Details of a Researcher - Fujii, Hideki

Papers - Fujii, Hideki

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

T Mishima, J Murata, M Toyoshima, H Fujii, M Nakajima, T Hayashi, T Kato, I Saiki

Clinical & experimental metastasis 16 ( 6 ) 541 - 50 1998.08

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0262-0898

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.

A new pseudo-peptide analogue of the Arg-Gly-Asp (RGD) sequence inhibits liver metastasis of colon 26-L5 carcinoma cells.

Y Ohnishi, H Fujii, K Murakami, T Sakamoto, K Tsukada, M Fujimaki, M Kojima, I Saiki

Cancer letters 124 ( 2 ) 157 - 63 1998.02

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0304-3835

We have investigated the effect of the pseudo-peptide analogue (FC-336) of the Arg-Gly-Asp (RGD) sequence in a liver metastasis model by the inoculation of a highly liver-metastatic cell line of colon 26 carcinoma (colon 26-L5) into the portal vein of BALB/c mice. The intraportal injection of colon 26-L5 cells with FC-336 resulted in a marked suppression of liver metastatic colonies in a dose-dependent manner and it reduced the liver weights to a normal level. However, the co-injection of tumor cells with a high dose of RGDS tetrapeptide led to a slight inhibition of liver metastasis. The multiple i.v. administration of FC-336 after tumor inoculation as well as the injection of FC-336 with tumor cells caused significant inhibition of experimental metastasis in the liver. The multiple i.v. administration of the RGDS peptide did not show any inhibitory activity. FC-336 significantly enhanced the survival rate of mice compared with untreated controls when injected intraportally with tumor cells or when intravenously administered after tumor inoculation. Zymography analysis showed that FC-336 inhibited the degradation of gelatin substrate by matrix metalloproteinases (MMPs) produced by colon 26-L5 cells, while RGDS peptide did not affect the enzymatic degradation. These findings clearly indicate that the pseudo-peptides of the RGD sequence (FC-336) have a potent inhibitory activity on liver metastasis of colon 26-L5 carcinoma cells.

Vaccination with B7-1+ tumor and anti-adhesion therapy with RGD pseudo-peptide (FC-336) efficiently induce anti-metastatic effect.

H Fujii, M Inobe, Y Hayakawa, F Kimura, M Murakami, Y Onishi, I Azuma, T Uede, I Saiki

Clinical & experimental metastasis 16 ( 2 ) 141 - 8 1998.02

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0262-0898

We have previously shown that expression of costimulatory ligand B7-1 on MHC class I+ tumor cells (B16-BL6 melanoma) resulted in marked reduction of lung metastasis caused by i.v. injection into immunocompetent syngeneic mice and led to induction of immunity to the challenge by the parental B7-1 negative tumor. Here we investigated the effectiveness of irradiated B7-1 transfected tumor cells as a vaccine on established tumor metastasis and whether or not expression of B7-1 molecule on tumor cells in combination with administration of anti-adhesion peptide FC-336 can augment the antimetastatic efficacy. Immunization with X-irradiated B7-1 transfectants after i.v. injection of B7-1- parental B16-BL6 cells was effective in inhibiting lung metastasis. We also found that vaccination with irradiated B7-1 transfectants after excision of primary tumor on day 21 resulted in significant inhibition of spontaneous lung metastasis by intrafootpad injection of viable parental B16-BL6 melanoma, as compared with the untreated control. However, immunizing twice with mock transfectants did not affect inhibition of spontaneous lung metastasis of wild-type tumors. On the other hand, multiple administration of a pseudo-peptide of RGD sequence (FC-336) after tumor inoculation inhibited spontaneous lung metastasis through the interference of tumor invasion, migration and adhesion. Combined treatment of B7-1 transfected tumor vaccine and anti-adhesive therapy with FC-336 led to the augmentation of the antimetastatic effect in both experimental and spontaneous metastasis models, as compared with either treatment alone. B7-1- and FC-336-mediated inhibition of tumor metastasis may be mediated by different mechanisms at various steps of metastasis, based on the regulation (promotion or inhibition) of tumor interaction with host cells and components.

