Effect of a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on invasion of human colorectal cancer cell line SL-174T

AIML To investigate the effect and mechanism of action of the nitric oxide synthase （NOS） inhibitor NG-nitro-L-arginine methyl ester （L-NAME） on invasion and metastasis of human colorectal cancer cell line SL-174T. METHODS： Human colorectal cancer cel4 line SL-174T was cultured and treated separately with four different dosages of L-NAME for 72 h, Nitric oxide （NO） production was measured with Griess reagent, The effect of L-NAME on invasion and migration of SL-174T cells were evaluated by using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane （Matrigel）, RT-PCR was performed to determine the mRNA levels of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor metalloproteinase-2 （TIMP-2）,RESULTS： L-NAME could significantly inhibit NO production of SL-174T in a dose-dependent manner. After being treated for 72 h with 0.2, 0.4, 0.8, and 1.0 mmol/L L- NAME, respectively, the ability of the L-NAME treated SL- 174T cells to invade the reconstituted basement membrane decreased significantly （t = 8.056, P〈0.05; t= 14.467, P〈0.01; t= 27.785, P〈0.01; and t= 29.405, P〈0.01, respectively） and the inhibition rates were 10.29%, 19.62%, 34.08%, and 42.23%, respectively. Moreover, L-NAME could inhibit migration of SL-174T cells, and the inhibition rates were 20.76%, 24.95%, 39.43%, and 46. 85% for L-NAME at 0.2, 0.4, 0.8, and 1.0 mmol/L, respectively （t = 15.116, P〈0.01）. In addition, after treatment with L-NAME, expression of MMP-2 mRNA was significantly decreased （t = 71.238, P〈0.01） and that of TIMP-2 mRNA was markedly increased （t = -13.020, P〈0.01）. CONCLUSION： L-NAME exerts anti-invasive and anti- metastatic effects on SL-174T cell line via downregulating MNP-2 mRNA expression and upregulating TIMP-2 mRNA expression.

CT and MR imaging for detecting neoplastic invasion of esophageal inlet

AIM: Direct neoplastic invasion of esophageal inlet is an uncommon but significant sequela of advanced head and neck carcinomas. The aim of this study was to seek an optimal CT or MRI criterion for determining the neoplastic esophageal inlet involvement in order to help tumor staging and surgical planning. METHODS: CT and MRI of 78 head and neck tumor cases were investigated retrospectively. At the level of the esophageal inlet on axial CT and MRI scans, the distance between the posterior aspect of cricoid cartilage and the anterior aspect of vertebra (d-CV) was measured by two senior radiologists who were unaware of clinical findings. Then, according to pathologie evidence and follow-up findings, these patients were divided into patients group, including 32 cases with neoplastic invasion of esophageal inlet and control group, including 46 cases without neoplastic esophageal inlet involvement. The statistical difference based on d-CV between the two groups was determined. The optimal criterion of d-CV on CT or MRI was assessed and lts accuracy, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were evaluated respectively. RESULTS: In control group, d-CV at the esophageal inlet level was 0.94±0.15 cm on axial CT and 0.91±0.18 cm on axial MRI, whereas in patient group, d-CV was 1.24±0.32 cm on CT and 1.31±0.36 cm on MRI. There was a statistical significance in d-CV between the two groups on CT and MRI modalities (P<0.01). d-CV greater than 1.0 cm was the typicall feature of neoplastic invasion of the esophageal inlet with 73% sensitivity, 83% specificity, 79% accuracy, 76% PPV, 80% NPV on CT and 84% sensitivity, 77% specificity, 80% accuracy, 70% PPV, 88% NPV on MRI respectively. CONCLUSION: Except for other CT and MR imaging features of neoplastic invasion of esophageal inlet, d-CV greater than 1.0 cm is an optimal adjunct criterion for esophageal inlet invasion by advanced head and neck carcinomas.

