Fusaric acid Fumonisin B1 CO -treatment regulates AMPK signalling and induces Apoptosis in HEPG2 cells.
Loading...
Date
2019
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Background/Aim: Fusaric acid (FA) and Fumonisin B1 (FB1) are mycotoxins produced by
Fusarium fungal species. These mycotoxins are major contaminants of maize and contribute
to toxicity in animals and humans. The main mechanisms of FA and FB1 toxicity involve the
induction of oxidative stress and apoptosis; however, FA was additionally found to chelate
divalent cations, whereas FB1 inhibits sphingolipid synthesis. AMPK is an energy sensor
involved in regulating cell proliferation. AMPK targets the transcription factors, p53 and
FOXO3a that play a major role in apoptosis. To date numerous studies have investigated the
individual effects of FA and FB1, however, their combined synergistic effects are unclear.
This study investigated the effect of FA and FB1 co-treatment on AMPK-induced apoptosis in
liver HepG2 cells.
Methods: HepG2 cells were cultured and co-treated with various concentrations (5, 27,
100μM and combined 104μM FA and 200μM FB1 IC50s) of FA and FB1 for 24 hrs. Cytotoxic
effects of FA and FB1 on HepG2 cells were determined using the MTT assay. The TBARS
assay was used to determine oxidative stress. Western blot was used to determine protein
expression of AMPK, p-AMPK and p53, whereas q-PCR was used to measure FOXO3a
mRNA expression. LDH assay was used to measure membrane integrity. ATP levels and
activity of caspases -3/7, -8 and -9 were measured using luminometry.
Results: A combination of FA and FB1 decreased cell viability in a dose dependant manner.
An IC50 of 27μM for FA and FB1 was obtained. ATP levels were significantly increased at
5μM and 27μM, whereas at 100μM and combined IC50s were significantly decreased
(p<0.0001). Oxidative stress was significantly increased in FA and FB1 treated cells in a
dose dependent manner (p<0.0001). The protein expression of total AMPK was decreased
at 5μM, but increased at 27μM, 100μM and combined IC50s in relation to control (p<0.0001).p-
AMPK showed a significant decrease (p<0.0001) in all FA and FB1 treated samples despite
the increase in the expression of total AMPK. FOXO3a mRNA expression was decreased at
5μM and at combined IC50s, with the decrease being significant at 5μM. The results also
indicated an increase at 27μM and 100μM (p<0.0001). p53 protein expressions were
significantly decreased in all samples (p<0.0001). Caspase -3/7, -8 and -9 were significantly
increased at 5-100μM and decreased at combined IC50s in HepG2 cells. In FA and FB1
samples, LDH levels were significantly decreased at 5μM and 27μM, and significantly
increased at 100μM and combined IC50s (p<0.0001).
Conclusion: FA and FB1 co-treatments suppressed AMPK signalling by downregulating p-
AMPK and induced apoptosis and/necrosis in HepG2 cells.
Description
Masters Degree. University of KwaZulu-Natal, Durban.