Growth-suppressive activities of serine protease inhibitors, FOY-305, ONO-3403 and FO-349 toward human carcinoma cells

FOY-305 is a synthetic serine protease inhibitor and ONO-3403 and FO-349 are its derivatives. The effects of these compounds on the proliferation of several human carcinoma cell lines were investigated in vitro by MTT colorimetric assay. ONO-3403 showed the most potent growth-inhibitory activity among these protease inhibitors. The half maximum inhibition concentrations of ONO-3403 toward BxPC-3 pancreatic carcinoma, T24 bladder carcinoma and A431 epidermoid carcinoma cells were 20-30 mu g/ml whereas those toward pancreatic carcinomas, PANC-1 and Mia PaCa-2, were 60-80 mu g/ml. Since FOY-305 has been shown to be effective in chemotherapy for human oral cancer, ONO-3403 is expected to be a more effective anticancer drug.     

Enhancement of anti-proliferative activities of 5-FU, HCFU, SN-38, THP and ACNU by a serine protease inhibitor, FOY-305

The effects of a synthetic serine protease inhibitor, FOY-305, on the proliferation of normal and transformed mouse fibroblasts were investigated in vitro by MTT colorimetric assay. FOY-305 inhibited the growth of normal NIH3T3 cells and their src- and erbB2-transformed cells with a half maximum inhibitory concentration (IC50) of 1-1.2 mg/ml whereas it suppressed the growth of ras-transformed cells more effectively (IC50 was 0.5-0.6 mg/ml). Flow cytometric analysis using synchronized NIH3T3 cells has shown that the growth-inhibitory activity of FOY-305 was due to the suppression of G(1)/S transition. The synergistic effects between FOY-305 and traditional anticancer drugs were also investigated by the MTT assay and the results showed that FOY-305 significantly enhanced the antiproliferative activities of 5-fluorouracil (5-FU), 1-hexylcarbamoyl-5-fluorouracil (HCFU), 7-ethyl-1-hydroxy-7-ethyl-10-[4-(1-piperidino)-1-piperidino] camptothecin (SN-38), pirarubicin (THP) and 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU).     

TGF-beta1 promotes liver metastasis of pancreatic cancer by modulating the capacity of cellular invasion

We investigated the effect of TGF-beta1 on liver metastasis of pancreatic cancer using surgical specimens of pancreatic cancer and human pancreatic cancer cell lines Capan-2 and SW1990. Immunostaining of TGF-beta1 showed that TGF-beta1 positivity was significantly related to venous invasion and tumor staging, and also relatively associated with liver metastasis. Cellular invasion and protease production of MMP-2 and u-PA, and in vivo liver metastasis were significantly enhanced after treatment of cells with TGF-beta1. These findings suggest that TGF-beta1 might play an important role in enhancing liver metastasis of pancreatic cancer.     

Plasminogen activator inhibitor-1 (PAI-1) gene transfection inhibits the liver metastasis of pancreatic cancer by preventing angiogenesis

Plasminogen activator inhibitor-1 (PAI-1) is a unique type of serine protease inhibitor and one of the key regulators of tumor invasion and metastasis. The purpose of this study was to elucidate the effect of PAI-1 gene transfection on liver metastasis and its mechanism by using the human high liver metastasis pancreatic cancer cell line, SW1990. PAI-1-transfected SW1990 (SW/PAI-1) produced a significantly higher level of PAI-1 in supernatant than parental cells. While no difference was observed for the production of u-PA and u-PA activity in the supernatant, cell proliferation of SW/PAI-1 was slightly suppressed on the 7th day of incubation compared to parental cells. Cellular invasion, in vivo tumorigenesis in xenograft and liver metastasis were significantly suppressed in SW/PAI-1 cells compared to parental cells. The angiogenesis of xenograft by detecting microvascular density and the production of metastasis-related factors, such as VEGF and TGF-beta1, were also decreased in SW/PAI-1 cells. These findings suggested that PAI-1 gene transfection might have the ability to prevent the liver metastasis of pancreatic cancer by modulating angiogenesis.     

