Inhibitory effects of lactoferrin on pulmonary inflammatory processes induced by lipopolysaccharide by modulating the TLR4-related pathway.

This study tested the ability of lactoferrin to modulate pulmonary inflammation. To construct in vitro and in vivo inflammatory lung models, cells from the human lung adenocarcinoma cell line (A549) were exposed to lipopolysaccharide (LPS, 1 µg/mL), and mice (CD-1) were intratracheally administered LPS [10 mg/kg of body weight (BW), tracheal lumen injection], respectively. The A549 cells were preincubated with lactoferrin (10 mg/mL), and the mice were intraperitoneally injected with lactoferrin (100 mg/kg of BW), followed by LPS treatment. The concentrations of proinflammatory cytokines (IL-1ß and TNF-α) in culture medium of A549 cells and in bronchoalveolar lavage fluid of the mice were determined using enzyme-linked immunosorbent assays. The toll-like receptor 4-related pathway (TLR4/MyD88/IRAK1/TRAF6/NFκB) was determined at gene and protein expression levels in A549 cells and mouse lung tissue. Results showed that LPS treatment significantly elevated the concentrations of IL-1ß and TNF-α in the A549 cell culture medium and in bronchoalveolar lavage fluid of the mice; it also elevated both the mRNA and protein expressions of TLR4 and the TLR4 downstream factors in A549 cells and mouse lung tissue. Nevertheless, lactoferrin apparently depressed the releases of IL-1ß and TNF-α from A549 cells and lung tissues stimulated by LPS, and significantly suppressed the TLR4 signaling pathway. Lactoferrin also promoted the enhancement of miR-146a expression in A549 cells and mouse lung tissue. Moreover, 100°C heating for 3 min caused total loss of the previously listed bioactivity of lactoferrin. Collectively, we proved that lactoferrin intervened in LPS-induced inflammation in the pulmonary cell model and in the mouse model, through inhibiting the TLR4-related pathway.

LncRNA RUSC1-AS1 promotes the proliferation of hepatocellular carcinoma cells through modulating NOTCH signaling.

The long non-coding RNA (lncRNA) RUSC1-AS1 has been reported to be dysregulated in the progression of many cancers. Also, RUSC1-AS1 had been detected to be highly expressed in laryngeal squamous cell carcinoma and breast cancer cells, suggesting that RUSC1-AS1 may be a biomarker for cancers. However, the biological role and regulatory mechanism of RUSC1-AS1 in hepatocellular carcinoma (HCC) remain unknown. In this study, we found that RUSC1-AS1 was upregulated in HCC tissues and cells, and predicted unfavorable prognosis of HCC patients. The function assays including colony formation, EdU, TUNEL assay revealed that RUSC1-AS1 facilitated HCC cell proliferation and inhibited HCC cell apoptosis. Furthermore, mechanism assays including luciferase reporter assay and RIP assay demonstrated that RUSC1-AS1 could directly bind to hsa-miR-7-5p. Besides, hsa-miR-7-5p targeted and negatively regulated NOTCH3 expression. Moreover, RUSC1-AS1 sponged hsa-miR-7-5p to upregulate NOTCH3 and to trigger the NOTCH signaling pathway. The rescue assays depicted that RUSC1-AS1 regulated HCC cell proliferation and apoptosis through modulating NOTCH signaling. In conclusion, lncRNA RUSC1-AS1 promoted the proliferation and reduced the apoptosis of HCC cells through activation of NOTCH signaling via hsa-miR-7-5p/NOTCH3 axis.

Investigation and comparison of the anti-tumor activities of lactoferrin, α-lactalbumin, and ß-lactoglobulin in A549, HT29, HepG2, and MDA231-LM2 tumor models.

To investigate the anti-tumor activities of lactoferrin, α-lactalbumin, and ß-lactoglobulin, 4 types of human tumor cells (lung tumor cell A549, intestinal epithelial tumor cell HT29, hepatocellular cell HepG2, and breast cancer cell MDA231-LM2) were exposed to 3 proteins, respectively. The effects on cell proliferation, migration, and apoptosis were detected in vitro, and nude mice bearing tumors were administered the 3 proteins in vivo. Results showed that the 3 proteins (20 g/L) inhibited viability and migration, as well as induced apoptosis, in 4 tumor cells to different degrees (compared with the control). In vivo, tumor weights in the HT29 group (0.84 ± 0.22 g vs. control 2.05 ± 0.49 g) and MDA231-LM2 group (1.11 ± 0.25 g vs. control 2.49 ± 0.57 g) were significantly reduced by lactoferrin; tumor weights in the A549 group (1.07 ± 0.19 g vs. control 3.11 ± 0.73 g) and HepG2 group (2.32 ± 0.46 g vs. control 3.50 ± 0.74 g) were significantly reduced by α-lactalbumin. Moreover, the roles of lactoferrin, α-lactalbumin, and ß-lactoglobulin in regulating apoptotic proteins were validated. In summary, lactoferrin, α-lactalbumin, and ß-lactoglobulin were proven to inhibit growth and development of A549, HT29, HepG2, and MDA231-LM2 tumors to different degrees via induction of cell apoptosis.

