The optimum marker for the detection of lymphatic vessels.

Podoplanin, lymphatic vessel endothelial hyaluronic acid receptor-1, prospero-related homeobox-1 and vascular endothelial growth factor receptor 3 have been demonstrated to have crucial roles in the development of the lymphatic system and lymphangiogenesis process by combining with their corresponding receptors. Thus, the four markers have been widely used in labelling lymphatic vessels for the detection of lymphangiogenesis and lymphatic vessel invasion. Numerous authors have aimed to identify the roles of these four markers in the lymphatic system and the mechanisms have been partly clarified at the molecular level. The aim of the present review was to comprehensively clarify the characteristics and latent action modes of the four markers in order to determine which is the best one for the detection of lymphangiogenesis and lymphatic vessel invasion.

Current progress and outcomes of clinical trials on using epidermal growth factor receptor-tyrosine kinase inhibitor therapy in non-small cell lung cancer patients with brain metastases.

Non-small cell lung cancer (NSCLC) continues to be one of the major causes of cancer-related deaths worldwide, and brain metastases are the major cause of death in NSCLC patients. With recent advances in understanding the underlying molecular mechanism of NSCLC development and progression, mutations in epidermal growth factor receptor (EGFR) have been recognized as a key predictor of therapeutic sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Using EGFR-TKI alone or in combination with standard treatments such as whole-brain radiotherapy and surgery has been an effective strategy for the management of brain metastasis. Particularly, a newer generation of EGFR-TKIs, including osimertinib and AZD3759, has been developed. These new EGFR-TKIs can cross the blood-brain barrier and potentially treat EGFR-TKI resistance and improve prognosis. In this article, current progress and outcomes of clinical trials on the use of EGFR-TKIs for treating NSCLC patients with brain metastasis will be reviewed.

Downregulation of LSD1 suppresses the proliferation, tumorigenicity and invasion of papillary thyroid carcinoma K1 cells.

The present study aimed to evaluate the effects of lysine-specific demethylase 1 (LSD1) downregulation, induced by small interfering RNA (siRNA) transfection, on the proliferation, colony formation, migration and invasion of the papillary thyroid carcinoma K1 cell line. The siRNA targeting LSD1 and scrambled non-targeting siRNA were each transfected into papillary thyroid carcinoma K1 cells. Downregulation of LSD1 mRNA and protein level was evaluated by reverse transcription-quantitative polymerase chain reaction, and immunocytochemical (ICC) analysis and western blotting, respectively. A Cell Counting kit-8 assay was applied to estimate the effect of LSD1-siRNA on cell growth. Migration and invasion abilities were estimated by Transwell chamber assay. A soft agar colony formation assay was performed to estimate the effect of LSD1-siRNA on tumorigenicity in vitro. ICC data showed that LSD1 protein was strongly expressed in the blank and control K1 cells compared with the LSD1-siRNA cells (F=15.192, P<0.01). Compared with the control cells, cells transfected with siRNA targeting LSD1 exhibited significant downregulation of LSD1 mRNA (t=6.845, P<0.01) and protein (F=53.764, P<0.01) levels. siRNA targeting LSD1 also downregulated cell proliferation following transfection for 24, 48 and 72 h (t=4.777, P<0.001; t=3.302, P=0.003; and t=3.017, P=0.006, respectively). Compared with the control group, the amount of cell invasion was gradually reduced in the LSD1-siRNA group (t=12.301, P<0.01). The number of migrating cells was significantly higher in the negative control group compared with the LSD1-siRNA group (t=7.911, P<0.01), and the ability of colony formation in the LSD1-siRNA cells was notably reduced in the soft agar formation assay (t=3.612, P=0.005). siRNA targeting LSD1 efficiently inhibits the proliferation, colony formation, migration and invasion of papillary thyroid carcinoma K1 cells.

siRNA targeting RBP2 inhibits expression, proliferation, tumorigenicity and invasion in thyroid carcinoma cells.

