| BackgroundMultiple steps and multiple factors are involved in gastric carcinogenesis and tumor progression. During these processes, chemokine receptors play key roles in tumor invasion and metastasis. Chemokine receptors are G protein-coupled receptors containing7transmembrane domains that are differentially expressed in diverse cell types. Gastric cancer (GC) cells have been shown to express various chemokine receptors, such as CXCR4and CCR7, and co-opt these molecules for metastasis. Recently, CCR4has been reported to be expressed in GC cells and is associated with a poor prognosis in patients with GC, while the expression reasons are unclear. Previous reports have indicated that the selective expression of CCR4on Th2cells and Treg cells is important for immunosuppression and maintaining immune balance. And CCR4-positive adult T-cell leukaemia/lymphoma cells from a subset of patients could function as Treg cells, thereby escaping host immunity. Although these studies showed evidence of CCR4-related immunosuppression, most of these have only focused on immune cells or hematological tumor cells, and very little is known about the immune regulatory roles of CCR4in solid tumor cells. In addition, several studies provide evidence that tumor cells could not only migrate, but also exert a function of invasion through up-regulation of matrix metalloproteinases via the interaction between chemokines and their receptors. To date, there have been no data available on the relationship between tumor invasion and CCR4. In the present study, we aimed to elucidate the roles of CCR4in GC progression by using GC samples and cancer cell lines, based on a serial experiments, including immunohistochemistry (IHC), immunohistofluorescence (IHF), real-time RT-PCR, flow cytometric analysis (FCM), gene transfection, bio-plex and invasive assays. Our study will reveal novel insights into the mechanisms of cancer development as well as insights into the development of novel therapeutic targets for GC Purpose:To dectect CCR4expression in dysplastic gastric mucosas, gastric adenocarcinomas with adjacent normal gastric tissues and paired metastatic lymph nodes from GC tissues, respectively, as well as GC cell lines. And to analysis the expression profiles of CCR4in GC and the reason for its modulation.Methods:1. IHC was performed on parrafined sections of56dysplastic gastric mucosas,63gastric adenocarcinomas with63adjacent normal gastric tissues and42paired metastatic lymph nodes from GC tissues, to detect the expression distribution of CCR4. Immunohistochemical analysis of tumor tissues was performed to evaluate expression of CCR4in relation to histopathologic features and its related molecule, tumor necrosis factor (TNF)-α.2. Flow cytometry (FCM) and real time RT-PCR were carried out to analyze CCR4expression in human gastric cancer cell lines MKN-28(well differentiated), AGS (moderately differentiated), SGC-7901(moderately differentiated) and MK.N-45(low differentiated)3. Stimulation of gastric cell lines with various cytokines was used to determine the modulatory factors affcting CCR4expression in GC cell lines. And we also explored the signal pathway regulates the expression of CCR4in GC cancer cells by using a specific inhibitor of NF-κB.Results:1. There was an extremely significant up-modulation of CCR4in gastric tumor tissues (27/63;42.9%) in comparison with adjacent normal gastric tissues (0/63;0) and premaligant gastric tissues (2/56;3.6%)(both P<0.001). And CCR4positive expression (15/42;35.7%) of primary gastric carcinomas was significantly lower than that of metastatic lymph nodes (35/42;83.3%)(P<0.01). A positive relationship between expression of CCR4and tumor necrosis factor-a (P<0.01) in GC tissue has been found. 2. Detectable levels of CCR4mRNA expression were examined in four human gastric cancer cell lines by real-time RT-PCR, while there were no signifcant differences in the mRNA expression levels of CCR4among them (all P>0.05). Flow cytometric analysis showed low levels of CCR4protein expression, also without signifcant differences between them (all P>0.05).3. Stimulation of gastric cell lines with various cytokines showed that TNF-a uniquely upmodulated CCR4expression through activation of nuclear factor-KB in a dose-dependent manner.Conclutions:1. We showed that CCR4was negative staining in nonneoplastic cells, slowly-intensifying in premaligant tissues, broadly expressed in gastric cancer cells and even more broadly expressed in lymph nodes metastasis cancer. CCR4expression tended to increase gradually with the development and progression of the tumor.2. GC cells cannot constitutively express high levels of CCR4, which was not related to the degree of differentiation of tumor, while might be upregulated by NF-KB-activating stimuli such as TNF-α in tumor microenvironment.3. Although it has been reported that CCR4was expressed in gastric cancer, it is unclear about the modulation reasons. In the current study, we analyzed the expression distribution of CCR4in several types of gastric tissues and used cytokine stimulation assays for further