Pro-inflammatory type-1 and anti-inflammatory type-2 macrophages differentially modulate cell survival and invasion of human bladder carcinoma T24 cells

Findings from numerous studies suggest that inflammation is likely to have an important role in bladder carcinogenesis and cancer disease progression. While macrophages (M?s) constitute a major inflammatory component of the stroma of human bladder carcinoma, the regulatory role of such inflammatory leukocytes in tumor cell survival and invasion remains elusive. Human urothelial bladder cancer (UBC) T24 cells and monocyte-derived macrophages were used to study the relative contribution of pro-inflammatory type-1 (M?-1) and anti-inflammatory type-2 (M?-2) macrophages in the regulation of UBC cell behaviour. Cell-to-cell studies indicated that the number of viable cells were considerable higher in T24 cell/M?-2 cocultures but lower in T24 cell/M?-1 cocultures when compared to cultures of T24 cells alone. M?-1-derived factors inhibit T24 cell growth but fail to induce caspase-3-mediated apoptosis. M?-2-derived factors have the ability to suppress the inhibitory effect of M?-1-derived factors on T24 cell growth. Exogenous interleukin (IL)-10 reverse M?-1-mediated arrest growth in T24 cell/M?-1 cell cocultures. Further analyses showed that M?-1-derived factors induced tumor necrosis factor (TNF)-α gene expression, promoted cellular invasiveness and increased phosphoinositide 3-kinase (PI 3-K)/Akt signaling pathway activity in T24 cells. Inhibition of PI 3-K activation in T24 cells or blockade of TNFα receptor in T24 cell/M?-1 cell cocultures decreased cellular invasiveness but did not affect T24 cell viability. Based on these observations, we propose that similar functional interactions between UBC cells and infiltrating macrophages can take place in vivo and influence tumor cell survival and invasion during bladder cancer progression.     

Cytoplasmic Redistribution of E-Cadherin-Catenin Adhesion Complex Is Associated with Down-Regulated Tyrosine Phosphorylation of E-Cadherin in Human Bronchopulmonary Carcinomas

The E-cadherin-catenin complex, by mediating intercellular adhesion, regulates the architectural integrity of epithelia. Down-regulation of its expression is thought to contribute to invasion of carcinoma cells. To investigate the involvement of the E-cadherin-catenin adhesion system in the progression of human bronchopulmonary carcinomas, we compared the immunohistochemical distribution of E-cadherin, α-catenin, and β-catenin in four human bronchial cancer cell lines with different invasive abilities and in 44 primary bronchopulmonary tumors. Although invasive bronchial cell lines did not express E-cadherin and α-catenin, complete down-regulation of cadherin-catenin complex expression was a rare event in vivo in bronchopulmonary carcinomas. Nevertheless, a spotty and cytoplasmic pattern of E-cadherin and catenins was observed in 32 primary tumors, only in invasive tumor clusters. Immunoprecipitation experiments showed that this redistribution was not related to a disruption of cadherin-catenin interaction but to down-regulated tyrosine phosphorylation of E-cadherin. We conclude that loss of E-cadherin and/or catenins is not a prominent early event in the invasive progression of human bronchopulmonary carcinomas in vivo. The decreased tyrosine phosphorylation of E-cadherin may reflect a loss of functionality of the complex and implicates a major role in tumor invasion.     

Genetic and phenotypic changes associated with the acquisition of tumorigenicity in human bladder cancer

