Secretory leukocyte protease inhibitor is associated with MMP-2 and MMP-9 to promote migration and invasion in SNU638 gastric cancer cells

Secretory leukocyte protease inhibitor (SLPI) protects tissue from proteases, and promotes cell proliferation and healing during inflammatory response. SLPI is also overexpressed in gastric, lung and ovarian cancers, which accelerates the metastasis of cancer cells. Matrix metalloproteinases-2, -9 (MMP-2 and MMP-9) are overexpressed in high metastatic cancers, and promote the migration of cancer cells through collagen degradation. SLPI and MMP-2, -9 are critical factors in stimulating the metastatic processes but there are no reports of a direct correlation between these molecules. Therefore, this study examined the role of SLPI related to MMP-2 and MMP-9 using two gastric cancer cell lines, such as characterized non-metastatic SNU484 and highly metastatic SNU638 cells. SLPI, MMP-2 and MMP-9 mRNA and protein expression were higher in SNU638 cells than in SNU484 cells. In addition, the rate of cell migration and invasion was higher in the SNU638 cells than in SNU484 cells. Interestingly, after treatment with SLPI, the rate of migration and invasion was higher in the SNU484 cells than in the positive control (PC) SNU484 cells. The rate of migration was also higher in the SNU638 cells after SLPI treatment than in the SNU638 cells (PC) but the invasion rate was not changed. The expression and secretion of MMP-2 and MMP-9 as well the rate of cell migration and invasion were significantly lower in SLPI-siRNA transfected SNU638 cells (si-SLPI/SNU638) but higher in SLPI-treated SNU484 cells (SNU484 + SLPI). Strong Elk-1 phosphorylation was detected in SNU484 + SLPI and SNU638 cells but was barely detectable in SNU484 and si-SLPI/SNU638 cells. These results show that SLPI promotes the metastasis of SNU638 gastric cancer cells by increasing MMP-2 and MMP-9 expression through Elk-1 signaling, indicating its role as a signaling molecule not a protease inhibitor.     

Upregulation of CD147 promotes cell invasion,epithelial-to-mesenchymal transition and activates MAPK/ERK signaling pathway in colorectal cancer

Colorectal cancer (CRC) is one of the most common cancers in the world. CD147, a transmembrane protein, has been reported to be correlated with various cancers. In this study, we aimed to investigate the mechanism of CD147 in regulating drug resistance, cell invasion and epithelial-to-mesenchymal transition (EMT) in CRC cells. qRT-PCR and western blotting were used to evaluated the expression of CD147 in 40 CRC cases and 4 cell lines. Increased expression of CD147 at both mRNA and protein levels was found in CRC samples, and the level of CD147 was correlated with lymph node metastasis. CD147 overexpression increased the 5-Fluorouracil (5-FU) resistance, enhanced the invasion and EMT of CRC cells by regulating EMT markers and MMPs. Adverse results were obtained in CD147 knockdown CRC cell line. Further investigation revealed that CD147 activated MAPK/ERK pathway, ERK inhibitor U0126 suppressed the CD147-induced cell invasion, migration and MMP-2, MMP-9 expression. Taken together, our study indicates that CD147 promotes the 5-FU resistance, and MAPK/ERK signaling pathway is involved in CD147-promoted invasion and EMT of CRC cells.     

