PPARγ对人胰腺癌细胞中抑癌基因PTEN表达的影响

AIM: To explore the relationship between the expression of PTEN gene and the expression of PPARgamma, and the human pancreatic cancer cells PANC-1 were cultured in vitro. METHODS: The effects of rosiglitazone and GW9662 on the expression of PTEN gene and PTEN protein in the human pancreatic cancer cells PANC-1 were detected by RT-PCR and immunohistochemistry respectively. In addition, the percentage of the expression of PTEN protein was analyzed by flow cytometry. RESULTS: The expression of PTEN gene and PTEN protein in human pancreatic cancer cells PANC-1 were all increased significantly after treated with rosiglitazone. While those were markedly reduced in GW9662 treated groups, and it has a dose-effect relationship between them. CONCLUSION: The expression of PTEN gene were paralleled with the expression of PPARgammain human pancreatic cancer cells PANC-1, which may be related to its inhibitory effects on pancreatic tumor cells.     

Alu-polymerase chain reaction genomic fingerprinting technique identifies multiple genetic loci associated with pancreatic tumourigenesis

DNA fingerprinting can be used to detect genetic rearrangements in cancer that may be associated with activation of oncogenes and inactivation of tumour suppressor genes. We have developed a fingerprinting strategy based on polymerase chain reaction (PCR) amplification of genomic DNA with primers specific for the Alu repeat sequences, which are highly abundant in the human genome. This has been applied to DNA from pancreatic cancer and paired normal samples to isolate and identify fragments of genomic DNA rearranged in the malignant cells. These fragments have been sequenced and used as probes to isolate hybridising clones from gridded bacteriophage PI, phage artificial chromosome, and cosmid libraries for fluorescent in situ hybridisation mapping and the identification of expressed sequences. Further characterisation has identified a putative novel gene (ART1) that is up-regulated specifically in pancreatic cancer as well as another sequence with similarity to genes involved in differentiation (POU domains). In conclusion, we suggest that Alu-PCR fingerprinting may be a useful technique for the identification of genes involved in tumourigenesis. Genes Chromosom. Cancer 18:30–41, 1997. © 1997 Wiley-Liss, Inc.     

Sensory epithelial cells acquire features of prosensory cells via epithelial to mesenchymal transition

Epithelial to mesenchymal transition (EMT) plays a critical role during normal development and in adult tissue repair. It is known that immortalized epithelial cells can undergo an EMT and become cancer stem cells, and that epithelial cells from mouse pancreatic islet and avian inner ear can acquire mesenchymal traits in vitro via EMT. However, it is unclear whether epithelial cells from mammalian sensory system can undergo an EMT and obtain features of stem/progenitor cells. In this study, we used mouse utricle sensory epithelial cells (MUCs) as a mammalian cell model to address this issue. When cultured on 2-dimensional substrates, dissociated MUCs gradually lost their columnar shape and started to expand on the substrate with downregulation of expression of epithelial junction markers and upregulation of genes and proteins that are widely shown in mesenchymal cells. Moreover, MUCs expressed genes and proteins that are usually presented in prosensory epithelial cells and stem cells. These MUCs showed potential to differentiate into epithelial cells via a reverse EMT when they were forced to suspend in culture medium. Our findings reveal that sensory epithelial cells from mammalian tissue can undergo an EMT to become cells expressing features of stem cells that can be induced to become epithelial cells via a reverse EMT. The outcomes of this study may provide a novel approach to generate epithelial progenitors for use in cell replacement therapy to treat a number of human diseases, such as hearing loss and vision loss.     

