Targeting MUC1 and JNK by RNA interference and inhibitor inhibit the development of hepatocellular carcinoma.

Mucin 1 (MUC1), as an oncogene, is overexpressed in hepatocellular carcinoma (HCC) cells and promotes the progression and tumorigenesis of HCC through JNK/TGF-ß signaling pathway. In the present study, RNA interference (RNAi) and JNK inhibitor SP600125, which target MUC1 and/or JNK, were used to treat HCC cells in vitro, and the results showed that both silencing the expression of MUC1 and blocking the activity of JNK inhibited the proliferation of HCC cells. In addition, MUC1-stable-knockdown and SP600125 significantly inhibited the growth of tumors in the subcutaneous transplant tumor models that established in BALB/c nude mice rather than MUC1 or JNK siRNAs transiently transfection. Furthermore, the results from immunohistochemical staining assays showed that the inhibitory effects of MUC1 gene silencing and SP600125 on the proliferation of HCC cells in vivo were through the JNK/TGF-ß signaling pathway. These results indicate that MUC1 and JNK are attractive targets for HCC therapy and may provide new therapeutic strategies for the treatment of HCC.

Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner.

Maltose-binding protein (MBP) is a critical player of the maltose/maltodextrin transport system in Escherichia coli. Our previous studies have revealed that MBP nonspecifically induces T helper type 1 (Th1) cell activation and activates peritoneal macrophages obtained from mouse. In the present study, we reported a direct stimulatory effect of MBP on RAW264.7 cells, a murine macrophage cell line. When stimulated with MBP, the production of nitric oxide (NO), IL-1ß, IL-6 and IL-12p70, and the expressions of CD80, MHC class II and inducible nitric oxide synthase (iNOS) were all increased in RAW264.7 cells, indicating the activation and polarization of RAW264.7 cells into M1 macrophages induced by MBP. Further study showed that MBP stimulation upregulated the expression of TLR2 and TLR4 on RAW264.7 cells, which was accompanied by subsequent phosphorylation of IκB-α and p38 MAPK. Pretreatment with anti-TLR2 or anti-TLR4 antibodies largely inhibited the phosphorylation of IκB-α and p38 MAPK, and greatly reduced MBP-induced NO and IL-12p70 production, suggesting that the MBP-induced macrophage activation and polarization were mediated by TLR2 and TLR4 signaling pathways. The observed results were independent of lipopolysaccharide contamination. Our study provides a new insight into a mechanism by which MBP enhances immune responses and warrants the potential application of MBP as an immune adjuvant in immune therapies.

Expression and localization of activin receptor-interacting protein 2 in mouse tissues.

Activin plays important roles in reproductive tissues as a stimulator of follicle-stimulating hormone (FSH) secretion. Activin receptor-interacting protein 2 (ARIP2) has been recently identified in mouse tissues as a regulatory protein of activin signal transduction. However, the localization and function of ARIP2 are not well characterized. In this study, we found that ARIP2 mRNA and protein were widely expressed in mouse tissues by reverse transcription-PCR (RT-PCR) and Western blotting. The immunoreactivities of ARIP2 were mainly localized at myocardial cells of heart, Leydig cells in testis, macrophages and epithelial cells of bronchus in lung, renal tubule and collecting tubule, pancreatic islet, adrenal gland, adenohypophysis and hypothalamus by immunohistochemical staining. Furthermore, ARIP2 overexpression down-regulated signal transduction induced by activin A in pituitary gonadotroph LbetaT2 cells and inhibited FSH secretion from primary cultured anterior pituitary cells induced by activin A. These findings suggest that ARIP2 is widely distributed in various tissues and may be a negative regulator of activin action in pituitary cells.

Inhibitory effect of activin A on activation of lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells.

