Relaxin promotes in vitro tumour growth, invasion and angiogenesis of human Saos-2 osteosarcoma cells by AKT/VEGF pathway.

OBJECTIVES: In the present study, we determine the role of relaxin on cellular growth, invasion and angiogenesis of osteosarcoma Saos-2 cells in vitro, and discuss the molecular mechanisms of this action. MATERIALS AND METHODS: Saos-2 cells were transfected with Akt1/2 siRNA or VEGF siRNA for 24 hours then treated with 10-100 ng/mL recombinant human relaxin-2 (rh-RLN) for 48 h. MTT, matrigel and bone marrow-derived endothelial cells (BMDECs) was used for cell proliferation, invasion and angiogenesis assay. Western blot was used for relaxin-2, pAKT and VEGF protein assay. RESULTS: The results showed treatment with 10-100 ng/mL rh-RLN resulted in 18%, 48%, 107%, 212% increase in cell proliferation, respectively (vs control, *p < 0.05;**p < 0.01), the relative invasive cells was 1.4;1.9;2.6;4.8 (control was defined to 1) (vs control, #p < 0.01; ##p < 0.001) and the relative anglogenic branch points in Saos-2 cells was 1.04;1.36;1.69;2.10 (control was defined to 1.00) (vs control, *p < 0.05; **p < 0.01). Furthermore, treatment with rh-RLN exhibited a significant increase in the expression level of pAKT and VEGF proterin in dose-dependent manner. Saos-2 cells were transfected with AKT1/2 siRNA for 24 h. No significant increase of VEGF protein expression was shown after rh-RLN treatment. CONCLUSIONS: These results suggested that rh-RLN could promoted proliferation, invasion and angiogenesis by upregulation pAKT-dependent VEGF expression.

RNAi-mediated knockdown of relaxin decreases in vitro proliferation and invasiveness of osteosarcoma MG-63 cells by inhibition of MMP-9.

PURPOSE: The purpose of this study is to determine the role of relaxin knowdown by siRNA transfection in cellular growth and invasion of osteosarcoma MG-63 cells, and discusses the molecular mechanisms of this action. MATERIALS AND METHODS: The expression of relaxin in MG-63 cell was examined by western blot or RT-PCR. To evaluate the biological role of relaxin, proliferation assay (MTT) and invasion assay (BD Matrigel™), apoptosis assay (TUNEL and ELISA) and cell cycle analysis (flow cytometer) were performed after silencing relaxin using siRNA. MMP-9 expressions were analyzed using RT-PCR, western blot and zymography after silencing relaxin. RESULTS: Results showed that the downregulation of relaxin expression by siRNA in human osteosarcoma MG-63 cells significantly inhibited cell proliferation and invasion in vitro. Furthermore, relaxin knockdown led to cell arrest in the G1/G0 phase of the cell cycle, and eventual apoptosis enhancement in MG-63 cells. We provide evidence in our cell model that the relaxin siRNA down-regulated the expression of MMP-9 and the MMP-9 activity, suggesting that relaxin may promote the proliferation, invasion and metastasis of osteosarcoma cells by regulating the expression of MMP-9 and facilitating ECM degradation. CONCLUSIONS: Therefore, siRNA-directed knockdown of relaxin may represent a viable clinical therapy for osteosarcoma.

Heterozygous Ins/Del genotype of the CASP8 rs3834129 polymorphism significantly decreases the risk of coal workers' pneumoconiosis in a Chinese Han population: a case-control study.

OBJECTIVE: Coal workers' pneumoconiosis (CWP) is a chronic inflammatory and fibrotic pulmonary disease that involves a complex interaction of multiple environmental and genetic factors. Polymorphism, as a genetic factor, may affect the onset of the disease in susceptible populations. The present study investigated the association between the polymorphisms of six genes and CWP risk in a Chinese Han population. PATIENTS AND METHODS: Six polymorphisms (CASP8 rs3834129, IL1A rs1800587, IL6 rs1800796, IL4 rs2070874, TNFA rs361525, and NLRP3 rs1539019) were examined in 222 CWP subjects and 247 dust-exposed control subjects. RESULTS: The CASP8 rs3834129 Ins/Del genotype significantly decreased CWP risk (p=0.040; adjusted odds ratio [OR] = 0.586; 95% confidence interval [CI] 0.367-0.935) compared with the Ins/Ins genotype. Stratification analyses revealed a significant interaction between the heterozygous Ins/Del genotype and age. Compared with the Ins/Ins + Del/Del genotype, this was particularly evident among subjects aged 41-60 (p<0.001; adjusted OR = 0.054; 95% CI 0.007-0.420) and those with an exposure time of 20-29 years (p=0.014; adjusted OR = 0.392; 95% CI 0.183-0.842). This decreased risk was also found in the group with former smokers (p=0.012; adjusted OR = 0.448; 95% CI 0.238-0.844). Findings revealed that the heterozygous Ins/Del genotype of CASP8 rs3834129 was related to a significantly decreased risk of stage I CWP (p=0.045; adjusted OR = 0.592; 95% CI 0.353-0.992), but not stage II or III CWP. CONCLUSIONS: Our study indicated that the heterozygous Ins/Del genotype of CASP8 rs3834129 significantly decreased CWP risk in a Chinese Han population.

