Tetradenia riparia (hochst.) Codd (lamiaceae) aqueous leaf extract enhanced oxidant production and nf-b mediated apoptosis in hepatocellular carcinoma (HEPG2) cells.

Abstract

Introduction: Cancer has become a global health problem, contributing almost 20 million new cases and 10 million deaths worldwide in 2020. Ranked third globally, liver cancer accounted for 8.3% of the global cancer-associated mortality. Hepatocellular carcinoma (HCC) accounts for more than 90% of diagnosed primary liver cancer cases. Treatment may include chemotherapy, radiation therapy and liver transplant, but these treatments are expensive and induce serious adverse effects that reduce the efficacy of treatment. Medicinal plants are the suggested alternate treatment option attributed to anti-microbial, antioxidant and anti-inflammatory effects that may ameliorate the harsh side effects, but also demonstrate anti-cancer activity. Tetradenia riparia is a readily available South African shrub with medicinal and anti-cancer potential, but the mechanisms require elucidation. This study aimed to investigate the antioxidant and cell death mechanisms activated by Tetradenia riparia aqueous leaf extract (TRALE) in HCC (HepG2) cells. Methods: HepG2 cells were treated with several concentrations of TRALE (0–3000 μg/ml) for a duration of 24 hours to assess the cell viability. The methylthiazol tetrazolium (MTT) assay was used to obtain an IC50, which was used to treat the HepG2 cells for all subsequent assays. After treatment, mitochondrial activity was assessed as a measure of cell viability using luminometry for the ATP and mitochondrial membrane potential (m) assays. The cells were assayed for oxidative stress by quantifying free radical mediated membrane damage (TBARS, NOS and LDH cytotoxicity assays, and iNOS gene expression), and antioxidant response using luminometry (GSH, GSSG, ratio of GSH/GSSG), qPCR (SOD2, catalase, GPx1, Nrf2) and western blotting (HSP70). Induction of apoptosis was determined by luminometrically quantifying caspase-3/7, caspase-8, and caspase-9 activities. Furthermore, the Annexin V apoptosis and necrosis assay elucidated the cell death pathway. Protein expression of cIAP (Western blot) and qPCR for NF-κB and BCL-2 were also evaluated. Results: After a 24-hour exposure, TRALE conferred a dose-dependent reduction in cell viability that was associated with a notable drop in ATP at the IC50. An increase in ΔΨM led to increased levels of ROS, which increased lipid peroxidation and RNS production despite downregulated iNOS. Elevated SOD2 in response to ROS production enhanced the HepG2 cell’s ability to convert superoxide radicals into H2O2, while catalase and Gpx1 were upregulated to prevent harmful ROS formation and protect macromolecules from oxidative damage. Increased GPx1 was associated with depletion of GSH and the GSH/GSSG ratio, but Nrf2 was downregulated and HSP70 was similar to the control. The downregulation of Nrf2 was associated with increased NF-κB. Initiator caspase-8 activation corresponded with downregulated cIAP, while downregulated BCL-2 contributed to caspase-9 activation. In addition, activation of caspase-3/7 facilitated phosphatidylserine externalisation. Necrotic markers and LDH were decreased, suggesting that TRALE induced apoptosis in HepG2 cells. Conclusion: The decreased cell viability was associated with depletion of ATP, while m contributed to ROS production that increased RNS and caused lipid peroxidation. Although GSH was depleted and Nrf2 was downregulated, the upregulated SOD2, catalase and Gpx1 suggest a response suggested a response to ameliorate oxidative stress as implied by unchanged HSP70. However, oxidants were still elevated and associated with increased NF-κB. Interestingly, iNOS expression was downregulated, thus NF-B contributed to initiation of apoptosis that was associated with decreased BCL-2 and cIAP, and increased caspase activity. In addition, necrosis was not evident. Therefore, the TRALE-treated HepG2 cells were more susceptible to apoptosis. Further studies are required to elucidate the role of NF-κB in TRALE-induced apoptosis.