Quantitative fluorescent microassay for identification of antiproliferative compounds.

A reproducible and convenient microassay was developed by us to measure the antiproliferative activity of biological materials and anticancer drugs. The assay involved the labeling of target cells after their incubation with anticancer agents with the fluorescent vital dye hydroethidine at a dose of 28 micrograms/ml for 30 minutes. Once internalized into live cells, hydroethidine is dehydrogenated to ethidium, which then intercalates into DNA. Hydroethidine labeled the cells uniformly. The cells were lysed with detergent, and the relative fluorescence of this dye was monitored in a microfluorimeter at the speed of 30 seconds per 96-well plate. Because this assay is accurate and reproducible, does not present problems of disposal of labeling agents, and is extremely rapid, it should prove very useful for the screening of many potential anticancer agents with antiproliferative activity.

Treatment of experimental lung metastasis with local thoracic irradiation followed by systemic macrophage activation with liposomes containing muramyl tripeptide.

The purpose of these studies was to determine whether the combination of a low-dose local thoracic irradiation (LTI) followed by systemic activation of macrophages with liposomes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE) would significantly decrease established experimental fibrosarcoma lung metastases. Male C3Hf/Kam mice were given i.v. injections of 1 X 10(5) fibrosarcoma cells. Five days later, groups of mice were treated with saline, with 8 Gy LTI, or with liposomes containing MTP-PE or first with 8 Gy LTI and followed by multiple i.v. injections of liposomes containing MTP-PE. Most of the mice in the groups treated with liposomes died by day 42 of the experiment. In contrast, 60% of the mice treated with the combination of LTI and liposomes containing MTP-PE were alive by day 140 of the study. These mice were killed and were found to be free of tumors. Control studies demonstrated that liposomes administered i.v. to mice given LTI were trapped in the capillary bed of the lungs and activated the tumoricidal properties of lung macrophages. We conclude that, in this combination, low-dose LTI, which can lead to both tumor cell death and inflammatory changes in the lung capillaries, could precede i.v. administration of liposomes containing MTP-PE. This combination of treatments can lead to destruction of tumor foci in the lung that cannot be achieved with either treatment alone.

Inhibition by sulfated chitin derivatives of invasion through extracellular matrix and enzymatic degradation by metastatic melanoma cells.

We have investigated the effects of sulfated chitin derivatives and heparin on the invasion of B16-BL6 melanoma cells through reconstituted basement membrane Matrigel which contains laminin, type IV collagen, heparin sulfate proteoglycan, and entactin. 6-O-sulfated chitin (S-chitin) and 6-O-sulfated and carboxymethyl chitin (SCM-chitin) significantly inhibited the penetration of tumor cells through Matrigel in parallel with the increased degree of sulfation. However, 6-O- and N-sulfated but partially N-deacetylated chitin derivative (SCM-chitosan) and CM-chitin had no effect. SCM-chitin with a high degree of sulfation (SCM-chitin III), which exhibited fairly low levels of anticoagulant activity, was more effective than intact heparin. SCM-chitin III and heparin were also shown to block the attachment and migration of tumor cells to laminin-coated substrates, which are considered to be involved in tumor invasion. The inhibition of cell attachment and migration by SCM-chitin III and heparin is likely to depend upon their specific binding to laminin molecules (possibly the heparin-binding domain). Degradation of heparan sulfate by heparanase was inhibited by SCM-chitin III and heparin in a dose-dependent manner. Surprisingly, SCM-chitin III could inhibit type IV collagenolytic activity of tumor cells more potently than heparin. Thus, nontoxic SCM-chitin III of low anticoagulant properties may provide a promising basis for the prevention of cancer metastasis.

Inhibition of tumor-induced angiogenesis by sulfated chitin derivatives.

The effect of antimetastatic sulfated chitin derivatives (SCM-chitin III) on angiogenesis induced by B16-BL6 cells was examined in syngeneic mice. SCM-chitin III caused a marked decrease of the number of vessels toward tumor mass (angiogenic response) without affecting the tumor cell growth when coinjected with tumor cells (on day 0), or injected into tumor site on day 1 or 3 after tumor inoculation. In contrast, carboxymethyl chitin as well as heparin had no effect. Invasion of endothelial cells through reconstituted basement membrane (Matrigel) toward tumor-conditioned media was significantly inhibited by SCM-chitin III in a Transwell chamber assay. SCM-chitin III also inhibited the haptotactic migration of endothelial cells to fibronectin-substrate, but did not inhibit the chemotactic activity in tumor conditioned media in vitro. SCM-chitin III did not directly affect the viability and the growth of tumor cells and endothelial cells in vitro. These results suggest that inhibition of lung tumor metastasis by SCM-chitin III may in part be due to the inhibition of tumor-associated angiogenesis.