Oral administration of a Kampo (Japanese herbal) medicine Juzen-taiho-to inhibits liver metastasis of colon 26-L5 carcinoma cells.

Y Ohnishi, H Fujii, Y Hayakawa, R Sakukawa, T Yamaura, T Sakamoto, K Tsukada, M Fujimaki, S Nunome, Y Komatsu, I Saiki

Japanese journal of cancer research : Gann 89 ( 2 ) 206 - 13 1998.02

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0910-5050

We have investigated the inhibitory effect of oral administration of Juzen-taiho-to, a Kampo Japanese herbal medicine, on liver metastasis by the inoculation of a liver-metastatic variant (L5) of murine colon 26 carcinoma cells into the portal vein. Oral administration of Juzen-taiho-to for 7 days before tumor inoculation resulted in dose-dependent inhibition of liver tumor colonies and significant enhancement of survival rate as compared with the untreated control, without side effects. We also found that liver metastasis of L5 cells was enhanced in BALB/c mice pretreated with anti-asialo GM1 serum or 2-chloroadenosine, and in BALB/c nu/nu mice, compared to normal mice. This indicates that NK cells, macrophages, and T-cells play important roles in the prevention of metastasis of tumor cells. Juzen-taiho-to significantly inhibited the experimental liver metastasis of colon 26-L5 cells in mice pretreated with anti-asialo GM1 serum and untreated normal mice, whereas it did not inhibit metastasis in 2-chloroadenosine-pretreated mice or T-cell-deficient nude mice. Oral administration of Juzen-taiho-to activated peritoneal exudate macrophages (PEM) to become cytostatic against the tumor cells. These results show that oral administration of Juzen-taiho-to inhibited liver metastasis of colon 26-L5 cells, possibly through a mechanism mediated by the activation of macrophages and/or T-cells in the host immune system. Thus, Juzen-taiho-to may be efficacious for the prevention of cancer metastasis.

A new pseudo-peptide of Arg-Gly-Asp (RGD) with inhibitory effect on tumor metastasis and enzymatic degradation of extracellular matrix.

H Fujii, N Nishikawa, H Komazawa, M Suzuki, M Kojima, I Itoh, A Obata, K Ayukawa, I Azuma, I Saiki

Clinical & experimental metastasis 16 ( 1 ) 94 - 104 1998.01

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0262-0898

A series of pseudo-peptide analogs of the Arg-Gly-Asp (RGD) sequence of fibronectin have been synthesized, and their anti-metastatic effects in mice and inhibitory effects on tumor cell invasion in vitro have been examined. The partially modified retro pseudo-peptide of RGD, Rrev-COCH2CO-D (FC-63), was more effective in inhibiting tumor metastasis than the original RGDS peptide. Replacement of the malonyl moiety of FC-63 with a carboxyethylene linkage (Rrev-COCH2CH2-D, FC-303 ) achieved more potent inhibition of lung metastasis of melanoma cells than FC-63. Among the analogs, FC-336, a p-xylylendiamine derivative having two FC-303 moieties, showed the most potent inhibitory effect on experimental lung metastasis produced by i.v. co-injection with B16-BL6 melanoma or colon 26 M3.1 cells in a dose-dependent manner. Multiple administrations of FC-336 after tumor inoculation also showed efficient therapeutic potency against spontaneous lung metastasis of B16-BL6 melanoma in mice. Furthermore, FC-336 effectively inhibited the invasion, migration and adhesion of tumor cells in vitro, but its inhibitory effects were not more than those of RGDS peptide. Zymography analysis revealed that FC-336 inhibited the degradation of gelatin substrate by matrix metalloproteinases (MMPs) produced by tumor cells, while the RGDS peptide did not affect the enzymatic degradation. These findings indicate that the pseudo-peptides of the RGD sequence, possessing the inhibitory property of the degradation by MMPs differently from original RGD-containing peptides, may be advantageous and useful in preventing tumor metastasis.

