Inhibition of K1735-M2 melanoma cell invasion in vitro by retinoic acid

Melanoma cell invasion in vitro was tested by means of confrontation cultures of melanoma multicellular spheroids with rounded fragments of embryonic chick heart tissue. Quantitative determination of invasion was performed using a computerized image analysis program, facilitating the evaluation of the efficacy of potentially anti-invasive compounds. Retinoic acid (RA; 1 µm) considerably impaired K1735-M2 melanoma cell invasion, as demonstrated by various measuring parameters. Parameter TUMAREA, expressing the amount of tumor tissue, indicates a growth inhibitory effect and the invasion parameter STRCSTR shows that after treatment with RA the stromal component was better preserved than in untreated controls. Besides the inhibitory effect of RA on melanoma cell invasion in confrontation cultures, RA increased the dynamics of adhesion of melanoma cells to the extracellular matrix components type I collagen and laminin, and slightly impaired melanoma cell directional migration. Fluorescence microscopy using rhodamine-labeled phalloidin showed that RA also modulated the organization of the actin cytoskeleton by inducing the formation of actin-containing stress fibers. Our data show that 1 µM RA exhibited a pronounced anti-invasive effect on highly metastatic melanoma cells in vitro. Impairment of host tissue degradation, altered adhesion abilities, changes in the actin cytoskeleton, as well as the antiproliferative effect may all account for inhibition of melanoma cell invasion.     

Differential effects of synthetic sphingosine derivatives on melanoma cell motility, growth, adhesion and invasion in vitro

Cancer cell surface glycosphingolipids are considered to play a critical role in tumor growth and metastasis. However, the implications of glycoconjugates in the control of cell motility, which is considered to be involved in tumor invasion, are not fully understood. In this study, the effects of a series of synthetic sphingosine derivatives, obtained by the chemical transformation of azidosphingosines, on directional migration of K1735-M2 melanoma cells grown on type I collagen-coated surfaces were investigated. Following the application of 60 µm (2R, 3S, 4E)-2,3-epimino-4-octadecen-3-ol (S4) the migration rate was 94 ± 10 m/day, compared with 377 ± 22 µm/day in the control experiment. Six other analogues were not as potent. S4 also considerably down-modulated melanoma single cell motility. Inhibition of motile activity was associated with changes in the actin filament organization as well as with changes in the number and distribution of vinculin plaques. Moreover, the compound reduced the attachment abilities of melanoma cells to basement membrane Matrigel. Tumor cell invasion, however, was less affected and proliferation remained unimpaired after treatment with S4. These data suggest at least one intracellular mode of action of this particular synthetic sphingosine derivative by modulation of cytoskeletal organization. Melanoma cell motility and growth may be controlled independently via glycosphingolipids.     

Down-regulation of focal adhesion kinase, pp125FAK , in endothelial cell retraction during tumor cell invasion

Although endothelial cell retraction is required before tumor cell invasion, its molecular mechanism still remains obscure. We previously demonstrated that conditioned medium (CM) derived from a human pan-creatic cancer cell line, PSN-1, induced endothelial cell retraction and facilitated tumor cell invasion. To investigate the molecular change of events in the transduction of extracellular signals during endothelial cell retraction, we examined the effect of the CM derived from PSN-1 cells on the tyrosine phosphorylation in endothelial cells. Immunoblot analyses revealed that the PSN-1 CM decreased tyrosine phosphorylation of a 120-130 kD protein, and induced the concomitant down-regulation of focal adhesion kinase, pp125^FAK , dur-ing endothelial cell retraction in time- and dose-dependent fashions. These changes preceded endothelial cell retraction and were reversible after removal of the CM. Further quantitative densitometric analyses demon-strated that the extent of decrea se in tyrosine phosphorylated 120-130 kD protein during the endothelial cell retraction was likely to be proportional to that of the down-regulation of pp125^FAK . A tyrosine phosphory-lated 120-130 kD protein immunoprecipitated by anti-phosphotyrosine antibody immunoreacted with anti-pp125^FAK antibody. These results suggested that decreased amount of a tyrosine phosphorylated 120-130 kD protein probably due to the down-regulation of pp125^FAK might be associated with the signal transduction pathway in the endothelial cells during their retraction. Furthermore, these findings were also observed in the CM from another four human cancer cell lines, suggesting the down-regulation of pp125^FAK in endothelial cells during tumor cell invasion. © Rapid Science 1998     

