Fungal antitumor protein D1 is internalized via endocytosis and inhibits non-small cell lung cancer proliferation through MAPK signaling pathway.

Lung cancer has the highest mortality among cancer-related deaths worldwide. Among lung cancers, non-small cell lung cancer (NSCLC) is the most common histological type. In the previous research, we isolated a protein (D1) from Boletus bicolor that inhibits the proliferation of NSCLC cell lines. In this study, we elucidated the internalization mechanism and antitumor mechanism of protein D1 in A549 cells. Protein D1 has a strong inhibitory effect on A549 cells. It binds to secretory carrier membrane protein 3 on the A549 cell membrane and enters A549 cells by clathrin-mediated endocytosis. In vitro, protein D1 activates mitogen-activated protein kinase (MAPK) signaling pathway. JNK and p38MAPK are the biological targets for protein D1. In vivo, protein D1 inhibits the tumor growth of NSCLC xenografts by inducing apoptosis and inhibiting cell proliferation. Protein D1 alters the expression of genes related to apoptosis, cell cycle, and MAPK signaling pathway in tumor cells.

Purification of a novel protein with cytotoxicity against non-small-cell lung cancer cells from Boletus bicolor.

A novel protein (D1 component) was purified from Boletus bicolor by ion-exchange chromatography and gel chromatography on a HiTrap™ Q HP column, a diethylaminoethanol cellulose-52 column, and a Sephadex G75 column, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the D1 component was a single protein band with a molecular weight of about 40 kDa. The sulforhodamine B assay showed that at concentrations as low as 25-75 µg/ml, protein D1 significantly inhibited the proliferation of human lung adenocarcinoma cell lines (A549 and H1299 cells) and had little effect on human normal kidney cells (HEK293 cells). After labeling protein D1, it was found that D1 could enter the cytoplasm of tumor cells and colocated with lysosomes. Flow cytometry results demonstrated that protein D1 induced apoptosis and G1 phase arrest of the cell cycle in A549 and H1299 cells. The Western blot analysis results showed that the expression of apoptosis and cell cycle-related proteins of cleaved caspase-3, cytochrome c, Bax, P16, and P21 was significantly upregulated, whereas the expression of Bcl-2, CDK4, cyclin D, p-Rb, and E2F was significantly downregulated after treatment with protein D1. Therefore, D1 exhibits potential to be developed into an antitumor agent.

Purification and characterization of a novel ubiquitin-like antitumour protein with hemagglutinating and deoxyribonuclease activities from the edible mushroom Ramaria botrytis.

A novel ubiquitin-like antitumour protein (RBUP) was isolated from fruiting bodies of the edible mushroom Ramaria botrytis. The protein was isolated with a purification protocol involving ion exchange chromatography on DEAE-Sepharose fast flow and gel filtration on Sephadex G-75. SDS-PAGE, Native-PAGE and ultracentrifugation analysis disclosed that RBUP was a monomeric protein with a molecular weight of 18.5 kDa. ESI-MS/MS demonstrated that it shared 69% amino acid sequence similarity with Coprinellus congregates ubiquitin (gi|136667). The protein exhibiting strong anticancer activity towards A549 cells. Analysis by employing AO/EB staining and Annexin V-FITC/PI detection indicated that the cytotoxic effect of RBUP was mediated through induction of apoptosis. Furthermore, RBUP displayed hemagglutinating and deoxyribonuclease activities. A temperature of 40 °C and pH of 7.0 were required for optimal DNase activity. Therefore, it was estimated that RBUP exerted its antitumour effect by inducing apoptosis, and its hemagglutinating and DNase activities were also thought to participate in this effect. These results demonstrated that RBUP was a multifunctional protein with potential medicinal applications.

Characterization of Avian Influenza A (H7N9) Virus Prevalence in Humans and Poultry in Huai'an, China: Molecular Epidemiology, Phylogenetic, and Dynamics Analyses.

OBJECTIVE: To trace the source of human H7N9 cases in Huai'an and elucidate the genetic characterization of Huai'an strains associated with both humans and birds in live poultry market. METHODS: An enhanced surveillance was implemented when the first human H7N9 case was confirmed in Huai'an. Clinical specimens, cloacal swabs, and fecal samples were collected and screened by real-time reverse transcription-polymerase chain reaction (RT-PCR) for H7N9 virus. The positive samples were subjected to further RT-PCR and genome sequencing. The phylodynamic patterns of H7N9 virus within and separated from Huai'an and evolutionary dynamics of the virus were analyzed. RESULTS: Six patients with H7N9 infection were previously exposed to live poultry market and presented symptoms such as fever (>38.0 °C) and headaches. Results of this study support the hypothesis that live poultry markets were the source of human H7N9 exposure. Phylogenetic analysis revealed that all novel H7N9 viruses, including Huai'an strains, could be classified into two distinct clades, A and B. Additionally, the diversified H7N9 virus circulated in live poultry markets in Huai'an. Interestingly, the common ancestors of the Huai'an H7N9 virus existed in January 2012. The mean nucleotide substitution rates for each gene segment of the H7N9 virus were (3.09-7.26)×10-3 substitutions/site per year (95% HPD: 1.72×10-3 to 1.16×10-2). CONCLUSION: Overall, the source of exposure of human H7N9 cases in Huai'an was live poultry market, and our study highlights the presence of divergent genetic lineage of H7N9 virus in both humans and poultry specimens in Huai'an.