There has been a general lack of human paired cell lines that both reproduce the in vivo spectrum of tumor progression of bladder cancer and have some of the genetic changes associated with progression in human tumor tissue. T24, a cell line established from an invasive human transitional cell carcinoma (TCC) of the bladder, has been used extensively in bladder cancer research. However, a significant limitation of this cell line is its lack of tumorigenicity when injected into immunocompromised mice. This characteristic was used to our advantage as we sought to characterize T24T, a highly tumorigenic variant that could then be used to elucidate the genes responsible for human bladder tumor progression. In culture, T24T has a faster doubling time, reaches a higher cell density in monolayer culture, and is more motile than T24 at higher cell densities. T24T is able to form colonies in soft agar, whereas T24 is not, and expresses HRAS, a gene associated with increased aggressiveness in human TCC, at higher levels than T24. Most importantly, T24T forms solid tumors when injected subcutaneously in SCID mice both with and without Matrigel (Sigma, St. Louis, MO), whereas T24 does not. Cytogenetically, the 2 cell lines contain at least 5 shared structural anomalies, as determined by detailed karyotyping. Interestingly, T24T has acquired 4 new structural changes, 3 of which [add(10)(p12), i(10)(q10), -15] have been observed in loss of heterozygosity (LOH) studies of tumor progression in human TCC. It appears that the T24/T24T model may be an excellent tool for the study of human TCC progression because of its relationship with known karyotypic changes associated with human bladder cancer progression. We are currently taking advantage of these paired cell lines to identify genes involved in human TCC progression. Genes Chromosomes Cancer 27:252-263, 2000.     

In vitro invasiveness of human bladder cancer from cell lines and biopsy specimens

Aggregates prepared from cell lines established from a human transitional cell carcinoma of the urothelium (Hu 456) or from apparently normal urothelium before (Hu 609) and after phenotypic transformation (Hu 609T) were confronted with fragments of embryonic chick cardiac muscle in organ culture. In this assay a correlation was found between in vitro invasiveness of animal cell lines and their capacity to produce invasive tumours in syngeneic animals [1, 10, 11]. The invasiveness of cells from established human urothelial lines was compared to the invasiveness of cells from fresh biopsy specimens of a normal urothelium, a non-invasive papilloma, and a metastasizing transitional cell carcinoma. Cells from all established lines (Hu 609, Hu 609T and HU 456) and from the biopsy specimens of the transitional cell carcinoma occupied and eventually replaced the cardiac muscle by contrast with cells from the normal urothelium or from the non-invasive papilloma. We concluded that the organ culture assay for invasiveness might be used to define malignancy of human bladder cell lines and to follow the various steps during the acquisition of invasiveness in vitro.     

肝癌衍生生长因子与膀胱癌转移的相关性及其对细胞侵袭能力影响

目的探讨肝癌衍生生长因子(HDGF)基因与膀胱癌转移的相关性,及其稳定表达对膀胱癌细胞侵袭能力的影响。方法应用荧光定量PCR检测原发未转移膀胱癌和原发伴转移膀胱癌组织中HDGF mRNA的表达。设计并合成HDGF特异性的siRNA,转染膀胱癌T24细胞系,检测转染后细胞侵袭能力的变化。结果原发伴转移膀胱癌组织中HDGF mRNA表达较原发未转移膀胱癌中明显上调(P0.05)。RNA干扰后,HDGF蛋白明显降低,细胞侵袭能力也随之明显降低(P0.05)。结论 HDGF基因与膀胱癌的转移相关,其表达下调能够抑制T24细胞的侵袭能力。     

Periostin表达对鼻咽癌细胞体外侵袭行为的影响

探讨Periostin表达对体外鼻咽癌（NPC）细胞侵袭行为的影响及其机制。 方法 采用Western blotting检测激光捕获显微切割纯化的鼻咽癌间质和正常鼻咽黏膜间质、鼻咽癌低转移潜能细胞系6-10B和高转移潜能细胞系5-8F及NIH 3T3成纤维细胞中Periostin的表达水平。应用阳性脂质体法将pCMV-neo-Periostin质粒瞬时转染到NIH 3T3细胞,运用Western blotting检测转染前后NIH 3T3细胞中Periostin的表达水平,利用Boyden小室穿膜侵袭实验检测Periostin蛋白对肿瘤细胞的体外侵袭能力的影响,明胶酶谱法检测其对基质金属蛋白酶（MMPs）活性的影响。 结果 Periostin在纯化的鼻咽癌间质中高表达,在纯化的正常鼻咽黏膜间质中及高转移细胞系5-8F中弱表达,在低转移细胞系6-10B及NIH 3T3细胞中不表达。瞬时转染过表达Periostin的NIH 3T3细胞中Periostin的表达明显增强。Boyden小室实验提示,分泌蛋白Periostin可明显增强鼻咽癌细胞6-10B的体外侵袭能力,转染组穿膜的细胞数与空白对照组及未转染对照组比较,明显增多(P<0.01);转染组与6-10B细胞共培养后,培养上清中MMPs的活性较对照组明显增强。 结论 Periostin 蛋白能增强鼻咽癌细胞的侵袭能力,其机制可能与上调MMPs的活性有关。     

