Selective inhibition of TNF-alpha-induced activation of mitogen-activated protein kinases and metastatic activities by gefitinib.

We have reported that the selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib ('Iressa', ZD1839), suppressed intrahepatic metastasis of hepatocellular carcinoma CBO140C12 cells. In this study, we focused on the tumour necrosis factor-alpha (TNF-alpha) signalling pathways. Real-time reverse transcription-polymerase chain reaction showed that TNF-alpha mRNA was expressed in large quantities in the implanted tumour. Gefitinib inhibited EGF- but not hepatocyte growth factor (HGF)-induced activation of mitogen-activated protein kinase (MAPK) cascades, suggesting selectivity of the inhibitor. However, gefitinib inhibited the TNF-alpha-induced activation of MAPKs and Akt. In addition, TNF-alpha-induced metastatic properties including adhesion to fibronectin, mRNA expression of integrin alpha v, production of matrix metalloproteinase-9 and invasion were inhibited by gefitinib without affecting cell proliferation. Furthermore, the TNF-alpha-induced responses except for NF-kappaB activation were blocked by metalloprotease inhibitors, suggesting that gefitinib inhibited the transactivation of EGFR induced by TNF-alpha. These results suggest that the TNF-alpha signalling pathway is a possible target of gefitinib in suppressing the intrahepatic metastasis of hepatocellular carcinoma.

Port-site metastasis after CO2 pneumoperitoneum: role of adhesion molecules and prevention with antiadhesion molecules.

BACKGROUND: Port-site metastasis is a continuing problem in laparoscopic cancer surgery. To clarify the role of adhesion molecules in the development of port-site metastasis, particularly with regard to prevention, we performed experiments in which port-site metastasis was inhibited using antibodies against extracellular matrix proteins or the active Arg-Gly-Asp (RGD) peptide after CO2 pneumoperitoneum in a murine model. METHODS: We examined the development of port-site metastasis under the following conditions: (1) CO2 pneumoperitoneum with or without hyaluronic acid and anti-integrin or anti-CD44 antibody and (2) CO2 pneumoperitoneum and a RGD peptide or pseudo-RGD sequence peptide (FC-336). BALB/c mice ( n = 130) were injected with 5 x 10(5) human gastric cancer cells (MKN45) and either antibody or peptide, treated with CO2 pneumoperitoneum, and injected intraperitoneally with antibody or peptide for 5 days. Three weeks after CO2 pneumoperitoneum, the frequency and weight of port-site metastatic tumors were determined. RESULTS: Anti-integrin antibody significantly decreased the weight of port-site metastatic tumors without hyaluronic acid (control vs anti-integrin: 8.2 +/- 7.1 vs 3.6 +/- 4.5 mg; p < 0.05) but not the frequency of port-site metastases. With hyaluronic acid, the frequency of port-site metastasis and the weight of port-site metastatic tumors were significantly decreased both by anti-integrin and by anti-CD44 antibody (control vs anti-integrin and anti-CD44; 95% and 8.5 +/- 7.2 mg vs 50% and 3.1 +/- 4.3 mg and 55% and 3.3 +/- 5.1 mg, respectively; p < 0.05). RGD peptide and FC-336 also inhibited port-site metastasis in a dose-dependent manner. CONCLUSION: Cell adhesion molecules integrin and CD44 play an important role in the development of port-site metastasis after laparoscopic cancer surgery. Intraperitoneal injection of RGD peptide or pseudo-RGD sequence peptide (FC-336) can prevent port-site metastasis.

Anti-metastatic and anti-angiogenic activities of a new matrix metalloproteinase inhibitor, TN-6b.

We investigated the anti-metastatic and anti-angiogenic effects of TN-6b, a new broad-spectrum inhibitor of matrix metalloproteinases (MMPs), against Lewis lung carcinoma (LLC) and hepatic sinusoidal endothelial (HSE) cells. TN-6b potently inhibited the activities of MMP-2 and -9 secreted by LLC and HSE cells in a zymogram assay. TN-6b, at non-cytotoxic concentrations, caused a marked inhibition of invasion and migration of LLC, and tube-like formation of HSE cells. In contrast, TN-6d, an inactive enantiomer of TN-6b, did not inhibit the invasion and tube-like formation. Daily subcutaneous (s.c.) administration of TN-6b at doses of 30 and 60 mg/kg in mice resulted in a potent inhibition of tumour-induced angiogenesis of B16 melanomas and lymph node metastasis of LLC cells. In conclusion, TN-6b effectively inhibited lymph node metastasis of LLC cells through its anti-invasive and anti-angiogenic properties. These findings suggest that the MMP inhibition correlates well with its anti-angiogenic and anti-metastatic efficacy and TN-6b has the therapeutic potential to inhibit angiogenesis and metastasis in vivo and in vitro.

