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This study focused on the inhibitory effect of rhodomyrtone, a bioactive compound isolated from the leaves of Rhodomyrtus tomentosa (Aiton) Hassk., on cancer metastasis in epidermoid carcinoma A431 cells and on the verification of the underlying related molecular mechanisms of this event. We demonstrated that rhodomyrtone at the subcytotoxic concentration (0.5 and 1.5 µg/ml) exhibited pronounced inhibition of cancer metastasis by reducing cell migration, cell adhesive ability and cell invasion of A431 cells in a dose-dependent manner. Data demonstrated that rhodomyrtone could inhibit the focal adhesion kinase (FAK) and phosphorylation of protein kinase B (AKT), c-Raf, extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK involved in the downregulation the enzyme activities and protein expression of matrix metalloproteinase-2 (MMP-2) and MMP-9. Moreover, we found that rhodomyrtone increased the expression of TIMP-1 and TIMP-2, which are inhibitors of MMP-9 and MMP-2, respectively. Rhodomyrtone also inhibited the expression of NF-κB and phosphorylation of NF-κB in a dose-dependent manner. These results suggested that rhodomyrtone inhibited A431 cell metastasis by reducing MMP-2/9 activities and expression through inhibiting ERK1/2, p38 and FAK/Akt signaling pathways via NF-κB activities. This finding suggested that rhodomyrtone may be a novel antimetastasis agent for treatment of skin cancer cells.

Gardeniae Fructus (GF, Zhi Zi in China), a fruit of Gardenia jasminoides Ellis, has been used in traditional medicine to reduce inflammation and headache and to treat hepatic disorders, hypertension, and icterus. In recent studies, extract of raw or stir-baked GF was shown to have pharmacological activities for viral infection, thrombosis, hyperlipidemia, convulsion, inflammation, oxidative stress, and others. In addition, baked GF extract suppressed the proteolytic activities and altered the cellular morphology of tumor cells. However, the effects of ethanol extract of baked GF (EBGF) on the metastatic and angiogenic capacities of malignant tumor cells and its detailed mechanism of action have not been reported. In this study, we found that EBGF significantly inhibited phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 and -13 and uPA expression via suppression of PMA-induced nuclear translocation of NF-κBp65. Metastatic potential, including migration, invasion, and colonization, was substantially reduced by EBGF with no cytotoxicity. In addition, EBGF attenuated tumor-induced angiogenesis, including microvessel sprouting, migration of endothelial cells (ECs), and tube formation of ECs, by inhibiting the release of pro-angiogenic factors from tumor cells. In C57BL/6 mice, we observed that daily administration of EBGF at 50 and 100 mg/kg suppressed metastatic colonization of B16F10 melanoma cells in the lungs. Furthermore, EBGF administration did not cause adverse effects, suggesting that EBGF is safe and may be a potential herbal medicine capable of controlling metastatic malignant cancers.

The neural guidance molecular, Slit2, and its cognate receptor, Robo1, play critical roles in the development of the nervous system, nevertheless, their functions are not limited to this system. Numerous studies have shown decreased Slit2 expression in a wide variety of cancers, highlighting its potential as a tumor suppressor. However, the Slit2/Robo1 signaling axis was reported to induce either suppressive or stimulatory effects on tumor growth and metastasis, depending on cellular context. There is a paucity of information on the effects of the Slit2/Robo1 signaling axis on the growth and metastasis of human hepatocellular carcinoma (HCC). Large-scale data mining of the Oncomine database has revealed heterogeneous expression of Slit2 in HCC. We screened the Sk-hep-1, a cell line showing a relatively high level of Slit2, and low level of Robo1 expression. After Slit2 knockdown and Robo1 overexpression in these cells, we found Slit2 and Robo1 exerted opposing effects on tumor growth and metastasis both in in vitro and in vivo models. Slit2 knockdown and Robo1 overexpression in Sk-hep-1 cells promoted tumor growth and metastasis, suggesting a negative and positive role for Slit2 and Robo1, respectively, in tumor progression. Robo1 overexpression upregulated matrix metalloproteinase (MMP)2, -9 and membrane-type1 MMP (MT1-MMP) expression, stimulated MMP2, but not MMP9 activation, and downregulated expression of TIMP1 and 2. The PI3K/Akt signaling pathway is of importance in regulating MMP2 expression in Sk-hep-1 cells, since Robo1 overexpression stimulated phosphorylation of Akt while the PI3K inhibitor LY294002, significantly inhibited the upregulation of MMP2 and also the enhanced cell invasion induced by Robo1 overexpression. We postulate that Robo1 promotes tumor invasion partly by the upregulation of MMP2 after activation of PI3K/Akt signaling pathway. Notably, Slit2 knockdown caused the upregulation of Robo1 expression both at the mRNA and protein levels. Thus, the stimulatory effects of Slit2 knockdown on tumor progression can be ascribed, at least in part, to the upregulation of Robo1 and its positive role in tumor progression.

A proton beam is a next generation tool to treat intractable cancer. Although the therapeutic effects of a proton beam are well known, the effect on tumor metastasis is not fully described. Here, we investigated the effects of a proton beam on metastasis in highly invasive 4T1 murine breast cancer cells and their orthotopic breast cancer model. Cells were irradiated with 2, 4, 8 or 16 Gy proton beam, and changes in cell proliferation, survival, and migration were observed by MTT, colony forming and wound healing assays. 4T1 breast cancer cell-implanted BALB/c mice were established and the animals were randomly divided into 4 groups when tumor size reached 200 mm3. Breast tumors were selectively irradiated with 10, 20 or 30 Gy proton beam. Breast tumor sizes were measured twice a week, and breast tumor and lung tissues were pathologically observed. Metastasis-regulating gene expression was assessed with quantitative RT-PCR. A proton beam dose-dependently decreased cell proliferation, survival and migration in 4T1 murine breast cancer cells. Also, growth of breast tumors in the 4T1 orthotopic breast cancer model was significantly suppressed by proton beam irradiation without significant change of body weight. Furthermore, fewer tumor nodules metastasized from breast tumor into lung in mice irradiated with 30 Gy proton beam, but not with 10 and 20 Gy, than in control. We observed correspondingly lower expression levels of urokinase plasminogen activator (uPA), uPA receptor, cyclooxygenase (COX)-2, and vascular endothelial growth factor (VEGF), which are important factors in cancer metastasis, in breast tumor irradiated with 30 Gy proton beam. Proton beam irradiation did not affect expressions of matrix metalloproteinase (MMP)-9 and MMP-2. Taken together, the data suggest that, although proton beam therapy is an effective tool for breast cancer treatment, a suitable dose is necessary to prevent metastasis-linked relapse and poor prognosis.