Rare cause of upper gastrointestinal bleeding owing to hepatic cancer invasion:A case report

Upper gastrointestinal bleeding refers to bleeding that arises from the gastrointestinal tract proximal to the ligament of Treitz.The primary reason for gastrointestinal bleeding associated with hepatocellular carcinoma is rupture of a varicose vein owing to pericardial hypotension.We report a rare case of gastrointestinal bleeding with hepatocellular carcinoma in a patient who presented with recurrent gastrointestinal bleeding.The initial diagnosis was gastric cancer with metastasis to the multiple lymph nodes of the lesser curvature.The patient underwent exploratory laparotomy,which identified two lesions in the gastric wall.Total gastrectomy and hepatic local excision was then performed.Pathological results indicated that the hepatocellular carcinoma had invaded the stomach directly,which was confirmed immunohistochemically.The patient is alive with a disease-free survival of 1 year since the surgery.Hepatocellular carcinoma with gastric invasion should be considered as a rare cause of upper gastrointestinal bleeding in hepatocellular carcinoma patients,especially with lesions located in the left lateral hepatic lobe.Surgery is the best solution.

PROXIMAL BRONCHIAL INVASION OF LUNG CANCER: ACLINICOPATHOLOGICAL STUDY

Objective: To investigate the characteristics of proximal bronchial invasion of lung cancer for different types. Methods: Proximal bronchus of 151 operatively resected specimens of hilar type lung cancer were selected for cross-sectional pathological study. Forty-one specimens were obtained from total pulmonectomy, and 110 from pulmonary lobectomy. Results: Evidence showed that the direct invasion of tumor cells through the submucosal layer or multiple layers was the major form of lung cancer spread; 96.6% of the cancerous invasion occurred at the proximal bronchial wall less than 1.5 cm apart from the margin of the cancer. The extension of invasion was correlated with the histopathologic type of cancer, mode of invasion and TNM classification (pT, pN). Besides, the invasion in the bronchial wall by metastatic lymph nodes was also an important way for the cancer to spread. Conclusion: In order to achieve the radical removal of a tumor, it is imperative to keep a distance of 1.5 cm or more between the excision margin of the bronchus and the tumor, and to completely resect the hilar and mediastinal lymph nodes.

Nomograms predicting long-term survival in patients with invasive intraductal papillary mucinous neoplasms of the pancreas: A population-based study

BACKGROUND There are few effective tools to predict survival in patients with invasive intraductal papillary mucinous neoplasms of the pancreas.AIM To develop comprehensive nomograms to individually estimate the survival outcome of patients with invasive intraductal papillary mucinous neoplasms of the pancreas.METHODS Data of 1219 patients with invasive intraductal papillary mucinous neoplasms after resection were extracted from the Surveillance,Epidemiology,and End Results database,and randomly divided into the training(n=853)and the validation(n=366)cohorts.Based on the Cox regression model,nomograms were constructed to predict overall survival and cancer-specific survival for an individual patient.The performance of the nomograms was measured according to discrimination,calibration,and clinical utility.Moreover,we compared the predictive accuracy of the nomograms with that of the traditional staging system.RESULTS In the training cohort,age,marital status,histological type,T stage,N stage,M stage,and chemotherapy were selected to construct nomograms.Compared with the American Joint Committee on Cancer 7th staging system,the nomograms were generally more discriminative.The nomograms passed the calibration steps by showing high consistency between actual probability and nomogram prediction.Categorial net classification improvements and integrated discrimination improvements suggested that the predictive accuracy of the nomograms exceeded that of the American Joint Committee on Cancer staging system.With respect to decision curve analyses,the nomograms exhibited more preferable net benefit gains than the staging system across a wide range of threshold probabilities.CONCLUSION The nomograms show improved predictive accuracy,discrimination capability,and clinical utility,which can be used as reliable tools for risk classification and treatment recommendations.

Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97

AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied. RESULTS: Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10). CONCLUSION: Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.

Relationship between phenotypes of cell-function differentiation and pathobiological behavior of gastric carcinomas