Effects of a new antiallergic drug, VUF-K-8788, on infiltration of lung parenchyma by eosinophils in guinea pigs and eosinophil-adhesion to human umbilical vein endothelial cells (HUVEC).

Airway inflammation and reversible airway obstruction are hallmarks of bronchial asthma. In this study, we investigated the effects of a new antiallergic drug, 7-[3-[4-(2-quinolinylmethyl)-1-piperazinyl]-propoxy]-2,3-dihydro-4H-1,4-benzothiazin-3-one (VUF-K-8788), on histopathological changes in lung parenchyma of guinea pigs during late-phase asthmatic reaction (LAR), and on eosinophil-adhesion to human umbilical vein endothelial cells (HUVEC). Repeated exposure to ovalbumin of sensitized guinea pigs induced inflammatory phenomena such as hyperplasia of airway epithelial cells, perivascular edema and infiltration of lung parenchyma by eosinophils. VUF-K-8788 inhibited these histopathological phenomena at 10 mg/kg p.o. Moreover, the eosinophil-adherence to HUVEC was inhibited by VUF-K-8788 at the concentration of 10-30 microM. In conclusion, this inhibitory effect of VUF-K-8788 on eosinophil-adherence might contribute to the prevention of LAR and infiltration by eosinophils in the experimental asthmatic model in guinea pigs.

New spirostanol steroids and steroidal saponins from roots and rhizomes of Dracaena angustifolia and their antiproliferative activity.

The MeOH extract of Nam ginseng (roots and rhizomes of Dracaena angustifolia) afforded nine new compounds, including three spirostanol sapogenins, named namogenins A-C (1-3), four spirostanol saponins, named namonins A-D (4-7), a furostanol saponin, named namonin E (8), and a pregnan glycoside, named namonin F (9), along with another eight known steroidal saponins (10-17). Their structures were determined on the basis of spectral analyses and chemical methods. All compounds were tested for their antiproliferative activity against murine colon 26-L5 carcinoma, human HT-1080 fibrosarcoma, and B-16 BL6 melanoma cells. Compounds 4, 5, and 10 showed potent antiproliferative activity against HT-1080 fibrosarcoma cells, having IC(50) values of 0.2, 0.3, and 0.6 microM, respectively, comparable to that of doxorubicin.

Social isolation stress impairs the resistance of mice to experimental liver metastasis of murine colon 26-L5 carcinoma cells.

Our previous study has demonstrated that the exposure of male BALB/c mice to social isolation stress caused a suppressed immune response and enhanced liver metastasis of colon 26-L5 carcinoma cells. To more precisely understand the influence of psychosocial factors on the metastatic process, here we have investigated the effect of social isolation stress on the vulnerability of the host to develop liver metastasis of colon 26-L5 cells, including the time span and incidence of metastatic formation, survival time and chemotherapy response. Isolation stress decreased the time period required for the metastasis formation relative to that in controls. On day 7 after the tumor injection, the 75% incidence of tumor metastasis in the stressed mice was 5 times the 15% incidence in the unstressed mice. When exposed to the challenge of lower cell numbers (0.025, 0.05, 0.1 x 10(4)/mouse) of colon 26-L5 cells, mice subjected to isolation stress developed an elevated incidence of metastasis (33.3, 66.6, and 100%, respectively) as compared with the controls (0, 33.3 and 50%, respectively). The survival time following the tumor inoculation was also shorter in the stressed mice (21.83 +/- 1.59d) than in the control mice (24.08 +/- 1.68 d). Furthermore, the response of liver metastasis to chemotherapy consisting of 2 mg/kg cisplatin (CDDP) was worse in the stressed mice than that in unstressed mice. These findings suggested that social isolation stress could significantly impair the resistance of mice to the development of metastasis.

Accumulation of extracellular matrix in the liver induces high metastatic potential of hepatocellular carcinoma to the lung.