Metastasis of colorectal cancer (CRC) depends critically on MMP-9. KiSS-1 is a human malignant melanoma metastasis-suppressor gene. Thus, the interaction between MMP-9 and KiSS-1 has drawn considerable attention in recent years. In the present study, it was hypothesized that KiSS-1 gene could repress the metastatic potential of colorectal cancer cells by inhibiting the expression of MMP-9. Stable transfection of KiSS-1 specific siRNA and KiSS-1 expression vector in human CRC cell line HCT-116 was achieved by lentivirus infection. Moreover, the cell proliferation, invasiveness, and apoptosis were evaluated by CCK-8 method, transwell experiment, and fluorescence activated cell sorter, respectively. We also investigated the expression of MMP-9, PI3K, Akt, pAKt, and NF-кB subunit p65 using western blotting. KiSS-1 overexpression significantly decreased the cell proliferation and invasiveness of HCT-119 cells, while apoptosis was enhanced. The result of western blotting showed that synthesis of MMP-9, PI3K, p65, and phosphorylation of Akt were significantly blocked by overexpression of KiSS-1. Concatenated treatment of KiSS-1 overexpression vector with PI3K and Akt agonists attenuated the effect of KiSS-1 on the biological activity of CRC cells and also released the expression of MMP-9, PI3K, p65, and phosphorylation of Akt from the influence of overexpression of KiSS-1. Overexpression of KiSS-1 suppressed the invasiveness of CRC cells, and the gene exerted its function by reducing the expression of MMP-9 via blocking of tge PI3K/Akt/NF-κB pathway.

The Wnt signaling pathway is activated in hepatocellular carcinoma (HCC). This study investigated the effects of FH535, an inhibitor of the Wnt signaling pathway, on the proliferation and motility of HCC cells. HLF cells and PLC/PRF/5 cells, HCC cells, were subjected to 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay with the addition of FH535. RNA was isolated from the cells and subjected to real-time quantitative PCR. Hematoxylin and eosin (H&E) staining was performed to analyze apoptosis. A scratch assay was performed to analyze cell motility. Cell proliferation significantly decreased (P<0.05). The expression levels of cyclin D1 significantly decreased in both cell lines (P<0.05). Pyknotic nuclei were observed in the cells cultured with FH535 (50 µM). In the scratch assay, the distance between the growing edges of cells and the scratched line significantly decreased with the addition of FH535 at 50 µM (P<0.05). The expression levels of matrix metalloproteinase 9 significantly decreased at 50 µM (P<0.05). FH535 suppressed the proliferation of HCC cells by downregulating the expression of cyclin D1 and by inducing apoptosis. Further, it suppressed cell motility by downregulating the expression of matrix metalloproteinase.

Short-hairpin RNA of frizzled-2 (shRNA-Fz2) is known to suppress the proliferation of hepatocellular carcinoma (HCC) cells; however, its effect on HCC cell motility is unknown. In this study, suppression of HCC cell motility by shRNA-Fz2 was analyzed, and introduction of shRNA-Fz2 into HCC cells was facilitated with ultrasound (US) irradiation generated from a diagnostic US device, which was enhanced by the contrast-enhanced US reagent Sonazoid. The HCC cell lines HLF and PLC/PRF/5 that were transfected with shRNA-Fz2 were plated to form monolayers, following which the cell monolayers were scratched with a sterile razor. After 48 h, the cells were stained with hematoxylin and eosin, and the distance between the growing edge of the cell layer and the scratch lines was measured. Total RNA from the cells was isolated and subjected to real-time quantitative PCR to quantify matrix metalloproteinase 9 expression at 48 h after transfection of shRNA-Fz2. Starch-iodide method was applied to analyze the generation of H2O2 following US irradiation with the addition of Sonazoid in the liquid, and cell proliferation was analyzed 72 h later. The distances between the growing edge of the cell layer and the scratch lines and MMP9 expression levels were significantly decreased with transfection of shRNA-Fz2 (P<0.05). In the starch-iodide method, absorbance significantly decreased with the addition of Sonazoid (P<0.05), which suggested that US irradiation with Sonazoid generated H2O2 and enhanced sonoporation. ShRNA-Fz2 suppressed cell proliferation of both cell lines at a mechanical index of 0.4. Motility of HLF cells and PLC/PRF/5 cells was suppressed by shRNA-FZ2. Sonazoid enhanced sonoporation of the cells with the diagnostic US device and the suppression of proliferation of both HCC cell lines by shRNA-Fz2.

Polysaccharides isolated from the fruiting body of Inonotus obliquus (PFIO) are known to possess various pharmacological properties including antitumor activity. However, the anti-metastatic effect and its underlying mechanistic signaling pathway involved these polysaccharides in human non-small cell lung carcinoma remain unknown. The present study therefore aimed to determine the anti-metastatic potential and signaling pathways of PFIO in the highly metastatic A549 cells. We found that PFIO suppressed the migration and invasive ability of A549 cells while decreasing the expression levels and activity of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, PFIO decreased the phosphorylation levels of mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) as well as the expression level of COX-2, and inhibited the nuclear translocation of nuclear factor κB (NF-κB) in A549 cells. These results suggested that PFIO could suppress the invasion and migration of human lung carcinoma by reducing the expression levels and activity of MMP-2 and MMP-9 via suppression of MAPKs, PI3K/AKT, and NF-κB signaling pathways.

FoxM1 is a specific transcription factor that has an important function in aggressive human carcinomas, including cervical cancer. However, the specific function and internal molecular mechanism in cervical cancer remain unclear. In this study, RNAi-mediated FoxM1 knockdown inhibited cell growth. This process also decreased the migration and invasion activities of HeLa cells in vitro. Downregulation of FoxM1 inhibited tumor growth and angiogenesis in vivo. In addition, the expressions of uPA, matrix metalloproteinase (MMP)-2, MMP-9 and VEGF were significantly decreased in vitro and in vivo. These results suggested that the inactivation of FoxM1 could be a novel therapeutic target for cervical cancer treatment.

Experimental and clinical studies have revealed the effectiveness of a specific nutrient synergy (SNS) mixture composed of ascorbic acid (AA), lysine, proline, arginine, epigallocatechin gallate (EGCG) and other micronutrients in targeting crucial physiological mechanisms involved in cancer progression and metastasis. HTLV-1 causes adult T-cell leukemia (ATL). The spread and metastases of ATL as well as other tumors has been associated with matrix metalloproteinases, especially the gelatinases MMP-2 and MMP-9. The objective of this study was to investigate whether SNS, AA and EGCG affects the gelatinolytic activity of MMP-2 and its transcriptional and translational levels in HTLV-1-positive and -negative malignant T-cells. The results indicated that SNS and EGCG caused a dose-dependent decline in the activity, transcription and translation of MMP-2 after treatment with SNS and EGCG, while AA was only able to inhibit the activity at maximum doses tested and to some extent, the protein expression levels of MMP-2, without affecting their transcriptional levels. The highest activity was noted in the case of SNS which is likely to be due to a synergistic effect of the different constituents in the formulation. These results point towards the potential integration of SNS in the anti-invasive treatment of ATL and related diseases.

