PAR-1- and PAR-3-type thrombin receptor expression in primary cultures of human renal cell carcinoma cells.

In this study, we report coexpression of proteinase-activated receptor (PAR)-1- and PAR-3-type thrombin receptors in primary cultures obtained from surgically resected specimens of renal cell carcinomas (RCCs). Receptor expression on RNA level was evaluated by using the RT-PCR technique. Results demonstrated the presence of mRNA encoding PAR-1 and PAR-3, but mRNA encoding PAR-4 could not be found in human RCC cells. The expression of PAR-1 and PAR-3 on protein level was investigated with confocal laser fluorescence and freeze-fracture electron microscopy. Both thrombin receptor types were localized on the cell membrane but were also found on intracellular compartments of RCC cells. On the outer cell membrane, clustering of PAR-1 and PAR-3 molecules was partly observed. This is the first study demonstrating presence of both PAR-1 and PAR-3 in human carcinoma cells.     

Presence of the proteinase-activated receptor-2 (PAR-2) in human brain tumor cells--trypsin- and SLIGRL-induced calcium response in primary cultured meningiomas.

This study focused on proteinase-activated receptor-2 (PAR-2) in primary cultured human meningioma cells. Stimulation of these cells with the serine proteinase trypsin resulted in a dose-dependent transient calcium response. Since the specific PAR-2 agonist peptide SLIGRL also induced [Ca2+]i mobilization in human meningioma cells and successive application of SLIGRL and trypsin elicited no new calcium signal we conclude that trypsin-induced calcium signaling is mediated by PAR-2 in human meningioma cells. To our knowledge, this is the first report describing functional PAR-2-type receptors in human brain tumor cells.     

PAR 1-type Thrombin Receptors are Involved in Thrombin-induced Calcium Signaling in Human Meningioma Cells

Thrombin is known to play a role as regulator in tumor spreading and tumor growth. Proteinase-activated receptor 1 (PAR 1)-type thrombin receptors were identified in different cancer cells including human glioblastoma cells. Thus a function of PAR 1 in brain tumors may be suggested. In this study, the presence of PAR 1-type thrombin receptors was investigated in primary cell cultures established from operated human meningiomas from two 59- and 79-year-old women. Characterization of PAR 1 on binding level was performed using immunofluorescence studies with the monoclonal anti-PAR 1 antibody Mab 61-1 directed against a domain in the NH2-terminus of PAR 1. These binding sites constitute functional thrombin receptors that are involved in thrombin-induced signaling in human meningioma cells as demonstrated by investigation of alpha-thrombin- and PAR 1-activating hexapeptide (TRAP-6)-induced [Ca2+]i mobilization. To our knowledge, this is the first report demonstrating thrombin-induced intracellular signaling in human meningioma cells mediated by the PAR 1-type thrombin receptor.     

Monoclonal antibody to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) induces apoptosis in primary renal cell carcinoma cells in vitro and inhibits tumor growth in vivo

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a variety of tumor cells through two of its receptors: TRAIL-R1 and TRAIL-R2. We investigate the susceptibility of human renal cell carcinoma (RCC) cells to TRM-1 and HGS-ETR2, 2 human monoclonal agonistic antibodies specific for TRAIL-R1 and TRAIL-R2, respectively. HGS-ETR2 effectively induced apoptotic cell death in 10 of 11 cell cultures, including 2 human RCC cell lines and 9 human primary RCC cell cultures, with a more pronounced effect after preincubation with anti-human IgG Fc. In contrast, TRM-1 was effective in only 1 primary RCC cell culture. The increased effectiveness of HGS-ETR2 for inducing cell death might have been affected by differences in the cell-surface expression of the 2 TRAIL receptors, namely that TRAIL-R2 but not TRAIL-R1 was frequently expressed in most of the RCC cells tested. The activities of caspase-9, -8, -6, and -3 were increased with HGS-ETR2-induced apoptosis, and cell death could be blocked by specific caspase inhibitors for caspase-9, -8, and -3, and the general caspase inhibitor. In vivo administration of HGS-ETR2 with or without cross-linker significantly suppressed tumor growth of subcutaneously inoculated human RCC xenografts in immunodeficient mice. These results suggest the potential utility of TRAIL-R2 antibody as a novel therapeutic agent in RCC.     