The liver undergoes pathogenic changes such as hepatitis, fibrosis and cirrhosis under continuous stimulation by hepatitis virus or alcohol intake, leading to the development of hepatocellular carcinoma. The metastatic potential of HCC can be positively or negatively regulated by pathogenic alterations of liver. We investigated whether the metastatic abilities of HCC after orthotopic implantation can be influenced in the fibrotic liver by continuous injection of carbon-tetrachloride (CCl4) for seven weeks. The incidence of lung metastasis after orthotopic implantation of murine HCC (CBO140C12) fragments into CCl4-treated livers was higher than into normal livers. The amount of mRNA for MMP-2 increased in the CCl4-treated livers as compared with normal livers, and CBO140C12 cells constitutively expressed mRNA for MT1-MMP in early amplification cycles by RT-PCR. In addition, we found that the culture of CBO140C12 cells on the substrates pre-coated with ECM components increased the expression of MMP-2 mRNA. Thus, enhanced incidence of lung metastasis in the fibrotic liver might be partly due to: i) over-expression of MMP-2 in the fibrotic liver in cooperation with MT1-MMP on the CBO140C12 cell surface, ii) over-expression of MMP-2 in CBO140C12 cells, possibly mediated by the interaction of tumor cells (surface integrins) with accumulated ECM in the fibrotic liver. This is the first report showing that increase of MMP-2 in the fibrotic liver can influence the metastatic potential of HCC cells.

Critical contribution of IFN-gamma and NK cells, but not perforin-mediated cytotoxicity, to anti-metastatic effect of alpha-galactosylceramide.

The glycolipid alpha -galactosylceramide (alpha -GalCer), which is presented by CD1d and specifically activates Valpha 14 NKT cells, exerts a potent anti-metastatic effect when administered in vivo. In this study, we demonstrated that alpha -GalCer administration led to rapid elimination of NKT cells by apoptosis in the liver and spleen, after they produced IFN-gamma and IL-4. In contrast, a more prolonged secretion of IFN-gamma was observed by liver and splenic NK cells after alpha -GalCer administration. Cytotoxic activity of liver mononuclear cells was not augmented 3h after alpha -GalCer administration, but was increased at 24 h when NKT cells were mostly depleted. The alpha -GalCer-induced cytotoxic activity was abolished in IFN-gamma -deficient and NK cell-depleted mice as well as CD1-deficient mice, suggesting that the alpha -Galcer-induced cytotoxicity was mainly mediated by IFN-gamma -activated NK cells. While the alpha -GalCer-induced cytotoxicity in vitro was mostly perforin dependent, anti-metastatic effect of alpha -GalCer was impaired in NK cell-depleted or IFN-gamma -deficient mice but not in perforin-deficient mice. Collectively, these results indicated that the anti-metastatic effect of alpha -GalCer is mainly mediated by NK cells, which are activated secondarily by IFN-gamma produced by alpha -GalCer-activated NKT cells, in a perforin-independent manner.

Differential regulation of Th1 and Th2 functions of NKT cells by CD28 and CD40 costimulatory pathways.

Valpha14 NKT cells produce large amounts of IFN-gamma and IL-4 upon recognition of their specific ligand alpha-galactosylceramide (alpha-GalCer) by their invariant TCR. We show here that NKT cells constitutively express CD28, and that blockade of CD28-CD80/CD86 interactions by anti-CD80 and anti-CD86 mAbs inhibits the alpha-GalCer-induced IFN-gamma and IL-4 production by splenic Valpha14 NKT cells. On the other, the blockade of CD40-CD154 interactions by anti-CD154 mAb inhibited alpha-GalCer-induced IFN-gamma production, but not IL-4 production. Consistent with these findings, CD28-deficient mice showed impaired IFN-gamma and IL-4 production in response to alpha-GalCer stimulation in vitro and in vivo, whereas production of IFN-gamma but not IL-4 was impaired in CD40-deficient mice. Moreover, alpha-GalCer-induced Th1-type responses, represented by enhanced cytotoxic activity of splenic or hepatic mononuclear cells and antimetastatic effect, were impaired in both CD28-deficient mice and CD40-deficient mice. In contrast, alpha-GalCer-induced Th2-type responses, represented by serum IgE and IgG1 elevation, were impaired in the absence of the CD28 costimulatory pathway but not in the absence of the CD40 costimulatory pathway. These results indicate that CD28-CD80/CD86 and CD40-CD154 costimulatory pathways differentially contribute to the regulation of Th1 and Th2 functions of Valpha14 NKT cells in vivo.

Antiproliferative activity of diarylheptanoids from the seeds of Alpinia blepharocalyx.