Invasion by esophageal cancer cells: functional contribution of the urokinase plasminogen activation system, and inhibition by antisense oligonucleotides to urokinase or urokinase receptor

Early metastasis contributes to the very poor prognosis of esophageal carcinoma. The recent immunohistochemical finding that invasive esophageal carcinomas express elevated levels of urokinase (uPA) and urokinase receptor (uPA-R) in vivo suggest that the plasminogen activation system may contribute to metastasis in esophageal cancer. The aim of our study was to functionally investigate, at the molecular level, the relative contribution of uPA and uPA-R to the invasiveness of esophageal cancer cells in vitro. The three esophageal cancer cell lines, OC1-3, generated in our laboratory, were analyzed for uPA and uPA-R expression by RT-PCR, immunoenzymatic staining, and quantitative ELISA. Invasiveness of all cell lines was quantified as percentage cellular invasiveness in a standardized Matrigel in vitro assay. OC1 and OC3, which were found to coexpress both uPA and uPA-R, displayed stronger invasiveness (44% and 32.5% respectively) relative to OC2 (19%) which expressed uPA-R but was negative for uPA. Transfection of OC2 cells with the uPA cDNA resulted in two variants, OC2.uPA1 and OC2.uPA2, stably expressing functional uPA. Both transfectants exhibited enhanced invasiveness (60% and 50% respectively) relative to the parent uPA-negative OC2 cells (19%). Antisense oligonucleotide inhibition of either uPA or uPA-R expression resulted in a similar, marked reduction in invasiveness of esophageal tumor cells which normally coexpress both molecules (OC1, OC3 and the uPA-expressing OC2-transfectant clones). Neither antisense treatment altered the basal invasiveness of OC2, which expresses uPA-R but not uPA. In conclusion, coexpression of uPA with its receptor, uPA-R, is required for functional involvement of the urokinase system in invasion by esophageal carcinoma cells. Our results suggest that these synergistic mediators of invasiveness are quantitatively major contributors to the invasiveness of esophageal carcinoma.     

High expression of Notch ligand Jagged2 is associated with the metastasis and recurrence in urothelial carcinoma of bladder

Background: The Notch signaling pathway is closely related with human organ development and tumorgenisis. Jagged2 is among the most popular topic in Notch studies currently. Recent studies found its vital role in tumor metastasis in breast cancer; however, its expression profile and its prognostic value in urothelial carcinoma of bladder have not been investigated. Methods: Immunohistochemistry was used to detect the expression of Jagged2 in 120 bladder urothelial carcinoma. Moreover, the expression of Jagged2 was analyzed by Western blot in 60 bladder urothelial carcinoma and 20 normal epithelial tissues. MTT assay and flow cytometry and transwell assay were used to examine the proliferative and invasive ability of bladder cancer cells with the treatment of GSIXX (the inhibitor of Jagged2). Prognostic value of Jagged2 expression and its correlation with tumor metastasis and recurrence were evaluated, and the proliferative and invasive ability and cell cycle process of the bladder cancer cells were detected as well. Results: There was a significantly higher Jagged2 expressions in bladder urothelial carcinoma and highly invasive bladder T24 cells than those in bladder normal tissues and the superficial bladder BIU-87 cells. Jagged2 expression was positively correlated with histological grade, p T stage, recurrence, and metastasis. With the increasing concentration of GSIXX, we found that not only the cell proliferation and invasion activity decreased significantly, but also the cell cycle was blocked at G₂/M stage. Conclusions: Jagged2 expression status was closely correlated with important histopathologic characteristics (grades and stages) and the recurrence and metastasis of bladder urothelial carcinomas. Furthermore, Jagged2 played an important function on the bladder cancer cells’ proliferation by regulating the cancer cell cycle from G₁/S to G₂/M and probably promoted the invasion and metastasis of bladder cancer.     