Direct antiproliferative effects of recombinant human interferon-alpha B/D hybrids on human tumor cell lines.

The purpose of these studies was to examine the antiproliferative properties of 16 recombinant human IFN-alpha B/D hybrids against various human tumor lines of different histological origin and to determine whether any of the hybrid molecules possessed immunomodulating activity that could active antitumor properties in peripheral blood monocytes of normal donors. Hybrids with the B domain at the NH2 terminal end exhibited higher activity for antiviral activity and a higher level of direct antitumor antiproliferative activities as compared with hybrids with the D domain at the NH2 terminal end. The positive hybrids were directly cytostatic to melanoma, glioblastoma, renal carcinoma, colon carcinoma, and prostatic carcinoma cells. Tumor cell sensitivity to IFN-alpha hybrids was independent of sensitivity to IFN-gamma or to Adriamycin. The growth of a normal cell line (human embryo fibroblast) was unaffected by IFN-alpha hybrids but was completely arrested by Adriamycin. Some of the IFN-alpha hybrids were also cytostatic to mouse melanoma, lung carcinoma, and fibrosarcoma cell lines, albeit at lower levels than they were to human cells. The incubation of monocytes with IFN-alpha hybrids with the B domain at the NH2 terminal end was also associated with marked antitumor cytotoxicity. Kinetic studies, however, indicated that this activity was attributable to IFN-alpha carried on monocytes and acting directly on tumor cells. We conclude that recombinant human IFN-alpha B/D hybrids possess potent direct antiproliferative activity against a large variety of human tumor lines.

Inhibition of the metastasis of murine malignant melanoma by synthetic polymeric peptides containing core sequences of cell-adhesive molecules.

We investigated that the antimetastatic and antiadhesive activities of peptides based on Arg-Gly-Asp adhesive signal in fibronectin could be augmented by their polymerization. Poly(Arg-Gly-Asp), which consists of a repetitive sequence of Arg-Gly-Asp, inhibited lung metastases in C57BL/6 mice more effectively than Arg-Gly-Asp tripeptide was able to do, when coinjected or separately injected with B16-BL6 cells. The adhesion of tumor cells to fibronectin was specifically inhibited by adding poly(Arg-Gly-Asp) but not unrelated peptides. In contrast, poly(Arg, Gly, Asp), in which three amino acids are randomly arranged, showed neither inhibition of lung metastases nor any adhesive ability to attach to tumor cells. The inhibitory effect of polymeric peptides containing the Arg-Gly-Asp sequence on lung metastases decreased according to the decreasing repeat units of the Arg-Gly-Asp core sequence. Polymeric peptides with Arg-Gly-Asp entrapped within the liposome membranes also caused a remarkable reduction of metastatic colonies. In a spontaneous metastasis model, multiple i.v. administrations of poly(Arg-Gly-Asp) after tumor inoculation caused the significant reduction of metastatic colonies in the lung but did not affect the growth (size) of primary tumor. We found that the polymerization (multivalency) of the Arg-Gly-Asp core sequence was able to augment the inhibition of tumor lung metastases in experimental and spontaneous metastasis models as well as the cell-adhesive property more effectively than a monovalent unit of Arg-Gly-Asp peptide.

Synergism between human recombinant gamma-interferon and muramyl dipeptide encapsulated in liposomes for activation of antitumor properties in human blood monocytes.

Highly purified human blood monocytes, isolated by centrifugal elutriation under endotoxin-free conditions, were activated in vitro by combining subthreshold amounts of human recombinant gamma-interferon (r-IFN-gamma) and muramyl dipeptide (MDP) to become tumor cytotoxic against allogeneic A375 melanoma cells. Only intact r-IFN-gamma and MDP produced synergism for human monocyte activation. Neither pH 2-treated r-IFN-gamma and intact MDP nor heat-treated IFN-gamma and intact MDP, nor intact IFN-gamma and the biologically inactive stereoisomer of MDP, N-acetylmuramyl-D-alanyl-D-isoglutamine, produced activation of blood monocytes. The encapsulation of intact r-IFN-gamma and MDP within the same preparation of multilamellar liposomes was synergistic for monocyte activation. These data show that synergism for monocyte activation can be produced by human r-IFN-gamma and MDP produced synthetically can be simultaneously delivered to monocytes. Because both r-IFN-gamma and MDP can now be produced in large standardized quantities their synergism for activation of tumoricidal properties in human monocytes could be of clinical significance.

