different levels of proteins were also investigated.