基因沉默PRDX1 表达对人结直肠癌SW480 细胞侵袭迁移的影响

目的:探讨RNA 干扰过氧化还原酶1(Peroxiredoxin 1,PRDX1)表达对人结直肠癌SW480 细胞侵袭转移能力的影响。方法:筛选RNA 干扰PRDX1 的慢病毒质粒,与阴性对照慢病毒质粒分组转染结直肠癌SW480 细胞,转染后的SW480 细胞可分为PRDX1 基因沉默组(si-PRDX1)和阴性对照组(Vector)。实时荧光定量PCR(qRT-PCR)和免疫印迹法(Western blot)分别检测两组细胞中PRDX1 mRNA 和蛋白表达;采用Transwell 侵袭和迁移实验检测基因沉默PRDX1 表达对结直肠癌细胞侵袭及迁移能力的影响;通过Western blot 检测两组细胞中基质金属蛋白酶(MMP)家族部分蛋白表达水平。结果:基因沉默PRDX1 表达可有效抑制结直肠癌SW480 细胞中PRDX1 mRNA 和蛋白水平的表达,与阴性对照组相比(Vector),差异均具有统计学意义(P<0.01),说明基因沉默PRDX1 的SW480 细胞系构建成功;Transwell 侵袭和迁移实验显示si-PRDX1组细胞的侵袭及迁移能力较对照组均明显降低(P<0.01);Western blot 结果显示,与Vector 组相比,si-PRDX1 组细胞中组织基质金属蛋白酶抑制剂2(TIMP-2)的表达明显增加,而MMP-2 及MMP-9 的表达显著下降,且差异均具有统计学意义(P<0.05)。结论:基因沉默人结直肠癌SW480 细胞的PRDX1 表达可有效抑制细胞的侵袭、迁移及转移能力,其机制可能会通过调控TIMP-2、MMP-2 及MMP-9 的表达介导。     

Effect of CD133 overexpression on the epithelial-to-mesenchymal transition in oral cancer cell lines

Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the world. In OSCC, CD133 promotes tumor invasion and metastasis by inducing the epithelial-to-mesenchymal transition (EMT). A small subset of cancer cells known as cancer stem cells (CSCs) are thought to give rise to differentiated tumor cells and to predict tumor recurrence and metastases, i.e., CSCs may be metastatic precursors. In this study, we show that ectopic overexpression of CD133 in OSCC cell lines KB, YD9, and YD10B cells significantly promotes the EMT and acquisition of stemness properties. CSC properties were analyzed by colony-formation assay and measurement of OCT4, SOX2, and NANOG expression, and the EMT was monitored by cell migration, a cell invasion assay, and analysis of E-cadherin, N-cadherin, and vimentin expression. CD133 overexpression led to formation of irregular spheroid colonies consistent with a stem cell phenotype and increased the expression of OCT4, SOX2, NANOG, N-cadherin, and vimentin. Taken together, these findings show that elevated levels of CD133 lead to OSCC invasiveness and metastasis, associated with the upregulation of EMT and stemness markers.     

eEF1A2 promotes cell migration, invasion and metastasis in pancreatic cancer by upregulating MMP-9 expression through Akt activation

eEF1A2 is a protein translation factor involved in protein synthesis that is overexpressed in various cancers, with important functions in tumor genesis and progression. We have previously showed that the ectopic expression of eEF1A2 is correlated with lymph node metastasis and perineural invasion in pancreatic cancer. In this study, we investigated the functional role of eEF1A2 in the regulation of cell migration, invasion, and metastasis in pancreatic cancer. Furthermore, we investigated the potential molecular mechanisms involved. By evaluating the invasive ability of a panel of pancreatic cancer cell lines with different metastatic potentials, eEF1A2 expression in cells was positively associated with their invasive ability. The knockdown of eEF1A2 by siRNA decreased the migration and invasion of PANC-1 cells. By contrast, the ectopic expression of exogenous eEF1A2 significantly promoted the migration and invasion of SW1990 cells. Stable eEF1A2 overexpression in a nude mouse model of peritoneal metastasis likewise dramatically enhanced the intraperitoneal metastatic ability of SW1990 cells. In addition, eEF1A2 overexpression could upregulate MMP-9 expression and activity. A significant positive correlation between the overexpression of both eEF1A2 and MMP-9 was observed in pancreatic cancer tissues. The inhibition of MMP-9 activity reduced the promoting effect of eEF1A2 on cell migration and invasion. Furthermore, eEF1A2-mediated cell migration and invasion, as well as MMP-9 expression and upregulation, were largely dependent on the eEF1A2-induced Akt activation. The findings suggested the potentially important role of eEF1A2 in pancreatic cancer migration, invasion, and metastasis. Thus, the results provide evidence of eEF1A2 as a potential therapeutic target in the treatment of aggressive pancreatic cancer.     

