Development and Validation of High-Content Analysis for Screening HDAC6-Selective Inhibitors.

Throughout recent decades, histone deacetylase (HDAC) inhibitors have shown encouraging potential in cancer treatment, and several pan-HDAC inhibitors have been approved for treating malignant cancers. Numerous adverse effects of pan-HDAC inhibitors have been reported, however, during preclinical and clinical evaluations. To avoid undesirable responses, an increasing number of investigations are focusing on the development of isotype-selective HDAC inhibitors. In this study, we present an effective and quantitative cellular assay using high-content analysis (HCA) to determine compounds' inhibition of the activity of HDAC6 and Class I HDAC isoforms, by detecting the acetylation of their corresponding substrates (i.e., α-tubulin and histone H3). Several conditions that are critical for HCA assays, such as cell seeding number, fixation and permeabilization reagent, and antibody dilution, have been fully validated in this study. We used selective HDAC6 inhibitors and inhibitors targeting different HDAC isoforms to optimize and validate the capability of the HCA assay. The results indicated that the HCA assay is a robust assay for quantifying compounds' selectivity of HDAC6 and Class I HDAC isoforms in cells. Moreover, we screened a panel of compounds for HDAC6 selectivity using this HCA assay, which provided valuable information for the structure-activity relationship (SAR). In summary, our results suggest that the HCA assay is a powerful tool for screening selective HDAC6 inhibitors.

Hepatitis B virus X protein promotes hepatocellular carcinoma invasion and metastasis via upregulating thioredoxin interacting protein.

Hepatitis B virus X protein (HBx), a multifunctional protein encoded by the X gene of the hepatitis B virus (HBV) is involved in the metastasis of HBV-associated hepatocellular carcinoma (HCC) through various pathways, including upregulating intracellular reactive oxygen species (ROS). Thioredoxin interacting protein (TXNIP) is a key mediator of intracellular ROS, but its function in HBx-mediated metastasis of HBV-associated HCC is elusive. In the present study, HBV-associated HCC tissues with or without metastasis and HepG2 cells were used to study the function of TXNIP in HBx-mediated metastasis of HBV-associated HCC. Initially, the expression levels of TXNIP and HBx in HBV-associated HCC tissues were detected by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. The results revealed that high expression of TXNIP may be an independent risk factor for metastasis of HBV-associated HCC, and the mRNA levels of TXNIP and HBx were positively associated. Secondly, the association between HBx and TXNIP was investigated using a HBx expression stable cell line, in which HBx expression was induced and controlled by doxycycline. The results demonstrated that HBx may upregulate TXNIP expression in HepG2 cells. Thirdly, the effects of TXNIP and HBx on HepG2 cell migration and invasion were studied by scratch and Matrigel invasion assays, respectively. The results demonstrated that TXNIP overexpression enhanced HepG2 cell migration and invasion. In addition, ectopic expression of HBx promoted HepG2 cell migration and invasion, and this effect may be attenuated by knockdown of TXNIP expression, which indicated that TXNIP may be involved in the process. In summary, the present results demonstrated that TXNIP may be involved in HBx-mediated metastasis of HBV-associated HCC.

Luteolin Inhibits Hepatitis B Virus Replication through Extracellular Signal-Regulated Kinase-Mediated Down-Regulation of Hepatocyte Nuclear Factor 4α Expression.

Whether luteolin inhibits HBV replication has not been validated and the underlying mechanism of which has never been elucidated. In this study, we show that luteolin reduces HBV DNA replication in HepG2.2.15 cells. Luteolin effectively inhibited the expression of hepatocyte nuclear factor 4α (HNF4α) and its binding to the HBV promoters in HepG2.2.15 cells. While the extracellular signal-regulated kinase (ERK) was activated by luteolin, inhibition of ERK abolished luteolin-induced HNF4α suppression. Consistently, blocking ERK attenuated the anti-HBV activity of luteolin. In a HBV replication mouse model, luteolin decreased the levels of HBsAg, HBeAg, HBV DNA replication intermediates, and the HBsAg and HBcAg expression. Taken together, our results validated the anti-HBV activity of luteolin in both in vitro and in vivo studies and established a signaling cascade consisting of ERK and HNF4α for inhibition of HBV replication by luteolin, which may be exploited for clinical application of luteolin for anti-HBV therapy.

[Advances in molecular mechanisms of tenascin-C in promoting tumor metastasis].

Tenascin-C (TNC) is an extracellular matrix glycoprotein, which is usually highly expressed in embryonic tissues and tumor tissues, but is not expressed or just lowly expressed in mature tissues. TNC is involved in various complex signaling pathways during tumor metastasis, especially through modulating FAK, RhoA, Wnt and Notch pathways by interacting with syndecan-4, integrin α5ß1, matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). As a result, TNC affects epithelial mesenchymal transition, tumor cell adhesion, proliferation and angiogenesis, which eventually enhances the invasion and metastasis ability of many tumors. Further studies have demonstrated that TNC could be used as prognosis or metastasis marker of patients with malignant tumor.

Tenascin-C expression is associated with poor prognosis in hepatocellular carcinoma (HCC) patients and the inflammatory cytokine TNF-α-induced TNC expression promotes migration in HCC cells.

Although tenascin-c (TNC) in inflammatory microenvironment contributes to progression in some tumors, its role in hepatocellular carcinoma (HCC) in metastasis and the mechanism by which TNC expression is regulated in HCC cells are elusive. In this study, we examined TNC expression in 100 HCC tissue samples by immunohistochemistry and compared which between the groups with or without metastasis. TNC expression was higher in metastatic HCC tissues than that in the non-metastatic HCC tissues, which was associated with the Knodell inflammation scores. Importantly, high level of TNC expression was associated with lower survival rate and shorter survival time in the HCC patients. We then investigated the mechanism by which TNC expression is regulated in HCC cells with an in vitro cell culture system. The recombinant TNF-α and conditioned medium from macrophages induced TNC expression at both mRNA and protein levels in HepG2 cells. The induction of TNC expression by conditioned medium from macrophages was suppressed by a TNF-α neutralizing antibody. TNF-α-promoted cell migration was inhibited by a TNC siRNA. In addition, TNF-α-induced TNC expression was blocked by a NF-κB pathway inhibitor. These results suggest that TNF-α in the tumor microenvironment induces TNC expression in HCC cells through the NF-κB pathway, which in turn, promotes HCC cell migration. Thus, TNC may play an important role in promoting HCC metastasis and TNC expression could be a predictive factor for poor prognosis in HCC patients.