XAF1 promotes colorectal cancer metastasis via VCP-RNF114-JUP axis.

Metastasis is the main cause of colorectal cancer (CRC)-related death, and the 5-year relative survival rate for CRC patients with distant metastasis is only 14%. X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a zinc-rich protein belonging to the interferon (IFN)-induced gene family. Here, we report a metastasis-promoting role of XAF1 in CRC by acting as a novel adaptor of valosin-containing protein (VCP). XAF1 facilitates VCP-mediated deubiquitination of the E3 ligase RING finger protein 114 (RNF114), which promotes K48-linked ubiquitination and subsequent degradation of junction plakoglobin (JUP). The XAF1-VCP-RNF114-JUP axis is critical for the migration and metastasis of CRC cells. Moreover, we observe correlations between the protein levels of XAF1, RNF114, and JUP in clinical samples. Collectively, our findings reveal an oncogenic function of XAF1 in mCRC and suggest that the XAF1-VCP-RNF114-JUP axis is a potential therapeutic target for CRC treatment.

[Inhibitory effect of iguratimod on TNFalpha production and NF-kappaB activity in LPS-stimulated rat alveolar macrophage cell line].

AIM: To investigate the effect of iguratimod (T-614), a non-steroidal anti-inflammatory drug, on TNFalpha mRNA expression and TNFalpha production, and on the activity of nuclear factor-kappaB (NF-kappaB) in the rat alveolar macrophage cell line (NR8383) activated by LPS. METHODS: NR8383 cells were pretreated with T-614 (13.4, 26.7, 53.4 micromol x L(-1)), then were stimulated with LPS. The production of TNFalpha in the supernatant of NR8383 was assayed by enzyme-linked immunosorbent assay (ELISA). The TNFalpha mRNA level was determined by a semi-quantitative PCR assay. Assessment of the NF-kappaB DNA binding activity was performed by an ELISA kit. RESULTS: T-614 inhibited LPS-stimulated mRNA expression and production of TNFalpha in a concentration-dependent manner, as well as the activity of NF-kappaB. The IC50 value of effect of T-614 on TNFalpha level was 26.2 micromol x L(-1). CONCLUSION: The inhibitory effect of T-614 on the production of TNFalpha in LPS-stimulated NR8383 cells may be mediated by suppression of NF-kappaB activity.

RTN4-C gene expression in hepatocellular carcinoma and its influence on SMMC7721 cell growth and apoptosis.

RTN4-C gene is a member of RTNs. To investigate its expression in human hepatocellular carcinoma tissues and study its function on SMMC7721 cells, RT-PCR was conducted to detect its expressions in human hepatocellular carcinoma tissues. RTN4-C-pcDNA3. 1 plasmid was reconstructed and stably transfected into SMMC7721 cells by Lipofect AMINE. Growth curve of SMMC7721 measured by MTT and apoptosis indentified with acridine orange staining were examined to demonstrate the effect of RTN4-C on SMMC7721. Immunocytochemical analysis for mutant p53, c-Fos, Hsp70 proteins were conducted. The results showed that the transfected SMMC7721 cells grew more slowly than control and the average inhibition rate at the 1st, 2nd and 3rd day were 33.8% +/- 0.026, 56.2% +/- 0. 094, 34.8% +/- 0.077 respectively. In transfected SMMC7721 cell line, the apoptosis was strengthened,mutant p53 protein tranferred from nucleus to cytoplasm and c-Fos, Hsp70 proteins were decreased. Our data indicated RTN4-C gene was expressed differently in hepatocellular carcinoma and its paracancerous tissues. By tranferring mutant p53 protein from nucleus to cytoplasm and decreasing c-Fos, Hsp70 protein expression, RTN4-C inhibited SMMC7721 cells growth and promoted its apoptosis.

Down regulation of DRET gene in human hepatocellular carcinoma tissues samples from Qidong area, China.

OBJECTIVE: To analyze the expression of DRET gene in hepatocellular carcinomas (HCCs) tissue specimens in comparison with normal liver tissue to evaluate the relationship between DRET gene and HCC. METHODS: 250 primary HCCs and 50 normal liver tissue samples from Qidong area, China, were studied for DRET mRNA and protein expression with the use of Northern blot analysis, in situ hybridization and immunohistochemistry. RESULTS: By Northern analysis, moderate to strong DRET mRNA expression was present in normal liver samples. In contrast, DRET mRNA expression in tissue samples of primary HCCs was markedly decreased compared with normal controls. Primary HCCs that gave rise to metastasis showed significantly lower DRET mRNA levels than nonmetastasizing HCCs. By in situ hybridization analysis, nonmetastatic HCCs samples didn't differ from controls. In contrast, most of the primary metastasizing HCC showed only faint or moderate DRET mRNA expression. Tissue sections of nonmetastatic HCC exhibited lower DRET immunoreactivity than control samples, but higher labeling index than metastatic HCC samples. CONCLUSIONS: Expression of DRET on mRNA and protein levels in HCC cells is related to HCC metastatic ability.

Expression of TN4 gene and its role in human hepatocarcinogenesis from Qidong, a liver cancer risk area.