LEA-1/ICAM-1 interaction is essentially involved in the pathogenesis of delayed-type hypersensitivity-induced liver injury to picryl chloride.

Q Xu, J Jiang, J Cao, F Wu, H Fujii, I Saiki

Life sciences 62 ( 15 ) 1281 - 92 1998

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0024-3205

The kinetics of lymphocyte function associated antigen 1 (LFA-1) expression on spleen cells (SPC) and liver non-parenchymal cells (NPC), and intercellular adhesion molecule 1 (ICAM-1) expression on hepatocytes (HC) was examined in acute liver injury mice induced by a DTH reaction to picryl chloride (PCl). The peak expression of LFA-1 on SPC was seen at 6 hr after eliciting liver injury, and then that of LFA-1 on NPC and ICAM-1 on HC appeared at 12 hr. Thereafter, the serum ALT elevation reached to a peak at 18 hr. A splenectomy before the PCl elicitation significantly reduced the ALT elevation. Both SPC and NPC from liver injury mice induced a remarkable release of ALT from HC in vitro, in parallel with their LFA-1 expression. The pre-treatment of NPC or SPC with anti-LFA-1 mAb, irrespective of the presence of complement, completely blocked the ALT release. Also, when HC was prebound with anti-ICAM-1 mAb, neither NPC nor SPC showed a cytotoxicity against the HC. Furthermore, the treatment of NPC with either anti-Thy1.2 or anti-CD4 mAb in the presence but not absence of complement, showed a complete abolishment of ALT release. Anti-CD8 mAb plus complement also tended to inhibit ALT release. The twofold increase in CD4+ LFA-1+ and mild increase in CD8+ LFA-1+ populations were also confirmed in NPC at 12 hr. These results suggest that PCl elicitation in liver may trigger an increased expression of LFA-1 on SPC and NPC and ICAM-1 on HC. LFA-1/ICAM-1 interaction between liver-infiltrating NPC, mainly including CD4+ and CD8+ T cells, and HC may be an essential step for the hepatocyte damage in PCl-DTH liver injury.

Anti-invasive activity of synthetic serine protease inhibitors and its combined effect with a matrix metalloproteinase inhibitor.

T Ikeda, K Murakami, Y Hayakawa, H Fujii, M Ohkoshi, I Saiki

Anticancer research 18 ( 6A ) 4259 - 65 1998

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0250-7005

Tumor invasion into the extracellular matrix (ECM) and basement membrane (BM) is a crucial step of tumor metastasis. In order to investigate the possible therapeutic procedure for the tumor invasion, we investigated the anti-invasive activities of several synthetic serine protease inhibitors. FOY-305, a serine protease inhibitor, showed no cytotoxic activity against human HT-1080 fibrosarcoma cells at concentrations ranging from 0.1 to 100 micrograms/ml, while its analogs ONO-3403 and FO-349 showed slight cytotoxic activities at the concentration of 100 micrograms/ml. These compounds inhibited the activity of urokinase-type plasminogen activator (u-PA) which is one of serine proteases and considered to be associated with tumor invasion and metastasis in fibrin zymography. FOY-305 more potently inhibited the invasion of HT-1080 cells into the reconstituted BM Matrigel, as well inhibited u-PA activity, compared with ONO-3403 and FO-349. These results suggest that the anti-invasive activity of these compounds is consistent with their anti-fibrinolytic activities. In addition, the combined treatment of FOY-305 with FC-336 processing anti-invasive and anti-MMP properties resulted in marked enhancement of anti-invasive activity. In conclusion, FOY-305 inhibited the invasion of tumor cells through interference with the u-PA activity of tumor cells, and this inhibitory activity was augmented by the combination with a MMP inhibitor.