clarifying the prolfiles of CCR4expression in gastric cancer, as well as exploring its modulation mechanisms. Purpose:Here we aimed to determine the difference of CCR4expression between ly-rich GC and conventional GC, establish co-culture systems and further explore the regulatory role of highly expressed CCR4in tumor immunity.Methods:1. The difference of CCR4expression between ly-rich GC and conventional GC was further explored by IHC.2. Generation of SGC-7901cells transiently overexpressing the full length CCR4was accomplished using the plasmid CCR4-EGFP with Lipofectamine. The CCR4expression was determined by a fluorescence microscope, real-time RT-PCR and FCM, respectively. We established co-culture systems of SGC-7901cells overexpressing CCR4and human PBMC isolated from heparinized whole blood by use of Ficoll density gradient centrifugation.3. Then, we detected the co-culture supernatant by using Bio-Plex cytokine assays and the production of GrzA, GrzB, and perforin in human PBMC by FCM.Results:1. More frequently moderate to strong positive staining for CCR4in lymphocyte-rich carcinomas than those in conventional carcinomas (P<0.05) was accompanied by the development of lymphoid aggregates with germinal centers that was related to Th2responses.2. The transfection efficiency was confirmed by a fluorescence microscope, RT-PCR and FCM at24or48hours after transfection, respectively. Coculture experiments showed the levels of IFN-y in the PBMC+CCR4groups were significantly lower than those in the PBMC+MOCK groups(P<0.01) and the PBMC+CCR4+compound39groups(P<0.05). While the IL-10levels in the CCR4groups were much higher than those in the other two groups (both P<0.01). Significantly, the CD56+NK cells in PBMC+CCR4groups displayed decreased GrzA production compared with those in the PBMC+MOCK groups (P<0.05) and the PBMC+CCR4+compound39groups (P<0.05), respectively. There were no significant changes in GrzB and perforin among each group.Conclusions:1. The highly and aberrantly expressed CCR4in GC cells might negatively regulate anti-tumor immunity, which caused a shift towards a Th2cytokine pattern and inhibited secretion of cytolytic effector molecules.2. The current study confirmed that CCR4itself has a regulatory effect on tumor immunity, which was different from previous studies reported that CCR4was indirectly involved in immunosuppression through its chemiotaxis. Our data provided important evidence that downmodulation of CCR4expression could be a promising immunotherapy for human GC. Purpose:In the present study, we aimed to investigate the expression profiles of CCR4in GC cells of histologically node-negative (pNO) patients and its role in GC cell invasion. Furthermore, we tried to explore the mechanism underlying that cancer cell invasion with the finding that the forced expression of CCR4in GC cells significantly enhanced MMP9expression.Methods:1. Tissue samples were obtained from70patients with pNO GC, and immunohistochemistry or immunohistofluorescence analysis was performed on GC tissues to detect CCR4expression and its relation to histopathologic features.2. Additionally, we overexpressed CCR4in GC cell lines SGC-7901and BGC-823using lipofectamine, and the transfection efficiency was confirmed by a fluorescence microscope, real-time RT-PCR and FCM, respectively.3. In vitro matrigel invasion assays were used to assess the effect of CCR4on gastric cancer cell invasion.4. Real-time RT-PCR and gelatin zymography were used to detect the effect of CCR4overexpression on production of MMP9.Results:1. The IHF or IHC analysis revealed that CCR4expression displayed different patterns. Both membrane and cytoplasm expression of CCR4was observed most frequently in well-differentiated tumor nests of intestinal type, while the less-differentiated cells tended to display a cytoplasmic pattern of CCR4expression in diffuse type gastric cancer. The highly invasive behaviour of tumour cells was linked to a cytoplasmic CCR4expression pattern. And overexpression of CCR4was positively associated with the depth of tumor invasion in pNO GC (P<0.05).2. Matrigel invasion assays indicated that over-expression of CCR4in GC cell lines SGC-7901and BGC-823significantly enhanced the invasive ability of these cells in a ligand-independent way compared with MOCK groups, respectively (both P<0.05). 3. Results from real-time RT-PCR and gelatin zymography showed a significant increase in MMP9prodution induced by the forced expression of CCR4in SGC-7901and BGC-823cells compared with MOCK groups, respectively (both P<0.05).Conclusions:1. CCR4was closely associated with the depth of tumor invasion in pNO GC. And GC cells with highly invasive behavior tended to display a cytoplasmic CCR4expression pattern, which indicated a lingand-independent activation of CCR4. Our data reveal novel insights into the molecular mechanisms of pNO gastric cancer development.2. Furthermore, our data indicated that CCR4-induced MMP9production may at least partially underlie the new mechanism for CCR4-mediated tumor invasion in GC. |
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