Chemically modified tetracyclines inhibit human melanoma cell invasion and metastasis

Recent work has shown that chemically modified tetracyclines (CMTs) are potent inhibitors of matrix metal-loproteinase (MMP) activity, both in vitro and in vivo, which is distinct from their antimicrobial activities (Golub et al. Crit Rev Oral Biol Med, 2, 297-321, 1991; Ryan et al. Curr Opin Rheumatol, 8, 238-47, 1996). The process of tumor cell invasion requires MMP-mediated degradation of extracellular matrix barriers as a key step in the metastasic cascade. In this study, we examined the effect(s) of doxycycline and CMTs on extracellular levels of gelatinase A and B activity from a highly invasive and metastatic human melanoma cell line C8161, and correlated these observations with changes in the cells' biological behavior in an in vitro invasion assay and in an in vivo SCID mouse model. The results indicate that coincident with the ability of these compounds to differentially suppress extracellular levels of gelatinase activity, C8161 cells treated with doxycycline, CMT-1, CMT-3, or CMT-6 were less invasive in vitro in a dose-dependent manner (3-50 mg/ml). Furthermore, data derived from the in vivo model indicate that SCID mice dosed orally with CMT-1 or CMT-3 contained a reduced number of lung metastases following i.v. injection of C8161 cells via tail vein inoculation. These observations suggest that careful screening of different CMTs could lead to the identification of compounds which suppress the formation and magnitude of metastases associated with certain cancers, and if used as an adjunct to other treatment regimes, lead to greater efficacy in the treatment of metastatic cancers. © Rapid Science 1998     

Low expression of KLF17 is associated with tumor invasion in esophageal carcinoma

Purpose: KLF17 belongs to the Sp/KLF zinc-finger protein family as a regulator in tumor development. However, its expression and biologic function has remained unclear in EC. Methods: The esophageal carcinoma tissue samples and adjacent normal tissues were obtained from the Second Hospital of Hebei Medical University. Immunohistochemistry, Western blot, and transfection were applied to evaluate the expression and clinical significance of KLF17 in esophageal cancer. Results: In this study, we showed that KLF17 was overexpressed in esophageal normal samples compared to the cancer. Moreover, KLF17 was upregulated at lymph node non-metastatic cancer tissues when compared to metastatic cancer tissues. KLF17 overexpression decreased EC cell proliferation, migration and invasion ability. In contrast, the knockdown of KLF17 increased EC cell proliferation, migration and invasion ability. Conclusion: These results suggest that KLF17 inhibits tumor development and may serve as a potential therapeutic target.     

Overexpression of L1 cell adhesion molecule correlates with aggressive tumor progression of patients with breast cancer and promotes motility of breast cancer cells