Antimetastatic effect by Juzen-taiho-to in the liver metastasis model of colon 26-L5 carcinoma

Ohnishi Y, Fujii H, Kimura F, Hayakawa Y, Sakukawa R, Murata J, Sakamoto T, Tazawa K, Fujimaki M, Saiki I

Biotherapy 12 ( 1 ) 187 - 189 1998

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted

Effect of anti-adhesion therapy by a new pseudo-peptide of arg-gly-asp (RGD) on tumor metastasis

Ohnishi Y, Fujii H, Murata J, Sakamoto T, Tazawa K, Fujimaki M, Saiki I

Jpn J Gastroenterological Surgery 31 ( 4 ) 1004 - 1009 1998

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted

Antimetastatic and antitumor effect of a recombinant human tissue inhibitor of metalloproteinases-2 in murine melanoma models.

T Oku, N Ata, K Yonezawa, H Tokai, H Fujii, A Shinagawa, E Ohuchi, I Saiki

Biological & pharmaceutical bulletin 20 ( 8 ) 843 - 9 1997.08

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0918-6158

Tumor metastasis into distinct organs and tissues, of which many patients with malignancies die, is regulated in multiple steps. Using a murine metastasis model, in which highly metastatic B16-BL6 melanoma cells were inoculated i.v. into syngeneic C57BL/6 mice, the administration of a recombinant human tissue inhibitor of metalloproteinases-2 (r-hTIMP-2) once a day on days -1 to 3 after the implantation significantly inhibited the formation of metastatic foci in the lungs. The antimetastatic effect of r-hTIMP-2 was detected irrespective of administration route [i.v., i.p., s.c., and i.m. routes] and in a dose-dependent manner. The i.m.-injection of r-hTIMP-2 during the early phase after tumor inoculation is suggested to be essential for antimetastasis. In another model using spontaneously metastasing B16-BL6 cells, multiple i.m.-injections of r-hTIMP-2 also resulted in a reduced but not statistically significant number of pulmonary metastases. In addition to these antimetastatic effects, a slight inhibitory effect on tumor cell growth was observed in vitro and in vivo. In conclusion, the antimetastasis by r-hTIMP-2 may be due to inhibition of the degradation of the extracellular matrix by matrix metalloproteinases (MMPs) and, in part, to the suppression of the tumor cell growth.

Alpha-melanocyte-stimulating hormone blocks invasion of reconstituted basement membrane (Matrigel) by murine B16 melanoma cells.

J Murata, K Ayukawa, M Ogasawara, H Fujii, I Saiki

Invasion & metastasis 17 ( 2 ) 82 - 93 1997

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0251-1789

We have examined the effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on invasive ability of murine melanoma cell lines with different metastatic potential in a Matrigel invasion assay. alpha-MSH potently blocked the invasion of B16-BL6 cells with highly metastatic potential in a concentration-dependent manner, whereas it was less effective in inhibiting the invasion of weakly metastatic B16-F1 cells. Pretreatment of B16-BL6 cells with alpha-MSH resulted in a decrease of the adhesiveness to fibronectin and laminin substrates in a time-dependent fashion. As assessed by zymographic analysis, alpha-MSH partially inhibited the production of matrix metalloproteinase (MMP)-2 and -9 from both cell lines to a similar degree without affecting the degradative activity of these MMPs. alpha-MSH was more potent in inhibiting the migration of B16-BL6 cells towards both fibronectin- and laminin-coated substrates than that of B16-F1 cells. The growth and morphology of B16-BL6 cells were not changed after a 7-day incubation with alpha-MSH. The number of lung tumor colonies markedly decreased when B16-BL6 cells were coinjected intravenously with 10(-6) M alpha-MSH. However, alpha-MSH had no effect on the experimental lung metastases by B16-F1 cells. These results suggest that alpha-MSH suppressed the invasive and metastatic properties of B16 melanoma cells, and the degree of inhibition was associated with metastatic potential of B16 melanoma cells.

Antimetastatic effect by vaccination of tumor cells transfected with B7- 1 (CD80) gene

Ohnishi Y, Fujii H, Inobe M, Kimura F, Murata J, Murakami M, Uede T, Sakamoto T, Tazawa K, Fujimaki M, Saiki I

Biotherapy 11 ( 5 ) 621 - 622 1997

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted

Characterization of a liver metastatic variant of murine colon 26 carcinoma cells.