In order to estimate the effects of small interfering RNA (siRNA) targeting retinoblastoma binding protein 2 (RBP2) on the proliferation, expression, invasion, migration and tumorigenicity abilities of papillary thyroid carcinoma K1 cells, siRNA targeting RBP2 (RBP2-siRNA) and negative control siRNA were transfected into K1 cells. The mRNA levels of RBP2 in the transfected cells were estimated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the protein levels of RBP2 in these cells were evaluated by western blot analysis and immunocytochemical (ICC) analyses. The growth, tumorigenicity, migration and invasion abilities of the transfected cells were measured by Cell Counting Kit-8 (CCK-8), soft agar colony formation and transwell chamber assay, respectively. The ICC results demonstrated that the protein expression levels of RBP2 were lower in the RBP2-siRNA-transfected cells than in the blank and control cells (analysis of variance, F=26.754, P<0.01). RBP2-siRNA downregulated RBP2 at the mRNA (t=8.869) and protein level (F=60.835) (P=0.000 vs. control cells). In addition, the transfection of RBP2-siRNA into K1 cells also suppressed cell proliferation at 24, 48 and 72 h post-transfection (t=7.650, P<0.01; t=2.606, P=0.016; and t=2.377, P=0.027, respectively). Compared with the control group, the number of invasive and migrated cells were significantly reduced in the RBP2-siRNA-transfected group (t=4.774 and t=6.366, respectively; P<0.01). Furthermore, the tumorigenic potential of the cells transfected with RBP2-siRNA was markedly reduced, as indicated by the soft agar formation assay (t=2.749, P=0.014 vs. control cells). In conclusion, the transfection of RBP2-siRNA into papillary thyroid carcinoma K1 cells suppressed the expression of RBP2 in these cells, and reduced their proliferation, invasion, migration and tumorigenic potential. Therefore, targeting RBP2 may be an efficient approach to control thyroid carcinoma.

Value of diffusion-weighted magnetic resonance imaging on the follow-up of nasopharyngeal carcinoma after radiotherapy.

PURPOSE: To explore the value of diffusion-weighted imaging (DWI) and magnetic resonance imaging (MRI) on the follow-up of nasopharyngeal carcinoma (NPC) after radiotherapy. MATERIAL AND METHODS: Eighty-three NPC patients after radiotherapy were divided into two groups: 4 cases of residual tumor and 33 cases of non-residual within 6 months, the cases of recurrent and non-recurrent were 5 and 41 over 6 months, respectively. MRI and DWI imaging of these cases were closely analyzed, and the apparent diffusion coefficient (ADC) of the nasopharyngeal residual mass and nasopharyngeal wall thickening, skull base destruction and lateral pterygoid muscle were measured. RESULTS: The ADC of the lateral pterygoid muscle was (1.501 ± 0.069) × 10^{ - 3} mm^{2}/s in 83 cases. The ADCs of the non-residual group, residual group, non-recurrent group and recurrent group were (1.843 ± 0.133.4) × 10^{ - 3} mm^{2}/s, (1.097 ± 0.183) × 10^{ - 3} mm^{2}/s, (1.884 ± 0.134) × 10^{ - 3} mm^{2}/s and (0.787 ± 0.067) × 10^{ - 3} mm^{2}/s respectively.The ADCs of the residual group and recurrent group were lower than that of the lateral pterygoid muscle, while the ADCs of the non-residual group and non-recurrent group were higher than that of the lateral pterygoid muscle.The differences were statistically significant (P < 0.05). CONCLUSION: DWI can provide qualitative and quantitative information about the tumor cellular level; it can also identify residual, local fibrosis and recurrence after NPC radiotherapy sensitively and accurately by measuring the ADC of the region of interest.

Evaluation of the geometric accuracy of anatomic landmarks as surrogates for intrapulmonary tumors in image-guided radiotherapy.