Identification and characterization of motility stimulating factor secreted from pancreatic cancer cells: role in tumor invasion and metastasis

Cell motility is an important factor in the process of invasion and metastasis of tumor. In this study, the relationship between cell motility and experimental metastatic potential was examined using two human pancreatic cancer cell lines, SW1990 and PANC-1. Serum-free conditioned medium from the highly metastatic cell line SW1990 was found to contain a factor that stimulated the migration of and induced a fibroblast-like morphological change in the weakly metastatic cell line PANC-1. Preincubation of PANC-1 cells with SW1990 conditioned medium (SW-C.M.) induced liver metastasis following splenic injection of PANC-1 cells in nude mice, although no liver metastasis was observed without pretreatment of SW-C.M. This factor, temporarily termed PDMF (pancreatic cancer-derived motility factor) is a heparin non-binding protein having a molecular weight of 40 kDa calculated by gel-filtration HPLC which acts not only chemotactically but also chemokinetically, and also acts mainly in a paracrine fashion. However, this factor had no effect on the proliferation of PANC-1 cells; it therefore appears to be a so-called motility factor. Only TGF-b1 and IL-6 were recognized in the SW-C.M. among cytokines thought to stimulate cell motility. These cytokines stimulated the motility of PANC-1 cells, but differed from PDMF in the neutralizing test with antibody against these cytokines. Results of characterization and preliminary purification suggest that this factor may be a novel motility factor. The above findings suggest that this motility factor may play an important role in the invasion and metastasis of pancreatic cancer, and complete purification of it will be useful in elucidating the mechanism of progression of cancer and designing a strategy for inhibition of invasion and metastasis of pancreatic cancer.     

Role of Cripto-1 during epithelial-to-mesenchymal transition in development and cancer

Epithelial-to-mesenchymal transition (EMT) is a critical multistep process that converts epithelial cells to more motile and invasive mesenchymal cells, contributing to body patterning and morphogenesis during embryonic development. In addition, both epithelial plasticity and increased motility and invasiveness are essential for the branching morphogenesis that occurs during development of the mammary gland and during tumor formation, allowing cancer cells to escape from the primary tumor. Cripto-1, a member of the epidermal growth factor-Cripto-1/FRL-1/Cryptic (EGF/CFC) gene family, together with the transforming growth factor (TGF)-β family ligand Nodal, regulates both cell movement and EMT during embryonic development. During postnatal development, Cripto-1 regulates the branching morphogenesis of the mouse mammary gland and enhances both the invasive and migratory properties of mammary epithelial cells in vitro. Furthermore, transgenic mouse models have shown that Cripto-1 promotes the formation of mammary tumors that display properties of EMT, including the down-regulation of the cell surface adherens junctional protein E-cadherin and the up-regulation of mesenchymal markers, such as vimentin, N-cadherin, and Snail. Interestingly, Cripto-1 is enriched in a subpopulation of embryonal, melanoma, prostate, and pancreatic cancer cells that possess stem-like characteristics. Therefore, Cripto-1 may play a role during developmental EMT, and it may also be involved in the reprogramming of differentiated tumor cells into cancer stem cells through the induction of an EMT program.     

Increased expression of matrix metalloproteinases-21 and -26 and TIMP-4 in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma is known for early aggressive local invasion, high metastatic potential, and a low 5-year survival rate. Matrix metalloproteinases (MMPs) play important roles in tumor growth and invasion. Earlier studies on pancreatic cancer have found increased expression of certain MMPs to correlate with poorer prognosis, short survival time or presence of metastases. We studied the expression of MMP-21, -26, and tissue inhibitor of matrix metalloproteinases (TIMP)-4 in 50 tissue samples, including 25 adenocarcinomas, seven other malignant pancreatic tumors, and 18 control samples of non-neoplastic pancreatic tissue with immunohistochemistry. The regulation of MMP-21, -26, and TIMP-4 mRNAs by cytokines was studied with RT-PCR in pancreatic cancer cell lines PANC-1, BxPC-3, and AsPC-1. MMP-21, -26, and TIMP-4 were detected in cancer cells in 64, 40, and 60% of tumors, respectively. MMP-21 expressed in well-differentiated cancer cells and occasional fibroblasts, like TIMP-4, tended to diminish in intensity from grade I to grade III tumors. Patients with metastatic lymph nodes had increased expression of MMP-26 in actual tumor samples. All cultured cancer cell lines expressed MMP-21 basally at low levels, and presence of the protein was confirmed immunohistochemically in cultured cells. MMP-21 expression was induced by epidermal growth factor (EGF) in PANC-1 cells. MMP-26 was neither expressed basally nor induced by tumor necrosis factor alpha, transforming growth factor beta-1 (TGFbeta1), EGF, or interferon gamma. Basal TIMP-4 expression was lowest in the poorly differentiated cancer cell line PANC-1 compared to better-differentiated BxPC-3 and AsPC-1 cells. TIMP-4 expression was induced by TGFbeta1 in PANC-1 cells and by EGF in BxPC-3 cells. Our findings suggest that MMP-21 is not a marker of invasiveness, but rather of differentiation, in pancreatic cancer and it may be upregulated by EGF. The putative role of MMP-26 as a marker of metastases warrants further studies. Unlike other TIMPs, TIMP-4 was not upregulated in relation to aggressiveness of pancreatic cancer.     