AIM: To reveal the correlation between the functional differentiation phenotypes of gastric carcinoma cells and the invasion and metastasis by a new way of cell-function classification.METHODS:Surgically resected specimens of 361 gastric carcinomas(GC) were investigated with enzyme-, mucin-, and tumor-related marker immunohistochemistry. According to the direction of cell-function differentiation, stomach carcinomas were divided into five functionally differentiated types. RESULTS: (1) Absorptive function differentiation type (AFDT): there were 82 (22.7%) patients including 76 (92.7%) aged 45 years. Sixty-nine (84.1%) cases belonged to the intestinal type. Thirty-eight (46.3%) expressed CD44v6 and 9 (13.6%) of 66 male patients developed liver metastasis.The 5-year survival rate of patients in this group (58.5%) was higher than those with the other types (P【0.01). (2) Mucin secreting function differentiation type (MSFDT): 54 (15%) cases. Fifty-three (98.1%) tumors had penetrated the serosa, 12 (22.2%) expressed ER and 22 (40.7%) expressed CD44v6. The postoperative 5-year survival rate was 28.6%. (3) Absorptive and mucin-producing function differentiation type (AMPFDT): there were 180 (49.9%) cases, including 31 (17.2%) aged younger than 45 years. The tumor was more common in women (62, 34.4%,) and expressed more frequently estrogen receptors (ER) (129, 81.7%) than other types (P【0.01). Ovary metastasis was found in 12 (19.4%) out of 62 female subjects. The patients with this type GC had the lowest 5-year survival rate (24.7%) among all types. (4) Specific function differentiation type (SFDT): 13 (3.6%) cases. Nine (69.2%) tumors of this type derived from APUD system, the other 4 (30.7%) were of different histological differentiation. Sixty per cent of the patients survived at least five years. (5) Non-function differentiation type (NFDT): 32 (8.9%) cases. Nineteen (59.4%) cases had lymph node metastases but no one with liver or ovary metastasis. The 5-year survival rate was 28.1%. CONCLUSION: This new cell-function classification of GC is helpful in indicating the characteristics of invasion and metastasis of GC with different cell-function differentiation phenotypes. Further study is needed to disclose the correlation between the cell-functional differentiation phenotypes and the relevant genotypes and the biological behavior of gastric carcinoma.

Effects of Taxotere on invasive potential and multidrug resistance phenotype in pancreatic carcinoma cell line SUIT-2

INTRODUCTIONDevelopment of drug-resistance to chemotherapyand subsequent metastasis of tumor are primarilyresponsible for treatment failure and the death fromcancer. There have been many previous studies onthe relationship between expression of multidrugresistance (MDR) phenotype P-glycoprotein (P-gp)and the malignant properties of tumors, but theresults are often conflicting[1-8]. The difference intumor types or MDR phenotype induced by specificagents might account for this discrepancy. Taxotere(TXT), a member of the family of taxanes, hasantitumor activity through its effect of promotingthe polymerization of tubulin[9,10].

TIMP-3 gene transfection suppresses invasive and metastatic capacity of human hepatocarcinoma cell line HCC-7721

BACKGROUND: Tissue inhibitor of metalloproteinases (TIMPs) can restrain the tumor growth by their protein property and matrix metalloproteinases (MMPs) inhibition. There are currently four known TIMP family members-TIMP-1, 2, 3, 4. We determined whether increasing levels of TIMP-3 expression could suppress the malignant phenotype of human hepatocarcinoma cell line HCC-7721. METHODS: A recombinant expression vector, which contained full-length cDNA of human TIMP-3, was constructed and transfected into the human hepatocarcinoma cell line HCC-7721 by the lipofectamine technique. In vitro and in vivo tests such as Western blotting, immunohistochemistry as well as xenografting in nude mice were used to analyze expression levels of TIMP and MMP, and changes in malignant phenotype after the gene transfection. RESULTS: TIMP-3 expression in TIMP-3 gene-transfected HCC-7721 cells was upregulated as assessed by Western blotting. The ability of in vitro invasion through a Boyden chamber was significantly decreased compared to controls. Following subcutaneous injection into nude mice, the TIMP-3 transfected cells suppressed primary tumor growth, as characterized by reduced tumor weight, size and microvasculature as well as maintaining the extracellular matrix. CONCLUSION: The results suggest that upregulation of TIMP-3 expression in HCC-7721 cells inhibits invasion capacity in vitro as well as tumorigenic and metastatic potential in nude mice.

