Induction of Human Hepatocellular Carcinoma HepG2 Cell Apoptosis by Naringin
Ratana Banjerdpongchai1, Benjawan Wudtiwai, Patompong Khawon
1Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chinag Mai, Thailand Email : ratana.b@cmu.ac.th, rbanjerd@gmail.com.
Abstract:
Naringin, a bioflavonoid found in Citrus seeds, inhibits proliferation of cancer cells. The objectives of this study were to investigate the mode and mechanism(s) of hepatocellular carcinoma HepG2 cell death induced by naringin. The cytotoxicity of naringin towards HepG2 cells proved dosedependent, measured by MTT assay. Naringintreated HepG2 cells underwent apoptosis also in a concentration related manner, determined by annexin Vfluorescein isothiocyanate (FITC) and propidium iodide (PI) employing flow cytometry. Mitochondrial transmembrane potential (MTP) measured using 3,3'dihexyloxacarbocyanine iodide (DiOC6) and flow cytometer was reduced concentrationdependently, which indicated influence on the mitochondrial signaling pathway. Caspase3, 8 and 9 activities were enhanced as evidenced by colorimetric detection of paranitroaniline tagged with a substrate for each caspase. Thus, the extrinsic and intrinsic pathways were linked in human naringintreated HepG2 cell apoptosis. The expression levels of proapoptotic Bax and Bak proteins were increased whereas that of the antiapoptotic BclxL protein was decreased, confirming the involvement of the mitochondrial pathway by immunoblotting. There was an increased expression of truncated Bid (tBid), which indicated caspase8 proteolysis activity in Bid cleavage as its substrate in the extrinsic pathway. In conclusion, naringin induces human hepatocellular carcinoma HepG2 cell apoptosis via mitochondriamediated activation of caspase9 and caspase8mediated proteolysis of Bid. Naringin anticancer activity warrants further investigation for application in medical treatment.
Insights
Naringin, a citrus bioflavonoid, induces cancer cell death through apoptosis. This study reveals naringin triggers both intrinsic and extrinsic pathways, impacting mitochondria and caspases in hepatocellular carcinoma cells.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Naringin is a citrus bioflavonoid with known anticancer properties.
- Hepatocellular carcinoma (HCC) remains a significant global health challenge.
- Understanding the mechanisms of natural compounds against cancer is crucial for therapeutic development.
Purpose of the Study:
- To elucidate the mode and mechanisms of naringin-induced cell death in HepG2 hepatocellular carcinoma cells.
- To investigate the specific signaling pathways involved in naringin's cytotoxic effects.
- To assess the role of apoptosis, mitochondrial dysfunction, and caspase activation.
Main Methods:
- Cytotoxicity was assessed using MTT assay.
- Apoptosis was quantified via flow cytometry using Annexin V-FITC and propidium iodide staining.
- Mitochondrial transmembrane potential (MTP) was measured using DiOC6 and flow cytometry.
- Caspase activity (caspase-3, -8, -9) was determined using colorimetric assays.
- Protein expression of Bax, Bak, BclxL, and truncated Bid (tBid) was analyzed by immunoblotting.
Main Results:
- Naringin exhibited dose-dependent cytotoxicity against HepG2 cells.
- Naringin induced apoptosis in a concentration-dependent manner.
- Mitochondrial transmembrane potential decreased significantly with increasing naringin concentration.
- Activities of caspase-3, -8, and -9 were elevated.
- Expression of pro-apoptotic proteins (Bax, Bak) increased, while anti-apoptotic protein (BclxL) decreased.
- Truncated Bid (tBid) expression indicated caspase-8 activation.
Conclusions:
- Naringin induces apoptosis in HepG2 cells through a mitochondriamediated intrinsic pathway involving caspase-9 activation.
- Naringin also activates the extrinsic pathway via caspase-8-mediated Bid cleavage.
- These pathways are interconnected, leading to overall apoptosis.
- Naringin's anticancer potential warrants further investigation for therapeutic applications.
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