Thrombin—unique coagulation system protein with multifaceted impacts on cancer and metastasis

The association between blood coagulation and cancer development is well recognized. Thrombin, the pleiotropic enzyme best known for its contribution to fibrin formation and platelet aggregation during vascular hemostasis, may also trigger cellular events through protease-activated receptors, PAR-1 and PAR-4, leading to cancer progression. Our pioneering findings provided evidence that thrombin contributes to cancer metastasis by increasing adhesive potential of malignant cells. However, there is evidence that thrombin regulates every step of cancer dissemination: (1) cancer cell invasion, detachment from primary tumor, migration; (2) entering the blood vessel; (3) surviving in vasculature; (4) extravasation; (5) implantation in host organs. Recent studies have provided new molecular data about thrombin generation in cancer patients and the mechanisms by which thrombin contributes to transendothelial migration, platelet/tumor cell interactions, angiogenesis, and other processes. Though a great deal is known regarding the role of thrombin in cancer dissemination, there are new data for multiple thrombin-mediated events that justify devoting focus to this topic with a comprehensive approach.     

Commutators of PAR-1 signaling in cancer cell invasion reveal an essential role of the Rho-Rho kinase axis and tumor microenvironment

We recently reported that proteinase-activated receptors type I (PAR-1) are coupled to both negative and positive invasion pathways in colonic and kidney cancer cells cultured on collagen type I gels. Here, we found that treatments with the cell-permeant analog 8-Br-cGMP and the soluble guanylate cyclase activator BAY41-2272, and Rho kinase (ROK) inhibition by Y27632 or a dominant negative form of ROK lead to PAR-1-mediated invasion through differential Rac1 and Cdc42 signaling. Hypoxia or the counteradhesive matricellular protein SPARC/BM-40 (SPARC: secreted protein acidic rich in cysteine) overexpressed during cancer progression also commutated PAR-1 to cellular invasion through the cGMP/protein kinase G (PKG) cascade, RhoA inactivation, and Rac1-dependent or -independent signaling. Cultured primary cancer cells isolated from peritoneal and pleural effusions from patients with colon cancer or other malignant tumors harbored PAR-1, as shown by RT-PCR and FACS analyses. These malignant effusions also contained high levels of activated thrombin and fibrin, and induced a proinvasive response in HCT8/S11 human colorectal cancer cells. Our data underline the essential role of the tumor microenvironment and of several commutators targeting cGMP/PKG signaling and the RhoA-ROK axis in the control of PAR-1 proinvasive activity and metastatic potential of cancer cells in distant organs and peritoneal or pleural cavities. We also add new insights into the mechanisms linking the coagulation mediators thrombin and PAR-1 in the context of blood coagulation disorders and venous thrombosis often observed in cancer patients, as described in 1865 by Armand Trousseau.     

Up-regulation of intercellular adhesion molecule 1 (ICAM-1) on human renal cell carcinoma cells by interleukin-4

