Synergisic effect of APRIL knockdown and Jiedu Xiaozheng Yin, a Chinese medicinal recipe, on the inhibition of hepatocellular carcinoma cell proliferation

Abstract

It is well documented that A proliferation-inducing ligand (APRIL), a member of the tumor necrosis factor superfamily, plays a crucial role in the occurrence and development of tumors. In the present study, we evaluated the synergistic effect of APRIL knockdown and Jiedu Xiaozheng Yin (JXY), a Traditional Chinese Medicinal recipe, on the inhibition of hepatocellular carcinoma (HCC) cell proliferation and elucidated the underlying mechanism. The results demonstrated that both APRIL knockdown using small interfering RNA (siRNA) and JXY treatment could trigger cell cycle arrest and cell apoptosis, and suppress HCC cell proliferation through an NF-κB-related pathway. Synergism was further demonstrated between APRIL knockdown and JXY treatment. In conclusion, these results indicate that APRIL is a target gene for HCC and combination of siRNA-APRIL and JXY application holds great promise as a novel approach for the treatment of APRIL-positive HCC.

Figure 1.

(A) JXY-containing serum at different concentrations inhibits the proliferation of HCC cells in vitro. (B) Comparison of the levels of cell viability after treatment of JXY at the indicated concentrations for 72 h. The viability of the HCC cells was analyzed using methyl thiazolyl tetrazolium (MTT) method. Although JXY treatment had no effect on HCC cell proliferation (P>0.05), the combination of APRIL knockdown and JXY treatment significantly suppressed cell proliferation in a dose-dependent manner (*P<0.05). Data shown are expressed as the mean ± standard deviation. JXY, Jiedu Xiaozheng Yin; HCC, hepatocellular carcinoma; APRIL, A proliferation-inducing ligand.

Figure 2.

Effect of APRIL knockdown and JXY treatment on HCC cell apoptosis as confirmed by TUNEL assay. (A) The TUNEL assay was carried out by One Step TUNEL Apoptosis Assay kit. The images of TUNEL-positive cells were captured by a fluorescence microscope (magnification, ×200). (B) The quantitative result of the TUNEL assay was analyzed. Data are represented as the mean ± standard deviation; *P<0.05 vs. the control cells; **P<0.05 vs. the cells treated with JXY (Herb) alone; ^#P<0.05 vs. the cells treated with shRNA alone. APRIL, A proliferation-inducing ligand; JXY, Jiedu Xiaozheng Yin; HCC, hepatocellular carcinoma; TUNEL, TdT-mediated dUTP nick end labeling.

Figure 3.

Effect of APRIL knockdown and JXY treatment on mRNA expression levels in HCC cells. The mRNA expression levels of APRIL, TRAF6, NF-κB and IκBα were determined by reverse transcription-polymerase chain reaction and quantified by densitometric analysis. Images are representative of 3 independent experiments. The data were normalized to the mean mRNA expression level of the untreated control; *P<0.05 vs. the control cells; **P<0.05 vs. the cells treated with Herb alone. APRIL, A proliferation-inducing ligand; JXY (Herb), Jiedu Xiaozheng Yin; HCC, hepatocellular carcinoma; TRAF6, tumor necrosis factor receptor-associated factor 6; NF-κB, nuclear factor-κB.

Figure 4.

Effect of APRIL knockdown and JXY treatment on protein expression levels in HCC cells. (A) The expression level of all proteins was quantified by densitometric analysis. (B) The expression levels of proteins associated with the NF-κB pathway were assessed by western blot analysis. Data shown are expressed as the mean ± standard deviation of 3 repeated experiments; *P<0.05 vs. the control cells; **P<0.05 vs. the cells treated with the Herb alone; ^#P<0.05 vs. the cells treated with shRNA alone. APRIL, A proliferation-inducing ligand; JXY (Herb), Jiedu Xiaozheng Yin; HCC, hepatocellular carcinoma; TRAF6, tumor necrosis factor receptor-associated factor 6; NF-κB, nuclear factor-κB.

Figure 5.

APRIL knockdown and JXY treatment synergistically decrease the activity of NF-κB in HCC cells. The NF-κB activity in HCC cells was analyzed by an NF-κB reporter luciferase assay. Data shown are from representative experiments where electroporations were performed in triplicate. Activation is indicated by the mean ± standard deviation; *P<0.05 vs. the control cells; **P<0.05 vs. the cells treated with the Herb alone; ^#P<0.05 vs. the cells treated with shRNA alone. APRIL, A proliferation-inducing ligand; JXY (Herb), Jiedu Xiaozheng Yin; HCC, hepatocellular carcinoma.