CD44 variants but not CD44s cooperate with beta1-containing integrins to permit cells to bind to osteopontin independently of arginine-glycine-aspartic acid, thereby stimulating cell motility and chemotaxis.

The expression of osteopontin (OPN), CD44 variants, and integrins has been correlated with tumorigenesis and metastasis. Here we show that these proteins cooperate to enhance cell motility. First, we demonstrate that several different CD44 variants bind to OPN in an arginine-glycineaspartic acid-independent manner, but that the standard form of CD44 does not. These CD44 variants bind to both the amino- and COOH-terminal portions of OPN independently of the arginine-glycine-aspartic acid sequence, suggesting that multiple domains on OPN can be bound by the CD44 variants. Antibodies directed against the integrin beta1 subunit are able to inhibit this binding. The binding of CD44 variants to OPN is significantly augmented by both anti-CD44s and anti-CD44v antibodies. This augmentation by anti-CD44 antibodies is OPN specific and, again, can be blocked by anti-beta1 antibodies. Finally, we show that OPN binding by CD44 variants/beta1-containing integrins promotes cell spreading, motility, and chemotactic behavior.

Inhibition of angiogenesis and intrahepatic growth of colon cancer by TAC-101.

We demonstrated in this study that inhibition of intra-hepatic growth of colon cancer by TAC-101 is mediated by inhibition of angiogenesis. In vitro experiments showed that TAC-101 inhibited the proliferation of murine hepatic sinusoidal endothelial (HSE) cells induced by coculture with murine colon 26-L5 (L5) cells. HSE cell proliferation was also enhanced by conditioned medium of L5 cells (CM-L5), and this enhancement of proliferation was abrogated by anti-vascular endothelial growth factor antibody. CM-L5 also induced in vitro tube formation of HSE cells on Matri-gel, and this activity of CM-L5 was abrogated by TAC-101 in a concentration-dependent manner. On the other hand, p.o. administration of TAC-101 inhibited tumor-induced angiogenesis in vivo and decreased the weights of L5 tumors in the mouse liver. Reverse transcriptase-PCR analysis using in vivo tumor tissue suggested that repression of vascular endothelial growth factor expression by TAC-101 was associated with the antiangiogenic activity. TAC-101 alone and 5-fluorouracil (5-FU)/D,L-leucovorin (LV) significantly inhibited the intrahepatic growth of L5 tumors (P = 0.002 and 0.001, respectively), whereas 5-FU alone did not (P = 0.088). When TAC-101 was administered with 5-FU/LV, marked enhancement of antitumor activity was observed (95% inhibition; P<0.001). This enhanced antitumor effect was also observed in experiments using Co-3 human colon adenocarcinoma. Concurrent treatment with TAC-101 and 5-FU/LV and sequential treatment with 5-FU/LV followed by TAC-101 resulted in significant augmentation of antitumor activity against Co-3 (overall P = 0.007 and 0.015, respectively). These findings indicate that TAC-101 inhibits tumor angiogenesis and suggest that it may be effective against hepatic metastasis of colon cancer.

Invasiveness of hepatocellular carcinoma cell lines: contribution of membrane-type 1 matrix metalloproteinase.

Intrahepatic metastasis is one of the malignant features of hepatocellular carcinoma (HCC). Matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (u-PA)/plasmin, are known to be associated with the invasive properties of various types of tumor cells. In this study, we examined which proteinases play a role in the metastatic invasion of human HCC cell lines. JHH-5 and JHH-6 cells constitutively expressed mRNAs for both membrane-type 1 matrix metalloproteinase (MT1-MMP) and u-PA and invaded through reconstituted MATRIGEL in vitro, whereas JHH-7 cells expressed u-PA mRNA but not MT1-MMP and did not invade. However, hepatocyte growth factor (HGF) induced MT1-MMP expression on the surface of JHH-7 cells and markedly increased invasiveness of JHH-7 in a concentration-dependent manner. Moreover, cleavage activity for pro-MMP-2 was induced in HGF-treated JHH-7 cells. MMP inhibitor, rather than serine proteinase inhibitor, potently inhibited HCC cell invasion. Intrahepatic injection of HCC cell lines into athymic nude mice caused visible intrahepatic metastases in vivo. Moreover, JHH-7 tumors showed expression of MT1-MMP mRNA, while in vitro cultured JHH-7 cells did not. These findings suggest that MT1-MMP plays an important role in the invasive properties of HCC cells, and that HGF modifies the invasive properties of noninvasive HCC cells.