Background and purpose: L1 cell adhesion molecule (L1CAM) has been observed to be aberrantly expressed and implicated in progression of several types of human cancers. However, its roles in breast cancer have not been fully elucidated. In this study, we aimed to investigate the clinical significance of L1CAM in human breast cancer and to validate whether it participates in cancer cell migration and invasion. Methods: Immunohistochemical analysis of 100 breast cancer and matched non-cancerous breast tissues was performed to detect the expression and sub-cellular localization of L1CAM protein. Its associations with clinicopathological characteristics of breast cancer patients were statistically analyzed and its phenotypic effects were also evaluated in vitro. Results: Of the 100 breast cancer patients, 89 (89.0%) were positive for L1CAM immunostaining localized in the membrane of cancer cells. The immunoreactive scores of L1CAM protein in breast cancer tissues were significantly higher than those in matched non-cancerous breast tissues (P<0.05). Chi-Square analysis showed the significant associations between L1CAM overexpression and high tumor stage (P=0.01), advanced tumor grade (P=0.03), positive lymph node metastasis (P=0.01) and tumor recurrence (P=0.01) in breast cancer patients. Moreover, we found that RNA interference-mediated knockdown of L1CAM could inhibit the migration and invasion abilities of breast cancer cells in vitro. Conclusions: Our results suggest that the overexpression of L1CAM may be related to several established markers of poor prognosis in breast cancer patients. L1CAM might be a potential therapeutic target against metastatic breast cancer.     

Expression of focal adhesion kinase in uveal melanoma and the effects of Hsp90 inhibition by 17-AAG

IntroductionFocal adhesion kinase (FAK) is implicated in tumor progression and metastatic cascade, and has been shown to be overexpressed in a variety of human cancers. However, the role of FAK in human uveal melanoma (UM) is not well defined. The purpose of this study was to evaluate the expression of FAK in UM tumors and normal eyes, and to determine the effect of Hsp90 inhibition on FAK expression in UM cells.MethodsFAK expression was assessed in 39 UM specimens, FAK[pY397] expression was assessed in 51 UM specimens, and both FAK and FAK[pY397] expression were assessed in 20 normal eyes. The expression of FAK and FAK[pY397] was detected by Western blot in five UM cell lines after treatment with 10 μmol/L of 17-AAG.ResultsFAK was positive in 87.2% and FAK[pY397] in 90% of UM specimens. Low FAK expression was detected in non-tumor structures and in normal eyes. The cell lines with the most proliferative, invasive phenotype (92.1, SP6.5 and MKT-BR) displayed high expression of FAK[pY397], and the levels of FAK and FAK[pY397] were decreased in the presence of 17-AAG starting with 24 h of exposure.ConclusionFAK and FAK[pY397] were overexpressed in human UM tumors compared to normal ocular tissue and high levels of FAK[pY397] were seen in the most aggressive UM cell lines. Hsp90 inhibition led to downregulation of FAK expression. We propose a role for FAK in the pathogenesis of UM. Future studies are needed to explore the use of Hsp90 inhibitors as a feasible approach for modulating FAK in UM.     

Neural cell adhesion molecule is upregulated in nerves with prostate cancer invasion

Currently postulated mechanisms of perineural invasion (PNI) include interaction between tumor cells and nerves. Neural cell adhesion molecule (N-CAM), one of the well-known members of the immunoglobulin super-family of adhesion molecules, was implicated in PNI and metastasis in various types of cancer. Tissue microarray technology was used to build 2 sets of tissue array (with versus without PNI) from 50 prostate cancers (PCa). The slides were stained immunohistochemically, and the results were evaluated semiquantitatively using a 0 to 3+ scoring system. N-CAM staining was observed in all nerves with variable intensity. N-CAM expression was upregulated in 73% (31 of 42) of the nerves with PNI compared with nerves without PNI (P >.001). The results suggested that N-CAM is probably involved in PNI in PCa. It is conceivable that cancer cells, through a yet-to-be-established paracrine loop, signal the nerve to increase N-CAM production and increase adhesion. N-CAM upregulation in nerves may also facilitate cancer cells to migrate toward nerves and promote the process of perineural spread through increased survival using the nuclear factor kappa B pathway.     

