Artemisia afra crude aqueous leaf extract indices oxidative stress and inflammation in human colon adenocarcinoma cells via the upregulation of the TNF-a,p38 and STAT3 pathway.
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Date
2022
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Abstract
ABSTRACT
Introduction: Artemisia afra (A. afra) is a widely used medicinal plant located in the southern
African region. It is traditionally used to alleviate medical conditions such as coughs. Literature
indicates a protective role by improving antioxidant capacity and reducing cell proliferation, which
suggests anti-cancer potential. Colorectal carcinoma (CRC) is a global public health crisis and the
second common cause of cancer-related fatalities. Current cancer treatment is deemed effective but
not easily accessible and expensive in the southern African region. Therefore, the need for naturally
derived anti-cancer agents remains to be investigated for accessible and affordable treatment. This
study investigates the antiproliferative and antioxidant effects of A. afra crude aqueous leaf extract
in the Caco-2 cell line.
Materials and Methods: Caco-2 cells were treated with a range of A. afra concentrations (0-5000
μg/ml) for 48 hours. An IC50 was derived from the MTT assay and all subsequent assays compared
the IC50
-treatement to an untreated control. Mitochondrial integrity was luminometrically assessed by
measuring JC-10 fluorescence and ATP. Free radical production (TBARS, NOS) and membrane
damage (LDH cytotoxicity), together with GSH quantitation were used to infer the presence of
oxidative stress; antioxidant enzymes (SOD2, GPx-1, catalase, Nrf2) were also detected by western
blotting. Apoptotic induction was verified by measuring phosphatidylserine externalisation,
quantifying caspase activities and detecting pro- and anti-apoptotic proteins (Bax, Bcl2, cIAP, xIAP)
by western blotting. Single strand DNA fragmentation was evaluated via the comet assay.
Additionally, relative expression of DNA repair, inflammation and stress markers were determined
using western blotting and qPCR.
Results: Crude aqueous leaf extract of A. afra induced a dose-dependent reduction in cell viability,
yielding an IC50 of 250 μg/ml. Decreased mitochondrial integrity (p = 0.697) was associated with
significant depletion of intracellular ATP (p = 0.0043) and increased ROS production as validated by
increased lipid peroxidation (p = 0.1638) and DNA oxidation (amplified OGG1). In addition, increased
iNOS contributed to the production of RNS. Artemisia afra induced an antioxidant response that
elevated Nrf2 at the mRNA and protein level, causing increased GSH (p = 0.0001), GPx-1 (p =
0.5067) and catalase, but SOD2 was decreased. Heightened levels of heatshock proteins (HSP27 and
HSP70) correlate with increased ROS and upregulated phosphorylated p38 protein, but ERK and
JNK protein expression was downregulated. Significant downregulation caspase-8 (p = 0.0252),
caspase-9 (p = 0.0099) and caspases-3/7 (p = 0.0232) was associated with reduced Annexin-V) and
extracellular LDH. In addition, the Bax/Bcl-2 ratio (p = 0.0033) and protein expression of inhibitors
of apoptosis protein such as cIAP-1 and xIAP indicated reduced apoptotic activity in this study.
Comet tail analysis indicated intact DNA, in congruence with decreased OGG1. Both TNF-α (p =
0.2323) and STAT-3 were upregulated, but NF-ĸB was decreased. In addition, cellular Myc and
phosphorylated retinoblastoma were upregulated.
Conclusion: The crude aqueous leaf extract of A. afra induced mitochondrial toxicity and ROS
production. Despite a heightened antioxidant defense, ROS-mediated upregulation of TNF-, p38
and STAT3 promoted cell proliferation and inhibited apoptosis in Caco-2 cells. Taken together, A.
afra is a cytotoxic and genotoxic agent that may induce cancer in human colorectal cells.
Description
Masters Degree. University of KwaZulu-Natal, Durban.