Solitary lung tumors and their spontaneous metastasis in athymic nude mice orthotopically implanted with human non-small cell lung cancer.

We examined the tumorigenic and metastatic potentials of three human non-small cell lung cancer (NSCLC) cell lines, PC-14, A549 or Lu-99 cell lines suspended in Matrigel-containing phosphate-buffered saline were orthotopically implanted into the lungs of nude mice. The formation of a solitary tumor nodule in the lung was observed after the implantation of all cell lines. Intrapulmonary implantation of PC-14 or Lu-99 cells resulted in spontaneous distant metastases. In contrast, A549 cells caused multiple intrapulmonary metastases to the right and left lobes of the lung without producing visible lymphatic metastasis. We also investigated the expression of matrix metalloproteinases (MMPs), urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and c-MET in these cell lines in vitro and in vivo. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that the expression of MMP-2 and membrane-type 1 MMP (MT1-MMP) was elevated in PC-14 as compared with the other two cell lines. In contrast, stronger expression of c-MET was observed in A549 than in PC-14 or Lu-99. These results indicate that differential patterns of metastasis of lung cancer might be associated with differential expression of metastasis-associated molecules. Our orthotopic implantation models display clinical features resembling those of NSCLC, and may provide a useful basis for lung cancer research.

Quercetin inhibits the invasion and mobility of murine melanoma B16-BL6 cells through inducing apoptosis via decreasing Bcl-2 expression.

Quercetin has been known to have anti-tumor and anti-oxidation activities. In the present study, we have investigated its in vitro anti-metastatic activity. Quercetin inhibited the invasion and mobility of murine melanoma B16-BL6 cells in a dose-dependent manner but did not affect their adhesion to either laminin, fibronectin, or type VI collagen. Moreover, quercetin significantly inhibited the proliferation of B16-BL6 cells only in the case of time incubation longer than 48 h. Quercetin dose-dependently decreased the cell rates in S and G2-M phases of cell cycle. The effect of quercetin to cause a remarkable apoptosis of B16-BL6 cells was also demonstrated by flow cytometric assay as well as DNA fragmentation with a typical 180-bp ladder band in agarose electrophoresis and a quantitative analysis. Furthermore, quercetin markedly inhibited the expression of anti-apoptotic protein Bcl-2 but hardly influenced Bcl-XL. These results suggest that the inhibition of quercetin on invasiveness and migration of B16-BL6 cells are closely associated with the arrest of cell cycle as well as the induction of apoptosis by decreasing the Bcl-2 expression.

Inhibition by vasoactive intestinal polypeptide (VIP) of angiogenesis induced by murine Colon 26-L5 carcinoma cells metastasized in liver.

We investigated the effect of VIP on the liver metastases and angiogenesis by Colon 26-L5 carcinoma cells in mice. Daily systemic administration of VIP, beginning 3 days after tumor inoculation into a portal vein of mice, inhibited significantly the development of their liver metastases. Immunohistochemical staining for factor VIII-related antigen in the sections of liver metastases showed that the systemic administration of VIP caused significant prevention of angiogenesis within tumor masses. VIP (10-(10) to 10(-6) M) inhibited the invasion of reconstituted basement membrane (Matrigel) by hepatic sinusoidal endothelial (HSE) cells in a concentration-dependent manner in a Transwell chamber assay in vitro and achieved approximately 50% reduction of control at 10(-6) M. VIP (10(-6) M) also significantly suppressed the haptotactic migration of HSE cells to fibronectin, laminin or type I collagen substrates with a similar inhibition rate to the invasion assay. Exposure of VIP to HSE cells induced accumulation of intracellular cAMP in a concentration-dependent manner. The inhibitory effect of VIP (10(-6) M) on HSE cell migration was significantly abrogated in the presence of 3 x 10(-6) M H-89, a cAMP-dependent protein kinase inhibitor. VIP (10(-6) M) inhibited the morphogenesis of HSE cells into capillary-like structures on Matrigel-coated wells. VIP did not affect the proliferation of HSE cells and the production of gelatinases in HSE cells in vitro at the concentrations used in the invasion assay. These observations suggest that the anti-metastatic effect of VIP on liver metastases by Colon 26-L5 carcinoma cells in mice is partly due to the prevention of tumor angiogenesis probably through suppression of the motility of endothelial cells.

