The Mechanisms Of Juglanthraquinone C-induced Apoptosis Via Activating The Akt/Foxo Signal Pathway In HCC Cells

Juglanthraquinone C(JC), a naturally occurring anthraquinone extracted from Juglans mandshurica, could induce cell apoptosis in vitro. However, the underlying mechanisms involved in JC-induced apoptosis were still not fully understood. Here, we aimed to clarify the molacular mechanism of JC-induced apoptosis in human hepatocellular carcinoma(HCC) HepG2 and BEL-7402 cells.In order to find the higher effective and lower toxicity compound, we analyzed the structure-activity relationship of JC and six structurally related compounds. The results showed that JC exhibited the stronger inhibitory effect on cell viability than all six structurally related compounds.In order to clarify the molacular mechanism of antitumor activity of JC, Affymetrix HG-U133 Plus 2.0 arrays were used to analyze the gene expression profile after HepG2 cells exposed to JC or DMSO. The data indicated that JC could enhance the expression of apoptosis-promoting gene and have effect on expression of gene related to cell proliferation and cell cycle in HepG2 cells.In order to verify the apoptosis inducing effect of JC, DAPI staining were used to examine cell apoptosis. The results revealed that HepG2 and BEL-7402 cells exposed to JC showed chromatin condensation and fragmented nuclei. These results confirmed that JC could induce apoptosis of HepG2 and BEL-7402 cells.In order to clarify the molecular mechanisms by which JC induced cell apoptosis. The gene expression profiles in HepG2 cells exposed to JC or DMSO were analyzed and the data showed that the mRNA levels of four genes that could positively regulate Akt signaling were significantly up-regulated after treated with JC in HepG2 cells. The Western blot analysis further revealed that PI3K/Akt signal was strongly activated by JC in HepG2 and BEL-7402 cells. Since PI3K/Akt signal is a well-known survival pathway, in order to investigate the relationship between apoptosis and Akt activation induced by JC, we first knocking down the expression of Akt by RNA interferance technique, and then tested the apoptosis inducing effect of JC, the result shown that knocking down the expression of Akt obviously inhibited the apoptosis inducing effect of JC, while overexpression of constitutively active mutant Akt greatly accelerated JC-induced apoptosis. These results suggested that activating the PI3K/Akt signaling pathway was required in JC-induced apoptosis in HepG2 and BEL-7402 cells.To clarify the detail mechanism by which Akt signal inducing apoptosis, we analyzed the downstream signal of Akt. The Foxo3 a is a key downstream target of the PI3K/Akt pathway. The previous results revealed that JC suppressed the nuclear localization of Foxo3 a in HepG2 cells. In this study, the results suggested that JC treatment increased the levels of phosphorylated Foxo3 a and inhibited the nuclear translocation of Foxo3 a. Furthermore, overexpression of Foxo3 a abrogated JC-induced apoptosis in HepG2 and BEL-7402 cells. Further results revealed that reduction of nuclear translocation of Foxo3 a further led to an increase of intracellular reactive oxygen species(ROS) levels by suppressing ROS scavenging enzymes catalase, and the antioxidant N-acetyl-L-cysteine and catalase successfully decreased JC-induced apoptosis in HCC cells. These results suggested that JC induced apoptosis of HCC cells by increasing intracellular ROS levels.Collectively, this study demonstrated that JC induces cell apoptosis via activation of Akt/Foxo signaling pathway and increasing intracellular ROS levels in HCC cells.