Combination of Wogonin and Artesunate Exhibits Synergistic anti-Hepatocellular Carcinoma Effect by Increasing DNA-Damage-Inducible Alpha, Tumor Necrosis Factor α and Tumor Necrosis Factor Receptor-Associated Factor 3-mediated Apoptosis

Abstract

Hepatocellular carcinoma (HCC) is difficult to treat, and is the second leading cause of cancer-related death worldwide. This study aimed to examine whether combination of wogonin and artesunate exhibits synergistic anti-HCC effect. Our data show that the combination treatment exhibits synergistic effect in reducing HCC cell viability by increasing apoptosis as indicated by the elevated cleavage of caspase 8, 3 and PARP. Interestingly, PCR array and the subsequent studies indicate that the combination treatment significantly increases the expression of DNA-damage-inducible, alpha (GADD45A), tumor necrosis factor (TNFα) and TNF receptor-associated factor 3 (TRAF3). Knockdown of GADD45A, TNFα or TRAF3 abolishes the combination treatment-enhanced apoptosis and the synergistic effect in reducing HCC cell viability. In the HCC-bearing xenograft mouse models, although the combination treatment increases the activity of NFκB in the tumor tissues, it exhibits a more potent anti-HCC effect than the mono-treatment, which may due to the enhanced apoptosis as indicated by the increased expression of GADD45A, TNFα, TRAF3 and apoptotic markers. Our study clearly demonstrates that the combination of artesunate and wogonin exhibits synergistic anti-HCC effect, and support the further development of this combination as alternative therapeutics for HCC management.

Keywords: GADD45; GADD45a; TRAF3; artesunate; hepatocellular carcinoma; tumor necrosis factor-α; wogonin.

FIGURE 1

Combination of artesunate and wogonin exhibits synergistic effect in inhibiting HCC cell proliferation. (A). HepG2 and (B). Hep3B cells were treated with different doses of ATN (5, 10, 20, 40 μM), WOG (5, 10, 20, 40 μM), or combinations of both. Cell viabilities were measured by MTT assay after 72 h treatment. ATN, artesunate; WOG, wogonin. Shown is mean ± SE, n = 3 independent experiments.

FIGURE 2

Combination of artesunate and wogonin significantly increases apoptosis in the HCC cells. (A). HepG2 cells and (B). Hep3B cells treated with ATN (10 μM), WOG (10 μM) or combination of both for 48 h were stained with Annexin V/PI. Vehicle served as control. Flow cytometry analysis of the percentages of early and late apoptosis in these cells (bottom panel). (C). Expression of cleaved-PARP, cleaved-caspase 8, cleaved-caspase 3, full length caspase 12, cleaved-caspase 12, full length caspase 10, cleaved-caspase 10, cleaved-caspase 9, full length caspase 7, cleaved-caspase 7, full length caspase 6, cleaved-caspase 6 and full length caspase 4, and quantitative analysis of the protein expressions, of the HepG2 cells treated with ATN (10 μM), WOG (10 μM) or the combination of both for 48 h. (D) Expression of cleaved-PARP, cleaved-caspase 8 and cleaved-caspase 3, and quantitative analysis of the protein expressions, of the Hep3B cells treated with ATN (10 μM), WOG (10 μM) or combination of both for 48 h. Shown is mean ± SE, n = 3 independent experiments. 1*p < 0.05, **p < 0.01, compared with control; a<0.05, aa<0.01 compared with ATN; b < 0.05, compared with WOG. ATN, artesunate; WOG, wogonin; PI, propidium iodide; Cleaved-PARP, cleaved-poly-(ADP-ribose) polymerase.

FIGURE 3

Combination of artesunate and wogonin changes the profiles of the apoptosis-related genes in HCC cells. (A). Heat map of the PCR array showing the expression levels of the apoptosis-related genes in the HepG2 cells treated with ATN (10 μM), WOG (10 μM) or the combination of both for 48 h. Red color demonstrated an increased, and green color demonstrated a reduced expression of the genes. A table (right panel) showing the genes in the PCR array in the corresponding position (gene A1 in the figure corresponds to the gene ABL1 in the table). (B). Analysis of the number of altered genes between different treatment groups. (C). The mRNA expression level of GADD45A, HRK, PYCARD, TNFRSF11B, TRAF3 and TNF in the HepG2 cells treated with ATN (10 μM), WOG (10 μM) or the combination of both for 48 h. qPCR showing the relative mRNA levels of (D). HRK, (E). GADD45A, (F). PYCARD, (G). TNF, (H). TNFRSF11 B and (I). TRAF3 in HepG2 cells treated with ATN (10 μM), WOG (10 μM) or the combination of both for 48 h. (J). Protein expression of TNFα, TRAF3 and GADD45α, and quantitative analysis of the protein expressions, of the HepG2 cells treated with ATN (10 μM), WOG (10 μM) or the combination of both for 48 h. Shown is mean ± SE, n = 3 independent experiments. *p < 0.05, **p < 0.01, compared with control; a<0.05, aa<0.01 compared with ATN; b < 0.05, bb < 0.01, compared with WOG. ATN, artesunate; WOG, wogonin; GADD45A, growth arrest and DNA-damage-inducible alpha; HRK, harakiri; PYCARD, apoptosis-associated speck-like protein containing a caspase recruitment domain; TNFRSF11B, tumor necrosis factor receptor superfamily member 11b; TRAF3, TNF receptor-associated factor 3; TNF, tumor necrosis factor.