Social isolation stress augments angiogenesis induced by colon 26-L5 carcinoma cells in mice.

We have previously shown that tumor necrosis factor-alpha (TNF-alpha), which is an important angiogenesis-related factor, was over-secreted in male BALB/c mice under social isolation stress as compared with the control, and closely associated with a remarkable elevation of tumor invasion and metastasis of colon 26-L5 carcinoma cells. In the present study, we explored the effect of isolation stress on the angiogenesis caused by colon 26-L5 carcinoma cells in vivo and in vitro. Social isolation lead to the enhancement of tumor growth after intrahepatic implantation with a fragment of colon 26-L5 tumor. Angiogenic response (number of vessels oriented towards tumor mass) and tumor growth (size) were significantly increased in the socially isolated mouse relative to that in the group-housed mice. Furthermore, higher protein level of hepatic TNF-alpha was found in the stressed mice than that in the control. Expression of mRNA for vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were also elevated in the tumor regions and liver tissues of the stressed mice in comparison with that in group-housed mice. On the other hand, hepatic sinusoidal endothelial (HSE) cells treated with TNF-alpha exhibited a marked promotion of the migration, invasion, expression of mRNA for matrix metalloproteinase (MMP)-9, and tube-like formation, but no cytotoxicity against the cells in vitro. The above data suggest that the social isolation stress augmented the tumor-induced angiogenesis probably by up-regulating the angiogenesis-related factors, including TNF-alpha, VEGF and HGF, and consequently mediating the functions of endothelial cells such as migration, invasion, and tube-like formation.

Inhibition of tumor invasion and extracellular matrix degradation by ubenimex (bestatin).

We have investigated the effect of the immunomodulator ubenimex (hereafter referred to as bestatin) on the enzymatic degradation of the extracellular matrix by human renal cell carcinoma SN12M cells during the invasive process. The invasion of SN12M cells into reconstituted basement membrane (Matrigel) was inhibited by the presence of bestatin in a concentration-dependent manner. However, bestatin did not have any effect on tumor cell adhesion and migration to the extracellular matrices which may be involved in tumor cell invasion. Bestatin inhibited the degradation of type IV collagen by tumor cells, but not by tumor-conditioned medium (TCM), in a concentration-dependent manner. We also found that bestatin inhibited hydrolysing activities towards substrates of aminopeptidases in SN12M cells. Since bestatin was found to inhibit aminopeptidase activity, the inhibition of tumor invasion by bestatin is likely to be associated with its action as an enzyme inhibitor. Bestatin only slightly inhibited tumor cell plasmin activity, which can lead to the conversion of the latent collagenase to the active form, but this slight effect was not significant. The zymography of TCM from SN12M cells showed that the treatment of tumor cells with bestatin resulted in the disappearance of the 68 kDa type IV collagenase-enzyme level (active form) and slight reduction of the 72 kDa type IV collagenase-enzyme level (latent form). These results indicated that bestatin may inhibit tumor cell invasion through a mechanism involving its inhibitory action on aminopeptidases in tumor cells, suggesting that the aminopeptidase may partly be associated with the conversion of a latent form of type IV procollagenase to an active form or the secretion of the collagenases from tumor cells.

4-[3,5-Bis(trimethylsilyl)benzamido] benzoic acid (TAC-101) inhibits the intrahepatic spread of hepatocellular carcinoma and prolongs the life-span of tumor-bearing animals.

We examined the in vivo anti-tumor activity of the benzoic acid derivative, TAC-101 (4-[3,5-bis(trimethylsilyl)benzamido] benzoic acid), for intrahepatic spread of JHH-7 human hepatocellular carcinoma (HCC) cells and its mechanism of action. Oral administration of TAC-101 markedly inhibited liver tumor of JHH-7 cells and prolonged the life-span of tumor-bearing mice without affecting the body weight. The life-prolonging effect of TAC-101 was more effective than that of other anti-cancer agents including CDDP, 5-FU, and CPT-11 (T/C (%) of life-span; 181 to 219, 128, 133, and 142%, respectively). In vitro, TAC-101 at the concentration of more than 10 microM showed direct cytotoxicity against JHH-7 cells caused by induction of apoptosis. Hepatocyte growth factor (HGF) enhanced the invasive ability of JHH-7 cells without affecting the cell viability. Non-cytotoxic concentrations of TAC-101 inhibited the JHH-7 invasion induced by HGF and down-regulated the expression of c-MET protein in a concentration-dependent manner. In summary, these results suggest that TAC-101 would be useful for a new class of therapeutic agents and that it may improve the prognosis of patients with liver-tumors including metastasizing tumor and HCC.