组织金属蛋白酶抑制剂TIMP—2基因转染对胃癌细胞体内外侵袭能 …

了解ＴＩＭＰ－２基因转地胃癌细胞侵袭行为的影响，进行阻断肿瘤细胞侵转移的筛选尝试。     

Down-regulating ribonuclease inhibitor enhances metastasis of bladder cancer cells through regulating epithelial–mesenchymal transition and ILK signaling pathway

Accumulating evidences implicate that ribonuclease inhibitor (RI) plays a suppressing role in cancer development. However, the mechanisms underlying antitumor of RI remain largely unknown. Epithelial–mesenchymal transition (EMT) is regarded as a key event in tumor progression. The reports have demonstrated that EMT was implicated in metastasis of bladder cancer. Therefore, we suppose that RI might involve regulating EMT of bladder cancer. Here bladder cancer T24 cells were transfected with pGensil-1-siRNA-RI vectors. HE staining, living cell observation, Phalloidine-FITC staining of microfilament, cell adhesion, scratch migration, and Matrigel invasion were examined respectively. RI expression and colocalization with ILK were detected using confocal microscope. Proteins associated with EMT were determined with Western blotting and immunohistochemistry in vivo and in vitro. Effects of RI expression on tumor growth, metastasis and EMT related proteins in BALB/C nude mouse and clinical human bladder cancer specimens were valued with histological, immunohistochemical and immunofluorescent examination respectively. We demonstrated that down-regulating RI increased cell proliferation, migration and invasion, changed cell morphology and adhesion, and rearranged cytoskeleton by inducing EMT and ILK signaling pathway in bladder cancer cells. In addition, we showed that down-regulating RI promoted tumorigenesis and metastasis of bladder cancer in vivo. Finally, we found that bladder cancer with invasive capability had higher Vimentin, Snail, Slug and Twist as well as lower E-cadherin and RI expression in clinical human specimens. Our results suggest that RI could play a novel role in inhibiting metastasis of bladder through regulating EMT and ILK signaling pathway.     

Expression and function of SIRT6 in muscle invasive urothelial carcinoma of the bladder

SIRT6, a member of the class III histone deacetylase, has been shown to inhibit glycolysis and promote DNA double strand break repairs. Despite of its proposed tumor suppressor role, no significant differences in SIRT6 mRNA levels among normal bladder urothelium, non-muscle invasive, and muscle invasive urothelial carcinoma were noted in the two largest bladder cancer gene expression datasets available in Oncomine^TM. We therefore studied the expression and function of SIRT6 in muscle invasive urothelial carcinoma of the bladder. Immunohistochemistry studies of SIRT6 on radical cystectomy samples showed a dramatic decline of SIRT6 expression when bladder cancer progressed from T2 to T4. Functional study with bladder cancer cell lines confirmed its role in inhibiting glycolysis and cell proliferation. Reducing SIRT6 with siRNA, however, did not sensitize bladder cancer cells to drug induced DNA damage. The differential expression patterns of SIRT6 amongst different T stages of muscle invasive bladder cancers indicate less reliance on glycolysis when urothelial carcinoma invades deeper through the bladder and into the adjacent tissues.     

N-Cadherin Expression in Human Prostate Carcinoma Cell Lines : An Epithelial-Mesenchymal Transformation Mediating Adhesion with Stromal Cells