Immunohistochemical evidence for macroautophagy in neurones and endothelial cells in Alzheimer's disease.

AIM: To determine the pathological structures associated with macroautophagy in Alzheimer's disease (AD) and any relationship to disease progression. METHODS: Immunohistochemistry using antibodies to beclin-1, Atg5 and Atg12, early macroautophagy markers and LC3, the mammalian homologue of the later macroautophagy marker Atg8, were localized in formalin-fixed, paraffin-embedded medial temporal lobe sections of AD cases at variable neuritic disease stages. Double immunofluorescence labelling was used to co-localize these macroautophagy markers with Abeta and phospho-tau (AT8) and correlations performed using Spearman rank tests. RESULTS: Atg12 immunoreactivity in AD was either dispersed in the soma and dendrites or concentrated in tau-immunoreactive dystrophic neurites and some neurofibrillary tangles. Fewer Atg12-immunopositive neurones were observed with longer disease durations. Atg12-immunoreactive endothelial cells were found spatially associated with Abeta-positive plaques, with more Atg12-immunoreactive capillary endothelial cells with higher neuritic disease stage. These findings were confirmed by the other autophagy markers beclin-1, Atg5 and LC3. CONCLUSION: The data confirm that macroautophagy occurs in neurones undergoing neuritic degeneration in AD, identified early macroautophagy markers in capillary endothelial cells in close proximity to Abeta plaques, and found that evidence for macroautophagy changes with disease progression.

Isoflurane and sevoflurane affects Wnt/ß-catenin signaling pathways in hippocampal formation of neonatal rats.

OBJECTIVE: General anesthesia impairs spatial learning and memory in neonatal rats. The aim of this study was to investigate whether the Wnt pathway was involved in neonatal isoflurane and sevoflurane exposure-induced neurocognitive impairment. MATERIALS AND METHODS: Sprague-Dawley rats were randomly assigned to administration isoflurane or sevoflurane for 6 hours at postnatal 7 days. Wnt inhibitor XAV 939 was administrated 30 min before anesthesia. Morris water maze was used to test the learning and memory at 5-week and 10-week. Hematoxylin and Eosin (H&E) stain was performed to evaluation the neuronal death in the hippocampus. Quantitative Real-time PCR (q-PCR) and Western blot assays were used to measure mRNA and proteins expression levels of the Wnt3a, GSK 3ß and ß-catenin, respectively. RESULTS: The results showed that isoflurane or sevoflurane could significantly increase neonatal death and cell lost in the developing brain and the Wnt inhibitor could improve the cell degeneration. It demonstrated that isoflurane or sevoflurane could impair the P7 rats learning and memory capability, while these effects were reduced over time. When rats treated Wnt inhibitor at 30 min before anesthesia, the impairment of brain could relieve. q-PCR and Western blot demonstrated that isoflurane or sevoflurane affects expression levels of Wnt3a, GSK 3ß and ß-catenin. These results suggested that impairment of learning and memory in P7 rats may be related to the Wnt signaling pathway. CONCLUSIONS: The results suggested general anesthesia treatment led to increased brain cell degeneration and impaired learning and memory in P7 rats via Wnt signaling pathway.

Genetic dissection of silicon uptake ability in rice (Oryza sativa L.).

The adequate presence of silicon (Si) in rice plants can enhance their yield and improve their tolerance to various biotic and abiotic stresses. In this study Si uptake abilities were compared between the japonica rice cultivar (cv.) Kinmaze and the indica rice cv. DV85 under three Si concentrations (0.16, 0.4, and 1.6mM) at different time points from 1 to 12h. The results showed that the phenotypic values of two traits-Si uptake by individual plants (SP, Si uptake by all roots of a plant) and Si uptake per unit root dry weight (SR=SP/root dry weight)-of Kinmaze were significantly higher than those of DV85 (P<0.01). Meanwhile, a kinetic study indicated that the Si transporters in Kinmaze and DV85 had the same affinity for silicic acid, but with different Vmax values, indicating that Kinmaze had more Si transporters in the roots than DV85. This may be the main reason for the difference in Si uptake ability between Kinmaze and DV85. In addition, a mapping population consisting of 81 recombinant inbred lines (RILs) derived from the cross between Kinmaze and DV85 was used to detect quantitative trait loci (QTLs) underlying SP and SR. The RILs follow a continuous one-peak distribution and show transgressive segregation in both directions for SP, SR, and root dry weight (RDW). Three QTLs for SP, four for SR, and three for RDW were detected. This can explain 7.16-17.15% of the phenotypic variation (PVE). Thus, the results obtained in this study provide a better understanding of the mechanism of rice Si uptake ability and the basis for fine-mapping the genes involved.