BACKGROUND: We investigated the expression and role of TN4 in the oncogenesis of human hepatocellular carcinoma (HCC) from Qidong which is a HCC risk area. METHODS: The expression of TN4 in HCC was observed using immunohistochemical staining (IHC). TN4 levels were manipulated in human liver cancer cell SMMC7721, using pcDNA3.1 eukaryotic expression constructs designed to express the complete TN4 cDNA. The biological changes of the cells were observed before and after transfection of TN4 and the change of gene expression was analysed by atlas cDNA expression array. RESULTS: Among 100 pairs of samples of HCC, TN4 down-regulation expression and up-regulation expression positive rate were 81% (81/100), 19% (19/100), respectively (P < 0.01). TN4 protein was mainly localized in cytoplasm and membrane. The positive rate of TN4 were 10% (3/30), 100% (70/70) in lymph node metastasis and no lymph node metastasis, respectively (P < 0.01). The growth rates of the derivative SMMC7721-TN4 cell lines were decreased in comparison with that of normal SMMC7721 cells and pcDNA-SMMC7721. Some gene expression was changed before and after transfection of TN4. At 30 days of post-implantation of SMMC7721-TN4, SMMC7721-pcDNA3, SMMC7721 group produced tumors of (301.9 +/- 143.4) mm(3), (2418.7 +/- 362.8) mm(3), (2317.4 +/- 587.8) mm(3), respectively, (P < 0.01). Tumor inhibiting rate was 82.4% in TN4 transfection group. Sections of tumors were observed for their degree of tissue necrosis and there was higher degree of necrosis in tumors of the TN4-SMMC7721 cell group than those of the SMMC7721, SMMC7721-pcDNA groups. CONCLUSIONS: TN4 may play an important role in the oncogenesis of human HCC, especially in Qidong, the HCC risk area and TN4 could be a candidate tumor suppressor gene for HCC.

Codon 249 mutation in exon 7 of p53 gene in plasma DNA: maybe a new early diagnostic marker of hepatocellular carcinoma in Qidong risk area, China.

AIM: One of the characteristics of hepatocellular carcinoma (HCC) in Qidong area is the selective mutation resulting in a serine substitution at codon 249 of the p53 gene (1, 20), and it has been identified as a "hotspot" mutation in heptocellular carcinomas occurring in populations exposed to aflatoxin and with high prevalence of hepatitis B virus carriers (2,3,9, 10,16,24). We evaluated in this paper whether this "hotspot" mutation could be detected in cell-free DNA circulating in plasma of patients with hepatocellular carcinoma and cirrhosis in Qidong, China, and tried to illustrate the significance of the detection of this molecular biomarker. METHODS: We collected blood samples from 25 hepatocellular carcinoma patients, 20 cirrhotic patients and 30 healthy controls in Qidong area. DNA was extracted and purified from 200 microl of plasma from each sample. The 249(Ser) p53 mutation was detected by restriction digestion analysis and direct sequencing of exon-7 PCR products. RESULTS: We found in exon 7 of p53 gene G-T transversion at the third base of codon 249 resulting 249(Arg) - 249(Ser) mutation in 10/25 (40 %) hepatocellular carcinoma cases, 4/20 (20 %) cirrhotics, and 2/30 (7 %) healthy controls. The adjusted odds ratio for having the mutation was 22.1(95 % CI, 3.2-91.7) for HCC cases compared to controls. CONCLUSION: These data show that the 249(Ser) p53 mutation in plasma is strongly associated with hepatocellular carcinoma in Qidong patients. We found this mutation was also detected, although it was at a much lower frequency, in plasma DNA of Qidong cirrhotics and healthy controls; We consider that these findings, together with the usual method of HCC diagnosis, will give more information in early diagnosis of HCC, and 249(Ser) p53 mutation should be developed to a new early diagnostic marker for HCC.

The relationship between metallothionein-1F (MT1F) gene and hepatocellular carcinoma.

To investigate the expression of MT1F gene in hepatocellular carcinoma tissue and the growth suppression effect of exogenous introduction of MT1F gene on liver cell line HepG2 and to explore the potential application of MT1F gene in gene therapy of tumor. Eukaryotic expression vector of pCMV-MT1F plasmid was introduced into HepG2 line which expressed no MT1F protein originally with lipofectamine transfection method. The cell growth curve, soft agar colony formation rate and tumorigenicity in SCID mice were examined to demonstrate the growth suppression effect of exogenous MT1F gene on HepG2 cell line. The MT1F mRNA and MT1F protein were also detected in 60 pairs of surgical specimens of hepatocellular carcinoma by in situ hybridization and immunohistochemistry. The transfected HepG2 cell line grew more slowly than control HepG2 as shown by cell growth curves, the soft agar colony formation rate (3.8 percent vs. 7.4 percent, p <.01) and the average growth rate of tumor in SCID mice (30.9 +/- 6.9 vs. 70.3 +/- 5.6, p <.01). The expression level of MT1F mRNA and protein significantly increased in paracancerous tissue, normal tissue than in cancer tissues (75 percent, 70 percent vs. 16.7 percent by ISH and 66.7 percent, 60 percent vs. 10 percent by IHC, p <.01). Exogenous MT1F gene shows the strong effect of growth inhibition on HepG2 cell line. In the liver cancer tissue, MT1F shows down-regulated expression that supports the inhibited function of MT1F in cancer growth and suggests MT1F may have an important role in gene therapy of hepatocellular carcinoma.