反义miR-196a对人胰腺癌PANC-1细胞侵袭和迁移的抑制作用

目的:探讨微小RNA-196a(miR-196a)在胰腺癌细胞系中表达状况及反义miR-196a对人胰腺癌PANC-1细胞生物学行为的影响。方法:miR-196a采用实时荧光定量PCR检测。人工化学合成反义miR-196a并转染PANC-1细胞。CCK-8法检测细胞增殖。流式细胞术检测细胞凋亡情况。通过划痕损伤实验和Transwell小室细胞侵袭、迁移实验检测PANC-1细胞侵袭和迁移能力。构建野生型及突变型核因子κB抑制蛋白α(NFKBIA)3'UTR的萤光素酶报告载体,检测海肾萤光素酶的相对活性以验证miR-196a对NFKBIAmRNA的作用靶点。结果:人胰腺癌细胞系中miR-196a呈高表达;转染反义miR-196a后,PANC-1细胞miR-196a表达下降,其细胞增殖和凋亡无显著改变(P>0.05);而转染反义miR-196a组中通过Transwell小室的细胞数明显低于各对照组(P<0.01)。与各对照组相比,共转染野生型NFKBIA的3'UTR和反义miR-196a可使海肾萤光素酶相对活性显著提高。结论:miR-196a可能是癌微RNA(oncomiR)之一,有望成为人胰腺癌基因治疗的候选靶点。     

转录因子HOXA5在乳腺癌细胞中的下游信号通路

目的 探究转录因子HOXA5在乳腺癌进展中的作用。方法 构建过表达HOXA5的稳定转染BT549和SUM159细胞系, 在稳转BT549细胞系中利用Transwell检测细胞的转移能力;Western blotting检测上皮-间质转化（EMT）相关蛋白表达水平的变化;平板克隆实验检测细胞的增殖能力;转录组测序技术（RNA-seq）进一步分析HOXA5的调控基因,利用软件Cluster 3.0,Tree View和DAVID生物信息数据库（DAVID Bioinformatics Resources）以及Enrichr分析京都基因与基因组百科全书（KEGG）信号通路,绘制热图（heatmap）。 结果 BT549细胞系中,稳定转染HOXA5后的实验组细胞迁移能力显著降低(P<0.001),且上皮标志物 E-cadherin蛋白表达水平显著上调,间质标志物 N-cadherin, Twist1、Slug蛋白表达水平显著下调;平板克隆结果表明,实验组形成的克隆数目明显减少(P<0.05),克隆大小明显降低。 结论 HOXA5通过促进细胞由间质向上皮转化,抑制乳腺癌细胞迁移,并且抑制细胞增殖;HOXA5通过调节糖脂代谢途径调节癌细胞增殖,并且还通过调节细胞运动和黏附相关的基因抑制肿瘤细胞的迁移,通过肿瘤坏死因子（TNF）信号通路发挥抗肿瘤作用。总之,转录因子HOXA5对乳腺癌的发展和恶化起着显著的抑制作用。     