Characterization of the invasive and metastatic phenotype in human renal cell carcinoma.

The purpose of these studies was to identify some characteristics of metastatic cells and deficiencies of non-metastatic cells in the heterogeneous SN12 human renal cell carcinoma. The SN12 parental line and several isolated variants with different metastatic potential were studied both in vivo and in vitro. We compared the ability of metastatic and non-metastatic cells to adhere to components of the extracellular matrix or to endothelial cells, to migrate and invade, to form multicell aggregates, to survive in the circulation, and to produce experimental and spontaneous lung metastases. In general, highly metastatic SN12 cells capable of producing spontaneous lung metastases demonstrated invasion through reconstituted basement membrane-coated filters; the cells also released diffusible collagenolytic activity into the culture medium that could enhance invasion by otherwise non-invasive and non-metastatic SN12 cells. In addition to enhanced invasion, metastatic cells produced more homotypic aggregation then non-metastatic cells and survived to produce experimental metastasis. Collectively, these data confirm that metastatic cells must complete all steps of the process; in this process, failure to produce metastasis is probably due to one or more deficiencies.

Inhibition of tumor invasion and metastasis by calcium spirulan (Ca-SP), a novel sulfated polysaccharide derived from a blue-green alga, Spirulina platensis.

We have investigated the effect of calcium spirulan (Ca-SP) isolated from a blue-green alga, Spirulina platensis, which is a sulfated polysaccharide chelating calcium and mainly composed of rhamnose, on invasion of B16-BL6 melanoma, Colon 26 M3.1 carcinoma and HT-1080 fibrosarcoma cells through reconstituted basement membrane (Matrigel). Ca-SP significantly inhibited the invasion of these tumor cells through Matrigel/fibronectin-coated filters. Ca-SP also inhibited the haptotactic migration of tumor cells to laminin, but it had no effect on that to fibronectin. Ca-SP prevented the adhesion of B16-BL6 cells to Matrigel and laminin substrates but did not affect the adhesion to fibronectin. The pretreatment of tumor cells with Ca-SP inhibited the adhesion to laminin, while the pretreatment of laminin substrates did not. Ca-SP had no effect on the production and activation of type IV collagenase in gelatin zymography. In contrast, Ca-SP significantly inhibited degradation of heparan sulfate by purified heparanase. The experimental lung metastasis was significantly reduced by co-injection of B16-BL6 cells with Ca-SP. Seven intermittent i.v. injections of 100 microg of Ca-SP caused a marked decrease of lung tumor colonization of B16-BL6 cells in a spontaneous lung metastasis model. These results suggest that Ca-SP, a novel sulfated polysaccharide, could reduce the lung metastasis of B16-BL6 melanoma cells, by inhibiting the tumor invasion of basement membrane probably through the prevention of the adhesion and migration of tumor cells to laminin substrate and of the heparanase activity.

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

Aminopeptidase N/CD13 is a Zn(2+)-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.

Regulation of activator protein-1 activity in the mediastinal lymph node metastasis of lung cancer.

Orthotopic implantation of a metastatic cell line of Lewis lung carcinoma (LLC-MLN), which was isolated by an in vivo selection method, resulted in greater metastatic growth in mediastinal lymph nodes as compared with that of the original LLC cells. LLC-MLN cells also had increased invasive ability and activator protein-1 (AP-1) transcriptional activity as compared with the original LLC cells. This is well consistent with the previously reported finding that overexpression of AP-1 is associated with lymphatic metastasis in lung cancer patients. Oral administration of curcumin, which downregulates AP-1 transcription, significantly inhibited the mediastinal lymph node metastasis of orthotopically implanted LLC cells in a dose-dependent manner, but did not affect the tumor growth at the implantation site. Combined treatment with curcumin and an anti-cancer drug, cis-diamine-dichloroplatinum (CDDP), resulted in a marked inhibition of tumor growth at the implanted site and of lymphatic metastasis, and a significant prolongation of the survival time. The downregulation of transcriptional AP-1 activity by curcumin as seen in the dual luciferase assay caused inhibition of LLC cell invasion through the repression of expression of the mRNAs for urokinase-type plasminogen activator (u-PA) and its receptor (u-PAR). Inhibition of AP-1 transcriptional activity may offer improved therapeutic efficacy for lung cancer patients with lymphatic metastasis.