Involvement of matrix metalloproteinase-7 in invasion-metastasis through induction of cell dissociation in pancreatic cancer

Epidermal growth factor receptor (EGFR) mediated mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway was isolated as invasion-metastasis related factor in pancreatic cancer in our previous studies. Matrix metalloproteinase-7 (MMP-7) and tight junction (TJ) proteins are indicated to be involved in cancer invasion-metastasis. To clarify the underlying mechanism of involvement of MMP-7 in cancer invasion, western blotting, invasion assay and immunohistochemistry were performed in dissociated (PC-1.0 and AsPC-1) and non-dissociated (PC-1 and Capan-2) pancreatic cancer cells, as well as pancreatic cancer tissues. Intracellular MMP-7 protein presented as pre-proenzyme and its expression was decreased by AG1478 (EGFR inhibitor) or U0126 (MEK inhibitor) treatment in pancreatic cancer cells. Activated MMP-7 protein was only detected in the medium of PC-1.0 and AsPC-1 cells, but not detected in the medium of PC-1 and Capan-2 cells. Moreover, MMP-7 treatment significant induced the dissociation of cell colonies in PC-1 and Capan-2 cells. Synchronously, TJ structure was apparently disrupted and translocation of TJ proteins to cytoplasm or extracellular medium was induced in PC-1 and Capan-2 cells. Furthermore, MMP-7 treatment markedly increased the in vitro invasion of PC-1 and Capan-2 cells. In addition, MMP-7 expression at the invasive front was obviously stronger than that at the center of pancreatic cancer tissues. Activation of MMP-7 protein is closely involved in disruption of TJ structure and consequent induction of cell dissociation as well as invasion in pancreatic cancer. EGFR mediated MEK/ERK signaling pathway is implied to be involved in regulation of MMP-7 expression in pancreatic cancer cells.     

Loss of hepatocyte growth factor activator inhibitor type 1 participates in metastatic spreading of human pancreatic cancer cells in a mouse orthotopic transplantation model

Hepatocyte growth factor activator inhibitor type 1 (HAI‐1) is a membrane‐bound serine protease inhibitor that is expressed on the surface of epithelial and carcinoma cells. On the cell surface, HAI‐1 regulates membrane‐anchored serine proteases, with matriptase being the most critical target. Matriptase is involved in pericellular processing of biologically active molecules, including protease‐activated receptor‐2 (PAR‐2). Previously we reported that S2‐CP8 cells, a metastatic variant of the SUIT‐2 human pancreatic adenocarcinoma cell line, showed markedly decreased HAI‐1 expression. To assess the significance of HAI‐1 loss in invasion and spontaneous metastasis of S2‐CP8 cells, we established stable S2‐CP8 sublines that expressed HAI‐1 under the control of a tetracycline‐regulated promoter. In vitro migration and invasion assays revealed inhibitory effects of HAI‐1 on S2‐CP8 cell migration and invasion. Matriptase activity was suppressed by the expression of HAI‐1. As the enhanced invasiveness in the absence of HAI‐1 was alleviated by knockdown of matriptase by 81% and of PAR‐2 completely, and PAR‐2 antagonist also suppressed the invasion, matriptase‐mediated PAR‐2 activation is involved in HAI‐1 loss‐induced invasion of S2‐CP8 cells. We then analyzed the effect of HAI‐1 expression on metastasis of S2‐CP8 cells in vivo using a nude mouse orthotopic xenograft model. Although approximately 50% of the control mice developed distant metastasis, mice treated with doxycycline to induce HAI‐1 expression did not develop metastasis. These data indicate that HAI‐1 loss contributes to invasion and dissemination of a highly metastatic subline of SUIT‐2, suggesting crucial roles for the balance of pericellular serine proteases/inhibitors in pancreatic cancer progression.     

