Curcumin Promotes Apoptosis of Activated Hepatic Stellate Cells by Inhibiting Protein Expression of the MyD88 Pathway.

Activation and proliferation of hepatic stellate cells (HSC) play an important role in the progress of liver fibrosis. HSC activation occurs in response to inflammatory cytokines, cellular interactions with immune cells, and morphogenetic signals. The literature hints to a role of the adaptor protein MyD88 in fibrosis. Although curcumin has been shown to exert inhibitory effects on the proliferation of HSC in vitro, its influence on the MyD88 pathway in HSC has remained unclear. Here, we investigated whether curcumin accelerates apoptosis of HSC through the MyD88 pathway. HSC (rat HSC T6) were divided into a control group, MyD88 small interfering RNA (siRNA) group, curcumin group, and curcumin + MyD88 siRNA group. The MyD88 siRNA groups were exposed to siRNA for 48 h. The curcumin groups were cultured in the presence of curcumin for 24 h. Apoptosis was detected by flow cytometry. For Toll-like receptor (TLR) 2 and 4 as well as MyD88 and the dependent factors NF-κB, TNF-α, and IL-1ß, mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR). For MyD88, protein expression was further observed by Western Blot. Both curcumin and MyD88 siRNA inhibited the mRNA expression of MyD88 pathway-related effectors (TLR2, TLR4, NF-κB, TNF-α, IL-1ß) in HSC. Furthermore, both treatments reduced the expression of MyD88 protein in HSC and promoted their apoptosis. These effects were more obvious in the curcumin + MyD88 siRNA group. This study demonstrates that curcumin promotes apoptosis of activated HSC by inhibiting the expression of cytokines related to the MyD88 pathway. It elucidates the possible mechanisms of curcumin in inducing apoptosis of HSC through the MyD88 pathway.

Lentivirus-mediated RNAi knockdown of insulin-like growth factor-1 receptor inhibits the growth and invasion of hepatocellular carcinoma via down-regulating midkine expression.

The insulin-like growth factor-1 receptor (IGF-1R) overexpression contributes to the development of a variety of cancers. The present study explored the role of IGF-1R in the development and progression of hepatocellular carcinoma (HCC) and the possibility of IGF-1R silencing by lentivirus-mediated RNA interference (RNAi) as a therapeutic target for HCC. We showed that IGF-1R mRNA was up-regulated in Huh7 and Hep3B cells and human HCC tissues, and that IGF-1R knockdown by RNAi led to decreased proliferation, apoptosis induction, and decreased migration and invasion of Huh7 and Hep3B cells. Further, the in vivo study indicated that IGF-1R knockdown markedly diminished the tumorigenesis and metastasis of Huh7 xenograft. Moreover, the intratumoral administration of lentivirus-IGF-1R siRNA led to significant tumor growth inhibition in an established Huh7 xenograft model. Mechanistic investigations showed that midkine was found to be the most significantly down-regulated protein in Huh7 cells with IGF-1R knockdown, and ectopic overexpression of midkine significantly rescued inhibition of Huh7 cell proliferation, migration, and invasion caused by IGF-1R suppression. Collectively, these data suggest that IGF-1R inhibition by RNAi can significantly suppress HCC growth and invasion at least partially through down-regulating midkine expression, and IGF-1R is a potential target for HCC gene therapy.

Induction of autophagy and apoptosis by miR-148a through the sonic hedgehog signaling pathway in hepatic stellate cells.

