The role of MT2-MMP in cancer progression.

The role of MT2-MMP in cancer progression remains to be elucidated in spite of many reports on MT1-MMP. Using a human fibrosarcoma cell, HT1080 and a human gastric cancer cell, TMK-1, endogenous expression of MT1-MMP or MT2-MMP was suppressed by siRNA induction to examine the influence of cancer progression in vitro and in vivo. In HT1080 cells, positive both in MT1-MMP and MT2-MMP, the migration as well as the invasion was impaired by MT1-MMP or MT2-MMP suppression. Also cell proliferation in three dimensional (3D) condition was inhibited by MT1-MMP or MT2-MMP suppression and tumor growth in the nude mice transplanted with tumor cells were reduced either MT1-MMP or MT2-MMP suppression with a prolongation of survival time in vivo. MT2-MMP suppression induces more inhibitory effects on 3D proliferation and in vivo tumor growth than MT1-MMP. On the other hand, TMK-1 cells, negative in MT1-MMP and MMP-2 but positive in MT2-MMP, all the migratory, invasive, and 3D proliferative activities in TMK-1 are decreased only by MT2-MMP suppression. These results indicate MT2-MMP might be involved in the cancer progression more than or equal to MT1-MMP independently of MMP-2 and MT1-MMP.

Evaluation of matrix metalloproteinase-2 (MMP-2) activity with film in situ zymography for improved cytological diagnosis of breast tumors.

BACKGROUND: Fine-needle aspiration (FNA) biopsy of breast tumors is a reliable diagnostic method for identifying breast carcinoma. However, it is sometimes difficult to definitely distinguish malignant from benign lesions. To improve the cytological diagnosis of breast tumors, we investigated the expression of active matrix metalloproteinase-2 (MMP-2), as detected by film in situ zymography (FIZ). METHODS: We evaluated 34 fresh breast tumors, 25 paraffin-embedded breast tissue specimens, and a human cancer cell line (HT1080). MMP-2 expression was determined by immunocytochemistry or immunohistochemistry. Frozen sections and aspiration cytology samples of breast cancer were incubated on gelatin-coated films for the detection of active MMP-2. RESULTS: Immunohistochemistry showed that MMP-2 was expressed in cancer cells and stromal cells, but not in most benign breast lesions. Active MMP-2, but not pro-MMP-2, in the conditioned medium of HT1080 cells showed gelatinolytic activity on FIZ analysis, while the expression of both forms of MMP-2 was detected by immunocytochemistry. Gelatinolytic activity was also detected by FIZ analysis of aspiration cytology samples and frozen sections from the breast cancers, and there was a significant correlation between this gelatinolytic activity and the detection of MMP-2 expression by immunocytochemistry. CONCLUSIONS: The present study demonstrated that measurement of gelatinolytic activity by FIZ analysis of aspiration cytology samples may be useful for improving the cytological diagnosis of breast tumors.

Transactivation of epidermal growth factor receptor after heparin-binding epidermal growth factor-like growth factor shedding in the migration of prostate cancer cells promoted by bombesin.

BACKGROUND: A pathway consisting of bombesin, G-protein coupling receptors (GPCRs), metalloproteases, pro-heparin-binding epidermal growth factor (proHB-EGF), and epidermal growth factor receptor (EGFR) has been reported in prostate cancer cells. The occurrence of HB-EGF shedding from proHB-EGF in this pathway, however, has not been proven directly. In addition, it is still unclear how much this pathway contributes to the migration of prostate cancer cells. In this study, we tried to directly elucidate HB-EGF shedding in this pathway and to determine its contribution to the migration of prostate cancer cells. METHODS: RT-PCR and indirect immunofluorescence staining for HB-EGF and its receptors, such as EGFR and HER4/erbB4, were performed on PC-3 cells. The influences of bombesin, anti-EGFR neutralizing monoclonal antibody, HB-EGF, and HB-EGF shedding inhibitor on the migration of PC-3 cells were studied by means of in vitro wound assays. The amount of HB-EGF shed from PC-3 cells with alkaline phosphatase-tagged HB-EGF in the presence of bombesin was determined by measuring AP activity. Immunoprecipitations and phosphotyrosine Western blotting were performed to detect EGFR transactivated by bombesin. RESULTS: PC-3 expressed HB-EGF and EGFR, but not HER4/erbB4. PC-3 migrated in the presence of bombesin, but its migration was partly inhibited by the neutralizing antibody against EGFR. PC-3 also migrated in the presence of HB-EGF, but HB-EGF shedding inhibitor partly inhibited this phenomenon. HB-EGF was shed from PC-3 cells in the presence of bombesin, and this shedding was inhibited by HB-EGF shedding inhibitor. In addition, the EGFR on PC-3 was activated in the presence of bombesin and inactivated in the presence of HB-EGF shedding inhibitor. CONCLUSIONS: These results indicated that HB-EGF shedding and the following transactivation of EGFR occurs in this pathway and that this pathway partly contributes to the migration of prostate cancer cells.

Heavy ion irradiation inhibits in vitro angiogenesis even at sublethal dose.

Angiogenesis is essential for tumor growth and metastasis. Because endothelial cells are genetically stable, they rarely acquire resistance to anticancer modalities, and could, thus, be a suitable target for radiation therapy. Heavy ion radiation therapy has attracted attention as an effective modality for cancer therapy because of its highly lethal effects, but the effects of heavy ion irradiation on in vitro cell function associated with angiogenesis have not been reported. Our study found that in vitro angiogenesis was inhibited by high linear energy transfer carbon ion irradiation even at sublethal dose (0.1 Gy). ECV304 and HUVEC human umbilical vascular endothelial cells were irradiated with 290 MeV carbon ion beams of approximately 110 keV/ micro m or 4 MV X-ray of approximately 1 keV/ micro m. Their adhesiveness and migration to vitronectin or osteopontin were inhibited, and capillary-like tube structures in three-dimensional culture were destroyed after carbon ion irradiation concomitant with the inhibition of matrix metalloproteinase-2 activity, down-regulation of alphaVbeta3 integrin, which is one of the adhesion molecules, slight up-regulation of membrane type1- matrix metalloproteinase, and significant up-regulation of tissue inhibitor of metalloproteinase-2. On the other hand, sublethal X-ray irradiation promoted migration of endothelial cells, and the capillary-like tube structure in three-dimensional culture progressed even after 16 Gy irradiation. These results provide an implication that heavy ion beam therapy could be superior to conventional photon beam therapy in preventive effects on in vitro angiogenesis even at sublethal dose, and might inhibit angiogenesis in vivo.