Activin A is a member of transforming growth factor beta (TGF-beta) superfamily, which is also named restrictin-P, and can inhibit the secretion of nitric oxide (NO) and interleukin-1beta (IL-1beta) from LPS-activated mouse macrophages. In this study, the regulation effect and possible mechanism of activin A as an anti-inflammatory factor on lipopolysaccharide (LPS)-activated macrophages were investigated in vitro. It was observed that activin A could not only decrease the secretion of IL-1beta and NO, as well as the mRNA expressions of IL-1beta and iNOS, but also suppress the pinocytosis of mouse macrophage cell line RAW264.7 cells induced by LPS. In addition, activin A could obviously reduce the expressions of CD68 and CD14, as well as Toll-like receptor 4 (TLR4) on RAW264.7 cells induced by LPS, but could not influence the proliferation of RAW264.7 cells. These findings suggest that activin A may play an important down-regulation role in inflammatory factor production and phagocytosis of the activated macrophages via suppressing the maturation of LPS-induced macrophages or LPS-TLR4 signal transduction.

Regulation of activin receptor-interacting protein 2 expression in mouse hepatoma Hepa1-6 cells and its relationship with collagen type IV.

AIM: To investigate the regulation of activin receptor-interacting protein 2 (ARIP2) expression and its possible relationships with collagen type IV (collagen IV) in mouse hepatoma cell line Hepal-6 cells. METHODS: The ARIP2 mRNA expression kinetics in Hepal-6 cells was detected by RT-PCR, and its regulation factors were analyzed by treatment with signal transduction activators such as phorbol 12-myristate 13-acetate (PMA), forskolin and A23187. After pcDNA3-ARIP2 was transfected into Hepal-6 cells, the effects of ARIP2 overexpression on activin type II receptor (ActRII) and collagen IV expression were evaluated. RESULTS: The expression levels of ARIP2 mRNA in Hapel-6 cells were elevated in time-dependent manner 12 h after treatment with activin A and endotoxin LPS, but not changed evidently in the early stage of stimulation (2 or 4 h). The ARIP2 mRNA expression was increased after stimulated with signal transduction activators such as PMA and forskolin in Hepal-6 cells, whereas decreased after treatment with A23187 (25.3% +/- 5.7% vs 48.1% +/- 3.6%, P < 0.01). ARIP2 overexpression could remarkably suppress the expression of ActRIIA mRNA in dose-dependent manner, but has no effect on ActRIIB in Hepal-6 cells induced by activin A. Furthermore, we have found that overexpression of ARIP2 could inhibit collagen IV mRNA and protein expressions induced by activin A in Hapel-6 cells. CONCLUSION: These findings suggest that ARIP2 expression can be influenced by various factors. ARIP2 may participate in the negative feedback regulation of signal transduction in the late stage by affecting the expression of ActRIIA and play an important role in regulation of development of liver fibrosis induced by activin.

Effects of activin A on the activities of the mouse peritoneal macrophages.

Activin A is a kind of pre-inflammatory factor that belongs to the transforming growth factor-beta (TGF-beta) superfamily. To investigate the effect and mechanism of activin A on the activities of mouse macrophages, the secretion of NO in the supernatant of cultured mouse peritoneal macrophages was examined by NO assay kit, and the expression of iNOS, ActRIIA and ARIP2 mRNA in mouse peritoneal macrophages was analyzed by RT-PCR. The results showed that activin A stimulated the secretion of NO and the expression of iNOS mRNA in non-activated mouse macrophages in a time- and dose-dependent manner. In contrast, activin A in the same concentration inhibited the secretion of NO in LPS-activated mouse macrophages in a dose-dependent manner. ActRIIA was highly expressed on macrophages, and activin A upregulated the ActRIIA mRNA expression in macrophages. Anti-ActRIIA antibody can block the secretion of NO from the macrophages stimulated by activin A. Furthermore, RT-PCR analysis revealed that activin A enhanced the ARIP2 mRNA expression in macrophages. These results indicated that Activin A may be a weak activator compared with LPS to mouse macrophages, and activin A may modulate the secretion of NO through ActRIIA-ARIP2 signal pathway in mouse macrophages.