Y Ohnishi, T Sakamoto, H Fujii, F Kimura, J Murata, K Tazawa, M Fujimaki, Y Sato, M Kondo, Y Une, J Uchino, I Saiki

Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 18 ( 2 ) 113 - 22 1997

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 1010-4283

Intraportal vein injection of highly metastatic L5 cells consistently resulted in liver metastases (increases in the number of tumor colonies in the liver), whereas inoculation of P cells rarely did. L5 cells invaded the basement membrane Matrigel in greater numbers than did P cells, suggesting that the metastatic potential of L5 cells is partly related to enhanced invasive properties. The enhanced adhesion of L5 cells to fibronectin-, laminin- and Matrigel-coated substrates, as well as their haptotactic migration to fribronectin, may be associated with the preferential expression of VLA-2 and VLA-4 integrins on the surface of these cells detected by flow cytometry. Gelatin zymograms showed that the degradative activity of 72-kD gelatinases was greater in L5 cells than P cells. These results indicate that, in addition to adhesiveness and motility, the invasive ability of L5 cells may also be attributed to enhanced gelatinolytic activity. L5 cells grew more rapidly than P cells in vitro. Thus, an experimental model using highly metastatic colon 26 L5 cells would be useful for analyzing the molecular mechanism of liver metastasis and for evaluating the efficacy of treatment of occult micrometastases which may already have been disseminated at the time of surgery.

Application of liposomes for cancer metastasis

Oku N, Koike C, Tokudome Y, Okada S, Nishikawa N, Tsukada H, Kiso M, Hasegawa A, Fujii H, Murata J, Saiki I

Advance Drug Delivery Review 24 215 - 223 1997

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted

Inhibitory effect of a traditional Chinese medicine, Juzen-taiho-to, on progressive growth of weakly malignant clone cells derived from murine fibrosarcoma.

Y Ohnishi, H Fujii, F Kimura, T Mishima, J Murata, K Tazawa, M Fujimaki, F Okada, M Hosokawa, I Saiki

Japanese journal of cancer research : Gann 87 ( 10 ) 1039 - 44 1996.10

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0910-5050

We have investigated the inhibitory effect of oral administration of Juzen-taiho-to, a Kampo (Chinese herbal) medicine, on progressive growth of a mouse fibrosarcoma. Spontaneously regressive QR-32 tumor cells were able to grow progressively in vivo when coimplanted s.c. with a foreign body, gelatin sponge, whereas QR-32 cells alone gradually grew for over 15 days after inoculation and thereafter regressed for up to 25 days. Oral administration of Juzen-taiho-to (40 mg/day/mouse) for 7 days after inoculation of QR-32 cells with gelatin sponge resulted in significant inhibition of tumor growth and prolongation of the survival of the tumor-bearing mice. This growth-inhibitory effect of Juzen-taiho-to observed on day 25 was dose-dependent over the dose range from 4 to 40 mg/day. Treatment with Juzen-taiho-to for 7 days before tumor inoculation with gelatin sponge also significantly suppressed tumor growth examined on day 25, as did the administration of bismuth subnitrate, which is well known to induce metallothionein, an antioxidant. On the other hand, inoculation of progressed tumor cells (QRsP) resulted in growth without gelatin sponge, leading to death in syngeneic mice. Administration of Juzen-taiho-to for 7 days after inoculation of QRsP cells resulted in a decrease of the tumor growth and prolongation of the survival of mice, but the effect was less than that on the growth of QR-32 regressor tumor after coimplantation with gelatin sponge. These results suggest that the inhibitory effect of Juzen-taiho-to is partly associated with prevention of gelatin sponge-elicited progressive growth, probably mediated by endogenous factors including antioxidant substances, in addition to the augmentation of host-mediated antitumor activity.