Overexpression of Integrin-linked Kinase Promotes Lung Cancer Cell Migration and Invasion via NF-κB-mediated Upregulation of Matrix Metalloproteinase-9

Integrin-linked kinase (ILK) is a highly conserved serine-threonine protein kinase which has been implicated in the regulation of various cellular processes. Previously, we have demonstrated that overexpression of ILK correlates with malignant phenotype in non-small cell lung cancer. Furthermore, forced overexpression of ILK promotes lung cancer cell invasion and migration. However, the molecular mechanisms by which ILK enhances the invasive phenotype of lung cancer cells are still not fully understood. In the present study, we found that overexpression of ILK stimulated matrix metalloproteinase-9 (MMP-9) expression and activity in lung cancer cells. ILK-induced cell migration and invasion were significantly inhibited by MMP inhibitor doxycycline as well as by anti-MMP-9 neutralizing antibody. In addition, overexpression of ILK induced phosphorylation and nuclear translocation of nuclear factor-κB (NF-κB) subunit p65. Finally, upregulation of MMP-9 was severely abolished by either BAY 11-7028, a specific NF-κB inhibitor, or small interfering RNA targeted to NF-κB p65 in ILK overexpression cells. Taken together, these findings suggest that ILK promotes lung cancer cell migration and invasion via NF-κB-mediated upregulation of MMP-9.     

GKN2在胃癌细胞增殖、转移中的作用及机制

目的探讨胃动蛋白2(gastrokine 2, GKN2)对胃癌细胞的生长增殖、侵袭、转移的影响及分子机制。方法利用qRT-pCR和Western blot法检测GKN2在胃癌组织中的表达;构建对照细胞株AGS-CON、SGC-7901-CON与GKN2过表达细胞株AGS-GKN2、SGC-7901-GKN2,应用qRT-pCR和Western blot法检测GKN2在各细胞株中的表达变化,RTCA系统和克隆形成实验检测细胞增殖功能,Transwell及划痕实验检测细胞迁移和侵袭功能,并通过Western blot观察GKN2表达变化对PI3K/AKT/PTEN/mTOR信号通路相关蛋白的作用。结果 GKN2在胃癌组织及多株胃癌细胞株中的表达明显低于癌旁组织和正常胃上皮细胞;RTCA系统显示GKN2过表达组的增殖活性明显低于对照组;克隆形成实验结果显示,GKN2过表达组的细胞克隆数和大小明显低于对照组;Transwell及划痕实验结果表明,GKN2过表达组纵向迁移/侵袭数和横向迁移数明显低于对照组。Western blot结果显示,GKN2过表达下调增殖相关蛋白PCNA、Survivin,抗凋亡蛋白BCL-2及转移相关蛋白MMP-7、MMP-9和MMP-2表达水平,而上调Timp2表达水平。Western blot结果亦显示,GKN2影响PI3K/AKT/PTEN/mTOR通路的激活水平,特别是明显上调PTEN的表达。结论 GKN2在胃癌组织及多株胃癌细胞株中呈低表达或表达丢失。GKN2影响胃癌细胞的增殖和转移,可能通过PI3K/AKT/PTEN/mTOR通路促进胃癌的发生、发展。     

褪黑素抑制胃癌细胞系SGC-7901的迁移和侵袭

目的 探讨褪黑素(MLT)对胃癌细胞迁移和侵袭的抑制作用及其分子机制。 方法 不同浓度(0.1、1、2 和4 mmol/L)的褪黑素干预人胃癌SGC-7901 细胞 24 h,应用划痕实验、Transwell小室法检测胃癌细胞迁移和侵袭能力的改变;ELISA试剂盒检测培养液上清中基质金属蛋白酶（MMP）-2、MMP-9的表达,Real-time PCR检测胃癌细胞MMP-2、MMP-9、细胞间黏附分子1(ICAM-1）和CD44基因表达;免疫印迹法检测ICAM-1、CD44、p-P38、P38和磷酸化丝裂原活化蛋白激酶激酶（p-MKK）3/6蛋白表达。 结果 褪黑素抑制胃癌细胞的迁移和侵袭,并呈剂量依赖性。与空白对照组比较,褪黑素降低胃癌细胞基质金属蛋白酶（MMP)-2、MMP-9和ICAM-1、CD44的表达,并抑制p-P38、P38和p-MKK3/6的表达。 结论 褪黑素通过抑制胃癌细胞MMP-2、MMP-9、ICAM-1和CD44的表达从而抑制胃癌细胞的迁移和侵袭能力,抑制作用可能与p38MAPK信号通路有关。     