In human prostate adenocarcinoma, an association between loss of E-cadherin, increased Gleason score, and extracapsular dissemination has been observed. Further characterization of the E-cadherin/catenin phenotype of human prostate carcinoma cell lines showed loss of E-cadherin and expression of N-cadherin in poorly differentiated prostate carcinoma cell lines (PC-3N derived from PC-3, PC-3, and JCA1). We showed that N-cadherin is concentrated at sites of cell-cell contact in PC-3N cellular extensions. N-cadherin was also expressed in prostate stromal fibroblasts both in vitro and in prostate tissue. Co-cultures of prostate stromal fibroblasts and PC-3N cells showed the immunolocalization of N-cadherin in intercellular contacts. In addition, the isoform expression of the cadherin binding protein p120(ctn) differed in relation to the expression of E- versus N-cadherin by the prostate carcinoma cell lines. The p100 isoform was more highly expressed in E-cadherin-positive carcinoma cell lines, whereas p120 was predominantly expressed only in N-cadherin-positive prostate carcinoma cell lines and prostate stromal fibroblasts. The N-cadherin-positive carcinoma cell line, PC-3N, displayed aggressive invasion into the surface of the diaphragm muscle after intraperitoneal injection of SCID mice. The gain of N-cadherin and loss of E-cadherin by invasive prostate carcinoma cell lines suggests a progression from an epithelial to a mesenchymal phenotype, which may allow for their interaction with surrounding stromal fibroblasts and facilitate metastasis.     

Fascin stain as a potential marker of invasiveness in carcinomas of the urinary bladder: a retrospective study with biopsy and cytology correlation

The evaluation of invasion in urothelial carcinomas of the urinary bladder cannot be determined on cytology and can be particularly challenging in biopsy cases with limited sampling. Recent studies of bladder resection specimens suggest that fascin overexpression may be a marker of aggressive urothelial carcinomas and can help facilitate the assessment of invasion. In this study, we evaluated urine cytology and corresponding biopsy specimens with proven invasive urothelial carcinoma for fascin expression by immunohistochemistry. Thirty‐five patients diagnosed with positive urine cytology and biopsy‐proven invasive urothelial carcinoma between January 2003 and February 2009 were identified. We found increased fascin expression in 100% (35/35) of SurePathTM&!trade; urine cytology preparations as well as 100% (35/35) of corresponding biopsy cases with invasive urothelial carcinoma. On urine cytology, cytoplasmic fascin staining was moderate to intense in malignant tumor cell clusters and single cells and not observed in benign urothelial cells. Staining in biopsy cases was generally intense and cytoplasmic and present in both the invasive (100%) and noninvasive (31%) components of the lesion. These findings uphold the association of increased fascin expression in invasive urothelial carcinomas of the urinary bladder. We furthermore demonstrate that fascin staining can be performed successfully on SurePathTM&!trade; urine cytology preparations in which increased fascin expression correlates with invasion on biopsy. While not a definitive marker of invasion, as it is observed in in situ carcinoma, we conclude that the utilization of fascin immunohistochemistry on urine cytology might serve as a useful adjunct in predicting invasiveness in subsequent biopsies. Diagn. Cytopathol. 2010. © 2010 Wiley‐Liss, Inc.     

An in vitro cell invasion assay: Determination of cell surface proteolytic activity that degrades extracellular matrix

Summary A correlative morphological and biochemical method is described that facilitates assaying the invasive phenotype of tumor cells in vitro. The method involves a labeled matrix substrata to measure the activity of cell surface proteases, and uses the covalent linkage of fluorescence-labeled fibronectin (or other purified extracellular matrix components) to the surface of crosslinked gelatin substrata. We have investigated the invasiveness of tissue culture cells based on two criteria: the ability of the cells to form invadopodia and create surface indentations into crosslinked gelatin films that can be visualized by differential interference contrast microscopy and transmission electron microscopy, as well as their ability to locally degrade fibronectin or other matrix components coated on loosely crosslinked gelatin films by fluorescence microscopy. In using this method, we have demonstrated the existence of cell surface proteases that may be important in the invasion of cells into the extracellular matrix. Also, we have identified various cell types including embryonic fibroblasts transformed by Rous sarcoma virus, malignant human melanoma cell lines, breast carcinoma cell lines, neural microglia, and neural crest cells that are invasive in culture.     