Interleukin-7 (IL-7) plays a pivotal role in the development and survival of lymphocytes, but its role in cancer cell responses remains unexplained. In this study, IL-7 treatment resulted in a significant induction in the wound-healing migration and Matrigel invasion of the 5637 bladder cancer cells, but it did not result in cell proliferation. In addition, IL-7 treatment strongly induced MMP-9 expression, and increased the binding activation of NF-κB and AP-1 motifs, the important transcription factors that regulate MMP-9 expression. Moreover, the treatment of 5637 cells with IL-7 stimulated the phosphorylation of ERK1/2. U0126, an ERK1/2-specific inhibitor, blocked IL-7-induced cell migration and invasion, and also suppressed the expression of MMP-9 in the presence of IL-7. Inhibition of the ERK1/2 function consistently reversed the binding activity of NF-κB without altering AP-1 activation in IL-7-stimulated cells. Among the cell cycle regulators examined, only the expression of the cell cycle inhibitor p27KIP1 was induced by IL-7. Moreover, the inhibition of p27KIP1 by small interfering RNA (siRNA) abolished the migration, invasion and phosphorylation of ERK1/2, the expression of MMP-9, and the binding activity of the NF-κB motif in IL-7-stimulated 5637 cells. These results demonstrated that the cell cycle inhibitor p27KIP1 is involved in ERK1/2-mediated MMP-9 expression via activation of the NF-κB binding motif, which leads to the migration and invasion of bladder cancer cells induced by IL-7. These novel results could help explain the migration and invasion of bladder tumor cells.

High mobility group box 1 (HMGB1) as a novel inflammatory molecule has been shown to be involved in a variety of cell physiological and pathological behaviors including immune response, inflammation and cancer. Evidence suggests that HMGB1 plays a critical role in the development and progression of multiple malignancies. However, the underlying molecular mechanisms for the HMGB1-mediated growth and invasion of gastric cancer have not yet been elucidated. The present study investigated the expression of HMGB1 in gastric adenocarcinoma (GAC) and the mechanisms by which it contributes to tumor growth and invasion. The correlation between HMGB1 expression and clinicopathological characteristics of GAC patients was assessed by immunohistochemical assay through tissue microarray procedures. The RNA and protein expressions of HMGB1 and downstream factors were detected by quantitative PCR and western blot assays; cell proliferation and invasion were determined by MTT, wound-healing and 3D-Matregel assays, subcutaneous SGC-7901 tumor models were established to verify tumor growth in vivo. We demonstrated that, the expression of HMGB1 was significantly increased in the nucleus of GAC tissues compared with that in adjacent non-cancer tissues (88.6 vs.70.5%, P<0.001), and correlated with the metastatic lymph node of GAC (P=0.018). Furthermore, knockdown of HMGB1 by shRNA inhibited cell proliferative activities and invasive potential, and downregulated the expression of NF-κB p65, PCNA and MMP-9 in GAC cells (SGC-7901 and AGS). The tumor volumes in SGC7901 subcutaneous nude mouse models treated with Lv-shHMGB1 was significantly smaller than those of the nonsense sequence group. Taken together, these findings suggest that increased expression of HMGB1 is associated with tumor metastasis of GAC, and knockdown of HMGB1 suppresses growth and invasion of GAC cells through the NF-κB pathway in vitro and in vivo, suggesting that HMGB1 may serve as a potential therapeutic target for GAC.

UVB-induced inflammation, in particular the overexpression of cyclooxygenase-2 (COX-2) and prostaglandin (PG) E2, has been implicated in photocarcinogenesis. UVB-induced COX-2 has been associated with β-catenin signaling in keratinocytes. However, a definitive role for COX-2 in the activation of β-catenin signaling as well as its role in UVB-induced skin tumors has not been established. We report that exposure of the skin to UVB resulted in a time- and dose-dependent activation of β-catenin in C3H/HeN mice. This response was COX-2-dependent as UVB-exposed COX-2-deficient mice exhibited significantly lower levels of UVB-induced activation of β-catenin. Moreover, treatment of mice with indomethacin, a COX-2 inhibitor, and an EP2 antagonist inhibited UVB-induced β-catenin signaling. Exposure of SKH-1 hairless mice to UVB radiation (180 mJ/cm2) 3 times a week for 24 weeks resulted in activation of β-catenin signaling in UVB-irradiated skin as well as UVB-induced skin tumors. Concomitantly, the levels of CK1α and GSK-3β, which are responsible for β-catenin signaling, were reduced while the levels of c-Myc and cyclin D1, which are downstream targets of β-catenin, were increased. To further verify the role of UVB-induced inflammation in activation of β-catenin signaling, a high-fat-diet model was used. Administration of high-fat diet exacerbated UVB-induced inflammation. Administration of the high-fat diet enhanced β-catenin signaling and the levels of its downstream targets (c-Myc, cyclin D1, cyclin D2, MMP-2 and MMP-9) in UVB-exposed skin and skin tumors in SKH-1 mice. These data suggest that UV-induced COX-2/PGE2 stimulates β-catenin signaling, and that β-catenin activation may contribute to skin carcinogenesis.

Strong clinical and experimental evidence demonstrates association of elevated levels of matrix metalloproteinase MMP-9 with cancer progression, metastasis and shortened patient survival, as it plays a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. MMP-9 is secreted in both the monomeric and dimeric form. Although there is little research on MMP-9 dimers, some studies have shown the dimer to be associated with more aggressive tumor progression. Our objective was to study the relative secretion patterns of MMP-9 monomer and dimer in a variety of cancer cell lines and the effect of a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract on MMP-9 secretion. The cancer cell lines were grown in their respective media, supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) in 24-well tissue culture plates. At near confluence, the cells were treated with NM at 0,10, 50, 100, 500 and 1000 µg/ml. Parallel sets of cultures were treated with PMA (100 ng/ml) for induction of MMP-9. Cell MMP-9 secretion was assayed by gelatinase zymography. MMP-9 dimer secretion patterns of cancer cells fell into different categories. We observed no MMP-9 dimer in prostate DU-145 and PC-3, pancreatic MIA-Pa-Ca2, colon HCT-116, bladder T-24, head and neck FaDu, glioblastoma A-172, T-98 and LN-18 and leukemia HL-60, Jurkat, and Raji cell lines. MMP-dimer secretion only with PMA induction was seen in breast MCF-7 and MDA-MB-231, uterine SK-UT-1, lung A-549, tongue SC-25, melanoma A2058, osteosarcoma U-2OS, rhabdomyosarcoma, fibrosarcoma HT-1080, chondrosarcoma SW-1350 and liposarcoma SW-872. Cervical HeLa and DoTc 2 4510, renal 786-0 and HCC SK-Hep-1 cells exhibited MMP-9 dimer without PMA treatment and increased secretion with PMA treatment. Sarcomas had the highest levels of MMP-9 monomer and dimer with and without PMA among these cancer cell lines. Cervical, uterine and male breast cancer cell lines showed the next highest levels of MMP-9, followed by breast cancer cell lines. Melanoma, renal, lung, head and neck and HCC showed lower levels and prostate, glioblastoma, bladder and leukemia cell lines the lowest. NM showed dose-dependent inhibition of MMP-9 monomer and dimer in all cell lines tested. In conclusion, high MMP-9 and dimer secretion levels correlated with the most aggressive cancer cell lines. NM was effective in inhibiting MMP-9 and dimer secretion in all cell lines tested, suggesting its therapeutic potential as an antimetastatic agent.