CNTN-1促进人食管癌EC9706细胞侵袭和迁移

目的探讨接触蛋白-1(CNTN-1)在食管癌转移中的作用。方法 q PCR和Western blot检测食管癌细胞系EC9706中CNTN-1的表达;RNA干扰和CNTN-1过表达质粒转染调整EC9706细胞CNTN-1的表达,并将细胞分为空白对照组、scrambled siRNA组、CNTN-1 siRNA组、pcDNA3.1-vector组和pcDNA3.1-CNTN-1组;Brd U和Transwell实验分别检测EC9706细胞增殖、侵袭和迁移能力;qPCR和Western blot检测基质金属蛋白酶MMP-2和MMP-9的表达。结果 CNTN-1在食管癌细胞EC9706中mRNA和蛋白水平较与正常食管上皮细胞显著上调(P0.05);转染CNTN-1siRNA后,EC9706细胞CNTN-1表达水平显著降低(P0.05),细胞增殖、侵袭和迁移能力显著下降(P0.05),同时细胞中侵袭转移相关蛋白MMP-2和MMP-9表达明显下降(P0.05);CNTN-1过表达质粒转染细胞后,EC9706细胞内CNTN-1表达水平上调(P0.05),细胞增殖、迁移和侵袭能力显著升高,同时MMP-2和MMP-9表达明显升高(P0.05)。结论 CNTN-1可能通过调节MMP-2和MMP-9表达促进食管癌细胞的侵袭转移。     

IMP3 expression is associated with epithelial-mesenchymal transition in breast cancer

IMP3 plays an important role in tumor invasion and metastasis, to which epithelial to mesenchymal transition (EMT) also contributes. The purpose of this study was to investigate whether IMP3 can regulate invasion and metastasis through EMT in breast cancers. The protein expression levels of IMP3 and EMT markers were analyzed by immunohistochemistry in 180 paraffin-embedded human breast tissue samples. There was an inverse correlation of IMP3 with E-cadherin protein expression (P = 0.042). IMP3 expression directly correlated with both Slug (P = 0.004) and vimentin (P < 0.001). Changes in E-cadherin, vimentin, and Slug mRNA and protein levels were examined by quantitative real-time reverse polymerase chain reaction (qRT-PCR) and western blotting. Overexpression of IMP3 reduced the expression of E-cadherin and upregulated Slug and vimentin in transfected cells. In contrast, knocking down IMP3 had the opposite expression of the three proteins. Ribo-immunoprecipitation qPCR revealed that IMP3 binds Slug mRNA directly. In a transwell assay, overexpression of Slug rescued the cell migration and invasion caused by silencing IMP3 in MDA-MB-231 cells. On the other hand, knockdown of Slug in T47D-IMP3 cells could also have the opposite change. Our results strengthen the association of IMP3 with the regulation of EMT. Slug is a functional target of IMP3. IMP3 could therefore promote invasion and migration through the EMT in breast cancer cells.     