激活和抑制Stat 3信号途径对胰腺癌细胞侵袭能力的影响

目的:通过激活和阻断人胰腺癌细胞中的Stat3信号转导通路,观察细胞株侵袭能力的变化并探讨其作用机制.方法:采用IL-6处理人胰腺癌细胞Capan-2,AG490处理人胰腺癌细胞SW1990后,MTT法检测细胞的增殖状态,免疫细胞化学法和Western 印迹法检测p-Stat3的表达,实时荧光定量PCR(real-time fluorogentic quantitative PCR, RFQ-PCR)和Western 印迹法检测血管内皮生长因子(vascular endothelial growth factor,VEGF)、基质金属蛋白酶-2(matrix metalloproteinase 2,MMP-2) mRNA及其蛋白的表达,体外侵袭实验检测细胞的侵袭能力.结果:IL-6可促进Capan-2细胞的增殖能力提高(P<0.05),p-Stat3的表达增强,VEGF和MMP-2 mRNA和蛋白表达明显升高(P<0.05),细胞侵袭能力增强.AG490可抑制SW1990细胞株的增殖(P<0.05),p-Stat3的表达下降,VEGF和MMP-2 mRNA和蛋白表达明显下降(P<0.05),侵袭能力减弱.结论:Stat3信号转导通路在胰腺癌侵袭过程中起着重要作用,以Stat3信号转导通路为靶点的基因治疗可能为胰腺癌治疗提供新的方向.     

Decrease in growth factor receptors after treatment with serine protease inhibitor ONO-3403

FOY-305 is a synthetic serine protease inhibitor and ONO-3403 is a derivative with a higher protease-inhibitory activity. The growth-suppressive effects of ONO-3403 were more prominent in Ha-ras-transformed NIH3T3 (ras-NIH) cells than in non-transformed NIH3T3 cells. After treatment of ras-NIH cells with ONO-3403 at 100-200 microg/ml, the percentage of cells found in G(1) phase decreased and, concomitantly, that in S phase increased. Molecular events caused by ONO-3403 were investigated by Western blot analysis using anti-phosphotyrosine antibody. The results showed a marked decrease in the tyrosine phosphorylation level of a 180-kDa protein after treatment with ONO-3403. This 180-kDa phosphotyrosine-containing molecule which was tentatively designated pY-p180 might be platelet-derived growth factor (PDGF) receptor since addition of PDGF to serum-starved NIH3T3 cells induced a marked tyrosine phosphorylation of the same size within 5 min. This was further confirmed by immunoprecipitation of cell extract with anti-PDGF-receptor antibody followed by Western blot analysis using anti-phosphotyrosine antibody. Treatment of T.Tn human esophageal carcinoma cells with ONO-3403 caused also decrease in pY-p180, which appeared to be epidermal growth factor receptor. Thus, ONO-3403 may induce growth suppression by down-regulation of cell surface growth factor receptors.     

Gabexate mesilate inhibits colon cancer growth, invasion, and metastasis by reducing matrix metalloproteinases and angiogenesis.

Gabexate mesilate (GM), a synthetic protease inhibitor, has an antiproteinase activity on various types of plasma serine proteases. However, its role on matrix metalloproteinases (MMPs) has not been identified. In this study, we investigated the effect of GM on MMPs and on the invasion and metastasis of human colon cancer cell lines and neoangiogenesis. The activities of MMPs secreted from these cells were significantly reduced by GM but unaffected by the serine protease inhibitor aprotinin. GM directly inhibited purified progelatinase A derived from T98G human glioblastoma cells. In vitro, GM significantly reduced the invasive ability of colon cancer cells but not cellular motility, whereas aprotinin affected neither. Liver metastatic ability and tumorigenic potential in nude mice were remarkably reduced on treatment with GM. Immunohistochemical analysis of GM-treated tumors in mice showed a marked increase in apoptosis and a significant reduction in tumor angiogenesis. Human umbilical vein endothelial cell proliferation, tube formation, and neoangiogenesis in the rabbit cornea and Matrigel implanted in mice were significantly inhibited by GM. These results suggest that GM is a novel inhibitor of MMPs and that it may inhibit the invasion and metastasis of human colon cancer cells by blocking MMPs and neoangiogenesis.     