Molecular determinants of human uveal melanoma invasion and metastasis

The molecular analysis of cancer has benefited tremendously from the sequencing of the human genome integrated with the science of bioinformatics. Microarray analysis technology has the potential to classify tumors based on the differential expression of genes. In the current study, a collaborative, multidisciplinary approach was utilized to study the molecular determinants of human uveal melanoma invasion and metastasis. Uveal melanoma is considered the most common primary intraocular cancer in adults, resulting in the death of approximately 50% of patients affected. Unfortunately, at the time of diagnosis, many patients already harbor microscopic metastases, thus underscoring a critical need to identify prognostic markers indicative of metastatic potential. The investigative strategy consisted of isolating highly invasive vs. poorly invasive uveal melanoma cells from a heterogeneous tumor derived from cells that had metastasized from the eye to the liver. The heterogeneous tissue explant MUM-2 led to the derivation of two clonal cell lines: MUM-2B and MUM-2C. Further morphological and functional analyses revealed that the MUM-2B cells were epithelioid, interconverted (expressing mesenchymal and epithelial phenotypes) highly invasive, and demonstrated vasculogenic mimicry. The MUM-2C cells were spindle-like, expressed only a vimentin mesenchymal phenotype, poorly invasive, and were incapable of vasculogenic mimicry. The molecular analysis of the MUM-2B vs. the MUM-2C clones resulted in the differential expression of 210 known genes. Overall, the molecular signature of the MUM-2B cells resembled that of multiple phenotypes – similar to a pluripotent, embryonic-like genotype. Validation of select genes that were upregulated and down-regulated was conducted by semiquantitative RT-PCR measurement. This study provides a molecular profile that will hopefully lead to the development of new molecular targets for therapeutic intervention and possible diagnostic markers to predict the clinical outcome of patients with uveal melanoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Matrix metalloproteinases in tumor invasion: role for cell migration

Matrix metalloproteinases (MMP) play a role in a wide range of tumorigenesis, including early carcinogenesis events, tumor growth and tumor invasion and metastasis. Given that the ability of tumor cells to infiltrate and disseminate widely is what makes the tumors malignant, a role of MMP in cell migration during this invasive and metastatic process is important. There are two types of cancer cell migration: single cell locomotion and cohort migration (cell movement en mass keeping cell-cell contact, which is frequently seen in better differentiated carcinomas). Cell surface localization and activation of MMP is essential for cells to migrate, through rearrangement of extracellular matrix (ECM) to suit cell migration. Certain MMP, such as gelatinases and membrane -type 1 MMP, have special mechanisms to localize at leading edges in both types of cell migration. Moreover, in cohort migration, expression of these MMP is regulated via cell-cell contact within migrating cell sheets and confined to the foremost pathfinder cells of the migrating cell sheets. New roles of cell surface MMP, such as cleavage of cell surface receptors or cofactors involved in cell-ECM interactions during cell migration, are also discussed.     

过表达细胞黏附分子1抑制卵巢癌细胞迁移及侵袭

目的:研究过表达细胞黏附分子1(cell adhesion molecule 1,CADM1)对卵巢癌增殖、迁移和侵袭的影响.方法:qRT-PCR测定CADM1mRNA在卵巢癌细胞系SKOV3及人正常卵巢上皮细胞hose中的表达,将SKOV3细胞分成两组,即CADM1过表达组和对照组,转染48 h后Western印迹测定两组CADM1蛋白表达量,采用lipofectamine 2000分别转染pcDNA3.1-CADM1及pcDNA3.1质粒,采用CCK-8、克隆形成、细胞划痕及Transwell实验分别检测两组细胞增殖、克隆形成、细胞迁移及侵袭能力.结果:CADM1mRNA在SKOV3中表达水平显著低于hose细胞系(1.54±0.34 vs.5.63±0.96,p＜0.05);转染48 h后,CADM1过表达组和对照组CADM1蛋白表达量分别为2.53±0.42,0.37±0.09,差异具有统计学意义(P＜0.05).CADM1过表达组和对照组在0,24,48,72 h 450 nm处的OD值差异无统计学意义(P＞0.05);CADM1过表达组与对照组克隆形成数相比(60.4±7.6 vs.58.3±8.2),差异无统计学意义(P＞0.05);CADM1过表达组细胞迁移率显著低于对照组(20.3％±3.5％vs.60.1％±4.2％,P＜0.05);CADM1过表达组侵袭细胞数显著少于对照组(24.5±5.3 vs.65.1±6.9,P＜0.05).结论:CADM1在卵巢癌细胞系中低表达,过表达CADM1对卵巢癌细胞增殖和克隆形成无影响,但可抑制迁移和侵袭,起抑癌基因的作用.     