[Contrast-enhanced ultrasonography in the diagnosis of acute experimental incomplete testicular torsion].

OBJECTIVE: To explore the application value of contrast-enhanced ultrasonography (CEUS) in the diagnosis of acute experimental incomplete testicular torsion. METHODS: Seven incomplete and 1 complete testicular torsion models were established in 8 healthy hybrid dogs by twisting unilateral spermatic cord. The twisted testes were included in the model group and the healthy ones in the control. All the dogs underwent CEUS before, immediately after, and/or 6 hours after the torsion, followed by time-intensity curve analysis. RESULTS: The arriving time (AT) and the time to peak (TTP) of the contrast agents in the bilateral testes were almost coincident, and the peak intensity (PI) and the area under the curve (AUC) in the bilateral testes basically the same before the torsion. The incompletely torsion testes showed delayed perfusion of contrast agents, prolonged AT and 'TP, and decreased PI and AUC as compared with the contralateral testes (P < 0.05), while the completely torsion one exhibited no perfusion all the time. CONCLUSION: CEUS has a very high application value in the diagnosis of acute incomplete testicular torsion.

[Ultrasonographic changes in the epididymis after long-term vasectomy].

OBJECTIVE: To evaluate the ultrasonographic changes in the epididymis after long-term vasectomy. METHODS: Sixty-four patients with a history of vasectomy for more than 10 years (vasectomy group) and another 60 without vasectomy (control group) were included in the study. The patients were referred to scrotal ultrasonography for epididymal indications. The shape, thickness and internal echoes of the head, body and tail of the epididymis were observed with high frequency ultrasonography (HFU), and the blood flow changes were observed with color Doppler flow imaging (CDFI) or color Doppler power imaging (CDPI). RESULTS: Significantly higher rates were found in the vasectomy group than in the control: thickened body (64.1% vs 15.0%) and tail (78.1% vs 51.7%) of the epididymis, thickened head, body and tail (42.2% vs 8.3%) of the epididymis, and epididymal tubular ectasia (54.7% vs 8.3%). However, increased blood flow in the epididymis was seen at a significantly lower rate in the vasectomy group than in the control (15.6% vs 61.7%). CONCLUSION: The ultrasonographic changes in the epididymis after long-term vasectomy were mainly epididymis thickening and epididymal tubular ectasia, mostly with no or diminished blood flow in the epididymis.

Systemic genetic transfer of p21WAF-1 and GM-CSF utilizing of a novel oligopeptide-based EGF receptor targeting polyplex.

Based on the fact that aberrant overexpression of some growth factor receptors was observed in a variety of human cancer cells, a novel nonviral gene delivery system GE7, which contains a 16-amino-acid ligand for identifying EGF receptor was constructed for tumor-targeted gene therapy. Intravenous administration of GE7 system revealed that it has the ability to target beta-galactosidase (beta-gal) reporter gene into murine hepatoma (Hepa) cells. Owing to the limited antitumor effects elicited by a single-gene transfer, recent efforts to treat malignancy using combined gene therapy have been accomplished with varying degrees of success. In this study, the human cyclin-dependent kinase inhibitor gene p21(WAF-1) and the murine cytokine gene granulocyte-macrophage colony-stimulating factor (GM-CSF) were used simultaneously for in vivo gene therapy through systemic injection of the EGF R targeted GE7/DNA complex into murine hepatoma-bearing mice. The results demonstrated that combined administration of p21(WAF-1) and GM-CSF could remarkably inhibit the growth of subcutaneously transplanted hepatoma Hepa cells, and significantly increase the survival rate of tumor-bearing mice. The activities of natural killer (NK) cells and specific cytotoxic T lymphocytes (CTL) were clearly enhanced after combined gene therapy. In vitro experiments showed that p21(WAF-1) gene transfer exhibited a suppressive function on the growth of Hepa cells and the expression of H-2K(b) and B7-1 molecules on Hepa cells increased significantly after combined genes delivery. All these results suggested that the GE7 system was able to target therapeutic genes efficiently to cancer cells, which showed high EGF R expression. The cotransfer of p21(WAF-1) and GM-CSF genes apparently inhibited the growth of tumors through (a) the arrest of tumor cell growth and (b) the enhancement of systemic antitumor immunity.