The highly aggressive pediatric sarcomas are characterized by high levels of matrix metalloproteinase (MMP)-2 and MMP-9, which play crucial roles in tumor invasion and metastasis by degradation of the extracellular membrane leading to cancer cell spread to distal organs. We examined the effects of cytokines, mitogens, inducers and inhibitors on MMP-2 and -9 expression in osteosarcoma (U2OS) and rhabdomyosarcoma (RD). The selected compounds included natural cytokines and growth factors, as well as chemical compounds applied in therapy of sarcoma and natural compounds that have demonstrated anticancer therapeutic potential. These cell lines were cultured in their respective media to near confluence and the cells were washed with PBS and incubated in serum-free medium with various concentrations of several cytokines, mitogens and inhibitors. After 24 h the media were removed and analyzed for MMP-2 and -9 by gelatinase zymography and quantitated by densitometry. Osteosarcoma and rhabdomyosarcoma showed bands corresponding to MMP-2 and -9 with dose-dependent enhancement of MMP-9 with phorbol 12-myristate 13-acetate (PMA) treatment. Tumor necrosis factor-α, interleukin-1β and LPS enhanced osteosarcoma U2OS MMP-9 secretion but had no effect on MMP-2 secretion. Tumor necrosis factor-α stimulated rhabdomyosarcoma MMP-2 expression, but had no effect on MMP-9 secretion. Doxycycline, epigallocatechin gallate, nutrient mixture (NM), actinomycin-D, cyclohexamide, retinoic acid and dexamethasone inhibited MMP-2 and -9 in U2OS osteosarcoma cells. PMA-treated RD cells showed dose-response inhibition of MMP-9 by doxycycline and epigallocatechin gallate and both MMPs by NM. Dexamethasone and actinomycin-D showed inhibition of MMP-2 secretion of RD cells. Our results show that cytokines, mitogens and inducers show variable upregulation of U2OS osteosarcoma and RD rhabdomyosarcoma MMP-2 and -9 secretion, and inhibitors demonstrate downregulation under stimulatory conditions, suggesting the application of these agents for the development of effective therapies in pediatric sarcomas.

Halofuginone (HF) is extracted from Dichroa febrifuga, a plant used in traditional medicine. We report that the HF extract inhibits the growth of breast cancer cells and induces the generation of reactive oxygen species (ROS) and apoptosis, an important feature of potential anticancer agents. In addition, HF significantly reduces the migration and invasion of MCF-7 and MDA-MB-231 human breast cancer cells after 12-O-tetraecanoylphorbol-13-acetate (TPA) stimulation. As matrix metalloproteinase-9 plays a critical role in tumor metastasis, we analyzed its expression with the HF extract treatment. Western blot analysis and gelatin zymography showed that HF suppresses MMP-9 expression and activity concentration-dependently. HF also decreases the nuclear protein levels of nuclear factor kappa B (NF-κB) and c-fos (AP-1), critical transcription factors regulating MMP-9 expression through binding the MMP-9 promoter region. Luciferase assays showed that HF decreases TPA-induced MMP-9 promoter binding activities of NF-κB and AP-1. Taken together, these are the first results indicating that halofuginone may represent a promising new agent for breast cancer chemotherapy.

The highly aggressive adult sarcomas are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9, which play crucial roles in tumor invasion and metastasis by degradation of the extracellular membrane leading to cancer cell spread to distal organs. We examined the effect of cytokines, mitogens, inducers and inhibitors on MMP-2 and MMP-9 secretion in chondrosarcoma (SW-1353), fibrosarcoma (HT-1080), liposarcoma (SW-872) and synovial sarcoma (SW-982) cell lines. The selected compounds included natural cytokines and growth factors, as well as chemical compounds applied in therapy of sarcoma and natural compounds that have demonstrated anticancer therapeutic potential. MMP-2 and MMP-9 secretions were analyzed by gelatinase zymography following 24-h exposure to the tested agents and quantitated by densitometry. Fibrosarcoma, chondrosarcoma, liposarcoma and synovial sarcoma showed bands corresponding to MMP-2 and MMP-9 with dose-dependent enhancement of MMP-9 with phorbol 12-myristate 13-acetate (PMA) treatment. In chondrosarcoma cells, tumor necrosis factor (TNF)-α had a stimulatory effect on MMP-9 and insignificant effect on MMP-2 and interleukin (IL)-1β stimulated MMP-9 and MMP-2. In fibrosarcoma and liposarcoma cells, TNF-α had a profound stimulatory effect on MMP-9, but no effect on MMP-2 and in synovial sarcoma an inhibitory effect on MMP-2 and no effect on MMP-9. IL-1β had a slight inhibitory effect on fibrosarcoma, liposarcoma and synovial sarcoma MMP-2 and MMP-9 except for MMP-9 in synovial sarcoma which showed slight stimulation. Lipopolysaccharide (LPS) stimulated expression of MMP-2 in fibrosarcoma and chondrosarcoma while inhibited it in liposarcoma. Doxycycline, epigallocatechin gallate and the nutrient mixture inhibited MMP-2 and MMP-9 in all cell lines. Actinomycin-D, cyclohexamide, retinoic acid, and dexamethasone inhibited MMP-2 and -9 in chondrosarcoma and fibrosarcoma cells. Our results show that cytokines, mitogens, inducers and inhibitors have an up or down regulatory effect on MMP-2 and MMP-9 expression in adult sarcoma cell lines, suggesting these agents may be effective strategies to treat these cancers.

Pediatric sarcomas are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the ECM and basement membrane, allowing cancer cells to spread to distal organs. Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of u-PA and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs). Our main objective was to study the effect of a nutrient mixture (NM) on activity of u-PA, MMPs and TIMPs in various human pediatric sarcomas. Human osteosarcoma MNNG-HOS, osteosarcoma U-2OS and rhabdomyosarcoma RD cell lines (ATCC) were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1,000 µg/ml. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. All sarcoma cell lines studied expressed u-PA, which was inhibited by NM in a dose-dependent manner. On gelatinase zymography, osteosarcoma MNNG-HOS showed a band corresponding to MMP-2 and induction of MMP-9 with PMA (100 ng/ml) treatment. U-2OS osteosarcoma cells showed strong bands corresponding to inactive MMP-2 and MMP-9 and faint bands corresponding to active MMP-2 and MMP-9 dimer; PMA treatment enhanced MMP-9 and MMP-9 dimer activity. Rhabdomyosarcoma showed MMP-2 and faint MMP-9 bands; PMA treatment enhanced MMP-9 expression. NM inhibited their expression in a dose-dependent manner. Activity of TIMPs was upregulated by NM in all cancer cell lines in a dose-dependent manner. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA/MMPs and TIMPs. These findings suggest the therapeutic potential of NM in treatment of pediatric sarcomas.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumours and it carries a poor prognosis due to a high rate of recurrence or metastasis after surgery. Bmi-1 plays a significant role in the growth and metastasis of many solid tumours. However, the exact mechanisms underlying Bmi-1-mediated cell invasion and metastasis, especially in HCC, are not yet known. In the present study, we sought to evaluate the expression of Bmi-1 in HCC samples and its relationship with clinicopathological characteristics and prognostic value, we also investigated related mechanisms underlying Bmi-1-mediated cell invasion in HCC. Our results showed that Bmi-1 is upregulated in HCC tissues compared to matched non-cancer liver tissues; and its expression is positively associated with tumour size, metastasis, venous invasion and AJCC TNM stage, respectively; multivariate analysis showed that high expression of Bmi-1 was an independent prognostic factor for overall survival. In addition, the shRNA-mediated inhibition of Bmi-1 reduced the invasiveness of two HCC cell lines in vitro by upregulating phosphatase and the tensin homolog deleted on chromosome 10 (PTEN) expression, inhibiting the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway and downregulating the expression and activities of matrix metalloproteinase (MMP)-2 and MMP-9 and vascular endothelial growth factor (VEGF). These data demonstrate that Bmi-1 plays a vital role in HCC invasion and that Bmi-1 is a potential therapeutic target for HCC.