慢性缺氧应激可能通过EMT增强乳腺癌MCF-7细胞恶性生物学行为

目的:探讨慢性缺氧应激对人乳腺癌MCF-7细胞恶性生物学行为的影响及可能机制。方法:将人乳腺癌MCF-7细胞分为缺氧组(1%O_2、5%CO_2和94%N_2)和正常对照组(常氧)进行培养。利用MTT法、CCK-8实验、细胞直接计数法及细胞侵袭和迁移实验对MCF-7细胞活力、增殖及侵袭和迁移能力进行检测;用软琼脂集落形成实验及Matrigel 3D培养技术检测MCF-7细胞非锚定生长能力及极性改变情况;利用MCF-7细胞构建裸鼠皮下种植瘤模型,检测慢性缺氧应激对体内肿瘤生长及肺转移的影响;利用倒置显微镜观察MCF-7细胞形态改变;Western blot检测低氧诱导因子1(hypoxia-inducible factor-1,HIF-1)和磷酸化的糖原合成酶激酶3β(glycogen synthase kinase-3β,GSK-3β)在缺氧环境下表达水平的改变,以及E-钙黏连蛋白(E-cadherin)、N-钙黏连蛋白(Ncadherin)、波形蛋白(vimentin)、基质金属蛋白酶3(matrix metalloproteinase-3,MMP-3)、MMP-9等上皮-间充质转化(epithelial-mesenchymal transition,EMT)相关蛋白的表达水平。结果:与正常对照组相比较,慢性缺氧组MCF-7细胞活力、增殖能力及侵袭迁移能力增强,细胞非锚定生长能力提高且在3D培养系统更容易发生极性改变,呈现侵袭样生长,体内生长及转移能力增强;除了HIF-1被缺氧诱导表达升高外,GSK-3β呈现活化趋势,且上皮样标志物E-cadherin蛋白表达水平明显下降,而间充质样标志物N-cadherin、vimentin、MMP-3和MMP-9蛋白表达水平明显升高。结论:慢性缺氧应激促进了乳腺癌细胞恶性生物学行为,且其机制可能与EMT有关。     

胃泌素促进胃癌细胞的迁移和侵袭

 目的：研究胃泌素在胃癌细胞迁移和侵袭中的作用。方法：用细胞划痕实验、Transwell小室迁移和侵袭实验检测10 nmol/L和100 nmol/L胃泌素处理后胃癌细胞AGS和SGC-7901迁移和侵袭能力的变化, MTT法检测细胞增殖能力, ELISA法检测细胞上清液中基质金属蛋白酶2（MMP-2）的含量。结果：10 nmol/L和100 nmol/L胃泌素处理胃癌细胞后,细胞迁移和侵袭能力增强。对照组、10 nmol/L和100 nmol/L胃泌素组AGS细胞平均迁移数分别为56.0、88.1和106.4 /view,SGC-7901细胞平均迁移数分别为52.8、91.0和113.3 /view, AGS细胞平均侵袭数分别为78.4、118.7和141.6 /view,SGC-7901细胞平均侵袭数分别为87.3、124.6和147.4/view（均P<0.01）;100 nmol/L胃泌素组细胞迁移和侵袭能力均高于10 nmol/L组（P<0.05）;胃泌素处理后细胞的增殖率及MMP-2的分泌量也显著增加（P<0.05）。结论：胃泌素通过促进MMP-2的分泌以剂量依赖的方式增强胃癌细胞的迁移和侵袭能力,可能是体内胃癌细胞增殖、侵袭和转移的重要机制之一。     

ELMO1在胃癌细胞侵袭和迁移中的作用

目的:探讨吞噬和细胞运动蛋白1(ELMO1)的表达在胃癌细胞侵袭和迁移中的作用和功能。方法:用Western blot和实时荧光定量PCR实验检测5种胃癌细胞和1种人正常胃黏膜上皮细胞ELMO1的蛋白和mRNA表达水平,并筛选出ELMO1表达量高的胃癌细胞株;用细胞转染实验沉默胃癌细胞株的ELMO1;用细胞划痕实验以及Trenswell小室迁移和侵袭实验检测抑制该基因的表达对胃癌细胞侵袭和迁移能力的影响。结果:胃癌细胞中ELMO1的表达量明显高于人正常胃黏膜上皮细胞(P0.01),其中SGC7901细胞的ELMO1表达量最高;在SGC7901细胞中ELMO1-siRNA可显著沉默ELMO1的表达(P0.05);沉默ELMO1可显著降低胃癌细胞侵袭和转移的能力(P0.01)。结论:ELMO1在胃癌细胞中高表达,并可促进胃癌细胞的侵袭和迁移。     