Role of urokinase-type plasminogen activator and inhibitory effect of protease inhibitor in invasion and metastasis of pancreatic cancer

We investigated the association of urokinase-type plasminogen activator (u-PA) with tumor cell invasion and hepatic metastasis using human pancreatic cancer cell lines (SW1990, PANC-1, and RWP-1). We also examined the effect of the protease inhibitor, gabexate mesilate. SW1990 cells showed a higher u-PA activity (41.2 U/ml) in substrate assay and invasiveness (21.6% IV) than PANC-1 (14.3 U/ml, 10.3% IV) and RWP-1 (22.1, 13.5), which correlated with in vivo hepatic metastasis. Gabexate mesilate significantly reduced the u-PA activity, invasiveness, and hepatic metastasis of SW1990 cells. These findings suggest the possible application of protease inhibitors to prevent tumor invasion and metastasis.     

Enhanced VEGF production and decreased immunogenicity induced by TGF-beta 1 promote liver metastasis of pancreatic cancer

TGF-betas are multifunctional polypeptides that regulate cell growth and differentiation, extracellular matrix deposition, cellular adhesion properties, angiogenesis and immune functions. In this study, we investigated the effect of TGF-beta1 on liver metastasis and its mechanism by using human pancreatic cancer cell lines Panc-1, Capan-2, and SW1990. Capan-2 and SW1990 cells demonstrated enhanced liver metastatic potential by in vivo splenic injection with TGF-beta1. Consequently, we examined the role of TGF-beta1 on in vitro angiogenesis and received cytotoxicity by peripheral blood mononuclear leukocytes (PBMLs). While TGF-beta1 slightly decreased cell proliferation, it also upregulated VEGF production in all cancer cells examined. The binding of PBMLs to cancer cells and cancer cell cytotoxicity during co-culture with PBMLs were remarkably decreased by treatment with TGF-beta1. Panc-1 cells revealed no liver metastasis despite their high immunogenetic and angiogenetic abilities, which was attributed to a lack of expression of the cell surface carbohydrates that induce attachment to endothelial cells. We concluded that the presence of TGF-beta1 in the microenvironment of tumour site might play an important role in enhancing liver metastasis of pancreatic cancer by modulating the capacity of angiogenesis and immunogenicity.     

maspin基因与肿瘤靶向治疗

maspin是一种抑癌基因,属于丝氨酸蛋白酶抑制剂超家族成员,能抑制肿瘤细胞生长、侵袭和转移,促进肿瘤细胞凋亡,影响肿瘤血管生成以及增加化疗敏感性.对maspin靶向治疗肿瘤的机制深入研究可为肿瘤治疗提供新的思路.     

Prognostic significance of tissue factor pathway inhibitor-2 in pancreatic carcinoma and its effect on tumor invasion and metastasis

Tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated kunitz-type serine proteinase inhibitor that plays an important role in plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor angiogenesis, invasion and metastasis. Earlier studies have shown that the production of TFPI-2 is downregulated during the progression of various tumors. To detect whether TFPI-2 can be expressed in human pancreatic carcinoma samples, to evaluate its prognostic significance on pancreatic carcinoma and to investigate its effect on tumor invasion and metastasis, we collected 9 normal pancreatic tissue samples and 41 pancreatic carcinoma samples and stably transfected the human pancreatic carcinoma cell line Panc-1 with a vector capable of expressing TFPI-2 gene. RT-PCR and Western blot analysis revealed that the expression of TFPI-2 in pancreatic carcinoma samples was markedly lower than that in normal pancreas samples, and there was no TFPI-2 expression in Panc-1 cell. Its expression was related with biological characters of pancreatic carcinoma. The results of Boyden chamber assay and orthotopic pancreatic carcinoma model showed that TFPI-2 could inhibit invasion and metastasis ability of pancreatic carcinoma in vitro and in vivo. Kaplan–Meier survival curve and Cox proportional hazards model assay identified TFPI-2 as an independent prognostic factor for pancreatic carcinoma. Our data suggest that TFPI-2 plays a significant role in the invasion and metastasis of pancreatic carcinoma cell in vitro and in vivo and is determined to be an important prognostic factor for pancreatic carcinoma patients.     