Effects of LY294002 on the invasiveness of human gastric cancer in vivo in nude mice

AIM: To investigate the effects of class phosphatidy-linositol 3-kinase (PI3K) inhibitor LY294002 on the invasiveness and related mechanisms of implanted tumors of SGC7901 human gastric carcinoma cells in nude mice. METHODS: Nude mice were randomly divided into model control groups and LY294002 treatment groups. On days 5, 10 and 15 after treatment, the inhibitory rate of tumor growth, pathological changes in tumor specimens, expression levels of matrix metalloproteinase (MMP)-2, MMP-9, CD34 [representing microvessel density (MVD)] and vascular endothelial growth factor (VEGF), as well as apoptosis indexes in tumor samples were observed.RESULTS: In this study, we showed that treatingthe tumors with LY294002 could significantly inhibit carcinoma growth by 11.3%, 29.4% and 36.7%, after 5, 10 and 15 d, respectively, compared to the control group. Hematoxylin & eosin staining indicated that the rate of inhibition increased progressively (23.51% ± 3.11%, 43.20% ± 3.27% and 63.28% ± 2.10% at 5, 10 and 15 d, respectively) along with apoptosis. The expression of MMP-2 was also downregulated (from 71.4% ± 1.6% to 47.9% ± 0.7%, 31.9% ± 0.9% and 7.9% ± 0.7%). The same effects were observed in MMP-9 protein expression (from 49.4% ± 1.5% to 36.9% ± 0.4%, 23.5% ± 0.9% and 7.7% ± 0.6%), the mean MVD (from 51.2% ± 3.1% to 41.9% ± 1.5%, 30.9% ± 1.7% and 14.9% ± 0.8%), and the expression of VEGF (from 47.2% ± 3.1% to 25.9% ± 0.5%, 18.6% ± 1.2% and 5.1% ± 0.9%) by immunohistochemical staining.CONCLUSION: The classPI3K inhibitor LY294002could inhibit the invasiveness of gastric cancer cells by downregulating the expression of MMP-2, MMP-9, and VEGF, and reducing MVD.

UROKINASE-TYPE PLASMINOGEN ACTIVATOR,ITS RECEPTOR AND INHIBITOR EXPRESSION IN HEPATOCELLULAR CARCINOMA RELATION TO CANCER INVASIVENESS AND PROGNOSIS

Objective: To study the relevance of uPA, uPAR and PAI 1 to hepatocellular carcinoma (HCC) Methods: The expression at protein level of uPA, uPAR and PAI 1 was determined in 48 cases of HCC and 12 cases of benign tumors of liver (as control) by immunohistochemistry Results: When compared to cancer adjacent liver tissue and the control, positive rate of immune staining for uPA, uPAR and PAI 1 on cell membrane were significantly higher in HCC cells ( P <0 05) Positive staining of uPA and uPAR was seen in 16 of 22 and 19 of 22 cases of HCC with invasion, respectively ( P <0 01 and P <0 001) In 8 of 8 cases with cancer embolus, and in 6 of 6 cases with lymph node metastasis was the expression of positive uPAR Compared with 2 of 17 cases without recurrence, uPAR was positive in 15 of 17 recurrent cases ( P <0 01) In 36 cases who survived, 17 was positive uPAR and 15 positive PAI 1, while in 12 cases who died 2 years after surgery, 12 were positive for uPAR and 9 positive PAI 1, respectively ( P <0 01 and P <0 05) In 15 positive cases for all three parameters, 11 had cancer invasion and 7 died within 2 years, while in negative cases, 2 had invasion and none died within 2 years ( P <0 05) Conclusion: Expression of uPA, uPAR and PAI 1 is increased in HCC, uPA and uPAR may contribute significantly to HCC invasion and metastasis uPAR and PAI 1 are associated with poor prognosis of HCC

Hematemesis as the initial complication of pancreatic adenocarcinoma directly invading the duodenum: A case report

Pancreatic carcinoma is a debilitating disease and carries a poor prognosis. It is a rare cause of upper gastrointestinal bleeding, even though pancreas, stomach, duodenum and jejunum are adjacent organs. The incidence of pancreatic adenocarcinoma directly invading the gastrointestinal tract leading to gastrointestinal hemorrhage is very low, and most of them present with melena and hematochezia. Here, we describe one unique case manifesting characteristically severe and unremitting hematemesis as an initial presentation of pancreatic adenocarcinoma. This tumor directly invaded the duodenal mucosa as a bleeding protruding tumor mass. Our MEDLINE search has confirmed that this is the first reported case with an initial manifestation of hematemesis from pancreatic adenocarcinoma in Asians.Pancreatic adenocarcinoma directly invading duodenum complicated by hemorrhage can be a rare cause of hematemesis, and clinicians should be reminded of it while they are making differential diagnosis.