全反维甲酸对黑素细胞与瘤细胞粘连性与移动性的影响

人表皮黑素细胞经全反维甲酸（ＲＡ）处理后，置于含有牛血清白蛋白、纤维连结蛋白与层粘连蛋白的培养板中检测其粘连性。结果发现经３×１０（－６）ｍｏｌ·Ｌ（－１）的ＲＡ处理的黑素细胞对上述３种蛋白质的粘连性及移动性均被抑制。人和小鼠黑素瘤细胞系经上述浓度ＲＡ处理后，其粘连性与移动性均被抑制     

血管细胞黏附分子-1增强人脑胶质瘤细胞系迁移和侵袭能力

目的探讨血管细胞黏附分子-1(VCAM-1)在脑胶质瘤细胞迁移侵袭能力中作用。方法用慢病毒p SGU6/GFP/Neo介导VCAM-1的shRNA、慢病毒EF1a-GFP/puro介导VCAM-1过表达载体、划痕迁移、Transwell侵袭、蛋白免疫印迹(Western blot)和细胞染色等实验技术和方法,观察了VCAM-1蛋白表达水平对人脑胶质瘤T98G和U251细胞系细胞迁移和侵袭能力的影响。其中,T98G细胞分为空白对照组、空载体对照组、乱序对照组和实验组(抑制VCAM-1蛋白表达水平组),U251细胞分为空白对照组、空载体对照组和实验组(过表达VCAM-1组),每组6个复孔。结果首先利用慢病毒介导VCAM-1的shRNA和过表达载体建立了稳定低表达VCAM-1的T98G细胞和稳定过表达VCAM-1的U251细胞。稳定低表达VCAM-1的T98G细胞划痕恢复能力(迁移能力)明显减弱(P0.01);而稳定过表达VCAM-1的U251细胞迁移能力明显提高(P0.05)。同样,稳定低表达VCAM-1的T98G细胞侵袭能力显著减弱(P0.05);而稳定过表达VCAM-1的U251细胞侵袭能力明显增强(P0.01)。结论VCAM-1可显著增强人脑胶质瘤细胞系细胞的迁移和侵袭能力。     

Expression and activation of pro-gelatinase A by human melanoma cell lines with different tumorigenic potential

The production of various proteolytic enzymes by tumor cells facilitate the invasion of solid tumors into surrounding tissues. We examined three cell lines (M1Dor, M4Be and M3Da) derived from malignant melanoma which exhibited different abilities to grow in nude mice following subcutaneous grafting. By in vitro invasion assay using Boyden-chambers technique, we found that none of those cell lines were able to invade the Matrigel. Several studies have substantiated the role of matrix metalloproteinases (MMP), mainly gelatinases MMP-9 and MMP-2, in melanoma cell invasion. Each cell line constitutively produced MMP-2 (but not MMP-9) in its latent form only, with stronger production for the most tumorigenic cell line in vivo (M3Da). Integrity of the MMP-2 activation process was studied since MMP-2 was also recovered as zymogen at the cell plasma membrane. All cell lines secreted TIMP-1 and TIMP-2 in a constitutive manner and again, but TIMP-2 production as well as MT1-MMP expression were found inversely related to their tumorigenic potential. Plating cells onto type I or type IV collagen did not trigger pro-MMP-2 activation; on the contrary, conversion of pro-MMP-2 to its active form could be evidenced when melanoma cell lines were seeded in a three dimensional type I collagen lattice. This revised version was published online in July 2006 with corrections to the Cover Date.     