Phosphorylation of ephrin-B1 regulates dissemination of gastric scirrhous carcinoma

Interaction of the Eph family of receptor protein tyrosine kinase and its ligand ephrin family induces bidirectional signaling via cell-cell contacts. High expression of B-type ephrin is frequently found in various cancer cells, and their expression levels are associated with high invasion of tumors and poor prognosis. However, whether ephrin-B1 actually promotes invasion of cancer cells in vivo has not been shown. We investigated the involvement of ephrin-B1 in regulating the invasiveness of scirrhous gastric cancer, which is a diffusely infiltrative carcinoma with high invasion potential. Reduction of ephrin-B1 expression by short inter-fering RNA or overexpression of phosphorylation-defective mutant suppressed migration and invasion of scirrhous gastric cancer cells in vitro without affecting tumor cell proliferation and apoptosis. Blocking of tyrosine phosphorylation of ephrin-B1 attenuates not only dissemination of cancer cells injected intraperitoneally but also local invasion and dissemination of orthotopically implanted cancer cells in the gastric wall of nude mice. Furthermore, blocking of ephrin-B1 phosphorylation attenuated the activation of Rac1 GTPase in these invasive gastric cancer cells. Our results suggest that tyrosine phosphorylation of ephrin-B1 promotes invasion of cancer cells in vivo and is a potential therapeutic target in some types of gastrointestinal cancers.     

Exposure to hypoxia, glucose starvation and acidosis: effect on invasive capacity of murine tumor cells and correlation with cathepsin (L + B) secretion

Cells in tumors may be exposed to adverse conditions such as nutrient deprivation, acidic pH and hypoxia. It has been shown previously that exposure to hypoxia, acidosis and glucose starvation in vitro increases the experimental metastatic ability of murine KHT-LP1 sarcoma, SCC-VII squamous carcinoma and B16 melanoma cells. This effect was most marked when cells were allowed to recover under normal in vitro growth conditions before injection. In the present study we examined whether the invasive capacity of the cells could be influenced by these modifications of the cell microenvironment. We used Matrigel, a basement membrane-like preparation in a two-chamber invasion assay to address this issue. Both KHT-LP1 and SCC-VII murine cell lines showed an increased ability to invade through Matrigel after hypoxia, and glucose starvation, but there was no consistent change in invasive capacity following acidosis exposure. The results for hypoxia and glucose starvation are in agreement with our previous studies of metastatic ability for these cell lines and we confirmed this for KHT-LP1 cells exposed to hypoxia in the current study. In parallel with the invasion assays, we compared cathepsin (L + B) content of the cells in treated and control suspensions. The effect observed varied according to the cell line and the treatment received (hypoxia, glucose starvation). There was an increase of cathepsin content for KHT-LP1 cells exposed to hypoxia and this increase correlated well with the increase of the invasion ability through Matrigel. We did not observe any increase of cathepsin for hypoxia-treated SCC-VII or for KHT-LP1 and SCC-VII cells treated with glucose starvation. These results suggest that transient hypoxia and glucose starvation can increase the invasive ability of tumor cell lines and thus may cause tumor progression by facilitating the invasive step of the metastatic process. The increased levels of cathepsin (L + B) in the KHT-LP1 cells treated with hypoxia, compared to control non-treated cells, may play a part in this increased invasive capacity.     

ETS-1 proto-oncogene as a key newcomer molecule to predict invasiveness in laryngeal carcinoma

ETS-1 protein is one of the key regulators in tumor invasion and progression. We aimed to evaluate the role of ETS-1 in the invasiveness and progression of laryngeal squamous carcinoma, as well as to determine the correlations between clinicopathological characteristics and expression of this molecule.We assessed the levels of ETS-1 in a total of 96 laryngeal specimens of varying degrees of dysplasia, microinvasive squamous carcinoma (8), and invasive squamous carcinoma (60), using normal mucosal epithelium (10) as a positive control. The relationship between ETS-1 expression and clinicopathological parameters of laryngeal carcinoma was also analyzed.We found a significantly higher ETS-1 expression in invasive laryngeal squamous cell carcinomas than in dysplasia (P < 0.001). A correlation between ETS-1 expression scores and grade was detected - T factor, stage, cartilage invasion, lymph node metastasis, as well as depth of invasion in laryngeal tumors.Our study is the first to demonstrate that ETS-1 expression is significantly increased in invasive carcinoma, but it is absent in low-moderate grade laryngeal dysplasia and non-neoplastic laryngeal mucosa. This data suggest that ETS-1 expression may play an important role in tumor invasion, and may function in the initiation of the invasive process in laryngeal squamous cell carcinoma.