Novel predictors for lymph node metastasis in submucosal invasive colorectal carcinoma

AIM To evaluate a novel grading system to predict lymph node metastasis(LNM) in patients with submucosal invasive colorectal carcinoma(SICRC).METHODS We analyzed the associations between LNM and various clinicopathological features in 252 patients with SICRC who had undergone radical surgery at the Seoul Saint Mary's hospital between 2000 and 2015.RESULTS LNM was observed in 31 patients(12.3%). The depth and width of the submucosal invasion, lymphatic invasion, tumor budding, and the presence of poorly differentiated clusters(PDCs) were significantly associated with the incidence of LNM. Using multivariate analysis, the receiver operating characteristic curvewas calculated and the area under curve(AUC) was used to compare the ability of the different parameters to identify the risk of LNM. The most powerful clinicopathological parameter for predicting LNM was lymphatic invasion(difference AUC = 0.204), followed by the presence or absence of tumor budding(difference AUC = 0.190), presence of PDCs(difference AUC = 0.172) and tumor budding graded by the Ueno method(difference AUC = 0.128). CONCLUSION Our results indicate that the tumor budding and the depth multiplied by the width measurements of submucosal invasion can provide important information for patients with SICRC.

Detection of Syndecan-1 and Heparanase-1 Genes in Esophageal Carcinoma by Quantitative RT-PCR

OBJECTIVE To quantitatively explore the expression of Syndecan-1 and heparanase-1 in esophageal cancer tissue as well as their relationship with the clinicopathological factors, in order to evaluate their roles in tumor invasion and metastasis.METHODS Real-time fluorescence quantitative PCR （Q-PCR） was used to analyze the expression levels of Syndecan-1 and heparanase-1 genes￡？participants included 67 cases with esophageal cancers and 32 healthy volunteers.RESULTS The expression of Heparanase-1 gene in esophageal cancers was higher than that in normal esophageal tissue （P 〈 0.001）, and the expression of Syndecan-1 gene in the normal esophageal tissue was higher compared with esophageal cancers （P 〈 0.001）. The positive rates of Syndecan-1 and Heparanase-1 gene in esophageal cancer were 13.4% （9/67） and 85.1% （57/67）.The expression of Syndecan-1 and Heparanase-1 genes was signifi cantly related to di. erentiation, depth of infi ltration, lymph node metastasis, vessel metastasis, and TNM stages of disease （P 〈 0.05）. In an attempt to measure the association between the 2 agents, this study found that the expression of Syndecan-1 mRNA had a significantly negative correlation with the expression of Heparanase-1 mRNA by using Spearman rank correlation test （OR = -0.572, P 〈 0.001）.CONCLUSION Syndecan-1 and Heparanase-1 play important roles in the invasion and metastasis of esophageal cancer. The reduction of Syndecan-1 and/or the increase of Heparanase-1 may influence the invasion and metastasis of malignant tumors.Thus the combination assay of Syndecan-1 and Heparanase-1 may contribute to the diagnosis and treatment of malignant tumors.

Advances in BRAF gene mutations in papillary thyroid carcinoma

Thyroid cancer is the most common endocrine system tumor.Ultrasound guided fine needle puncture(FNA)can identify benign and malignant thyroid nodules.However,due to the limitation of cytological detection,some thyroid nodules are difficult to distinguish benign and malignant.BRAF gene mutation is a common human oncogenic mutation and the highest mutation frequency in papillary thyroid carcinoma.The combination of FNA and BRAF gene detection can significantly improve the diagnostic rate of benign and malignant thyroid nodules and make up for the deficiency of single diagnosis of cytology.Moreover,while the incidence of thyroid cancer is growing rapidly worldwide,its mortality remains stable.The problem of overdiagnosis and overtreatment of thyroid cancer is becoming more and more obvious.However,due to the limitations of current studies on BRAF genes,its prognostic value for papillary thyroid carcinoma remains controversial.Therefore,in order to reduce the adverse effects of overdiagnosis and treatment,the relationship between gene and tumor biological behavior needs further study in the future.