FIGURE 4

The combination treatment-enhanced apoptosis is mediated by TNFα, TRAF3 and GADD45α in HCC cells. Relative mRNA level of (A). TRAF3, (D). TNFα, and (G). GADD45A in HepG2 cells after the respective siRNA-mediated knockdown of the gene, negative control (NC)-siRNA served as control. Protein expression levels of cleaved-PARP, cleaved-caspase 8 and cleaved-caspase 3 in (B). TRAF3-knockdown cells (si-TRAF3-HepG2) and control cells (si-TRAF3-NC), (E). TNFα-knockdown cells (si-TNFα-HepG2) and control cells (si-TNF-NC), (H). GADD45a-knockdown cells (si-GADD45A-HepG2) and control cells (si-GADD45A-NC), after the treatment with ATN (10 μM), WOG (10 μM) or the combination of both for 48 h. Flow cytometry analysis of the percentage level of early and late apoptosis in (C). si-TRAF3-HepG2, (F). si-TNFα-HepG2 and (I). si-GADD45A-HepG2 cells after the treatment with ATN (10 μM), WOG (10 μM) or the combination of both for 48 h. Shown is mean ± SE, n = 3 independent experiments. **p < 0.01 compared to control, a<0.05, aa<0.01 compared with ATN; b < 0.05 compared with WOG. ATN, artesunate; WOG, wogonin; TRAF3, TNF receptor-associated factor 3; TNF, tumor necrosis factor; GADD45A, growth arrest and DNA-damage-inducible alpha; PI, propidium iodide; Cleaved-PARP, cleaved-poly-(ADP-ribose) polymerase.

FIGURE 5

Knockdown of TNFα, TRAF3 or GADD45α abolishes the synergistic effect of the combination treatment in reducing the HCC cell viability. Cell viability of TRAF3-knockdown cells (si-TRAF3-HepG2), TNFα-knockdown cells (si-TNFα-HepG2) and GADD45a-knockdown cells (si-GADD45A-HepG2) after treating with (A). ATN (5 μM), WOG (5 μM) or the combination of both for 48 h; or treated with (B). ATN (10 μM), WOG (10 μM) or the combination of both; or (C). ATN (20 μM), WOG (20 μM) or the combination of both for 48 h. (D). Expressions of phosphorylated NFκB (p-NFκB) in HepG2 cells after treating with ATN (10 μM), WOG (10 μM) or the combination of both for 48 h. Shown is mean ± SE, n = 3 independent experiments.

FIGURE 6

The combination treatment significantly reduces tumor growth and exhibits a more potent anti-HCC effect than sorafenib in vivo. Six-week old male nude mice were inoculated subcutaneously with injections of HepG2 cells and randomly divided into 6 groups: (1) vehicle control (control 1) (i.p. 0.5% NaHCO₃ and 0.2% NaOH in saline), (2) artesunate (ATN) group (i.p. 60 mg/kg of ATN), (3) wogonin (WOG) group (i.p. 60 mg/kg of WOG), (4) ATN combined with WOG group (i.p. 60 mg/kg of ATN and 60 mg/kg of WOG), (5) vehicle control group for sorafenib (control 2) (i.p. 1% DMSO in soybean oil), (6) sorafenib group (daily i. p. 10 mg/kg of sorafenib). (A). Tumor size during the course of treatment, (B). tumor size on day 21, and (C). tumor weight of the mice after the treatments. (D). Expressions of cleaved PARP, caspase 8 and 3, and quantitative analysis of the expressions, in the tumor tissues after treatments. (E). Relative mRNA and (F). protein expressions and quantification of TNFα, TRAF3 and GADD45α in the tumor tissues after treatments. (G). Protein expression and quantification of phosphorylated NFκB (p-NFκB) in the tumor tissues after treatments. Shown is mean ± SE, n = 3-5 mice in each group. *p < 0.05, **p < 0.01 compared with control group, a<0.05, aa<0.01 compared with ATN group; b < 0.05, bb < 0.01 compared with WOG group. ATN, artesunate; WOG, wogonin; GADD45A, growth arrest and DNA-damage-inducible alpha; HRK, Harakiri; PYCARD, apoptosis-associated speck-like protein containing a caspase recruitment domain; TNFRSF11B, tumor necrosis factor receptor superfamily member 11b; TRAF3, TNF receptor-associated factor 3; TNF, tumor necrosis factor; PI, propidium iodide; Cleaved-PARP, cleaved-poly-(ADP-ribose) polymerase.