Motowo Nakajima

Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation

We have investigated the effect of monoclonal antibodies (MAbs) specific for aminopeptidase N/CD13 on the invasion of human metastatic tumor cells into reconstituted basement membrane (Matrigel). The invasion of human metastatic tumor cells (SN12M renal‐cell carcinoma, HT1080 fibrosarcoma and A375M melanoma) into Matrigel‐coated filters was inhibited by an anti‐CD 13 MAb, WM15, in a concentration‐dependent manner. However, this MAb did not have any effect on tumor‐cell adhesion and migration to the extracellular matrices, which may be involved in tumor‐cell invasion. MAb WM15 inhibited the degradation of type‐IV collagen by tumor cells in a concentration‐dependent manner. We also found that WM15 inhibited hydrolysing activities towards substrates of aminopeptidases in 3 different tumor cells. Since our previous study indicated that bestatin, an aminopeptidase inhibitor, was able to inhibit tumor‐cell invasion, as well as aminopeptidase activities of murine and human metastatic tumor cells, cell‐surface aminopeptidase N/CD13 may be partly involved in the activation mechanism for type‐IV collagenolysis to achieve tumor‐cell invasion, and anti‐CD13 MAb WM15 may inhibit tumor‐cell invasion through a mechanism involving its inhibitory action on the aminopeptidase N in tumor cells.

Human heparanase. Purification, characterization, cloning, and expression.

Heparan sulfate and heparan sulfate proteoglycans are present in the extracellular matrix as well as on the external cell surface. They bind various molecules such as growth factors and cytokines and modulate the biological functions of binding proteins. Heparan sulfate proteoglycans are also important structural components of the basement membrane. Heparanase is an endo-β-d-glucuronidase capable of cleaving heparan sulfate and has been implicated in inflammation and tumor angiogenesis and metastasis. In this study, we report the purification of a human heparanase from an SV40-transformed embryonic fibroblast cell line WI38/VA13 by four sequential column chromatographies. The activity was measured by high speed gel permeation chromatography of the degradation products of fluorescein isothiocyanate-labeled heparan sulfate. The enzyme was purified to homogeneity, yielding a peptide with an apparent molecular mass of 50 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Using the amino acid sequences of the N-terminal and internal heparanase peptides, a cDNA coding for human heparanase was cloned. NIH3T3 and COS-7 cells stably transfected with pBK-CMV expression vectors containing the heparanase cDNA showed high heparanase activities. The homology search revealed that no homologous protein had been reported.

Cell–matrix interaction via CD44 is independently regulated by different metalloproteinases activated in response to extracellular Ca2+ influx and PKC activation

CD44 is an adhesion molecule that interacts with hyaluronic acid (HA) and undergoes sequential proteolytic cleavages in its ectodomain and intramembranous domain. The ectodomain cleavage is triggered by extracellular Ca2+ influx or the activation of protein kinase C. Here we show that CD44-mediated cell–matrix adhesion is terminated by two independent ADAM family metalloproteinases, ADAM10 and ADAM17, differentially regulated in response to those stimuli. Ca2+ influx activates ADAM10 by regulating the association between calmodulin and ADAM10, leading to CD44 ectodomain cleavage. Depletion of ADAM10 strongly inhibits the Ca2+ influx-induced cell detachment from matrix. On the other hand, phorbol ester stimulation activates ADAM17 through the activation of PKC and small GTPase Rac, inducing proteolysis of CD44. Furthermore, depletion of ADAM10 or ADAM17 markedly suppressed CD44-dependent cancer cell migration on HA, but not on fibronectin. The spatio-temporal regulation of two independent signaling pathways for CD44 cleavage plays a crucial role in cell–matrix interaction and cell migration.

CD44 cleavage induced by a membrane-associated metalloprotease plays a critical role in tumor cell migration

CD44 is a cell surface receptor for hyaluronate, a component of the extracellular matrix (ECM). Although CD44 has been implicated in tumor invasion and metastasis, the molecular mechanisms remain to be elucidated. Here we find that CD44 expressed in cancer cells is cleaved at the membrane-proximal region of the ectodomain and the membrane-bound cleavage product can be detected using an antibody against the cytoplasmic domain of CD44. Furthermore, we report that CD44 cleavage is mediated by a membrane-associated metalloprotease expressed in cancer cells. A tissue inhibitor of metalloproteases-1 (TIMP-1), as well as metalloprotease inhibitors, inhibit CD44 cleavage in the cell-free assay. Contrary, serine protease inhibitors enhance CD44 cleavage, and the enhancement can be prevented by pretreatment with a metalloprotease inhibitor. Thus, CD44 cleavage is regulated by an intricate balance between some proteases and their inhibitors. Interestingly, treatment with the metalloprotease blocker 1,10-phenanthroline, which strongly prevent the CD44 cleavage, suppressed RERF-LC-OK lung cancer cell migration on a hyaluronate substrate, but not on several other substrates. These results suggest that CD44 cleavage plays a critical role in an efficient cell-detachment from a hyaluronate substrate during the cell migration and consequently promotes CD44-mediated cancer cell migration. Our present data indicate that CD44, not only ECM per se, is one of the targets of pericellular proteolysis involved in tumor invasion and metastasis.