Synthesis and biological properties of partially modified retro and retro-inverso pseudo peptides of Arg-Gly-Asp (RGD)

Nishikawa N, Komazawa H, Orikasa A, Yoshikane M, Yamaguchi J, Kojima M, Ono M, Itoh I, Azuma I, Fujii H, Murata J, Saiki I

Bioorg. Med. Chem. Lett. 6 ( 22 ) 2725 - 2728 1996.06

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted

Vaccination of tumor cells transfected with the B7-1 (CD80) gene induces the anti-metastatic effect and tumor immunity in mice.

H Fujii, M Inobe, F Kimura, J Murata, M Murakami, Y Onishi, I Azuma, T Uede, I Saiki

International journal of cancer 66 ( 2 ) 219 - 24 1996.04

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0020-7136

The present study demonstrates that the transfection of B7-1 or its variant MB7-2 genes into MHC class I+ tumor cells (B16-BL6 or K1735-M2 melanoma) resulted in the remarkable reduction of lung metastasis caused by i.v. injection into immunocompetent syngeneic mice. However, i.v. injection of the transfectants into T cell-deficient nude mice did not affect reduction of lung tumor colonies as compared with parental wild-type tumors, suggesting that such an inhibitory effect was closely associated with T cell-mediated responses. The reduced metastasis of B7+ tumor cells consequently led to the significant prolongation of survival. Expression of B7 on tumor cells did not influence the tumorigenicity in vivo and tumor cell invasion into basement membrane Matrigel in vitro. We also found that immunization of X-irradiated B7 transfectants was effective as a tumor vaccine for preventing lung metastasis caused by i.v. injection of B7- parental B16-BL6 cells but not against other syngeneic 3LL tumors. Thus, the B7-mediated anti-metastatic effect was tumor-specific. Vaccinations of irradiated B7+ tumor cells before and after surgical excision of the s.c. inoculated primary B7- tumors on day 21 achieved effectively the prevention of spontaneous lung metastasis. Our report that vaccination of irradiated B7+ tumor cells led to a therapeutic effect in an established tumor metastasis model clearly expands and confirms previous related observations.

Functional role of sialyl Lewis X and fibronectin-derived RGDS peptide analogue on tumor-cell arrest in lungs followed by extravasation.

I Saiki, C Koike, A Obata, H Fujii, J Murata, M Kiso, A Hasegawa, H Komazawa, H Tsukada, I Azuma, S Okada, N Oku

International journal of cancer 65 ( 6 ) 833 - 9 1996.03

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0020-7136

Our study demonstrates that synthetic sialyl Lewis X (SLex) as a ligand for selectins and fibronectin-derived RGDS peptide analogue [Ar(DRGDS)3] inhibits lung metastases produced by i.v. co-injection of B16-BL6 melanoma cells. To investigate the inhibitory mechanisms in a living animal, we performed positron-emission tomography (PET) analysis after i.v. injection of [2-18F]2-fluoro-2v-deoxy-D-glucose-labeled tumor cells with or without liposomal SLex or Ar(DRGDS)3. The real-time PET measurement for the first 120 min, started immediately after injection, showed that tumor-cell arrest, i.e., accumulation in the target organ (lung) was remarkably inhibited by liposomal SLex, but not inhibited by Ar(DRGDS)3 or liposomal Me-SLex, which is not recognized by selectins. In contrast, Ar(DRGDS)3 inhibited the invasion of B16-BL6 cells into reconstituted basement membrane (Matrigel) following tumor arrest, whereas SLex- or Me-SLex-entrapped liposomes did not affect tumor invasion. In the metastatic processes containing tumor-cell lodgement and arrest in the target organ followed by extravasation (invasion), SLex resulted in the inhibition of initial arrest of tumor cells, presumably tumor-endothelium interaction, while Ar(DRGDS)3 achieved inhibition of tumor invasion into basement membrane at later steps of the cascade, consequently leading to inhibition of metastasis. Thus, tumor-cell arrest in lungs in the metastatic processes must be precisely and properly controlled by different adhesion molecules at different stages, which are similar to those observed in leukocyte-endothelium interaction.