We have previously demonstrated that human renal cell carcinoma (RCC) cells express high-affinity IL-4 receptors (IL-4R). To study the functions of these receptors, we have examined the effect of IL-4 on the expression of intercellular adhesion molecule-1 (ICAM-1 or CD54) on human RCC cells. Following incubation with various concentrations of IL-4, RCC cells were examined for ICAM-1 expression by flow cytometric analysis. The 2 primary RCC cell cultures and the 2 cell lines examined expressed varying basal levels of ICAM-1 on the cell surface. IL-4 treatment increased ICAM-1 expression in a time-dependent manner and maximum augmentation of ICAM-1 expression was observed after a 48 hr incubation. The increase in ICAM-1 expression was specific because anti-hIL-4 antibody blocked this effect. No enhancement of ICAM-1 expression was observed when RCC cells were incubated with IL-4 in the presence of cycloheximide, indicating that the IL-4 effect requires new protein synthesis. Up-regulation of ICAM-1 expression was also observed at the mRNA level and maximum increase in message occurred 8 hr post-IL-4 treatment. Both IL-4 and IFN-γ also increased soluble ICAM-1 levels in WS-RCC culture supernatant. The significance of enhanced soluble and surface ICAM-1 expression was investigated by examining the lymphokine activated killer (LAK) cell-mediated lysis of IL-4-treated WS-RCC cells. LAK cells lysed WS-RCC cells very effectively, but lysis observed in target cells pre-treated with IL-4 did not correlate with the increased expression of ICAM-1 antigen. Our results indicate a previously unknown function of IL-4 on RCC and further demonstrate that IL-4R on RCC are functional. © 1995 Wiley-Liss, Inc.     

Favorable prognosis of renal cell carcinoma with increased expression of chemokines associated with a Th1-type immune response

The potential role of chemokines in clinical tumors remains poorly understood. Recent investigations have shown the differential expression of chemokine receptors on lymphocytes mediating Th1- and Th2-type immune responses. We examined Th1- and Th2-associated cytokines and chemokines, as well as the expression of their receptors in tumor-infiltrating lymphocytes in renal cell carcinoma (RCC). Sixty-seven patients with sporadic RCC were analyzed for the expression of Th1- and Th2-associated genes using real-time polymerase chain reaction. Tumor infiltration by CXC chemokine receptor 3 (CXCR3)-positive and CC chemokine receptor 5 (CCR5)-positive cells was detected by immunohistochemistry and by flow cytometry. The expression of Th1-associated genes was significantly increased in tumors compared to normal kidney tissues. The expression of interferon-gamma correlated positively with that of Th1 chemokines. Tumors expressing higher Th1 chemokines did not recur after curative surgery. Multivariate analysis showed that increased monokine induced by interferon (IFN)-gamma (MIG) expression was an independent favorable prognostic factor. Immunohistochemistry showed that the degree of CXCR3-positive cell infiltration significantly correlated with IFN-gamma inducible protein 10, MIG and IFN-gamma-inducible T cell a chemoattractant expression (I-TAC). Flow cytometric analysis showed increased expression of CXCR3 and CCR5 in tumor-infiltrating T lymphocytes compared to that in peripheral blood T cells. These results suggest that upregulation of the Th1-type immune response in RCC tumors with a favorable prognosis may be mediated by Th1-associated chemokines. Integrity of the Th1-type immune response seems to be required for tumor regression, suggesting that detection and correction of a defect in the Th1-type response cascade would thus be one of the main targets for tailor-made immunotherapy and gene therapy in RCC.     

Up-regulation of functional chemokine receptor CCR3 in human renal cell carcinoma.

There is increasing evidence that chemokines and chemokine receptors are causally involved in tumorigenesis by facilitating tumor proliferation and metastasis. Little is known about the possible function of chemokine receptors in the development and progression of renal cell carcinoma (RCC). We, therefore, analyzed the expression of chemokine receptors in tumor specimens and adjacent healthy kidney tissues [normal kidney cell (NKC)] from 10 RCC patients. We also characterized the permanent RCC cell line A-498. CCR6, CXCR2, and CXCR3 were consistently expressed by both malignant cells and NKCs. A-498 displayed additional expression of CXCR4. Importantly, the expression of CCR3 was almost absent on NKCs but clearly enhanced in a substantial proportion of RCC specimens. The primary CCR3 ligand, eotaxin-1/CCL11, induced intracellular Ca2+ mobilization, receptor internalization, and proliferation in A-498 cells confirming signaling competence of RCC-associated CCR3. In addition, we screened tumor tissue sections of 219 patients and found that 28% (62 of 219) expressed the CCR3 receptor. The presence of CCR3 in tumor samples seemed to correlate with the grade of malignancy. Previous work has established that eotaxin-1 expression is induced by tumor necrosis factor-alpha, a cytokine known to be present in RCC tissue. Our data, therefore, supports a scenario in which eotaxin-1 as part of tumor-associated inflammation promotes progression and dissemination of CCR3-positive RCC.     

