Di-2-picolylamine triggers caspase-independent apoptosis by inducing oxidative stress in human liver hepatocellular carcinoma cells.

Di-2-picolylamine (DPA) is an organic compound that has been shown to possess antioxidant properties when conjugated to form a metal complex. The basis of this study was to determine the effects of DPA on the proliferation and apoptosis of human hepatocellular carcinoma cells and elucidate the possible mechanisms. The methylthiazol tetrazolium assay served to measure cell viability and generated an IC50 of 1591 µM. Luminometry was used to investigate caspase activity and ATP concentration. It was observed that the decreased cell viability was associated with reduced ATP levels. Despite increased Bax and caspase 9 activity, cell death was caspase independent as indicated by the reduction in caspase 3/7 activity. This was associated with the downregulation poly(ADP-ribose) polymerase cleavage (Western blotting). However, the Hoescht assay depicted nuclear condensation and apoptotic body formation with elevated DPA levels suggesting DNA damage in HepG2 cells. DNA damage assessed by the comet assay confirmed an increased comet tail formation. The presence of oxidative stress was investigated by quantifying reactive species (malondialdehyde and nitrates concentration) and Western blotting to confirm the expression of antioxidant proteins. The DPA increased lipid peroxidation (RNS), a marker of oxidative stress, consequently causing cell death. The accompanying upregulation of stress-associated proteins superoxide dismutase (SOD2), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and Hsp70 verifies oxidative stress.

Toxicogenicity and mechanistic pathways of aflatoxin B1 induced renal injury.

The study investigated the toxicogenic effects, molecular mechanisms and proteomic assessment of aflatoxin B1 (AFB1 ) on human renal cells. Hek293 cells were exposed to AFB1 (0-100 µM) for 24 h. The effect on cell viability was assessed using the methylthiazol tetrazolium (MTT) assay, which also produced the half maximal inhibitory concentration (IC50 ) used in subsequent assays. Free radical production was evaluated by quantifying malondialdehyde (MDA) and nitrate concentration, while DNA fragmentation was determined using the single cell gel electrophoresis (SCGE) assay and DNA gel electrophoresis. Damage to cell membranes was ascertained using the lactate dehydrogenase (LDH) assay. The concentration of ATP, reduced glutathione (GSH), necrosis, annexin V and caspase activity was measured by luminometry. Western blotting and quantitative PCR was used to assess the expression of proteins and genes associated with apoptosis and oxidative stress. The MTT assay revealed a reduction in cell viability of Hek293 cells as the AFB1 concentration was increased, with a half maximum inhibitory concentration (IC50 ) of 32.60 µM. The decreased viability corresponded to decreased ATP concentration. The upregulation of Hsp70 indicated that oxidative stress was induced in the AFB1 -treated cells. While this implies an increased production of free radicals, the accompanying upregulation of the antioxidant system indicates the activation of defense mechanisms to prevent cellular damage. Thus, membrane damage associated with increased radical formation was prevented as indicated by the reduced LDH release and necrosis. In addition, cytotoxic effects were evident as AFB1 activated the intrinsic pathway of apoptosis with corresponding increased DNA fragmentation, p53 and Bax upregulation and increased caspase activity, but externalization of phosphatidylserine (PS), a major hallmark of apoptosis, did not occur in AFB1 treated renal cells. The results suggest that AFB1 induced oxidative stress leading to cell death by the intrinsic pathway of apoptosis in renal cells.

The molecular effect of 1,4,7-triazacyclononane on oxidative stress parameters in human hepatocellular carcinoma (HepG2) cells.

Antibiotic resistance poses a great threat to human, animal and environmental health. ß-Lactam antibiotics have been successful in combating bacterial infections. However, the overuse, inappropriate prescribing, unavailability of new antibiotics and regulation barriers have exacerbated bacterial resistance to these antibiotics. 1,4,7-Triazacyclononane (TACN) is a cyclic organic tridentate inhibitor with strong metal-chelating abilities that has been shown to inhibit ß-lactamase enzymes and may represent an important breakthrough in the treatment of drug-resistant bacterial strains. However, its cytotoxicity in the liver is unknown. This study aimed to determine the effect of TACN on oxidative stress in HepG2 cells. The HepG2 cells were treated with 0 to 500 µM TACN for 24 hours to obtain an IC50 for use in subsequent assays. Free radicals were measured using the thiobarbituric acid reactive substance and nitric oxide synthase assays, respectively, while antioxidant levels were assessed using luminometry (glutathione [GSH] and adenosine triphosphate [ATP]) and Western blot analysis (SOD, catalase, GPx-1, HSP70 and Nrf2). Percentage survival fluctuated as TACN concentration increased with a calculated IC50 of 545 µM. A slight increase in HSP70 and Nrf2 expression indicated the presence of stress and a response against it, respectively. However, free radical production was not increased as indicated by decreased malondialdehyde levels and reactive nitrogen species. Glutathione levels increased slightly, while ATP levels were marginally altered. The results suggest that TACN does not induce oxidative stress in HepG2 cells and can be exploited as a potential inhibitor.