The 95% EtOH extract of the seeds of Alpinia blepharocalyx (Zingiberaceae) showed significant antiproliferative activity towards human HT-1080 fibrosarcoma and murine colon 26-L5 carcinoma cells. Chemical investigation of the extract led to the isolation of forty-four new (1-44) and one known (45) diarylheptanoids, eleven phenolic compounds (46-56) together with beta-sitosterol glucoside (57). Almost all the isolated compounds showed significant antiproliferative activity in a concentration-dependent manner. Among the compounds, epicalyxin F (17) exhibited the most potent activity against the proliferation of colon 26-L5 carcinoma cells with an ED50 value of 0.89 microM, while calyxin B (2) exhibited the most potent activity against human HT-1080 fibrosarcoma cells with an ED50 value of 0.69 microM. Moreover, calyxins B (2) and K (11), epicalyxins F (17), I (20) and K (22), 6-hydroxycalyxin F (25), blepharocalyxin B (27) and mixtures of 7 and epicalyxin G (18) and of calyxin J (10) and epicalyxin J (21) possessed more potent activity than a clinically used anticancer drug, 5-fluorouracil, towards HT-1080 fibrosarcoma cells. Analysis of the structure activity relationship suggested that the position of the attachment of a chalcone or a flavanone moiety does not affect the activity, although their presence in association causes a substantial enhancement of the antiproliferative activity. Moreover, the conjugated double bond of the chalcone moiety and the phenolic hydroxyl group potentiate the antiproliferative activity of the compounds.

Inhibitory effect of berberine on the mediastinal lymph node metastasis produced by orthotopic implantation of Lewis lung carcinoma.

We examined the effect of berberine, a major component with anti-fungal properties contained in Coptidis Rhizoma and Phellodendri Cortex, on the lymph node metastasis of murine lung cancer. Oral administration of berberine for 14 days significantly inhibited the spontaneous mediastinal lymph node metastasis produced by orthotopic implantation of Lewis lung carcinoma (LLC) into the lung parenchyma in a dose-dependent manner, but did not affect the tumor growth at the implantation site of the lung. Combined treatment with berberine and an anti-cancer drug, CPT-11, resulted in a marked inhibition of tumor growth at the implantation site and of lymphatic metastasis, as compared with either treatment alone. Anti-activator protein-1 (anti-AP-1) transcriptional activity of non-cytotoxic concentrations of berberine caused the inhibition of the invasiveness of LLC cells through the repression of expression of urokinase-type plasminogen activator (u-PA).

Intrahepatic metastasis by orthotopic implantation of a fragment of murine hepatoma and its related molecules.

Intrahepatic metastasis is a major modality in the recurrence of hepatoma. Establishment of the intrahepatic metastasis model would be useful for evaluating new anticancer therapies and analyzing the molecular mechanisms of tumor metastasis. Orthotopic implantation of a fragment of CBO140C12 hepatoma into the liver resulted in the formation of a solitary tumor nodule and its intrahepatic metastasis. In contrast, implantation of ADras3 cancer cells did not show any metastasis on day 21. CBO140C12 cells showed enhancement of the invasive, adhesive and migratory capabilities, as compared with ADras3 cells. Furthermore, mRNA expression and gelatinolytic activity of MMP-9 were detected in CBO140C12 cells, and the expression of mRNA for MT1-MMP in CBO140C12 cells was greater than that in ADras3 cells. Thus, intrahepatic metastasis of CBO140C12 tumor might be involved in the enhancement of the invasiveness of tumor cells via marked expression of MMP-9 and MT1-MMP.

Effect of prostatic neuropeptides on migration of prostate cancer cell lines.

BACKGROUND: A previous study by the same authors demonstrated that among various neuropeptides in the prostate, calcitonin gene-related peptide (CGRP) and gastrin-releasing peptide (GRP) increased the invasive capacity of PC-3 prostate cancer cells through enhancement of cell motility, while substance P (SP) inhibited the invasiveness through suppression of motile response. METHODS: The effect of 10 kinds of neuropeptides were investigated, including CGRP, GRP, SP, neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), calcitonin (CT), leucine-enkephalin (L-ENK), methionine-enkephalin (M-ENK), glucagon and parathyroid hormone-related protein (PTH-rP), on the invasion of DU-145 prostate cancer cells through a reconstituted basement membrane (Matrigel) and the haptotactic migration of DU-145, TSU-pr1 and LNCaP prostate cancer cells using a Transwell cell culture chamber assay. RESULTS: It was found that GRP, CGRP and PTH-rP increased the invasive capacity of tumor cells. In contrast, SP, VIP, CT, L-ENK, M-ENK, NPY and glucagon had no significant effect. These three neuropeptides also increased the haptotactic migration of tumor cells to fibronectin. In addition VIP, CGRP and GRP increased the haptotactic migration of LNCaP prostate cancer cells and GRP and PTH-rP increased the migration of TSU-pr1 cells. CONCLUSION: The results indicated that some prostatic neuropeptides increased the invasive potential of prostate cancer cells partially through enhancement of cell motility.