OBJECTIVES: The purpose of this study was to evaluate the geometric accuracy of thoracic anatomic landmarks as target surrogates of intrapulmonary tumors for manual rigid registration during image-guided radiotherapy (IGRT). METHODS: Kilovolt cone-beam computed tomography (CBCT) images acquired during IGRT for 29 lung cancer patients with 33 tumors, including 16 central and 17 peripheral lesions, were analyzed. We selected the "vertebrae", "carina", and "large bronchi" as the candidate surrogates for central targets, and the "vertebrae", "carina", and "ribs" as the candidate surrogates for peripheral lesions. Three to six pairs of small identifiable markers were noted in the tumors for the planning CT and Day 1 CBCT. The accuracy of the candidate surrogates was evaluated by comparing the distances of the corresponding markers after manual rigid matching based on the "tumor" and a particular surrogate. Differences between the surrogates were assessed using 1-way analysis of variance and post hoc least-significant-difference tests. RESULTS: For central targets, the residual errors increased in the following ascending order: "tumor", "bronchi", "carina", and "vertebrae"; there was a significant difference between "tumor" and "vertebrae" (p=0.010). For peripheral diseases, the residual errors increased in the following ascending order: "tumor", "ribs", "vertebrae", and "carina". There was a significant difference between "tumor" and "carina" (p=0.005). CONCLUSIONS: The "bronchi" and "carina" are the optimal surrogates for central lung targets, while "ribs" and "vertebrae" are the optimal surrogates for peripheral lung targets for manual matching of online and planned tumors.

H3K27me3 protein is a promising predictive biomarker of patients' survival and chemoradioresistance in human nasopharyngeal carcinoma.

Trimethylation of lysine 27 on histone H3 (H3K27me3) is an epigenetic change which plays a critical role in tumor development and/or progression. However, the molecular status of H3K27me3 and its clinicopathologic/prognostic significance in nasopharyngeal carcinoma (NPC) have not been elucidated. In this study, the methods of Western blotting and immunohistochemistry (IHC) were utilized to examine the expression of H3K27me3 protein in NPC tissues and nonneoplastic nasopharyngeal epithelial tissues. Receiver operating characteristic (ROC) curve analysis was used to determine the cutpoint for H3K27me3 high expression. High expression of H3K27me3 could be observed in 127/209 (60.8%) of NPCs and in 8/50 (16.0%) normal nasopharyngeal epithelial tissues (P < 0.001). Further correlation analysis demonstrated that high expression of H3K27me3 was positively associated with tumor later T classification, tumor metastasis, advanced clinical stage and chemoradioresistance (P < 0.05). Moreover, high expression of H3K27me3 was closely associated with NPC patient shortened survival time as evidenced by univariate and multivariate analysis (P < 0.05). Consequently, a new clinicopathologic prognostic model with three poor prognostic factors (H3K27me3 expression, distant metastasis and treatment regimen) was constructed. The model could stratify risk significantly (low, intermediate and high) for overall survival and progression-free survival (P < 0.0001). These findings provide evidence that H3K27me3 expression, as examined by IHC, has the potential to be used as an immunomarker to predict NPC chemoradiotherapy response and patient prognosis. The combined clinicopathologic prognostic model may become a useful tool for identifying NPC patients with different clinical outcomes.

Family-based association study of Tim-1 and Tim-3 gene polymorphisms with childhood asthma in Chinese trios.

BACKGROUND: Asthma is a complex genetic disease, caused by the interaction of multiple genetic and environmental factors. T cell immunoglobulin domain and mucin domain (Tim) genes are located in chromosome 5q31-33, a region repeatedly linked to asthma or asthma-related phenotypes in several populations. Two members of Tim families, Tim-1 and Tim-3, which are expressed on T cell surface and potentially involved in T cell proliferation and differentiation, are good candidate genes for asthma. We investigated whether genetic variants or haplotypes in Tim-1 and Tim-3 genes confer susceptibility to asthma in a Chinese population. METHODS: A total of 9 polymorphisms were selected by using the HapMap Han Chinese population data and a haplotype-tagging single nucleotide polymorphism approach. Polymerase chain reaction fragment length polymorphism was adapted to determine the genotype in 118 complete Chinese trios of asthma. Then, transmission disequilibrium test (TDT), haplotypic relative risk (HRR), linkage disequilibrium and haplotype analysis were performed. RESULTS: The single locus TDT and HRR analysis showed the 9 polymorphisms were not transmitted preferentially to asthma-affected children (all p > 0.05). However, in both the haplotypic TDT and HRR analysis, the haplotype G-A-ins-C-G consisting of 5 Tim-1 polymorphisms was found to be overtransmitted to affected offspring (chi(2) = 4.51, p = 0.03) and the haplotype G-G-G consisting of 3 Tim-3 polymorphisms was found to be undertransmitted to asthma children (chi(2) = 8.24, p = 0.004). CONCLUSIONS: We conclude that it is unlikely that the Tim-1 or Tim-3 polymorphisms influence asthma susceptibility individually, but that the haplotypes of variants may be functional or may be in linkage disequilibrium with other functional single nucleotide polymorphisms.