Pentosan polysulfate, a sulfated oligosaccharide, is a potent and selective anti-HIV agent in vitro

Several sulfated oligo- or polysaccharides such as pentosan polysulfate, fucoidan, dextran sulfate, heparin and iota-, kappa- and lambda-carrageenans proved to be potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) in vitro. The most potent anti-HIV-1 activity was recorded for the oligosaccharide pentosan polysulfate, its 50% antiviral effective dose (ED50) being 0.19 microgram/ml in MT-4 cells. It inhibited HIV-1 antigen expression in HUT-78 cells at an ED50 of 0.02 microgram/ml, and complete inhibition of HIV-1 antigen expression was obtained at a concentration of 4.0 micrograms/ml. No toxicity for MT-4 cells was observed with pentosan polysulfate at a concentration of 2500 micrograms/ml. The anticoagulant activity of pentosan polysulfate was more than ten-fold lower than that of heparin (14.4 and 177 U/mg, respectively). In fact, pentosan polysulfate achieved its anti-HIV-1 activity at a concentration that is 370-fold below its anticoagulant threshold (1 U). Pentosan polysulfate inhibits virus adsorption to the cells, as was demonstrated by monitoring the association of radiolabeled HIV-1 virions with MT-4 cells.

The receptor for advanced glycation end‐products (RAGE) directly binds to ERK by a D‐domain‐like docking site

The receptor for advanced glycation end‐products (RAGE)‐mediated cellular activation through the mitogen‐activated protein kinase (MAPK) cascade, activation of NF‐κB and Rho family small G‐proteins, cdc42/Rac, is implicated in the pathogenesis of inflammatory disorders and tumor growth/metastasis. However, the precise molecular mechanisms for the initiation of cell signaling by RAGE remain to be elucidated. In this study, proteins which directly bind to the cytoplasmic C‐terminus of RAGE were purified from rat lung extracts using an affinity chromatography technique and identified to be extracellular signal‐regulated protein kinase‐1 and ‐2 (ERK‐1/2). Their interactions were confirmed by immunoprecipitation of ERK‐1/2 from RAGE‐expressing HT1080 cell extracts with anti‐RAGE antibody. Furthermore, the augmentation of kinase activity of RAGE‐bound ERK upon the stimulation of cells with amphoterin was demonstrated by determining the phosphorylation level of myelin basic protein, an ERK substrate. In vitro binding studies using a series of C‐terminal deletion mutants of human RAGE revealed the importance of the membrane‐proximal cytoplasmic region of RAGE for the direct ERK–RAGE interaction. This region contained a sequence similar to the D‐domain, a ERK docking site which is conserved in some ERK substrates including MAPK‐interacting kinase‐1/2, mitogen‐ and stress‐activated protein kinase‐1, and ribosomal S6 kinase. These data suggest that ERK may play a role in RAGE signaling through direct interaction with RAGE.

Heparanase expression correlates with invasion and poor prognosis in gastric cancers

Degradation of basement membrane and extracellular matrix structures are important features of the metastatic process of malignant tumors. Human heparanase degrades heparan sulfate proteoglycans, which represent the main components of basement membranes and the extracellular matrix. Because of the role of heparanase in tumor invasion and metastasis, we examined heparanase expression in primary gastric cancers and in cell lines derived from gastric cancers by immunohistochemistry and RT-PCR, respectively. Four of seven gastric cancer cell lines showed heparanase mRNA expression by RT-PCR. Heparanase protein was detected in both the cytoplasm and the nucleus of heparanase mRNA-positive cells by immunohistochemical staining. Heparanase expression was confirmed in 35 (79.5%) of 44 gastric tumor samples by immunohistochemical staining. However, no or weak heparanase expression was detected in normal gastric mucosa. In situ hybridization showed that the mRNA expression pattern of heparanase was similar to that of the protein, suggesting that increased expression of the heparanase protein at the invasive front was caused by an increase of heparanase mRNA in tumor cells. Analysis of the clinicopathologic features showed stronger heparanase expression in cases of huge growing tumors, extensive invasion to lymph vessels, and regional lymph node metastasis. In gastric cancer, patients with heparanase expression showed significantly poorer prognosis than those without such expression (p = 0.006). In conclusion, our findings suggest that high expression of heparanase in gastric cancer is a strong predictor of poor survival.