Analysis of genomic DNA alterations and mRNA expression patterns in a panel of human pancreatic cancer cell lines

Genomic alterations influencing the expression and/or activity of tumor suppressors or oncogenes such as KRAS2, CDKN2A, TP53, and DPC4 have been directly implicated in the initiation and progression of human pancreatic adenocarcinoma. In an effort further to systematically characterize the genomic alterations that occur in this disease, we conducted a genome wide analysis of alterations in gene copy number using array-based comparative genomic hybridization (CGH). For this analysis, we utilized a panel of 25 human pancreatic cancer cell lines derived from either primary or metastatic tumors. This panel also included a metastatic progression series of cell lines derived from COLO 357 cells. Array CGH permitted the identification of alterations in the copy number of genes that might participate in the aberrant behavior of pancreatic cancer cells. In addition, the acquisition of invasive and metastatic potential by derivatives of COLO 357 cells was accompanied by additional focal genomic alterations including point mutations and amplification of KRAS2. To complement the array CGH analysis, we also conducted an analysis of mRNA expression patterns in a subset of these cells using cDNA microarrays. By this means, we identified a set of candidate genes, including those regulated by RAS signaling, that may contribute to the process of cancer cell invasion and metastasis. Supplementary material for this article can be found on the Genes, Chromosomes, and Cancer website at http://www.interscience.wiley.com/jpages/1045-2257/suppmat/index.html.     

Mapping of the porcine urate oxidase and transforming growth factor beta 2 genes by fluorescencein situ hybridization

We have mapped two genes from human chromosome 1, urate oxidase (UOX) and transforming growth factor beta 2 (TGFB2), by fluorescencein situ hybridization (FISH) in the pig genome. Porcine-specific polymerase chain reaction (PCR) primers for both genes were designed from the porcine cDNA sequence. With the help of these primers yeast artificial chromosome (YAC) clones forUOX andTGFB2 were isolated from a pig YAC library. These DNA probes were used for FISH analysis.TGFB2 was localized to SSC 10p16. With the YAC probe forUOX two porcine chromosome regions 6q26 and 6q32, revealed specific signals. These results, help to refine the comparative mapping data between human and pig.accepted for publication by H. Schmidt     

Pancreas microenvironment promotes VEGF expression and tumor growth: novel window models for pancreatic tumor angiogenesis and microcirculation

Pancreatic cancer has a poor prognosis, and treatment strategies based on preclinical research have not succeeded in significantly extending patient survival. This failure likely stems from the general lack of information on pancreatic tumor physiology, attributable to the difficulties in developing relevant, orthotopic models that accurately reflect pancreatic cancer in the clinic. To overcome this limitation, we developed abdominal wall windows suitable for intravital microscopy that allowed us to monitor angiogenesis and microvascular function noninvasively during tumor growth in vivo. We used two complementary tumor models in mice: orthotopic (human ductal pancreatic adenocarcinoma, PANC-1, grown in the pancreas), and ectopic (PANC-1 grown in the abdominal wall). We found that orthotopic PANC-1 tumors grew faster than the ectopic tumors and exhibited metastatic spread in the late stage similar to advanced pancreatic cancer in the clinic. Orthotopic PANC-1 tumors expressed vascular endothelial growth factor (VEGF)(121) and VEGF(165), contained higher levels of tumor cell-derived VEGF protein, and maintained vascular density and hyperpermeability during exponential tumor growth. Orthotopic PANC-1 tumors showed lower leukocyte-endothelial interactions in the early stage of growth. In addition, both VEGF(121) and VEGF(165) promoted the growth of PANC-1 cells in vitro. Finally, Anti-VEGF neutralizing antibody inhibited angiogenesis and tumor growth of PANC-1 tumors in both sites. We conclude that the orthotopic pancreas microenvironment enhances VEGF expression, which stimulates growth of PANC-1 tumors (compared with ectopic tumors). The mechanism is autocrine and/or paracrine and also is involved in the maintenance of blood vessels. This comparative system of orthotopic and ectopic pancreatic cancer will provide the rigorous understanding of pancreatic tumor pathophysiology needed for development of novel therapeutic strategies.     