Cigarette smoke, specifically the nicotine contained within, has been shown to correlate closely with cell invasion and strategies to downregulate their expression may ultimately be of clinical utility. Matrix metalloproteinase-9 (MMP-9) is critically involved in the cell invasion and metastasis processes. Since nicotine plays a crucial role in the regulation of MMP-9 expression, the investigation of plant-derived compounds capable of modulating nicotine-induced signaling is an issue of concern. In this study, the effects of (-)-epigallocatechin-3-gallate (EGCG), a major green tea catechin, on nicotine-induced cell invasion and MMP-9 activity in ECV304 human endothelial cells were examined. EGCG treatment was found to reduce the MMP-9 expression and transcriptional activity in a dose-dependent manner. EGCG inhibited nicotine-activated production of reactive oxygen species (ROS), which are known as important signaling molecules to activate MMP-9. To further study the mechanisms for the EGCG-mediated regulation of MMP-9, the transcription factors NF-κB and AP-1 activities were examined. EGCG suppressed the nicotine-induced NF-κB and AP-1 activation. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated MMP-9 expression. EGCG also abrogated the nicotine-induced activation of AP-1 subunits c-fos and c-jun. The above studies demonstrate that EGCG may exert at least part of its anti-invasive effect in ECV304 human endothelial cells by controlling MMP-9 expression through the suppression of ROS, NF-κB and AP-1.

Phenethyl isothiocyanate (PEITC) is a natural compound that is involved in chemoprevention as well as inhibition of cell growth and induction of apoptosis in several types of cancer cells. Previous studies have revealed that PEITC suppresses the invasion of AGS gastric and HT-29 colorectal cancer cells. However, the effects of PEITC on the metastasis of SAS oral cancer cells remain to be determined. Our results showed that PEITC treatment inhibited the invasion of EGF-stimulated SAS cells in a concentration-dependent manner, but appeared not to affect the cell viability. The expression and enzymatic activities of matrix metalloprotease-2 (MMP-2) and matrix metalloprotease-9 (MMP-9) were suppressed by PEITC. Concomitantly, we observed an increase in the protein expression of both tissue inhibitor of metalloproteinase-1 (TIMP-1) and -2 (TIMP-2) in treated cells. Furthermore, PEITC treatments decreased the protein phosphorylation of epidermal growth factor receptor (EGFR) and downstream signaling proteins including PDK1, PI3K (p85), AKT, phosphorylated IKK and IκB to inactivate NF-κB for the suppression of MMP-2 and MMP-9 expression. In addition, PEITC can trigger the MAPK signaling pathway through the increase in phosphorylated p38, JNK and ERK in treated cells. Our data indicate that PEITC is able to inhibit the invasion of EGF-stimulated SAS oral cancer cells by targeting EGFR and its downstream signaling molecules and finally lead to the reduced expression and enzymatic activities of both MMP-2 and MMP-9. These results suggest that PEITC is promising for the therapy of oral cancer metastasis.

Adult sarcomas are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the ECM and basement membrane, allowing cancer cells to spread to distal organs. Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of u-PA and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs). Our main objective was to study the effect of a nutrient mixture (NM) on the activity of u-PA, MMPs and TIMPs in various human adult sarcomas. Human fibrosarcoma (HT-1080), chondrosarcoma (SW-1353), liposarcoma (SW-872), synovial sarcoma (SW-982) and uterine leimyosarcoma (SK-UT-1) cell lines (ATCC) were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1,000 µg/ml. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. Fibrosarcoma, chondrosarcoma, liposarcoma and leiomyosarcoma cancer cell lines expressed u-PA, which was inhibited by NM in a dose-dependent manner. However, no bands corresponding to u-PA were detected for synovial sarcoma cells. On gelatinase zymography, fibrosarcoma, chondrosarcoma, liposarcoma and synovial sarcoma showed bands corresponding to MMP-2 and MMP-9 with enhancement of MMP-9 with PMA (100 ng/ml) treatment. Uterine leiomyosarcoma showed strong bands corresponding to inactive and active MMP-9 and a faint band corresponding to MMP-9 dimer induced with PMA treatment, but no MMP-2 band. NM inhibited their expression in a dose-dependent manner. Activity of TIMPs was upregulated by NM in all cancer cell lines in a dose-dependent manner. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA/MMPs and TIMPs. These findings suggest the therapeutic potential of NM in treatment of adult sarcomas.

Quinazolinone compounds have been shown to have antitumor activity in many human cancer cell lines. In the present study, we investigated the anti-metastatic activity of MJ-33 (2-(3-ethoxyphenyl)-6-pyrrolidinylquinazolinone), a novel quinazolinone derivate, and the signaling pathway of MJ-33 in human prostate cells. MJ-33 exhibited a growth inhibitory effect on DU145, LNCaP and PC-3 cells by MTT assay. DU145 cells showed greater sensitivity to the growth inhibition of MJ-33 than that of LNCaP and PC-3 cells. MJ-33 also had an inhibitory effect on the invasion, migration and adhesion of DU145 cells using Boyden chamber transwell assays, wound-healing and adhesion assay. In addition, MJ-33 inhibited cell metastasis through the reduction of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator (u-PA) enzyme activities and protein levels by gelatin zymography assay and western blot analysis, respectively. MJ-33 reduced the protein levels of p-JNK, p-p38, p-ERK, p-AKT and nuclear NF-κB (p65), c-fos and c-Jun protein levels by western blotting. Using electrophoretic mobility-shift assay (EMSA), we demonstrated that MJ-33 blocked the activation of transcription factor AP-1 (activator protein-1) and NF-κB, which led to the inhibition of MMP-2 and MMP-9 expression. Collectively, our data showed that MJ-33 decreased protein levels of MAPKs (mitogen-activated protein kinases), AKT, AP-1 and NF-κB, resulting in the inhibition of matrix metalloproteinases. Downregulation of MMP-2 and MMP-9 reduces the invasion, migration and adhesion activities of DU145 cells. MJ-33 may be a promising agent against prostate cancer metastasis.