Effects of bone sialoprotein on pancreatic cancer cell growth, invasion and metastasis

Bone sialoprotein (BSP) is an acidic glycoprotein that plays an important role in cancer cell growth, migration and invasion. The expression, localization and possible function of BSP in chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC) were analyzed by QRT-PCR, laser capture microdissection, DNA microarray analysis, immunoblotting, radioimmunoassays and immunohistochemistry as well as cell growth, invasion, scattering, and adhesion assays. BSP mRNA was detected in 40.7% of normal, in 80% of CP and in 86.4% of PDAC samples. The median BSP mRNA levels were 6.1 and 0.9copies/microl cDNA in PDAC and CP tissues, respectively, and zero copies/microl cDNA in normal pancreatic tissues. BSP was weakly present in the cytoplasm of islet cells and ductal cells in 20% of normal pancreatic tissues. BSP was localized in the tubular complexes of both CP and PDAC, as well as in pancreatic cancer cells. Five out of 8 pancreatic cancer cell lines expressed BSP mRNA. Recombinant BSP (rBSP) inhibited Capan-1 and SU8686 pancreatic cancer cell growth, with a maximal effect of -46.4+/-12.0% in Capan-1 cells and -45.7+/-14.5% in SU8686 cells. rBSP decreased the invasion of SU8686 cells by -59.1+/-11.2% and of Capan-1 cells by -13.3+/-3.8% (P<0.05), whereas it did not affect scattering or adhesion of both cell lines. In conclusion, endogenous BSP expression levels in pancreatic cancer cells and low to absent BSP expression in the surrounding stromal tissue elements may indirectly act to enhance the proliferation and invasion of pancreatic cancer cells.     

Correlation of translocation of tight junction protein Zonula occludens-1 and activation of epidermal growth factor receptor in the regulation of invasion of pancreatic cancer cells

Mitogen-activated protein kinase signal transduction pathway was isolated as a potential factor related to cancer cell dissociation in dissociated (PC-1.0 and AsPC-1) and non-dissociated (PC-1 and Capan-2) pancreatic cancer cells in our previous works. On the other hand, changes of structure and function of tight junction (TJ) are reported to be correlated with carcinogenesis and tumor development. In this study, the translocation of TJ protein Zonula occludens-1 (ZO-1) and the activation of epidermal growth factor receptor (EGFR) were examined to demonstrate the involvement and correlation of TJ protein translocation and EGFR activation in the cell dissociation and subsequent invasion of pancreatic cancer. Immunocytochemistry, fluorescence intensity analysis and in vitro invasion assay were performed in dissociated and non-dissociated pancreatic cancer cells. The obvious translocation of cell-cell junction localized ZO-1 protein to the cytoplasm and nucleus, simultaneous activation of EGFR, as well as the dissociation of cell colonies of non-dissociated pancreatic cancer cells were induced by dissociation factor treatment. However, EGFR inhibitor, AG1478, treatment significantly induced the redistribution of ZO-1 protein to the sites of cell-cell junction and the cell aggregation, as well as simultaneous suppression of EGFR activation in both the dissociated and the non-dissociated pancreatic cancer cells. In addition, AG1478 treatment markedly enhanced the in vitro invasion of non-dissociated pancreatic cancer cells. Translocation of TJ protein ZO-1 is closely involved in the induction of invasion through EGFR activation in pancreatic cancer cells.     

Medroxyprogesterone acetate inhibits human pancreatic carcinoma cell growth by inducing apoptosis in association with Bcl-2 phosphorylation