Autophagy is an evolutionarily conserved biological process that is activated in response to stress. Increasing evidence indicate that dysregulated miRNAs significantly contribute to autophagy and are thus implicated in various pathological conditions, including hepatic fibrosis. MiR-148a, a member of the miR-148/152 family, has been found to be downregulated in hepatic fibrosis and human hepatocellular carcinoma. However, the role of miR-148a in the development of hepatic fibrosis remains largely unknown. In this study, we describe the epigenetic regulation of miR-148a and its impact on autophagy in hepatic stellate cells (HSCs), exploring new targets of miR-148a. We found that miR-148a expression was significantly increased under starvation-induced conditions in LX-2 and T-6 cells. In addition, dual-luciferase reporter assays showed that miR-148a suppressed target gene expression by directly interacting with the 3'-untranslated regions (3'-UTRs) of growth arrest-specific gene 1 (Gas1) transcripts. Intriguingly, Gas1, which encodes a Hedgehog surface binding receptor and facilitates the Hedgehog (Hh) signaling pathway, inhibited autophagosome synthesis. Furthermore, we demonstrated a novel function for miR-148a as a potent inducer of autophagy in HSCs. Overexpressing of miR-148a increased autophagic activity, which inhibited proliferation and promoted apoptosis in HSCs. In conclusion, these data support a novel role for miR-148a as a key regulator of autophagy through the Hh signaling pathway, making miR-148a a potential candidate for the development of novel therapeutic strategies.

Curcumin, the main active constituent of turmeric (Curcuma longa L.), induces apoptosis in hepatic stellate cells by modulating the abundance of apoptosis-related growth factors.

In order to elucidate the mechanism of action of curcumin against hepatic fibrosis, cultured rat hepatic stellate cells (HSC) (HSC-T6) were incubated with curcumin for 24 h, after which apoptosis was measured by flow-cytometry. The protein levels of the pro-apoptotic factors Fas and p53b as well as of the anti-apoptotic factor Bcl-2 were monitored by immunocytochemical ABC staining after incubation with curcumin for 24 h. In the case of 20 µM curcumin, not only was the respective apoptosis index increased, but also the abundance of the pro-apoptotic factors Fas and p53 were amplified, whereas that of the anti-apoptotic factor Bcl-2 decreased. All these effects were highly reproducible (P<0.05). Consequently, curcumin has an up-regulating effect on pro-apoptotic factors like Fas and p53 as well as a down-regulating effect of the anti-apoptotic factor Bcl-2, thus inducing apoptosis in HSC.

Epigenetic modulation of insulin-like growth factor-II overexpression by hepatitis B virus X protein in hepatocellular carcinoma.

Hepatitis B virus X protein (HBx) is involved in the pathogenesis of hepatocellular carcinoma (HCC). Overexpression of the transcripts from the P3 and P4 promoters of the insulin-like growth factor-II (IGF-II) gene is observed in HCC. The present study investigated the involvement of HBx in IGF-II overexpression and its epigenetic regulation. Firstly, the effects of HBx on P3 and P4 mRNA expression, the methylation status of the P3 and P4 promoters, and MBD2 expression were analyzed in human HCC cells and HCC samples. Next, interaction between HBx and MBD2 or CBP/p300 was assessed by co-immunoprecipitation, and HBx-mediated binding of MBD2 and CBP/p300 to the P3 and P4 promoters and the acetylation of the corresponding histones H3 and H4 were evaluated by quantitative chromatin immunoprecipitation. Finally, using siRNA knockdown, we investigated the roles of MBD2 and CBP/p300 in IGF-II overexpression and its epigenetic regulation. Our results showed that HBx promotes IGF-II expression via inducing the hypomethylation of the P3 and P4 promoters, and that HBx increases MBD2 expression, directly interacts with MBD2 and CBP/p300, and elevates their recruitment to the hypomethylated P3 and P4 promoters with increased acetylation levels of the corresponding histones H3 and H4. Further results showed that endogenous MBD2 and CBP/p300 are necessary for HBx-induced IGF-II overexpression and that CBP/p300 presence and CBP/p300-mediated acetylation of histones H3 and H4 are partially required for MBD2 binding and its demethylase activity. These data suggest that HBx induces MBD2-HBx-CBP/p300 complex formation via interaction with MBD2 and CBP/p300, which contributes to the hypomethylation and transcriptional activation of the IGF-II-P3 and P4 promoters and that CBP/p300-mediated acetylation of histones H3 and H4 may be a rate-limiting step for the hypomethylation and activation of these two promoters. This study provides an alternative mechanism for understanding the pathogenesis of HBx-mediated HCC.