Serum matrix metalloproteinase-2 and its density in men with prostate cancer as a new predictor of disease extension.

We examined whether the serum matrix metalloproteinase‐2 (MMP‐2) level and MMP‐2 density could be predictors of the development and extension of prostate cancer. Serum samples were collected before any clinical treatment from 98 patients with prostate cancer and from 76 patients with benign prostatic hyperplasia (BPH). Control sera were obtained from 70 healthy men. The serum level of MMP‐2 was determined by 1‐step enzyme immunoassay. A newly defined MMP‐2 density parameter was determined by dividing the serum level of MMP‐2 by the prostate volume, which was measured by ultrasonography. The mean serum level of MMP‐2 in prostate cancer patients was significantly higher than in the control and BPH groups. Furthermore, the serum MMP‐2 levels in prostate cancer patients with metastasis were highly elevated compared with those without metastases. The MMP‐2 density in pathologically organ‐confined prostate cancer was significantly higher than that in BPH. There was a statistically significant difference in the MMP‐2 density between pT2N0M0 and pT1N0M0 prostate cancers. Moreover, the serum MMP‐2 level correlated well with the clinical course of prostate cancer with bone metastasis. Our results suggest that MMP‐2 plays an important role in the development and extension of prostate cancer and that the serum level of MMP‐2 and the MMP‐2 density indicate prostate cancer extension and are, therefore, useful for the followup of prostate cancer patients. Int. J. Cancer (Pred. Oncol.) 79:96–101, 1998.

EXPRESSION OF ENDOTHELIN RECEPTOR A ASSOCIATED WITH PROSTATE CANCER PROGRESSION

PURPOSE We determined the role of endothelin receptors in prostate cancer progression. MATERIALS AND METHODS We examined 51 prostate cancer specimens obtained at surgery or biopsy for the relationship of endothelin receptor expression determined by immunohistochemical staining with malignant potential. RESULTS The positive staining rate of endothelin receptor A in the 51 specimens was significantly higher than of endothelin B (71% versus 24%, p <0.0001). The staining rate of receptor A in Gleason score 5 to 10 disease was significantly higher than in Gleason 2 to 4 disease (91% versus 29%, p <0.0001). The overall staining rate of endothelin receptor A in nonorgan confined disease without bone metastasis but with extraprostatic disease was 87% in 23 cases, including 16 of 19 stage T3 (84%) and all 4 stage T4 (100%) cases. This rate was significantly higher than that of organ confined cancer (29%, p = 0.0003). All patients with bone metastasis had positive staining for endothelin receptor A. An especially high rate of intensely positive staining was observed for endothelin receptor A in biopsy specimens with bone metastasis or Gleason sum 8 to 10. Moreover, positive staining was stronger in cancer cells penetrating the prostatic capsule than in those at the primary foci. However, the positive staining rate of endothelin receptor B was not significantly different in organ and nonorgan confined cancer without bone metastasis (12% versus 26%, p = 0.4284), bone metastatic and nonmetastatic cancer (20% versus 36%, p = 0.2619) or the Gleason sum groups (p = 0.0874). CONCLUSIONS Our results indicate that endothelin receptor A expression may serve as a marker for and have an important role in prostate cancer progression.

Novel development of 5-aminolevurinic acid (ALA) in cancer diagnoses and therapy.

Early detection and intervention are needed for optimal outcomes in cancer therapy. Improvements in diagnostic technology, including endoscopy, photodynamic diagnosis (PDD), and photodynamic therapy (PDT), have allowed substantial progress in the treatment of cancer. 5-Aminolevulinic acid (ALA) is a natural, delta amino acid biosynthesized by animal and plant mitochondria. ALA is a precursor of porphyrin, heme, and bile pigments, and it is metabolized into protoporphyrin IX (PpIX) in the course of heme synthesis. PpIX preferentially accumulates in tumor cells resulting in a red fluorescence following irradiation with violet light and the formation of singlet oxygen. This reaction, utilized to diagnose and treat cancer, is termed ALA-induced PDD and PDT. In this review, the biological significance of heme metabolites, the mechanism of PpIX accumulation in tumor cells, and the therapeutic potential of ALA-induced PDT alone and combined with hyperthermia and immunotherapy are discussed.