BACKGROUND: A previous study found that medroxyprogesterone acetate (MPA) delayed the in vivo growth of three (AsPC-1, Capan-2, and MiaPaCa-2) of nine human pancreatic carcinoma cell lines transplanted into nude mice (Cancer 1995; 75:1263-72). The current study was undertaken to evaluate the basis for this inhibitor. METHODS: The estrogen receptor (ER) and progesterone receptor (PgR) status in nine human pancreatic carcinoma cell lines, AsPC-1, BxPC-3, Capan-1, Capan-2, Hs-700T, Hs-766T, MiaPaCa-2, PANC-1, and SUIT-2, was assessed using an enzyme immunoassay (EIA). The authors tested the growth inhibitory activity of MPA and the morphologic changes in these nine pancreatic carcinoma cell lines. Cell cycle progression and DNA fragmentation also were evaluated in these cell lines. Immunoblot analysis was used to determine bcl-2 expression and phosphorylation. RESULTS: In the EIA assay, ER was detected in three cell lines (BxPC-3, Capan-2, and MiaPaCa-2), and PgR was also detected in three (AsPC-1, Capan-2, and MiaPaCa-2). Medroxyprogesterone acetate inhibited the growth of three cell lines (AsPC-1, Capan-2, and MiaPaCa-2) with IC50 values ranging from 2.3 x 10(7) to 6.1 x 10(-7) M. In these three responsive cell lines, MPA caused cell detachment and decreased cell density. The nuclei of the MPA-treated cells were condensed and often fragmented. Cell cycle analysis of these three cell lines showed that MPA induced the appearance of a sub-G1 peak, which is characteristic of early apoptotic cells. DNA degradation assay after MPA treatment showed a typical DNA ladder pattern consistent with apoptosis. Immunoblot analysis of MPA-treated cells that overexpressed bcl-2 revealed a pattern consistent with bcl-2 phosphorylation. CONCLUSIONS: Clinically attainable concentrations of MPA can inhibit the growth of some human pancreatic carcinoma cells in vitro by inducing apoptosis, probably through their PgR, in association with the phosphorylation of bcl-2. This agent may be useful for treating patients with pancreatic carcinoma.     

Maspin在肿瘤中表达水平与抑瘤特性关系的研究进展

Maspin（mammary serine protease inhibitor）基因是一种丝氨酸蛋白酶抑制剂（serpin）基因,其编码的蛋白对肿瘤具有多方面的抑制作用：抑制新生血管的生成、增加细胞间的黏附性、抑制肿瘤细胞的侵袭和转移能力、诱导肿瘤细胞凋亡。在一些肿瘤如乳腺癌、前列腺癌组织中,Maspin表达水平随肿瘤的发展逐渐降低,在另一些肿瘤如胰腺癌组织呈高表达。Maspin在不同组织中的表达情况及生物学功能,仍需进一步研究。     

LRG1 in pancreatic cancer cells promotes inflammatory factor synthesis and the angiogenesis of HUVECs by activating VEGFR signaling

BackgroundThis study aimed to investigate the roles of leucine-rich alpha-2-glycoprotein 1 (LRG1) in regulating angiogenesis during pancreatic cancer (PC) pathogenesis.MethodsLRG1 expression in tissues was detected by qRT-PCR and immunohistochemistry. LRG1 in BxPC-3 and Capan-2 cells was knocked down or overexpressed. Cell viability and the migration and invasion abilities of cells were analyzed using the Cell Counting Kit-8 (CCK-8) assay and Transwell system, respectively. Interleukin-1 beta (IL-1β), IL-18, and vascular endothelial growth factor A (VEGFA) contents in cell culture were measured by ELISA, and the angiogenesis of HUVECs was assessed by the in vitro tube formation assay. In vitro LRG1 expression in BxPC-3 and Capan-2 cells was determined using immunofluorescence.ResultsThe results showed that LRG1 expression was significantly increased in pancreatic cancer tissues and cell lines. LRG1 knockdown inhibited the viability, migration, invasion, and IL-1β and IL-18 synthesis of BxPC-3 and Capan-2 cells. VEGFA synthesis in BxPC-3 and Capan-2 cells was also inhibited by LRG1 knockdown, which caused impaired tube formation of co-cultured HUVECs. LRG1 overexpression enhanced the viability, migration, and invasion of BxPC-3 and Capan-2 cells, also causing elevated tube formation of HUVECs and IL-1β and IL-18 synthesis in co-cultures of HUVECs and BxPC-3 or Capan-2 cells. Silencing of VEGF receptor (VEGFR) abrogated the enhanced tube formation and IL-1β and IL-18 synthesis in HUVECs co-cultured with BxPC-3 or Capan-2 cells overexpressing LRG1.ConclusionsIn conclusion, LRG1, which is highly expressed in pancreatic cancer cells, promotes inflammatory factor synthesis and the angiogenesis of HUVECs though activating the VEGFR signaling pathway.