百里醌对血管生成及胰腺癌生长的影响

目的: 探讨百里醌对血管生成和胰腺癌生长的影响及其机制。方法: 采用贴壁选择法培养人脐血内皮祖细胞(EPCs),细胞免疫组化检验细胞中血管内皮生长因子受体-2(VEGFR-2)、VIII因子和 CD34表达,证实细胞属性;观察不同浓度(10 nmol/L、20 nmol/L和40 nmol/L)百里醌对EPCs小管形成的影响;不同浓度(20 μmol/L、40 μmol/L和80 μmol/L)百里醌作用人胰腺癌细胞株PANC-1后,Western blotting检测胰腺癌细胞中血管内皮生长因子(VEGF)表达的变化;建立裸鼠胰腺癌原位移植瘤模型,分成对照组和百里醌组,实验结束后观察百里醌对裸鼠胰腺癌生长的抑制作用;免疫组织化学法检测裸鼠胰腺肿瘤组织中Ki-67、CD34和VEGF的阳性表达。结果: 体外成功培养出人脐血EPCs,百里醌可显著抑制体外EPCs小管形成;并抑制体外人胰腺癌PANC-1细胞中VEGF的表达;与对照组相比较,百里醌可明显抑制荷瘤裸鼠中胰腺肿瘤生长,并下调Ki-67、CD34和VEGF在胰腺肿瘤组织中的阳性表达。结论: 百里醌可抑制体内外血管生长,有望作为治疗胰腺癌的血管抑制药物。     

Claudin-1 expression is induced by tumor necrosis factor-alpha in human pancreatic cancer cells

Claudin-1 is a membrane protein with four transmembrane domains, that is exclusively localized at cellular tight junctions. Recent studies have reported that claudin-1 plays an important role in cancer invasion and metastasis. However, the significance of claudin-1 in pancreatic cancer is still unknown. In the present study, we investigated the role of claudin-1 expression in pancreatic cancer growth using the PANC-1 human pancreatic cancer cell line. Treatment with tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, resulted in increased detection of 89 kDa products of poly-(ADP-ribose) polymerase (PARP), a marker of apoptosis, and decreased PANC-1 cell proliferation by 23%. Expression of claudin-1 was up-regulated by TNF-alpha in a concentration-dependent manner in PANC-1 cells. PANC-1 cells treated with TNF-alpha and siRNA against claudin-1 showed a 15% increase in proliferation; i.e. the cells transfected with siRNA against claudin-1 showed resistance to TNF-alpha-induced apoptosis. These results suggest that claudin-1 expression is responsible for TNF-alpha-dependent growth signals and the proliferation of pancreatic cancer cells.     

Cloning of TPA-inducible early (TIE) genes by differential hybridization using TPA-Nonresponsive variant of mouse 3T3-L1 cells

The tumor promoter 12-O -tetradecanoylphorbol-13-acetate (TPA) induces DNA synthesis in quiescent 3T3-L1 cells but not in its variant VT-1 cells. A gt10 cDNA library was constructed using poly(A)⁺ RNA from 3T3-L1 cells that were stimulated by TPA for 20 min. Radioactive cDNA probes were prepared from mRNAs of TPA-treated 3 T3-L1 and VT-1 cells and used for screening of the 3T3-L1 cDNA library by differential hybridization. Nine of 6000 phage plaques hybridized only to the 3T3-L1 cDNA probe. Analysis of the nucleotide sequence of five of these clones indicated a high degree of homology with human or mouse type I and type III collagen genes. Three other independent clones showed no homology with any known DNA sequences. These isolated clones of TPA-inducible early (TIE) genes may be useful to study the signal transduction pathway of phorbol esters.