Inhibition by galloylglucose (GG6-10) of tumor invasion through extracellular matrix and gelatinase-mediated degradation of type IV collagens by metastatic tumor cells.

N Ata, T Oku, M Hattori, H Fujii, M Nakajima, I Saiki

Oncology research 8 ( 12 ) 503 - 11 1996

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0965-0407

Tumor invasion into extracellular matrix (ECM) and basement membrane (BM) is a crucial step in the complex multistage process that leads to metastasis formation. GG6-10 galloylglucose, isolated from Galla Rhois, inhibited the invasion of metastatic HT-1080 cells into a reconstituted BM, such as a Matrigel/fibronectin (FN)-coated filter, in a concentration-dependent fashion. GG6-10 affected neither the tumor cell adhesion and haptotactic migration to ECM components (Matrigel and FN), nor the growth of HT-1080 cells. The gelatin zymography revealed that GG6-10 was able to inhibit not only the degradation of gelatin mediated by matrix metalloproteinases (MMP)-2 and -9 in conditioned medium of HT-1080 tumor cells but also the production of MMP from the tumor cells in a concentration-dependent manner. MMP production is well known to be positively regulated by various cytokines, such as tumor necrosis factor-alpha (TNF-alpha). Thus, we examined the effect of GG6-10 on the TNF-alpha-mediated translation of the MMP-9 gene using HT-1080 cells transfected with the MMP-9 promoter linked to the luciferase gene as a reporter. Similarly to prednisolone, GG6-10 was found to inhibit the TNF-alpha-inducible promoter activity. In keeping with these results, GG6-10 might inhibit tumor cell invasion by inhibiting the gelatinolysis mediated by MMP-2 and -9 and interfering with the production of MMP via inhibiting transcription of the promoter for MMP.

Inhibition of tumor invasion and metastasis by peptidic mimetics of Arg-Gly Asp (RGD) derived from the cell recognition site of fibronectin.

H Fujii, N Nishikawa, H Komazawa, A Orikasa, M Ono, I Itoh, J Murata, I Azuma, I Saiki

Oncology research 8 ( 9 ) 333 - 42 1996

Research paper (scientific journal), Joint Work, Lead author, Last author, Corresponding author, Accepted, ISSN 0965-0407

The partially modified retro- and retro-inverso peptides of the Arg-Gly Asp (RGD) sequence of fibronectin, in which the direction of the Arg residue is reversed and/or the chirality of the amino acid residue is inverted, i.e., mainly R(rev)-COCH2CO-D and DR(rev)-COCH2CO-D, have been synthesized to examine their antimetastatic effects in murine lung or liver metastasis models, as well as their inhibitory effect on tumor cell invasion in vitro. R(rev)-COCH2CO-D inhibited lung metastasis produced by i.v. coinjection with B16-BL6 melanoma more potently than did other pseudo-peptides or the original RGDS peptide. Rrev-COCH2CO-D also showed antimetastatic effects against several different types of tumor cells such as B16-BL6 melanoma, Colon26 M3.1 carcinoma, and L5178Y-ML25 lymphoma cells, in a dose-dependent manner, and multiple administrations had a therapeutic effect on spontaneous lung metastasis. The invasion of melanoma cells into reconstituted basement membrane Matrigel in vitro was suppressed by R(rev)-COCH2CO-D more effectively than by RGDS. These results indicate that the antimetastatic effect by R(rev)-COCH2CO-D was in part due to the inhibition of tumor invasion. The RGDS peptide decomposed when incubated with fresh plasma in vitro, whereas R(rev)-COCH2CO-D was not affected by this treatment. Thus, the reversion of the Arg-Gly linkage in the RGD sequence resulted in protease resistance leading to the retardation of the clearance of the peptide in vivo, and consequently augmented its antimetastatic and antiinvasive properties. Designed peptide analogues may provide various advantages and be useful for preventing cancer metastasis.

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