LY2109761 inhibits metastasis and enhances chemosensitivity in osteosarcoma MG-63 cells.

OBJECTIVE: Studies have shown that transforming growth factor-beta (TGF-ß) is associated with metastasis and chemoresistance of osteosarcoma. The TGF-ß kinase inhibitor LY2109761 could inhibits metastasis and enhances chemosensitivity in several cancers, but its role and mechanisms in osteosarcoma (OS) is unclear. Here, we investigated the role and mechanism of LY2109761 on metastasis and chemosensitivity of OS MG-63 cells. MATERIALS AND METHODS: MG-63 cells were treated with LY2109761 or/and cisplatin. The cell viability and apoptosis of MG-63 cells were detected by MTT and ELISA. Matrigel invasion assay was used to detect cell invasion in vitro. pSMAD2 and S100A4 was detected by western blot assay. Furthermore, the efficacy of LY2109761 combined with S100A4 cDNA plaismid transfection on cell viability, apoptosis and chemosensitivity to cisplatin in OS MG-63 cells was further examined. RESULTS: LY2109761 was sufficient to induce apoptosis and inhibited growth of MG-63 cells in vitro. Combination with LY2109761 significantly augmented the cytotoxicity of cisplatin in MG-63 cells. LY2109761 significantly inhibited invasion of MG-63 cells in vitro. The LY2109761-induced increase in cell apoptosis and the cytotoxicity of cisplatin, and decrease in cell invasion was blocked completely when S100A4 expression was restored in the MG-63 cells by S100A4 cDNA plasmid transfection. CONCLUSIONS: Our data indicate that LY2109761 suppresses OS metastasis and enhanced chemosensitivity by targeting S100A4. LY2109761 may have important implications for the development of strategies for inhibiting metastasis and overcoming OS cell resistance to chemotherapy.

RLN2 regulates in vitro invasion and viability of osteosarcoma MG-63 cells via S100A4/MMP-9 signal.

OBJECTIVE: Relaxin-2 (RLN2) increases cell migration, invasiveness and proliferation in vitro of osteosarcoma cells, but the molecular mechanisms of this action are still unknown. In the present study, we identified S100A4 /MMP-9 signaling as a major mediator of the actions of RLN2 in osteosarcoma cells in vitro. MATERIALS AND METHODS: We have established stable transfectants of osteosarcoma MG-63 cells using small interfering RNA (siRNA) targeting RLN2. The stable transfectants (MG-63/RLN2 siRNA cells) were treated with 20 mM BB94 (a kind of MMP-9 activator) or 100 mM recombinant Human RLN2 (B-29/A-24) for 24 hs or transfected with S100A4 cDNA plasmid for 48 hrs or MMP-9 siRNA for 48 hrs then treated 100 mM recombinant Human RLN2 (B-29/A-24). Western blot assay was used to detect RLN2, S100A4 and MMP-9 expression. Matrigel invasion assay and wound healing assay was used to detect invasion in vitro. MTT was used to detect cell viability. RESULTS: Knockdown of RLN2 using small interfering RNA decreases S100A4 and MMP-9 expression and inhibits invasion and cell viability in vitro. in MG-63 cells. Treatment with 100 mM recombinant Human RLN2 (B-29/A-24) for 24 hrs in MG-63/ RLN2 siRNA cells increases S100A4 and MMP-9 expression, and increases the invasion and cell viability in vitro in MG-63 cells. Transfection with S100A4 cDNA plasmid in MG-63/RLN2 siRNA cells for 48 hrs increases MMP-9 expression, and increase the invasion and cell viability of MG-63/RLN2 siRNA cells. Treatment with 20 mM BB94 (MMP-9 activator) for 24 hrs in MG-63/RLN2 siRNA cells increases MMP-9 expression, and increases the invasion and cell viability in vitro. in MG-63 cells. CONCLUSIONS: Our results indicate that RLN2 regulats cell migration, invasiveness and proliferation of osteosarcoma cells in vitro, which may be mediated through S100A4/MMP-9 signaling.

Knockdown of neuropilin-1 suppresses invasion, angiogenesis, and increases the chemosensitivity to doxorubicin in osteosarcoma cells - an in vitro study.