An inhibitor of stress-activated MAP-kinases reduces invasion and MMP-2 expression of malignant melanoma cells

A lot of parallels have been described between invasion of malignant tumor cells and leukocyte movement during inflammatory responses. Concerning these similarities, we investigated the function of cytokine-suppressive anti-inflammatory drugs (CSAIDs), which act via inhibition of stress-activated MAP-kinases, in regulation of expression of proteolytic enzymes and in vitro invasion of malignant melanoma cells. The p38MAPK inhibitor SB203580 reduced matrigel invasion of MeWo cells by 60%, while the MEK-1 inhibitor PD98059 did not have any effect on invasion. Active p38MAPK was detected in MeWo cells by immunoblotting and confocal microscopy. Cells showed a constitutive expression of matrix-metalloproteinase (MMP)-2 as well as tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 mRNAs. Expression of MMP-1 or urokinase-type plasminogen activator (uPA) was not detected by Northern blot. Inhibition of p38MAPK by the specific inhibitor SB203580 resulted in downregulation of MMP-2 mRNA and protein levels as well as gelatinolytic activity, while expression levels of TIMP-1 and TIMP-2 mRNAs were not changed. The specific MEK-1 inhibitor PD98059 did not change expression of MMP-2 or TIMPs. Neither SB203580 nor PD98059 changed proliferation of cells. The results suggest that stress-activated protein kinases like p38MAPK are involved in regulation of expression of MMP-2 as well as in vitro invasion of malignant melanoma cells. Inhibitors of p38MAPK may be promising substances to interfere with a signaling cascade associated with invasion of malignant tumor cells.     

Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion

Malignant melanomas are characterized by their high propensity to invade and metastasize, but the molecular mechanisms of these traits have remained elusive. Our DNA microarray analyses of benign nevi and melanoma tissue specimens revealed that the genes encoding extracellular matrix proteins tenascin-C (TN-C), fibronectin (FN), and procollagen-I (PCOL-I) are highly upregulated in invasive and metastatic melanomas. The expression and distribution of these proteins were further studied by immunohistochemistry in benign nevi, radially and vertically growing melanomas, sentinel node micrometastases, and macrometastases. TN-C was increased in all invasive tumours and metastases, especially at invasion fronts, but not in benign nevi or non-invasive melanomas. Significantly, the intensity of TN-C staining correlated with metastasis to sentinel lymph nodes, better than tumour thickness (Breslow). Moreover, TN-C, FN, and PCOL-I appeared to co-localize in the tumours and form tubular meshworks and channels ensheathing the melanoma cells. Our data suggest that melanoma invasion is associated with the formation of special channel-like structures, providing a new concept, structured tumour cell spreading. Altogether, these data provide potential new prognostic markers and therapeutic targets/strategies for preventing melanoma dissemination.     

Amygdalin-mediated inhibition of non-small cell lung cancer cell invasion in vitro

Lung cancer is a common malignant tumor claiming the highest fatality worldwide for a long period of time. Unfortunately, most of the current treatment methods are still based on the characteristics of cancer cells in the primary lesion and the prognosis is often much poorer in patients with metastatic cancers. Amygdalin, a natural product of glycosides and lots of evidence shows that amygdalin can inhibit the proliferation of some kinds of cancer cells. In this study, we first obtained the highly metastatic NSCLC cell lines H1299/M and PA/M and further treated these cells with amygdalin. We found that the in vitro proliferability of H1299/M and PA/M was inhibited, but such inhibition required higher concentration of amygdalin. When lower concentration of amygdalin was used for the experiments, we observed that the in vitro invasive and migration capacities of H1299/M and PA/M were significantly inhibited. These results strongly suggested that amygdalin was likely to have anti-metastatic NSCLC effect. This study offers information of the role of amygdalin that may be useful as a therapeutic target in lung tumors.     