Long noncoding RNA SNHG15, a potential prognostic biomarker for hepatocellular carcinoma.

OBJECTIVE: The purpose of this study was to investigate the expression of lncRNA SNHG15 and its prognostic value in hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Expression levels of lncRNA SNHG15 in 152 pairs of HCC tissues and adjacent normal tissues were detected by quantitative real-time PCR. Associations between clinicopathological parameters and lncRNA SNHG15 expression were evaluated using chi-square tests. The overall survival was analyzed by log-rank test, and survival curves were plotted according to Kaplan-Meier. Univariate and multivariate Cox regression analyses were conducted to determine whether lncRNA SNHG15 was an independent predictor of survival. RESULTS: The lncRNA SNHG15 expression was significantly upregulated in tumor tissues compared with that in adjacent non-tumor tissues (p < 0.01). It is also proved that lncRNA SNHG15 expression was associated with histological grade (p = 0.000), TNM stage (p = 0.015), and vein invasion (p = 0.000). In addition, Kaplan-Meier analysis showed that increased LncRNA SNHG15 expression was associated with poor overall survival of patients (p = 0.0011). Moreover, the results of the multivariate analysis showed that high lncRNA SNHG15 expression was a significant independent predictor of poor survival in HCC (p < 0.05). CONCLUSIONS: Our findings suggest that lncRNA SNHG15 may serve as an efficient clinical biomarker and a therapeutic target for HCC patients.

Dendritic cell-directed lentivector vaccine induces antigen-specific immune responses against murine melanoma.

Lentivectors are potential vaccine delivery vehicles because they can efficiently transduce a variety of non-dividing cells, including antigen-presenting cells, and do not cause expression of extra viral proteins. To improve safety while retaining efficiency, a dendritic cell (DC)-specific lentivector was constructed by pseudotyping the vector with an engineered viral glycoprotein derived from Sindbis virus. We assessed the level of anti-tumor immunity conferred by this engineered lentivector encoding the melanoma antigen gp100 in a mouse model. Footpad injection of the engineered lentivectors results in the best antigen-specific immune response as compared with subcutaneous and intraperitoneal injections. A single prime vaccination of the engineered lentivectors can elicit a high frequency (up to 10%) of gp100-specific CD8(+) T cells in peripheral blood 3 weeks after the vaccination and this response will be maintained at around 5% for up to 8 weeks. We found that these engineered lentivectors elicited relatively low levels of anti-vector neutralizing antibody responses. Importantly, direct injection of this engineered lentivector inhibited the growth of aggressive B16 murine melanoma. These data suggest that DC-specific lentivectors can be a novel and alternative vaccine carrier with the potential to deliver effective anti-tumor immunity for cancer immunotherapy.

Determination of serum and urinary aluminum by HPLC with fluorometric detection of Al-lumogallion complex.

We describe a simple and sensitive HPLC method for quantifying aluminum (Al) in biological fluids by measuring the fluorescence of the Al-lumogallion complex (excitation wavelength 500 nm, emission wavelength 575 nm). Serum samples are deproteinized with 0.83 mol/L perchloric acid and centrifuged; the supernates are mixed with lumogallion reagent. Urine samples are pretreated with sodium hydroxide (2 mol/L) and methanol, kept for 1 h at -20 degrees C, and then centrifuged; the precipitate is resuspended in perchloric acid and mixed with lumogallion reagent, as for serum. The maximal fluorescence complex is formed after 1 h at pH 5 +/- 0.5. The HPLC mobile phase consists of (per liter) 100 mL, of 0.2 mol/L potassium hydrogen phthalate, 220 mL of acetonitrile, and distilled deionized water. The flow rate is 1 mL/min, and the injection volume is 5 microliters. The major aluminum species is eluted at 3.5 min, the lowest detection limit being 0.45 pg. We validated the method with samples collected from normal subjects and from workers occupationally exposed to aluminum. Comparing the results with those by traditional atomic absorption spectrometry of urinary aluminum suggests that the proposed method is reliable.