Introduction: Natural phenolic compounds are abundant secondary metabolites of plants. Over the last decades, polyphenols have attracted attention due to their potent antioxidant, anti-infection, anti-inflammation, immunity enhancement, and anticancer properties (1). Kuromanin chloride, gallic acid, and resveratrol are natural polyphenols of the flavonoids, phenolic acids, and stilbenes classes (2-4). Current evidence suggests that polyphenols exert their anticancer effects through several mechanisms including, inhibition of cell cycle arrest, apoptosis, induction of antioxidant enzymes, and autophagy (5). Several studies indicated that polyphenols modulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Nrf2 is a crucial transcription factor involved in cell protection against oxidative stress (6). Indeed, induction of various cytoprotective genes by Nrf2 leads to the protection of cells against exogenous or endogenous oxidative stress (7). However, Nrf2 activation was found to promote cancer development and malignant progression (8). The present study compared the effect of three polyphenols (kuromanin chloride, gallic acid, and resveratrol) on Nrf2 gene expression and cell viability in the HepG2 liver cancer cell line.
Methods: HepG2 hepatoma cell line was grown in RPMI-1640 medium with 10% FBS, and 1% Penicillin-Streptomycin and incubated under standard conditions. Cells were treated for 72h with an increasing concentration of each of the three polyphenols. After a 72h incubation period, cells were harvested and total RNA was extracted using RNX- Plus reagent (Sinaclon, Iran). The RNA concentration and purity were assessed by NanoDrop ND-2000. The cDNAs were synthesized using EURX one-Step RT-PCR Kit (Gdansk, Poland) and amplified by qPCR SYBR Green Master Mix (AddBio, Korea) on StepOn Real-Time PCR System. In addition, cell viability was determined by trypan blue exclusion assay.
Results: The results of the present study showed that kuromanin chloride and resveratrol significantly upregulated expression of Nrf2 gene dose-dependently in HepG2 cells, compared with the control group. The highest concentration of resveratrol (40 μM) increased the expression of Nrf2 by 5.5 fold (p< 0.001) compared to the non-treated control group. Concentrations of 10, 20, and 40 μM kuromanin chloride increased Nrf2 gene expression by 2.6, 3.6, and 4.9 fold, respectively. Low concentrations of gallic acid (10 and 20 μM) dramatically increased Nrf2 gene expression by 4.4 and 3 fold, respectively. However, no significant change was observed in 40 μM gallic acid. As shown by the results, all polyphenols significantly reduced cell viability and cell number compared with the control group. Gallic acid reduced cell viability and cell number more efficiently in comparison to resveratrol and kuromanin chloride. Compared to the control group, the highest concentration of gallic acid (40 μM) reduced cell number and cell viability by 73% (p< 0.001) and 13% (p< 0.001), respectively.
Conclusion: Our current study showed that all three polyphenols reduced the cell proliferation and viability of HepG2 cells. All three polyphenols increased Nrf2 gene expression, except the highest tested concentration of gallic acid (40 μM). Gallic acid is a dietary polyphenol with antioxidant and pro-oxidant characteristics (1). Gallic acid exhibited a stronger inhibition effect on the cell proliferation of HepG2 cells compared to resveratrol and kuromanin chloride. This effect of gallic acid may be due to its pro-oxidant properties. Many studies indicated the contradictory role of Nrf2 in cancers. Nrf2 expression protects both cancer and normal cells against oxidative stress. Indeed, Nr2 overexpression leads to enhancement of cancer cell proliferation through upregulation of metabolic genes (2). Further in-vivo and in vitro studies are required to shed light on the anticancer properties of different polyphenols against hepatocellular carcinoma through the induction of the Nrf2 pathway.