Keratinocyte growth factor (KGF), also known as fibroblast growth factor-7, and KGF receptor (KGFR) play important roles in the growth of epithelial cells and are overexpressed in a variety of malignant epithelial tumors, including pancreatic ductal adenocarcinoma (PDAC). We previously reported that co-expression of KGF and KGFR in PDAC is associated with venous invasion, enhanced vascular endothelial growth factor A expression and poor prognosis. Matrix metalloproteinase-9 (MMP-9) is known to participate in the degradation of type IV collagen, which is a primary component of extracellular matrices in the vascular basement membrane. In the present study, we examined the expression and roles of KGF, KGFR and MMP-9 in human PDAC cell lines and tissues. Quantitative real-time polymerase chain reaction analysis demonstrated the expression of MMP-9 mRNA in all eight PDAC cell lines. KGF, KGFR and MMP-9 were, respectively, expressed in 27 (43%), 23 (37%) and 35 (56%) of 63 patients. Each expression of KGF, KGFR or MMP-9 correlated positively with venous invasion. Furthermore, expression of KGF or MMP-9 correlated positively with liver metastasis. KGF-positive cases exhibited shorter survival than KGF-negative cases, while KGFR and MMP-9 expression were unrelated to prognosis. Administration of recombinant human KGF increased MMP-9 expression in PDAC cells, while transient transfection with short hairpin RNAs targeting KGF transcripts reduced MMP-9 expression in PDAC cells. Moreover, recombinant human KGF significantly enhanced migration and invasion of PDAC cells. These findings suggest that KGF and KGFR promote venous invasion via MMP-9 in PDAC, and closely correlate with liver metastasis. The KGF/KGFR pathway may be a critical therapeutic target for PDAC metastasis.

Glycyrrhetinic acid is an active triterpenoid metabolite of glycyrrhizin abundantly present in licorice roots. Glycyrrhetinic acid exists as α and β stereo-isomeric forms. Both stereo-isomeric forms are known to have anti-inflammatory and anticancer activity. However, the effects and anticancer mechanism of α glycyrrhetinic acid in prostate cancer cells has not yet been evaluated. Therefore, we investigated the growth inhibition, induction of apoptosis and the anticancer mechanisms of 18α-glycyrrhetinic acid (AGA), on the androgen-independent metastatic prostate cancer cell line DU-145. Our results showed that AGA inhibited proliferation and growth of these cells by inducing apoptosis as determined by Annexin V and flow cytometry analyses. Our studies also showed that HUVEC tube formation was drastically reduced when cultured in conditioned medium of AGA-treated DU-145 cells. In addition, AGA treatment prevented the invasion of DU-145 prostate cancer cells on matrigel coated transwells via down-regulation of NF-κB (p65), VEGF and MMP-9 expression. Furthermore, AGA treatment also down-regulated the expression of pro-inflammatory cytokine/growth factor genes HMGB1, IL-6 and IL-8 in DU-145 cells. Interestingly, AGA simultaneously upregulated the expression of non-steroidal anti-inflammatory gene-1 (NAG-1) in DU-145 cells suggesting its anti-inflammatory activity on prostate cancer cells. Taken together, the results of this study suggest that AGA may be a promising anticancer agent that merits further investigation for the chemoprevention and treatment of prostate cancer.

Snail and Slug play important roles in cancer progression by promoting epithelial-mesenchymal transition (EMT). Although Snail is well studied, its role in oral squamous cell carcinoma (OSCC) and its regulatory relationship with Slug remain unclear. We aimed to determine whether Snail and Slug interplay occurs in transforming growth factor-beta1 (TGF-beta1)-initiated EMT of OSCC cells, and to assess the expression of matrix metalloproteinases (MMPs) in this process. Three OSCC cell lines, SCC9, SCC15 and SCC25 were treated with recombinant TGF-beta1 for 0-6 days. Activities of the EMT regulators, Snail and Slug, and of extracellular hallmarks, MMP2 and 9, were detected using real-time PCR and Western blots. An in vitro wound healing assay then assessed short-term EMT, whilst phenotypic changes associated with the above gene expressions were evaluated by immunocytochemistry. Results showed that Slug and MMP9 were up-regulated at both mRNA and protein levels, while Snail expression rose and fell, in concert with expression of MMP2. In the wound healing assay, Snail was seen alone at the invasive front in SCC9 and SCC15 cultures, while both Snail and Slug appeared at wound margins in SCC25. Both MMP2 and MMP9 were detected at wound edges in all cell lines, with MMP2 localised in cell nuclei, MMP9 was restricted to the cytoplasm. The study suggests that both Snail and Slug act as regulators of TGF-beta1-trigged EMT in OSCC cells. Snail may up-regulate MMP2 or MMP9 initiating EMT, while Slug may share a role with Snail in maintaining longer-term EMT, by stimulating MMP9 expression.

Emodin, aloe-emodin and rhein are major compounds in rhubarb (Rheum palmatum L.), used in Chinese herbal medicine, and found to have antitumor properties including cell cycle arrest and apoptosis in many human cancer cells. Our previous studies also showed that emodin, aloe-emodin and rhein induced apoptosis in human tongue cancer SCC-4 cells. However, the detail regarding emodin, aloe-emodin and rhein affecting migration and invasion in SCC-4 cells are not clear. In the present study, we investigated whether or not emodin, aloe-emodin and rhein inhibited migration and invasion of SCC-4 cells. Herein, we demonstrate that emodin, aloe-emodin and rhein inhibit the protein levels and activities of matrix metalloproteinase-2 (MMP-2) but did not affect gene expression of MMP-2, however, they inhibited the gene expression of MMP-9 and all also inhibited the migration and invasion of human tongue cancer SCC-4 cells. MMP-9 (gelatinase-B) plays an important role and is the most associated with tumor migration, invasion and metastasis in various human cancers. Results from zymography and Western blotting showed that emodin, aloe-emodin and rhein treatment decrease the levels of MMP-2, urokinase plasminogen activator (u-PA) in a concentration-dependent manner. The order of inhibition of associated protein levels and gene expression of migration and invasion in SCC-4 cells are emodin >aloe-emodin >rhein. Our results provide new insight into the mechanisms by which emodin, aloe-emodin and rhein inhibit tongue cancers. In conclusion, these findings suggest that molecular targeting of MMP-9 mRNA expression by emodin, aloe-emodin and rhein might be a useful strategy for chemo-prevention and/or chemo-therapeutics of tongue cancers.

We have previously reported that intercellular adhesion molecule-3 (ICAM-3) is associated with an increase of cellular radio-resistance and cancer cell proliferation. In this study, we hypothesized that ICAM-3 has an additional effect on cancer cell migration and invasion because molecules induced by ICAM-3 are known as regulators of cell migration and invasion. To examine this hypothesis, we used NCI-H1299 non-small cell lung cancer (NSCLC) cell line (p53 and PTEN null cell) and constructed an ICAM-3-over-expressing stable transfectant, which exhibited increased cell migration and invasion. The increased migration and invasion resulted from up-regulation of expression and activities of MMP-2 and MMP-9. ICAM-3 also increased Akt phosphorylation, which caused an increase in cellular migration/invasion and MMP activities. Activity of several transcriptional factors located downstream in the Akt pathway was also tested, and constitutive activation of adenosine 3', 5'-monophosphate response element-binding protein (CREB) by ICAM-3 was detected. Blockage of the Akt pathway attenuated CREB activation, and a decrease in CREB expression reduced cellular migration/invasion and activity of MMPs. This result indicates that CREB functions in the signaling pathway between Akt and MMP. We also showed ICAM-3-induced cell migration and invasion in NCI-H460 NSCLC cells (wild-type p53 and PTEN cell) through the same signaling pathway. Taken together, our findings suggest that ICAM-3 stimulates cancer cell migration/invasion via ICAM-3/Akt/CREB/MMP pathway regardless of p53 and PTEN status, and this reflects the possibility that ICAM-3 could be considered as a candidate for anti-cancer drug development and as a cancer diagnostic marker.