The emerging role of the thrombin receptor (PAR-1) in melanoma metastasis--a possible therapeutic target

Melanoma remains as the deadliest form of skin cancer with limited and inefficient treatment options available for patients with metastatic disease. Within the last decade, the thrombin receptor, Protease Activated Receptor-1, has been described as an essential gene involved in the progression of human melanoma. PAR-1 is known to activate adhesive, invasive and angiogenic factors to promote melanoma metastasis. It is overexpressed not only in metastatic melanoma cell lines but is also highly expressed in metastatic lesions as compared to primary nevi and normal skin. Recently, PAR-1 has been described to regulate the gap junction protein Connexin 43 and the tumor suppressor gene Maspin to promote the metastatic melanoma phenotype. Herein, we review the role of PAR-1 in the progression of melanoma as well as utilizing PAR-1-regulated genes as potential therapeutic targets for melanoma treatment.     

凝血酶诱导非小细胞肺癌血管内皮生长因子分泌的实验

 目的 探讨凝血酶对非小细胞肺癌细胞株A549分泌血管内皮生长因子（Vascular endothelial growth factor，VEGF）的促进作用及其机制。方法 采用ELISA法检测凝血酶对AS49细胞分泌VEGF的浓度以及水蛭素对其阻断作用。采用RT-PCR法检测凝血酶受体PAR-1在A549细胞中的表达。结果 凝血酶在0．5～3U／ml的浓度范围内可以明显地增加VEGF的分泌，而〈0．5U／ml时这种促进作用则不明显，水蛭素能够完全阻断凝血酶的这种促进作用。PAR-1在A549细胞中表达，而在正常的胎肺细胞（KM13-17）中并不表达。结论 在一定浓度范围内，凝血酶能有效地促进A549细胞VEGF的分泌，而这种促进作用可能与PAR-1受体的表达有关。     

Role of low nuclear grading of renal carcinoma cells in the functional profile of tumor-infiltrating T cells

Tumor-infiltrating lymphocytes (TILs) from biopsies of 9 selected patients with pT1pN0M0 renal cell carcinoma (RCC) were analyzed at the clonal level for phenotypic distribution, cytokine secretion profile and antitumor cell proliferation and cytotoxicity. T-cell clones generated from RCCs were able to produce higher amounts of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) than the corresponding clones derived from peripheral blood mononuclear cells, thus suggesting a recruitment into tumors of T cells with peculiar functions. Moreover, CD4+ T-cell clones generated from TILs of nuclear grading 2 (G2)-type RCC patients produced significantly higher amounts of IL-4 and IL-10 and lower amounts of IFN-gamma than the corresponding clones generated from G1-type RCC and 2 renal angiomyolipoma (AML) patients. In addition, T-cell clones generated from lymphocytes infiltrating the peritumoral areas of G2-type, but not those from G1-type, RCC patients produced higher and lower amounts of IL-4 and IFN-gamma, respectively, than the corresponding clones derived from intratumoral T cells of the same patients. The proportion of T-cell clones derived from G2-type tumors and proliferating to autologous tumor cells (ATCs) was significantly lower than that of clones generated from G1-type RCC or AML patients. However, irrespective of their source, they exhibited similar cytokine profiles and produced comparable amounts of IL-4, IL-10 and IFN-gamma. Furthermore, the proportion and the production of both IL-4 and IFN-gamma of G2-type RCC-derived T-cell clones with cytotoxic activity against ATC were significantly lower than those of cytolytic clones generated from AML and G1-type RCCs. The concentrations of IL-4, IL-10 and IFN-gamma produced by the cytolytic clones from G2-type RCC were also lower than those produced by their noncytolytic counterparts obtained from the same patients. These data address the association of the nuclear grading of neoplastic cells with different local tumor-specific T-cell responses in RCC.