N, N Bis (2-Pyridylmethyl)-1, 2-Ethylenediamine Tetrahydrochloride Stimulates Intrinsic Apoptosis Mediated by Oxidative and Nitrosative Stress Induction of the NF-B/STAT3 Pathway in Human Hepatocellular Carcinoma (HepG2) Cells.

Abstract

Introduction: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Its incidence is rising, and this trend is expected to continue for decades. Several cancer therapeutics have already been discovered and are being used to treat HCC, however, most of them cause severe side effects which decrease the treatment's effectiveness. Metal chelators such as ethylenediaminetetraacetic acid (EDTA) have previously demonstrated anti-cancer potential. N, Nbis (2-pyridylmethyl)-ethylenediamine tetrahydrochloride (H2pmen) is a tetradentate ligand that forms stable complexes with Fe, Cr, Cu(II), and Zn (II), and it has been shown to be a potentially effective reagent for metal chelation. This study investigated the antiproliferative and cytotoxic effects of H2pmen in the HepG2 cell line. Methods: The cell viability was determined by treating HepG2 cells with different concentrations (0–1000 μM) of H2pmen over 24h. MTT assay was used to obtain an IC50, which was then used in all subsequent assays. The cells were then assayed for oxidative stress and membrane damage (TBARS, NOS, GSH, and LDH cytotoxicity), apoptotic induction (ATP assay, JC-10 assay, Annexin v, Caspases), cytochrome P450 3A4 activity (Luminometry). Protein expression of iNOS, SOD2, Bax, Caspase-2, and STAT3 were identified using western blot analysis. The gene expression of GPx1, Nrf2, NF-κB, p53, and OGG1 was determined using qPCR. Results: H2pmen induced a dose-dependent decrease in cell viability (IC50 of 209 μg/ml), a significant increase in CYP34A activity (p0.05 at IC20 and IC50), a decrease in ATP production (at IC20 p0.05 and at IC50), a significant decrease in m (p0.05 at IC20 and at IC50). The ROSassociated membrane was induced, indicated by an increase in lipid peroxidation (p0.05 at IC20 and p≥0.05 at IC50), an increase in RNS production (p≥0.05 at IC20 and at IC50), an upregulation in iNOS protein expression (at IC20 where p0.05 and at IC50) and NF-κB gene expression (at IC20 where p0.05 and at IC50). Oxidative stress occurred due to a decrease in GSH levels (at IC20 and p≥0.05 at IC50), a significant downregulation in SOD2 protein expression, and upregulation in gene expression of GPx-1 (at IC20 where p≥0.05 and at IC50) and Nrf2 (at IC20 and at IC50 where p0.05). H2pmen initiated caspase-dependent apoptosis that was indicated by a decrease in Caspase-2 (p0.05at IC20 and at IC50), caspase-8 (at IC20 and p≥0.05 at IC50), a slight insignificant decrease at IC20 and an increase at the IC50 in caspase-9, a significant upregulation in Bax (p0.05 at IC20 and at IC50) protein expression and p53 (at IC20 where p0.05 and at IC50) gene expression. The significant increase in caspase-3/7 (p≥0.05 at IC20 and IC50), Annexin V levels (p≥0.05 at IC20 and at IC50), LDH (p≥0.05 at xviii IC20 and IC50), STAT3 (p0.05at IC20 at IC50), PARP1 (p0.05 at IC20 and at IC50), and OGG1 (p0.05 at IC20 and at IC50) shows that apoptosis was executed by H2pmen in HepG2 cells. Conclusion: H2pmen reduced cell viability of HepG2 cells, exerting a cytotoxic effect associated with decreased m and ATP, and increased LDH leakage. The chelating properties of H2pmen was linked to the induction of oxidative and nitrosative stress that affected lipids and DNA. The HepG2 cells mounted an antioxidant defense involving Nrf2 to counteract the depletion of SOD2 and GSH, with evidence of its effect associated with upregulation of GPx. The prevailing oxidative stress activated DNA repair enzymes (PARP1 and p53), while NF-κB and STAT3 pathways were also induced. Bax-induced MOMP and caspase-2 invoked VDAC triggered caspase-dependant apoptosis via the intrinsic pathway.