[Expression of MMP-2 and its clinical significance in cervical squamous carcinoma].

OBJECTIVE: To investigate the relationship of the expression of matrix metalloproteinase-2 (MMP-2) with tumorigenesis, development and metastasis of cervical squamous carcinoma. METHODS: The expression and distribution of MMP-2 protein were detected by using immunohistochemical SP method. The active protein and mRNA of MMP-2 were determined by using gelatin Zymography and RT-PCR, respectively. The relationships between those indexes and the factors related to clinical pathology of cervical carcinoma were analyzed statistically. RESULTS: Immunohistochemical studies demonstrated that MMP-2 was expressed in 81.13% of squamous cell carcinomas (SCC), but was less frequently expressed in high-grade squamous intraepithelial lesions (HSIL, 47.62%, 10/21) and low-grade squamous intraepithelial lesions (LSIL, 10.00%, 1/10, P<0.01). In SCC, MMP-2 protein was expressed in 91.30%(21/23) of patients, which positively correlated with lymph node metastasis significantly (P<0.05). And the expression of MMP-2 was not significantly related to the pathological grade, or stage status. By direct analysis of enzyme activities we found that the gelatinolytic activity of MMP-2 was significantly higher in SCC [(5.81 +/- 2.17)x10(4)] than in HSIL [(2.28 +/- 0.83) x10(4), P<0.01] and LSIL [(1.94 +/- 0.71)x10(4), P<0.01]. Expression of MMP-2 mRNA was significantly higher in carcinoma(0.87 +/- 0.44) than in HSIL(0.46 +/- 0.22, P<0.01) and in LSIL(0.37 +/- 0.20, P<0.01). CONCLUSION: The positive expression of MMP-2 can be used to estimate the metastatic potentiality and help the adjuvant treatment. MMP-2 is related well with the occurrence, invasion and metastasis of cervical squamous carcinoma.

[The relationship between the haplotype of T cells immunoglobulin mucin-3 gene and allergic asthma in the Han population from Hubei province of China].

OBJECTIVE: To investigate the single nucleotide polymorphisms (SNPs) of -1516G/T in the promoter region and 4259G/T in the exon-3 region of the T cells immunoglobulin mucin-3 (TIM-3) and their linkage disequilibrium, and therefore to detect their haplotype relationship with allergic asthma of the Han population from Hubei province of China. METHODS: The two polymorphisms were detected with allelic specific polymerase chain reaction (ASPCR). In the 175 asthmatic subjects and in the 202 healthy controls collected from June, 2004 to October 2007 in the Han population from Hubei province. The genotype and allele frequencies, the D' value between the two SNPs sites, the haplotype and their frequencies were calculated and analyzed, respectively. RESULTS: The genotype frequencies of GG, GT and TT in -1516G/T polymorphism of TIM-3 gene were 82.7% (167/202), 17.3% (35/202), 0 (0/202) respectively in the 202 controls, and 82.9% (145/175), 17.1% (30/175), 0 (0/175) respectively in the 175 asthmatic subjects. The genotype frequencies of GG, GT and TT in 4259G/T polymorphism of TIM-3 gene were 0.5% (1/202), 2.5% (5/202), 97.0% (196/202) respectively in the 202 controls and 0.6% (1/175), 5.7% (10/175), 93.7% (164/175) respectively in the 175 asthmatic subjects. The control group: D' = 1.0, the asthma group D' = 0. 9. The 3 haplotypes were G-G, G-T, T-T in the Han population from Hubei province of China, and their haplotype frequencies were distributed similarly in asthma 3.4% (12/350), 88.0% (308/ 350), 8.6% (30/350) and in the controls 1.7% (7/404), 89.6% (362/404), 8.7% (35/404). None of these differences were statistically significant (chi2 = 2.15, 0.47, 0.003 respectively, all P > 0.05). CONCLUSION: There are strong linkage disequilibrium between the two SNPs sites in TIM-3 gene in Han population from Hubei province, but the haplotypes G-G, G-T and T-T are not associated with asthma susceptibility of this population. We cannot exclude the possibility that the haplotypes of TIM-3 may be associated with asthma susceptibility in other ethnic populations or the susceptibility to other atopic diseases and autoimmunity diseases.