OBJECTIVES: Neuropilin-1 (NRP-1) is a novel co-receptor for vascular endothelial growth factor (VEGF). NRP-1 expression in osteosarcoma tissues was significantly higher, and high NRP-1 expression was more frequently occurred in osteosarcoma tissues with advanced clinical stage, positive distant metastasis and poor response to chemotherapy. We tested a hypothesis that the NRP-1 gene plays a role in the invasiveness, angiogenesis and chemoresistance of human OS. MATERIALS AND METHODS: To determine the role of NRP-1 in OS, NRP-1 was stably transfected into the human OS cell line MG-63 to increase the NPR-1 level, and NRP-1 siRNA was stably transfected into the human OS cell line SaOS-2 to knockdown of NRP-1. The effect of NRP-1 on invasion and angiogenesis was assessed by Matrigel invasion assay and in vitro angiogenesis assay. Chemosensitivity to doxorubicin was assessed by MTT assay in the MG-63 and SaOS-2 cells following NRP-1 overexpression or siRNA-induced downregulation of NRP-1. RESULTS: The NRP-1 transfected MG-63 cells showed a markedly higher level of invasion in Matrigel invasion assay. The capillary-like structure formation of endothelial cells was also increased by coculture with the NRP-1 transfected MG-63 cells. On the contrary, the NRP-1 siRNA transfected SaOS-2 cells showed a markedly lower level of invasion in Matrigel invasion assay. The capillary-like structure formation of endothelial cells was also repressed by coculture with the NRP-1 siRNA transfected SaOS-2 cells. NRP-1 overexpression in MG-63 cells increased survival of cells after exposure to doxorubicin. In contrast, downregulation of NRP-1 expression in SaOS-2 cells markedly increased chemosensitivity after exposure to doxorubicin. CONCLUSIONS: We suggest that NRP-1 could be used as a biomarker for OS progression and a novel therapeutic or chemopreventive target for human OS treatment.

Rapid diagnosis in early stage renal tuberculosis by real-time polymerase chain reaction on renal biopsy specimens.

OBJECTIVE: To determine the early rapid diagnosis of renal tuberculosis (RTB) by real-time polymerase chain reaction (PCR) on renal biopsy specimens. METHODS: Ninety patients were selected for this study. The patients were divided into the following three groups: RTB, non-RTB (N-RTB) and clinically suspected RTB (CS-RTB). The renal biopsy specimens of these patients were used for Mycobacterium tuberculosis DNA detection by real-time PCR, using 35 and 40 as cycle threshold (C(T)) cut-off values. Morning urine samples were collected for M. tuberculosis culture. RESULTS: In the RTB group, 25 C(T)35 and 28 C(T)40 patients were PCR-positive, seven of whom were urine M. tuberculosis culture-positive. In the N-RTB group, four C(T)35 and 13 C(T)40 patients were PCR-positive, none of whom were urine M. tuberculosis culture-positive. In the CS-RTB group, nine C(T)35 and 14 C(T)40 patients were PCR-positive, two of whom were urine M. tuberculosis culture-positive during 12 months of follow-up. The sensitivity and specificity of real-time PCR (C(T)40) were respectively 93.3% and 56.7%. The sensitivity and specificity of real-time PCR (C(T)35) were respectively 83.3% and 86.7%. The sensitivity and specificity of the urine M. tuberculosis culture were respectively 23.3% and 100%. CONCLUSIONS: The detection of M. tuberculosis DNA in renal biopsy tissue by real-time PCR is highly sensitive. Real-time PCR can increase diagnostic accuracy and provide valuable information regarding the early diagnosis of RTB.

Effect of Lactobacillus isolates on the adhesion of pathogens to chicken intestinal mucus in vitro.

AIMS: The aims of this study were to investigate in vitro the effects of Lactobacillus isolates from a chicken on adhesion of pathogenic Salmonella and Escherichia coli to chicken intestinal mucus obtained from different intestinal regions. METHODS AND RESULTS: Bacteria were labelled by using methyl-1,2-[(3)H]-thymidine. The bacterial adhesion was assessed by measuring the radioactivity of bacteria adhered to the mucus. The results showed that the abilities of Lactobacillus spp. to bind to the same intestinal mucus were higher than those of pathogenic Salmonella and E. coli. Pretreatment of intestinal mucus with Lactobacillus fermentum and Lactobacillus acidophilus, alone or in combination, reduced the adhesion of the tested pathogens, but the reductive extent of pathogenic adhesion by Lactobacillus spp. in combination was relatively high. CONCLUSIONS: The tested bacteria had different adhesions to mucus glycoproteins isolated from different intestinal regions of chicken. Lactobacillus acidophilus and Lact. fermentum in combination revealed a better ability to inhibit attachments of Salmonella and E. coli to chicken intestinal mucus than Lactobacillus sp. alone. SIGNIFICANCE AND IMPACT OF THE STUDY: A mixture of intestinal Lactobacillus spp. from a chicken may play a protective role in excluding pathogenic Salmonella and E. coli from the intestine of chicken.