Cell softness reveals tumorigenic potential via ITGB8/AKT/glycolysis signaling in a mice model of orthotopic bladder cancer

Background:Bladder cancer,characterized by a high potential of tumor recurrence,has high lifelong monitoring and treatment costs.To date,tumor cells with intrinsic softness have been identified to function as cancer stem cells in several cancer types.Nonetheless,the existence of soft tumor cells in bladder tumors remains elusive.Thus,our study aimed to develop a microbarrier microfluidic chip to efficiently isolate deformable tumor cells from distinct types of bladder cancer cells.Methods:The stiffness of bladder cancer cells was determined by atomic force microscopy(AFM).The modified microfluidic chip was utilized to separate soft cells,and the 3D Matrigel culture system was to maintain the softness of tumor cells.Expression patterns of integrinβ8(ITGB8),protein kinase B(AKT),and mammalian target of rapamycin(mTOR)were determined by Western blotting.Double immunostaining was conducted to examine the interaction between F-actin and tripartite motif containing 59(TRIM59).The stem-cell-like characteristics of soft cells were explored by colony formation assay and in vivo studies upon xenografted tumor models.Results:Using our newly designed microfluidic approach,we identified a small fraction of soft tumor cells in bladder cancer cells.More importantly,the existence of soft tumor cells was confirmed in clinical human bladder cancer specimens,in which the number of soft tumor cells was associated with tumor relapse.Furthermore,we demonstrated that the biomechanical stimuli arising from 3D Matrigel activated the F-actin/ITGB8/TRIM59/AKT/mTOR/glycolysis pathways to enhance the softness and tumorigenic capacity of tumor cells.Simultaneously,we detected a remarkable up-regulation in ITGB8,TRIM59,and phospho-AKT in clinical bladder recurrent tumors compared with their non-recurrent counterparts.Conclusions:The ITGB8/TRIM59/AKT/mTOR/glycolysis axis plays a crucial role in modulating tumor softness and stemness.Meanwhile,the soft tumor cells become more sensitive to chemotherapy after stiffening,that offers new insights for hampering tumor progression and recurrence.

Activated vascular endothelia regulate invasion of glioma cells through expression of fibronectin

Background Previous researches have indicated that glioma invasion may occur within a tumor-host microecology, and that fibronectin may be involved in glioma invasion as an important component of the extracellular matrix. However, how the interaction between tumor cells and vascular endothelial cells affects glioma invasion is poorly understood. The aim of this study was to investigate the effects of the interaction between tumor cells and vascular endothelial cells on glioma invasion, and the relationship of this interaction to fibronectin. Methods The localization of fibronectin in different brain astrocytoma tissues was determined by immunohistochemistry Then, vascular endothelial cells and glioma cells were co-cultured in a Transwell co-culturing system. Fibronectin expression was detected by reverse transcriptase-polymerase chain reaction, immunocytochemistry, and enzyme-linked immunosorbent assay. Additionally, the influence of the interaction between tumor cells and vascular endothelial cells on glioma cell invasion was determined by an in vitro rapid invasion test. Results In brain astrocytoma tissues, fibronectin was present on the endothelial cells, in the extracellular matrix. Fibronectin expression was greater in higher grade tumors than in lower grade tumors. The interaction of glioma cells and vascular endothelial cells in vitro induced fibronectin release from vascular endothelial cells, which in turn stimulated glioma cell migration. This effect was inhibited by fibronectin blocking antibody. Conclusion Glioma cells may induce vascular epithelial cells to express fibronectin, and in turn fibronectin could promote glioma cell invasion.

Macrophage migration inhibitory factor enhances neoplastic cell invasion by inducing the expression of matrix metalloproteinase 9 and interleukin-8 in nasopharyngeal carcinoma cell lines