Gli1 enhances migration and invasion via up-regulation of MMP-11 and promotes metastasis in ERα negative breast cancer cell lines

Gli1 is an established oncogene and its expression in Estrogen Receptor (ER) α negative and triple negative breast cancers is predictive of a poor prognosis; however, the biological functions regulated by Gli1 in breast cancer have not been extensively evaluated. Herein, Gli1 was over-expressed or down-regulated (by RNA interference and by expression of the repressor form of Gli3) in the ERα negative, human breast cancer cell lines MDA-MB-231 and SUM1315. Reduced expression of Gli1 in these two cell lines resulted in a decrease in migration and invasion. Gli1 over-expression increased the migration and invasion of MDA-MB-231 cells with a corresponding increase in expression of MMP-11. Silencing MMP-11 in MDA-MB-231 cells that over-expressed Gli1 abrogated the Gli1-induced enhancement of migration and invasion. Sustained suppression of Gli1 expression decreased growth of MDA-MB-231 in vitro by increasing apoptosis and decreasing proliferation. In addition, silencing of Gli1 reduced the numbers and sizes of pulmonary metastases of MDA-MB-231 in an in vivo experimental metastasis assay. In summary, Gli1 promotes the growth, survival, migration, invasion and metastasis of ERα negative breast cancer. Additionally, MMP-11 is up-regulated by Gli1 and mediates the migration and invasion induced by Gli1 in MDA-MB-231.     

Frequent copy number variations of PI3K/AKT pathway and aberrant protein expressions of PI3K subunits are associated with inferior survival in diffuse large B cell lymphoma

Background

XB130 has been reported to be expressed by various types of cells such as thyroid cancer and esophageal cancer cells, and it promotes the proliferation and invasion of thyroid cancer cells. Our previous study demonstrated that XB130 is also expressed in gastric cancer (GC), and that its expression is associated with the prognosis, but the role of XB130 in GC has not been well characterized.

Methods

In this study, we investigated the influence of XB130 on gastric tumorigenesis and metastasis in vivo and in vitro using the MTT assay, clonogenic assay, BrdU incorporation assay, 3D culture, immunohistochemistry and immunofluorescence. Western blot analysis was also performed to identify the potential mechanisms involved.

Results

The proliferation, migration, and invasion of SGC7901 and MNK45 gastric adenocarcinoma cell lines were all significantly inhibited by knockdown of XB130 using small hairpin RNA. In a xenograft model, tumor growth was markedly inhibited after shXB130-transfected GC cells were implanted into nude mice. After XB130 knockdown, GC cells showed a more epithelial-like phenotype, suggesting an inhibition of the epithelial-mesenchymal transition (EMT) process. In addition, silencing of XB130 reduced the expression of p-Akt/Akt, upregulated expression of epithelial markers including E-cadherin, α-catenin and β-catenin, and downregulated mesenchymal markers including fibronectin and vimentin. Expression of oncoproteins related to tumor metastasis, such as MMP2, MMP9, and CD44, was also significantly reduced.

Conclusions

These findings indicate that XB130 enhances cell motility and invasiveness by modulating the EMT-like process, while silencing XB130 in GC suppresses tumorigenesis and metastasis, suggesting that it may be a potential therapeutic target.     

CUB-domain-containing protein 1 regulates peritoneal dissemination of gastric scirrhous carcinoma