A Kampo formulation: Byakko-ka-ninjin-to (Bai-Hu-Jia-Ren-Sheng-Tang) inhibits IgE-mediated triphasic skin reaction in mice: the role of its constituents in expression of the efficacy.

We have demonstrated that oral administration of a Kampo formulation, Byakko-ka-ninjin-to (Bai-Hu-Jia-Ren-Sheng-Tang), inhibited IgE-mediated triphasic skin reaction, including immediate phase response (IPR), late phase response (LPR) and very late phase response (vLPR), in passively sensitized mice with anti-DNP IgE antibody. Variant formulations of Byakko-ka-ninjin-to without Gypsum Fibrosum (Sekko), Glycyrrhizae Radix (Kanzo) or Oryzae Semen (Kobei) attenuated the inhibitory effect as compared with that of Byakko-ka-ninjin-to. The decreased effect of Byakko-ka-ninjin-to without Kanzo was restored by the addition of Kanzo to the variant formulations before oral administration, while the decreased effect of Byakko-ka-ninjin-to without Sekko could not be recovered by the addition of Sekko. Comparison of HPLC profiles of variant formulations without one crude drug with that of original Byakko-ka-ninjin-to revealed that some peaks could be detected only when five constituent crude drugs were simultaneously present during the preparation of Byakko-ka-ninjin-to formulation. Since elimination of Sekko from the Byakko-ka-ninjin-to constituents attenuated the efficacy although it did not show any activity per se, mutual interaction of Sekko with other constituents during the preparation may result in the production of new components. These findings suggest that the effect of Byakko-ka-ninjin-to formulation on cutaneous inflammatory disease can differ from the sum of the effect of the individual constituents.

Effect of a matrix metalloproteinase inhibitor (ONO-4817) on lung metastasis of murine renal cell carcinoma.

We examined the anti-metastatic effect of a newly developed inhibitor of synthetic matrix metalloproteinase (MMP), ONO-4817, on experimental pulmonary metastasis of murine renal cell carcinoma (Renca) cells and on tumor cell invasion, through reconstituted basement membrane (Matrigel) in vitro using the same cells. Oral administration of ONO-4817 (50-200 mg/kg/day) to Renca-bearing mice resulted in a dose-dependent inhibition of lung metastasis without a loss of body weight. ONO-4817 at the high dose of 200 mg/kg showed a tendency to prolong the survival of the mice. We also found that oral administration of ONO-4817 significantly inhibited the angiogenic response (number of vessels oriented towards the tumor mass) and the growth of tumors inoculated i.d. in syngeneic mice. In addition, ONO-4817, at non-cytotoxic concentrations of less than 10 microM, caused a marked inhibition of the invasion of Renca cells as compared to the vehicle control. Gelatin zymography revealed that ONO-4817 inhibited the enzymatic activity of MMP-2 produced by Renca cells in a concentration-dependent manner. In conclusion, ONO-4817 effectively inhibited lung metastasis of Renca cells through its anti-invasive and anti-angiogenic properties. These results suggest that use of the MMP inhibitor (MMPI) ONO-481 7 may provide a therapeutic basis for preventing lung recurrence and metastasis of renal cell carcinoma.

Effect of interferon-alpha A/D in combination with the Japanese and Chinese traditional herbal medicine juzen-taiho-to on lung metastasis of murine renal cell carcinoma.