Background Nasopharyngeal carcinoma (NPC) shows highly invasive and metastatic features. This study aims to investigate macrophage migration inhibitory factor (MIF)-induced invasion of NPC cells in vitro and the effects on matrix metalloproteinases (MMPs) and interleukin-8 (IL-8),and to study the mechanism of tumor cell invasion and metastasis in the early stage of NPC. Methods Two nasopharyngeal carcinoma cell lines,CNE-1 and CNE-2,were adopted in this study. The NPC cell invasion and migration were evaluated by microinvasion assay. The variation of expression percentages of MMP2- or MMP9-positive cells was detected by flow cytometry in two cell lines with or without MIF treatment. Western blotting and RT-PCR were used to assay the protein and mRNA expressions of MMP2 and MMP9. The IL-8 concentration secreted by NPC cells was compared with the cells with different treatments using ELISA. Results After treating with MIF for 48 hours,the cell numbers of CNE-1 and CNE-2 which went through the 8-μm filter membrane were increased. Compared with non-MIF treated NPC cells,significant difference could be found both in CNE-1( P =0.005) and CNE-2 cells ( P =0.001) . The percentages of MMP9-positive cells were significantly increased in both CNE-1 [from (28.5±2.5)% to (82.4±3.5)%, P =0.001] and CNE-2 [from (32.8±3.5)% to (86.1±1.6)%, P =0.002]. The relative intensity of MMP9 protein expression was also enhanced in both cell lines (CNE-1: from 83.1±6.0 to 242.9±22.9, P =0.002;CNE-2: from 84.4±4.3 to 278.9±29.7, P =0.003). Correspondingly,the increased MMP9 mRNA expression level was significantly detectable in both cell lines.The concentration of IL-8 in the supernatant of CNE-2 was higher [(1201.8±593.3) pg/ml] after treatment. It was also remarkably higher than that in the supernatant of CNE-2 without treatment ( P =0.026). However,there was no significant difference in the concentration variation of IL-8 in CNE-1 ( P =0.581), while the IL-8 mRNA level was only enhanced in CNE-2. Conclusions MIF can induce potent invasion of NPC cell lines in vitro , and the infiltrating lymphocytes in NPC might be responsible for the invasion and metastasis of tumor cells. MIF cytokine which is secreted by these infiltrating lymphocytes might contribute to the invasion as well as metastasis of NPC in the early stages by induction of MMP9 and IL-8 in an indirect pathway.

Relations of proliferative activities of gastric carcinoma cells to lymphatic involvement, venous invasion and prognosis

Background This study was to evaluate bivariate bromodeoxyuridine(BrdUrd)/DNA flow cytometric analysis in detection of gastric carcinoma and to study the relations of cellular BrdUrd labeling indices (LI), G_2/M-phase fraction(G_2/MPF) and DNA ploidy pattern to lymphatic involvement, venous invasion and prognosis.Methods Fresh tumor samples from 60 patients with gastric carcinoma were analyzed by bivariate BrdUrd/DNA flow cytometry. The results were correlated with lymphatic vessel invasion, lymphatic node metastasis, the number of matastatic lymphatic nodes, and venous invasion. Propidium iodide (PI) was used as a fluorescent probe for total cellular DNA, and a monoclonal antibody against BrdUrd was used as a probe for BrdUrd incorporated into DNA. Fluorescent-labeled goat anti-mouse antibody was used as a second antibody. S-phase fractions were measured by in vitro BrdUrd labeling, and DNA ploidy and G_2/MPF were also measured. Comparison of survival was performed with the log-rank test, the Chi-square test for qualitative data, and Student’s t test for quantu data. Results BrdUrd LI and G_2/MPF values were significantly higher in tumors with lymphatic vessel invasion than in those without invasion respectively (P<0.01); the patients who had tumors with lymphatic vessel invasion showed a significantly poor prognosis (P<0.01). Both BrdUrd LI and G_2/MPF values were significantly higher in tumors with lymphatic node metastasis than in those without metastasis (P<0.01). A statistical significant difference was noted in the 5-year survival rates between the patients with lymph node metastasis and those without metastasis. Compared with diploid carcinoma, the incidence of lymph node metastasis was significantly higher in aneuploid carcinoma (P<0.05), and the patients with aneuploid carcinoma showed a significantly poor prognosis (P<0.05). BrdUrd LI was significantly higher in patients with more than 5 metastatic lymph nodes than those with 1-4 metastatic lymph nodes (P<0.05) and those without metastasis (P<0.01). G_2/MPF values in those patients either with more than 5 metastatic lymph nodes or 1-4 metastatic lymph nodes were higher than those without metastasis (P<0.01 and P<0.05). A statistical significance was seen in the 5-year survival rates among the patients with no metastatic lymph node, 1-4 metastatic nodes and more than 5 metastatic nodes (P<0.01). G_2/MPF values were significantly higher in patients with venous invasion than in those without invasion (P<0.01).Conclusions Positive correlations exist between cellular BrdUrd LI, G_2/MPF with lymphatic involvement and prognosis, and DNA aneuploid with lymphatic involvement and prognosis. The same was true between G_2/MPF value and venous invasion in gastric carcinoma.