We have previously demonstrated the effectiveness of simultaneous RNA interference (RNAi)-mediated downregulation of urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9) in inhibiting tumor invasion in vitro and in vivo. In particular, we have shown that the downregulation of uPAR and MMP-9 inhibits intracranial tumor growth. The mechanism of the inhibition of tumor growth has not yet been determined. In this study, we have attempted to explain the mechanisms involved in the inhibition of invasiveness and tumor growth in vitro. SNB19 glioma cells were transfected with scrambled vector plasmid (pSV) and a siRNA-expressing plasmid targeting either uPAR (pU) or MMP-9 (pM) singly or in combination (pUM). Untransfected cells were also used as a control. Western blotting and RT-PCR analyses showed the downregulation of uPAR in pU-transfected cells and MMP-9 in pM-transfected cells. In cells transfected with pUM, we observed down-regulation of both uPAR and MMP-9, thereby indicating the specificity of the siRNA-expressing plasmids. An increase in caspase 9 expression was observed in cells transfected with pUM whereas no change in the level of caspase 9 was observed in pU or pM-transfected cells. Additionally, no change in the expression level of caspase 8 was observed. However, an increase in the expression level of cleaved PARP was observed in the case of cells transfected with pU, pM and pUM. Cells transfected with pUM showed the highest levels of cleaved PARP expression. Expression levels of APAF-1 were also higher in pUM-transfected cells with no change in expression levels of controls and in pU and pM-transfected cells. Total CAD expression levels did not change under any of the transfection conditions. However, immunohistochemical studies demonstrated that CAD was translocated to the nucleus, thereby indicating DNA damage. As determined by Western blot analysis of subcellular fractions, cytoplasmic levels of cytochrome c were also increased. We determined the extent of DNA damage using the TUNEL assay (poly-A termination of free -OH ends of degraded nuclear DNA). Based on our results we conclude that the simultaneous downregulation of uPAR and MMP-9 induces apoptosome-mediated apoptosis.

Recent changes in the lifestyle of young women have led to an increase in the rate of uterine cervical cancer. We investigated the clinicopathological characteristics of uterine cervical cancer in young women, and examined the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). Tumor samples from 439 patients with uterine cervical cancer, who were initially treated at Osaka City University Medical School Hospital, Japan between 1995 and 2004, were stained immunohistochemically. The patients were classified into two groups according to age at onset: group Y included women aged < or =35 years, and group O included women aged > or =36 years. Group Y had more cases of squamous cell carcinoma, while group O had more advanced cases (P<0.05). Advanced cases (beyond stage Ib2) had a significantly worse prognosis in group Y than in group O (P<0.05). There were no differences between the two groups in the expressions of VEGF, MMP-2 and COX-2. However, in advanced cases (beyond stage Ib2), the expression of VEGF, MMP-2 and COX-2 was significantly greater in group Y than in group O (P<0.05). The above findings suggest that the expression of VEGF, MMPs and COX-2 is related to a worse prognosis for advanced uterine cervical cancer in young women.

Neuroblastomas are the most common extra-cranial tumors of childhood and well known for their heterogeneous clinical behavior associated with certain genetic aberrations. Radiation therapy is an important modality for the treatment of high-risk neuroblastomas. In this study, we investigated whether ionizing irradiation modulate the migration and invasiveness of human neuroblastoma cells and expression of proangiogenic molecules known to be involved in tumor progression and metastasis. Irradiation of neuroblastoma cells resulted in increased migration and invasion as measured by spheroid migration and matrigel invasion assay respectively. Zymographic analysis revealed an increase in enzyme activity of MMP-9 and uPA in conditioned medium of irradiated neuroblastoma cells compared with non-irradiated cells. An increase in VEGF levels was also found in lysates of irradiated neuroblastoma cells. The up-regulation of uPA, MMP-9 and VEGF transcripts was also confirmed by RT-PCR analysis. Next, we examined the irradiated tumor cell-mediated modulation of endothelial cell behavior. Conditioned media from irradiated neuroblastoma cells enhanced capillary-like structure formation of microvascular endothelial cells. In a coculture system, irradiation of neuroblastoma cells enhanced endothelial cell invasiveness through Matrigel matrix. Endothelial cells treated with irradiated tumor cell conditioned medium were also analyzed for expression of uPA, MMP-9 and VEGF and compared to cells treated with non-irradiated tumor cell conditioned medium. These findings suggest that the irradiation effects of tumor cells could influence endothelial angiogenesis present in non-irradiated fields.

Secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human gliomas where it promotes invasion and delays tumor growth, both in vitro and in vivo. SPARC, which interacts at the cell surface, has an impact on intracellular signaling and downstream gene expression changes, which might account for some of its effects on invasion and growth. Additionally in vitro studies demonstrated that SPARC delays growth, increases attachment, and modulates migration of tumor cells in an extracellular matrix-specific and concentration-dependent manner. Because the signaling aspect of this migration is neither well understood nor characterized, we overexpressed SPARC in both the minimally-invasive U87 cell line and in the most aggressive invasive cell line, SNB19. We first performed RT-PCR analysis and observed an upregulation of uPA and its receptor, uPAR. We also observed increased expression levels of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9). Western blot analysis confirmed these results, and the enzymatic activity of the metalloproteinases and uPA was further supported by zymography. Downstream of the uPA-uPAR interaction, upregulation of PI3-K occurred in cells overexpressing SPARC. Using GST-TRBD, we showed the upregulation of active GTP-bound RhoA, but neither Rac1 nor Cdc42 were activated. The inhibition of uPA and uPAR downregulated PI3-K activity and cell migration, as shown by matrigel invasion assay. A dorsal skin-fold chamber model revealed the high angiogenic activity of SPARC, though the proliferation of SPARC overexpressing cells was unaffected. Our results show that the small GTPase RhoA was a critical mediator of invasion or migration in the uPA-uPAR/PI3-K signaling pathway.

To clarify the potential involvement of plasmin(ogen) cascade proteins in the cell dissociation and subsequent invasion of pancreatic cancer cells, western blot analysis, immunocytochemistry, immunohistochemistry, and in vitro invasion assay were performed in the cell lines or tissue of pancreatic cancer. The strong expression of plasmin(ogen), urokinase type plasminogen activator (uPA) and uPA receptor (uPAR), and apparently weak expression of the relevant proteins were found in the conditioned medium of dissociated (PC-1.0) and non-dissociated (PC-1) pancreatic cancer cells, respectively. Furthermore, uPA-treatment significantly induced the expression of plasmin(ogen) and uPAR in the conditioned medium of non-dissociated (PC-1) pancreatic cancer cells. Moreover, the expression of plasmin(ogen) and uPAR was stronger at the invasive front than at the center of human pancreatic cancer tissue. On the other hand, plasmin-treatment induced the expression of matrix metalloproteinase-2 (MMP-2), MMP-7 and MMP-9 in PC-1 cells. Simultaneously, plasmin- or uPA-treatments obviously induced the dissociation of cell colonies and in vitro invasiveness in PC-1 cells. The plasmin(ogen) cascade is closely involved in the invasion of pancreatic cancer cells and, especially in its early stage, cell dissociation. Targeting the plasmin(ogen) cascade may provide a new insight into molecular target therapy based on anti-invasion and anti-metastasis for pancreatic cancer.