Several studies have shown that the Kampo medicine Juzen-taiho-to (Si-Quan-Da-Bu-Tang in Chinese) has various biological activities, including anti-tumor effects when combined with surgical excision or with chemotherapeutic drugs. Here we investigated the effect of combined therapy with interferon (IFN)-alpha A/D and Juzen-taiho-to on experimental lung metastasis of murine renal cell carcinoma (Renca) cells. Five consecutive administrations of IFN-alpha A/D to Renca-bearing mice resulted in dose-dependent inhibition of lung metastasis. IFN-alpha A/D at the dose of 100,000 IU/mouse significantly inhibited the metastasis, but a marked loss of body weight was observed during and after the administration. In contrast, oral administration of Juzen-taiho-to (50 mg/mouse) alone tended to inhibit the metastasis, but the effect was not statistically significant. The combination treatment of suboptimal doses of IFN-alpha A/D and Juzen-taiho-to markedly augmented the antimetastatic effect without causing any loss of body weight, as compared with either treatment alone. Similar results were also obtained by treatment with IFN-gamma in combination with Juzen-taiho-to. Clinically, immunotherapy with IFNs has been primarily approved for the treatment of patients with metastatic renal cell carcinoma, but sufficient efficacy has not yet been obtained. Therefore, the combination of IFNs with Juzen-taiho-to may provide a means to increase the therapeutic potential of IFNs and to decrease their toxicity for the treatment of metastatic renal cell carcinoma.

Constituents of the Vietnamese medicinal plant Orthosiphon stamineus.

From the MeOH extract of the aerial part of Vietnamese Orthosiphon stamineus, five new isopimarane-type diterpenes [orthosiphols F-J (1-5)] and two new diterpenes [staminols A (6) and B (7)] with a novel carbon-framework, to which we proposed the name "staminane", and three new highly-oxygenated staminane-type diterpenes [staminolactones A (8) and B (9) and norstaminol A (10)1 were isolated. Moreover, staminolactone A (8) is 8,14-secostaminane-type and staminolactone B (9) is 13,14-secostaminane-type, while norstaminol A (10) is 14-norstaminen-type. Together with these new diterpenes, sixteen known compounds were also isolated and identified to be: 7,3',4'-tri-O-methylluteolin (11), eupatorin (12), sinensetin (13), 5-hydroxy-6,7,3',4'-tetramethoxyflavone (14), salvigenin (15), ladanein (16), tetramethylscutellarein (17), 6-hydroxy-5,7,4'-trimethoxyflavone (18), vomifoliol (19), aurantiamide acetate (20), rosmarinic acid (21), caffeic acid (22), oleanolic acid (23), ursolic acid (24), betulinic acid (25), and beta-sitosterol (26). All the isolated compounds were tested for their cytotoxicity towards highly liver metastatic murine colon 26-L5 carcinoma cells, and the new diterpenes, except for 4, and flavonoids (11, 12, 16, 18) showed cytotoxicity with an ED50 value between 10 and 90 microg/ml.

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.

Tumor invasion is inhibited by phosphorylated ascorbate via enrichment of intracellular vitamin C and decreasing of oxidative stress.

Tumor metastasis and invasion were shown to be inhibited by the 2-O-phosphorylated form (Asc2P) of L-ascorbic acid (Asc); intact Asc did not inhibit tumor invasion when added once, but appreciably inhibited it upon repeated addition. The anti-metastatic effect is attributable to a marked enrichment of intracellular Asc by Asc2P, subsequently dephosphorylated. Asc2P scavenged most of the intracellular reactive oxygen species (ROSin), and notably inhibited production of matrix metalloproteases and cell motility. ROSin was decreased by Asc2P more markedly than by Asc added once. Thus, involvement of ROSin in tumor invasion and a potent anti-metastatic therapy by ROSin-decreasing agents are suggested.

Characterization of a liver metastatic variant of murine K1735M2 melanoma cells.

Intraportal vein injection of highly metastatic K1735M2L5 cells consistently resulted in liver metastases (increases in the number of tumor nodules in the liver), whereas inoculation of K1735M2 cells rarely did so. K1735M2L5 cells invaded the basement membrane Matrigel in greater numbers than did K1735M2 cells, suggesting that the metastatic potential of K1735M2L5 cells is partly related to enhanced invasive properties. The adhesion to Matrigel- and laminin-coated substrates was enhanced in K1735M2L5 cells. The up-regulated expression of VLA-4 and VLA-6 on the surface of K1735M2L5 cells was detected by flow cytometry. The RT-PCR and gelatin zymography study revealed that the secretion of MMP-2 was higher in K1735M2L5 cells than in K1735M2 cells. These results indicate that the invasive ability of K1735M2L5 cells may also be attributed to enhanced gelatinolytic activity as well as adhesiveness. K1735M2L5 cells grew more rapidly than K1735M2 cells and showed fibroblastoid morphology with loose cell-cell contacts as compared with K1735M2 cells. Thus, experimental models using highly metastatic K1735M2L5 cells would be useful for analyzing the molecular mechanism of tumor metastasis and for evaluating the efficacy of treatments for metastases which may have already occurred at the time of the diagnosis.