Diffractaic acid exerts anti-cancer effects on hepatocellular carcinoma HepG2 cells by inducing apoptosis and suppressing migration through targeting thioredoxin reductase 1

Abstract

Hepatocellular carcinoma (HCC) represents one of the most common malignant tumors worldwide. Due to the limited number of available drugs and their side effects, the development of new chemotherapeutic strategies for HCC treatment has become increasingly important. This study is aimed at investigating whether diffractaic acid (DA), one of the secondary metabolites of lichen, exhibits a potential anticancer effect on HepG2 cells and whether its anticancer effect is mediated by inhibition of thioredoxin reductase 1 (TRXR1), which is a target of chemotherapeutic strategies due to overexpression in tumor cells including HCC. XTT assay results showed that DA exhibited strong cytotoxicity on HepG2 cells with an IC₅₀ value of 78.07 µg/mL at 48 h. Flow cytometric analysis results revealed that DA displayed late apoptotic and necrotic effects on HepG2 cells. Consistent with these findings, real-time PCR results showed that DA did not alter the BAX/BCL2 ratio in HepG2 cells but upregulated the P53 gene. Moreover, the wound healing assay results revealed a strong anti-migratory effect of DA in HepG2 cells. Real-time PCR and Western blot analyses demonstrated that DA increased TRXR1 gene and protein expression levels, whereas enzyme activity studies disclosed that DA inhibited TRXR1. These findings suggest that DA has an anticancer effect on HepG2 cells by targeting the enzymatic inhibition of TRXR1. In conclusion, DA as a TRXR1 inhibitor can be considered an effective chemotherapeutic agent which may be a useful lead compound for the treatment of HCC.

Keywords: Cytotoxicity; Diffractaic acid; Expression; Hepatocellular carcinoma; Inhibition; Thioredoxin reductase 1.

Fig. 1

Antiproliferative effect of DA against HepG2 cells. A Microscopic images of DA-treated HepG2 cells in a dose (10–250 µg/mL) and time (24 and 48 h) dependent manner. B Viability of HepG2 cells assessed by XTT assay after treatment with DA in a dose and time-dependent manner. C The IC₅₀ values of DA on HepG2 cells. Data are representative of three independent experiments and are presented as means ± SDs. *p < 0.05 (significant), **p < 0.01 (very significant), and ****p < 0.0001 (extremely significant). Scale bar 100 µm

Fig. 2

Induction of apoptosis in HepG2 cells by DA. A Flow cytometry results of hydrogen peroxide as a positive control (750 µM) and DA-treated HepG2 cells after 48 h incubation time. The living, early apoptotic, late apoptotic, and necrotic cells were represented by the lower left quadrant (Annexin V-FITC-/PI-), lower right quadrant (Annexin V-FITC + /PI-), upper right quadrant (Annexin V-FITC + /PI +), and upper left (Annexin V-FITC-/PI +) quadrant, respectively. B Representative bar graphs of BAX/BCL2 ratio and the relative expression of P53 gene in HepG2 cells after treatment with DA for 48 h. Data are representative of three independent experiments and are presented as means ± SEMs. ns p > 0.05 (not significant, ns) and ****p < 0.0001 (extremely significant)

Fig. 3

Suppression of migration of HepG2 cells by DA. A Representative images and quantitative analysis of wound healing assay in HepG2 cells after treatment with DA. B The area of the wound was measured in each well using ImageJ software analysis. Data are representative of the mean of six measurements of each wound area in three independent experiments (n = 18) and are presented as mean ± SEMs. *p < 0.05 (significant), ***p < 0.0005 (extremely significant), and ****p < 0.0001 (extremely significant). Scale bar 100 µm

Fig. 4

The effect of DA on TRXR1 at gene and protein expressions, and enzymatic activity level in HepG2 cells. A, B Changes in quantitative mRNA and protein expression levels of TRXR1 by DA in HepG2 cells. C Changes in the enzymatic activity level of TRXR1 by DA in HepG2 cells. Data are representative of three independent experiments and presented as means ± SEMs. ***p < 0.0005 (extremely significant) and ****p < 0.0001 (extremely significant)