CUB-domain-containing protein 1 (CDCP1) is a type-I transmembrane protein that is highly expressed in colon, breast, and lung cancers. We recently revealed that CDCP1 is associated with and phosphorylated by Src family kinases and is involved in the regulation of anchorage independence of certain lung cancer cell lines. In this study, we examined whether CDCP1 is involved in the regulation of tumor progression of scirrhous gastric cancer, which is a diffusely infiltrative carcinoma with high invasion potential. Expression and phosphorylation levels of CDCP1 correlated with the invasive potential of scirrhous gastric cancers. Reduction of CDCP1 expression by siRNA suppressed migration, invasion, and anchorage independence without affecting the proliferation of highly invasive scirrhous gastric cancer cells. However, CDCP1 overexpression promoted gastric cancer cell migration with low potential of invasion. Loss of CDCP1 suppressed invasion and dissemination of cancer cells that were orthotopically implanted in the gastric wall of nude mice. Expression and phosphorylation of CDCP1 were also detected in cancer cells of surgically resected tissues of human scirrhous gastric cancer by immunohistochemical analysis. Our results suggest that CDCP1 promotes invasion and peritoneal dissemination of cancer cells through the regulation of cell migration and anchorage independence. Therefore, it is both a potential prognostic and therapeutic target in certain types of gastrointestinal cancers, and suppression of its phosphorylation might be a useful strategy for modulating cancer metastasis.     

Transforming growth factor-β1 treatment of oral cancer induces epithelial-mesenchymal transition and promotes bone invasion via enhanced activity of osteoclasts

This study investigates relationships between EMT and bone invasion by OSCC. Three OSCC cell lines, SCC25, HN5, and Tca8113 were artificially induced to display EMT by adding 5 ng/mL of TGF-β1 to culture media for 1–3 days. Cell morphology and phenotypic changes was examined by immunocytochemical staining of CK and VIM. EMT markers, cell-invasion factors, and osteoclast-related molecules were analysed at mRNA, gelatine and protein levels by real-time PCR, gelatine zymography and Western blotting respectively. Mature osteoclasts differentiated from Raw264.7 cells were treated by conditioned medium (CM) of OSCC cells with/without TGF-β1. Immunohistochemistry was performed to validate proteins of CK, VIM, E-cad and Snail1 in OSCC tissue samples with bone invasion. Results showed minimal staining of VIM was found in SCC25 and HN5, while Tca8113 cells stained strongly. EMT markers Twist1 and N-cad were up-regulated; Snail1 and E-cad down-regulated in all cells. Of factors associated with invasion, MMP-2 was unchanged and MMP-9 increased in SCC25 and Tca8113, while MMP-2 was increased and MMP-9 unchanged in HN5. For osteoclast-related molecules, both MT1-MMP and RANKL were up-regulated, while OPG was down-regulated in all cells. CM of OSCC cells pre-treated with TGF-β1 showed to prolong survival of osteoclasts up to 4 days. All target molecules were validated in OSCC samples of bone invasion. These findings suggest that TGF-β1 not only induces EMT to increase the capacity of OSCC for invasion, but also promotes factors which prolong osteoclast survival. TGF-β1 may enhance the ability of MMP2/9 in resorbing bone and favouring invasion of cancer cells.     

Transient RNA silencing of tissue factor pathway inhibitor-2 modulates lung cancer cell invasion

Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates metalloproteinases (MMPs) involved in extracellular matrix (ECM) degradation. Its secretion in ECM makes TFPI-2 a potential inhibitor to regulate tumour invasion and metastasis. Moreover, TFPI-2 is frequently downregulated, particularly in aggressive cancers. In this study, we silenced TFPI-2 in the NCI-H460 non-small cell lung cancer cell line and evaluated the role of TFPI-2 in cell invasion and its impact on MMPs expression. As the effects of siRNA are transient, the consequences of both gene silencing and restoration to normal expression could be studied kinetically in the same cells. We showed that TFPI-2 expression by NCI-H460 cells was effectively downregulated using specific small interfering RNA and this silencing was associated with an increase in the invasive potential of tumour cells while migration was not affected. We also showed that mRNA levels and protein expression of MMP-2, -3, -9, -14 were not influenced by TFPI-2 silencing. Moreover, the gelatinase activity of MMP-2 and MMP-9 was unmodified. In contrast, MMP-1 mRNA levels and protein were significantly and similarly increased in cells transfected with TFPI-2 siRNA. In conclusion, this study confirms that TFPI-2 downregulation can contribute to tumour invasion of lung cancer cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.