Mechanistic Insights into Oxidative Stress and Apoptosis Mediated by Tannic Acid in Human Liver Hepatocellular Carcinoma Cells.

The study investigated the cytotoxic effect of a natural polyphenolic compound Tannic acid (TA) on human liver hepatocellular carcinoma (HepG2) cells and elucidated the possible mechanisms that lead to apoptosis and oxidative stress HepG2 cell. The HepG2 cells were treated with TA for 24 h and various assays were conducted to determine whether TA could induce cell death and oxidative stress. The cell viability assay was used to determine the half maximal inhibitory concentration (IC50), caspase activity and cellular ATP were determined by luminometry. Microscopy was employed to determine deoxyribonucleic acid (DNA) integrity, while thiobarbituric acid (TBARS) and nitric oxide synthase (NOS) assays were used to elucidate cellular reactive oxygen species (ROS) and reactive nitrogen species (RNS), respectively. Western blotting was used to confirm protein expression. The results revealed that tannic acid induced caspase activation and increased the presence of cellular ROS and RNS, while downregulating antioxidant expression. Tannic acid also showed increased cell death and increased DNA fragmentation. In conclusion, TA was able to induce apoptosis by DNA fragmentation via caspase-dependent and caspase-independent mechanism. It was also able to induce oxidative stress, consequently contributing to cell death.

Molecular mechanisms underlying the renoprotective effects of 1,4,7-triazacyclononane: a ßeta-lactamase inhibitor.

Broad-spectrum ß-lactam antibiotics such as penicillin are routinely used against both Gram-negative and Gram-positive bacteria. However, bacteria that produce ß-lactamase have developed resistance against these antibiotics by cleaving the ß-lactam ring and rendering the antibiotic inactive. To combat this effect, 1,4,7- Triazacyclononane (TACN), a cyclic organic compound derived from cyclononanes has been shown to preserve the activity of ß-lactam antibiotics by inhibiting ß-lactamase. However, its cytotoxic effects require elucidation. Given that the cytotoxic target for many therapeutics is the kidney, this study investigated the effects of TACN on human embryonic kidney cells (Hek293) cells. Hek293 cells were treated with TACN (0-500 µM) for 24 h and the cytotoxicity was assessed (MTT and LDH assay). Apoptosis was luminometrically detected by measuring phosphatidylserine externalisation and caspase activity and fluorescently detecting necrosis. DNA fragmentation was visualised using fluorescent microscopy. Expression of the apoptosis-related protein were determined by western blot. The results generated indicate that TACN does not initiate necrosis as LDH was decreased. Likewise, decreased apoptosis was supported by the decreased phosphatidylserine, caspases, Bax, cleaved PARP, IAP and NF-kB. However, increased DNA fragmentation was associated with increased p53. Therefore, effects of TACN at the nucleus, produced a p53 response to initiate DNA repair and did not culminate in cell death. The findings show that TACN is not cytotoxic to Hek293 cells via the apoptotic route. Since TACN did not induce cell death, its potential as a metallo-ß-lactamase inhibitor (MBLI) may be exploited to counteract the effect of MBL-producing bacteria. Restoring ß-lactam activity will curb the global menace of antibiotic resistance.

Cytoproliferative and Anti-Oxidant Effects Induced by Tannic Acid in Human Embryonic Kidney (Hek-293) Cells.

Tannic acid (TA) portrays a myriad of beneficial properties and has forthwith achieved incessant significance for its cytoprotective qualities in traditional and modern-day medicine. However, TA displays an ambiguous nature demonstrating anti-oxidant and pro-oxidant traits, beckoning further research. Although vast literature on the anti-proliferative effects of TA on cancer cell lines exist, the effects on normal cells remain unchartered. Herein, the cytoproliferative and anti-oxidant effects induced by TA in human embryonic kidney (Hek-293) cells were investigated. Data obtained from the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay demonstrated that TA increased the cell viability and cellular proliferation rate at higher concentrations. Hoechst assay, examining proliferation marker Ki67 supported these findings. DNA fragmentation and oxidative stress-inducers were specifically noted at IC25 and IC50 treatments via biochemical assays. This alluded to TA's pro-oxidant characteristics. However, the countervailing anti-oxidant defence mechanisms as the endogenous anti-oxidants and phase2 detoxification enzymes were significantly upregulated. Luminometry fortified the anti-oxidant capacity of TA, whereby executioner caspase-3/7 were not activated subservient to the activation of initiator caspases-8 and -9. Thus, proving that TA has anti-apoptotic traits, inter alia. Therefore, TA proved to harbour anti-oxidant, anti-apoptotic, and proliferative effects in Hek-293 cells with its partial cytotoxic responses being outweighed by its cytoprotective mechanisms.