Numerous studies have demonstrated a correlation of matrix metalloproteinase (MMP) overexpression with the prognosis of various kinds of cancer. The current study investigated whether the expression of MMPs is correlated with the prognosis of osteosarcoma. Expression levels of MMP-2, -9, MT1-MMP and TIMP-2 were examined immunohistochemically in samples from 47 patients with osteosarcoma. Correlation of the positivity of staining with prognosis was analyzed with the Kaplan-Meier method, and statistically analyzed with log-rank test. Co-localization of MMP-2, MT1-MMP and TIMP-2 was determined by double staining with fluorescence-conjugated antibodies. Activities of gelatinases in representative tissues were examined with gelatin zymography. MMP-2 was expressed strongly in 60% of cases (28/47), and MMP-9, MT1- MMP and TIMP-2 was strongly positive in 61% (29/47), 45% (21/47), and 91% (43/47), respectively. Increased MT1-MMP expression was associated significantly with poor prognosis in overall survival (P=0.0480). In cases of overexpression for both MMP-2 and MT1-MMP, there was a tendency for poor prognosis (P=0.0969). In 36 cases who underwent neoadjuvant chemotherapy, wide resection of the tumors and post-operative adjuvant chemotherapy, increased expression of MT1-MMP resulted in a significant negative prognostic factor for disease-free survival (P=0.0143). Also, co-overexpression of MT1-MMP and MMP-2 showed a tendency to correlate to the reduced disease-free survival (P=0.0502). Increased gelatinase activity was observed in tissues of co-overexpression of MT1-MMP and MMP-2. The results of this study demonstrate the correlation of MT1-MMP expression and the oncological outcome of osteosarcoma patients, suggesting the prognostic significance of these proteins in osteosarcoma patients.

The expression of matrix metalloproteinases (MMPs), particularly MMP-9, is significantly increased during tumor progression and is thought to play a major role in mediating angiogenic process. Since microvasculature plays an important role in controlling tumor growth, we investigated the effects of MMP-9 inhibition on endothelial cell migration and tube formation, two determinants of angiogenesis. Adenoviral-mediated MMP-9 downregulation inhibited endothelial cell migration in cell wounding and spheroid migration assays. To determine the effects of MMP-9 reduction in glioblastoma/endothelial co-cultures, we used a three-dimensional co-culture assay of glioblastoma spheroids and endothelial spheroids. Untreated controls showed invasion of both cell populations into each other whereas treatment of the co-cultures with adenoviral antisense MMP-9 particles resulted in reduced invasion. Next, inhibition of MMP-9 by adenoviral vectors in endothelial cells was assessed for in vitro capillary-like structure formation either by co-culture with glioblastoma cells or exposure to glioblastoma-conditioned medium. Addition of conditioned medium from human glioblastoma cells to endothelial cells treated with antisense MMP-9 adenoviral vectors or co-cultures of glioblastoma cell lines with MMP-9-reduced endothelial cells resulted in reduced capillary-like tube formation demonstrating the key role of MMP-9 in endothelial cell network organization. Examination of in vitro capillary-like tube structure formation using Matrigel showed a significant decrease in MMP-9 downregulated endothelial cells as compared to controls. In conclusion, the inhibition of MMP-9 is required for inhibition of endothelial cell migration and tube formation and is likely to be of importance in cerebral angiogenesis for therapeutic targets.

Transforming growth factor (TGF)-beta has been reported to exert growth inhibitory activity in normal epithelial cells whereas it induces cell proliferation and invasive phenotypes in advanced carcinomas. Our previous study showed that MCF10A, a spontaneously immortalized "normal" breast epithelial cell line, is resistant to TGF-beta-induced growth inhibition, suggesting that conversion of TGF-beta growth inhibitory signaling into an oncogenic pathway may occur at the early stage of tumor development/progression. To address this issue, we investigated the TGF-beta signaling pathway and its role in phenotypic transformation of MCF10A cells. TGF-beta treatment induced changes in the MCF10A cell morphology from cuboidal to an elongated spindle-like shape, accompanied with down-regulation of epithelial cell marker E-cadherin. TGF-beta treatment was sufficient to induce migrative and invasive phenotypes in these cells, an important phenotypic conversion during tumor progression. We also showed that TGF-beta treatment rapidly activated ERK-1/2 and p38 MAPK leading to upregulation of matrix metalloproteinase (MMP)-2 and MMP-9. Using chemical inhibitors and dominant negative mutants of MAPKs, we provide evidence that while both p38 MAPK and ERKs are required for TGF-beta-induced MCF10A cell migration and invasion, TGF-beta-induced MMP-2 and MMP-9 expression depends on p38 MAPK signaling, but is independent of ERK activity. This study demonstrates the roles of TGF-beta signaling pathways for induction of oncogenic signaling in preneoplastic human breast epithelial cells and will deepen our understanding of TGF-beta signaling in the progress of breast cancer.

MMP-2, MMP-9, and uPA have been previously described as important to the invasive and metastatic potential of human tumors, including breast, lung, glioblastoma, and prostate. We examined the activity of these proteases and the levels of their inhibitors (TIMP-1 and TIMP-2) in a series of human meningioma tissue samples. Normal brain tissue did not show elevated levels of uPA, MMP-2 or MMP-9 activity. Meningiomas showed a mild, to moderate to significantly high level of uPA, MMP-2, and MMP-9. However, no increase in TIMP-1 or TIMP-2 levels was detected. Immunohistochemistry confirmed the assay findings and localized these molecules to the cell surface. The findings provide evidence for elevated levels of uPA and MMPs in meningiomas and suggest a therapeutic target for minimizing the malignant propensity of meningiomas using protease inhibitors.

MMP-9, which degrades extracellular matrix, is believed to play a crucial role in tumor invasion and metastasis. Angiogenesis is also perceived as an important step in tumor growth and metastasis. The aim of this study was to investigate the expression of MMP-9 in tumor samples of HNSCC patients and to study a possible correlation to angiogenic markers. Cryostat sections of 52 HNSCC tumors were immunostained for MMP-9, bFGF and VEGF using a standard streptavidin-biotin complex procedure for light microscopic investigation. Microvessel density (MVD) was determined by staining of endothelial cells immunohistochemically using anti-vWF monoclonal antibody. MMP-9 positive staining was detected in 27/52 (52%) of the tumors. MMP-9 immunoreactivity did not correlate with the main clinicopathological characteristics of the patients (localisation, T-stage, N-stage, histological grading), but correlated with worse survival of the patients. MMP-9 negative tumors showed a significant lower mean MVD per microscopic field than MMP-9 positive tumors (p<0. 001). There was a significant association of MMP-9 and VEGF expression (p<0.05). The presence of MMP-9 in HNSCC cancer and the positive correlation with MVD and VEGF expression supports the theory that MMP-9 functions as a regulator of tumor angiogenesis supporting endothelial cell invasion. MMP-9 and VEGF might act co-operatively in the process of neovascularization in human head and neck cancer.