Atypical E-cadherin expression in cell clusters overlying focally disrupted mammary myoepithelial cell layers: Implications for tumor cell motility and invasion

Our recent studies showed that cell clusters overlying focal myoepithelial cell layer disruptions (FMCLD) had a significantly higher rate of ER negativity, genetic instabilities, and expression of invasion-related genes than adjacent cells within the same duct. This study attempted to determine if these cells would show aberrant E-cadherin expression, which imparts greater propensity for cell motility and invasion. Consecutive sections from breast tumors with a high frequency of FMCLD were double-immunostained for E-cadherin and a panel of related markers. The E-cadherin mRNA levels in cells overlying FMCLD and adjacent cells within the same duct were compared using real-time PCR. Nearly all the cell clusters overlying FMCLD were strongly immunoreactive for E-cadherin, whereas their adjacent counterparts within the same duct were largely negative. Cell clusters overlying FMCLD were generally arranged as tongue-like projections, “puncturing” deep into the stroma or tube-like structures that often contained red blood cells. The sub-cellular localization of E-cadherin in the above structures, however, was primarily cytoplasmic. The mRNA level of E-cadherin in cell clusters overlying FMCLD was significantly higher than that in adjacent cells within the same duct. These findings suggest that aberrant expression of E-cadherin may contribute to cell motility and invasion.     

Role of tissue factor pathway inhibitor-2 (TFPI-2) in amelanotic melanoma (C-32) invasion

Human tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein (PP5) and matrix-associated serine protease inhibitor (MSPI), is a 32-kDa extracellular matrix (ECM) protein consisting of three tandomly arranged Kunitz-type domains that inhibits plasmin, trypsin, chymotrypsin, cathepsin G and plasma kallikrein but not urokinase and tissue-type plasminogen activators or thrombin. Earlier studies in our laboratory revealed that the production of TFPI-2 is reduced or absent during the tumor progression of human gliomas. In the present study, we investigated the role of TFPI-2 in the invasiveness of the amelanotic melanoma cell line C-32. We stably transfected C-32 cells with a vector capable of expressing TFPI-2 in a sense orientation (0.7 kb). TFPI-2 protein production was then determined by western blotting and the mRNA level by northern blotting in parental and stably transfected (vector and sense) clones. The levels of TFPI-2 protein and mRNA were significantly higher in the sense clones, but neither was detected in parental and vector control clones. In addition, in vitro Matrigel invasion/migration assays revealed that the invasive behavior of sense clones was inhibited compared with the behavior of parental and vector clones. This is the first study to show that the upregulation of TFPI-2 plays a significant role in reducing the invasive behavior of human amelanotic melanomas. This revised version was published online in July 2006 with corrections to the Cover Date.     

Human melanoma invasion and metastasis enhancement by high expression of aminopeptidase N/CD13

Aminopeptidase N/CD13 is a Zn²⁺-dependent exoprotease present on the cell surface as a transmembrane protein. Our previous studies using aminopeptidase inhibitors and antibodies demonstrated that aminopeptidase N is involved in the degradation and invasion of the extracellular matrix (ECM) by metastatic tumor cells. In the present study we transfected human A375M melanoma cells with eukaryotic plasmid expression vectors that contained full length cDNA of aminopeptidase N/CD13 and examined their characteristics. The transfectants that expressed extremely high levels of aminopeptidase N/CD13 degraded type IV collagen and invaded ECM more actively than the parental and control vector-transfected cells. Furthermore, the aminopeptidase N/CD13-transfected A375M cells had significantly augmented lung colonizing potential in nude mice. The results show that the aminopeptidase N/CD13 plays an active role in degradation and invasion of ECM and may be involved in the molecular mechanisms of blood-borne metastasis.