It is AMPA receptor, not kainate receptor, that contributes to the NBQX-induced antinociception in the spinal cord of rats.

Studies demonstrated that intrathecal 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo[f]quinoxaline-7-sulfonamide disodium (NBQX), an antagonist of AMPA/kainate receptors, induced antinociception in the spinal cord of rats. The present study demonstrated that the NBQX-induced increases in hindpaw withdrawal latencies (HWLs) were dose-dependently attenuated by intrathecal pretreatment of the AMPA receptor desensitization inhibitor, diazoxide. The effect was unrelated to the opening of K+ channels by diazoxide. On the other hand, intrathecal pretreatment of concanavalin A, which selectively inhibits the desensitization of kainate receptor, produced no significant influence on the NBQX-induced antinociception. The results suggest that the NBQX-induced antinociception was mediated by AMPA receptors, not by kainate receptors, in the spinal cord of rats.

Involvement of mu- and delta-opioid receptors in the antinociceptive effects induced by AMPA receptor antagonist in the spinal cord of rats.

The present study was performed to explore the involvement of opioid receptors in the antinociception induced by a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist in rats. The hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation was assessed by hot plate test and the Randall Selitto Test. Intrathecal injection of 20 nmol of 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo[f]quinoxaline-7-sulfonamide (NBQX) disodium, a competitive AMPA receptor antagonist, increased significantly the HWLs to both thermal and mechanical stimulation in rats. The increased HWLs induced by NBQX were dose-dependently attenuated by the opioid receptor antagonist naloxone, while naloxone itself had no marked influences on the HWL of rats. Furthermore, the increased HWLs induced by NBQX were inhibited by the mu-opioid antagonist beta-funaltrexamine (beta-FNA) or the delta-opioid antagonist naltrindole, but not by the kappa-opioid antagonist nor-binaltorphimine (nor-BNI). The results suggest that mu- and delta-opioid receptors, not kappa-opioid receptor, are involved in the antinociception induced by AMPA antagonist in the spinal cord of rats.

[Down-regulation of metastatic phenotype in human melanoma cells by controlled expression of anti-sense matrix metalloproteinase 9].

OBJECTIVE: To investigate the correlation between matrix metalloproteinase 9 (MMP-9) expression and tumor invasion and metastasis as well, and to explore the potential application of controlled expression of target gene in tumor gene therapy. METHODS: One self-contained tetracycline-regulated retroviral vector containing anti-sense cDNA of MMP-9 was constructed and transfected into a metastatic human melanoma cell line WM451 which expressed a high level of MMP-9. Techniques such as growth rate measurment, MTT assay, (3)H-thymidine incorporation, colony forming ability in soft agar, invasion assay in Boyden chamber, as well as zymography and Western blot were applied to analyze the expression of MMPs and behaviors of tumor cells in vitro before and after gene transfection. Tumorigenecity and spontaneous metastasis were tested in nude mice. RESULTS: In the presence of exogenous tetracycline, the transfected antisense MMP-9 did not affect the endogenous level of MMP-9 in WM451 cells, and showed no significant changes in cell behaviors in comparison with that of the vector-transfected control cells. Nevertheless, withdrawal of tetracycline from the medium caused a significant down-regulation of expression and activity of MMP-9. The capacity of cell growth in vitro, colony forming ability in soft agar, invasion through Matrigel all were inhibited remarkably when compared with the controls. Spontaneous metastasis in nude mice was significantly inhibited. CONCLUSIONS: Transfection of anti-sense MMP-9 can down-regulate the invasion and metastasis of melanoma cells both in vitro and in vivo, further clarifying the important role of MMP-9 in tumor progression.