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OBJECTIVES: Tumour-associated macrophages participate in tumour development and progression. The aim of this study was to assess the interactions of pancreatic cancer cells and pro-inflammatory M1 and anti-inflammatory M2 macrophages, specifically their effect on pancreatic cancer cell migration and the changes in STAT-signalling. METHODS: Monocytes were isolated from healthy subjects and differentiated into macrophages with M-CSF. The macrophages were polarized towards M1 by IL-12 and towards M2 by IL-10. We studied also the effect of pan-JAK/STAT-inhibitor P6. Macrophage polarization and STAT and NFkB-activation in both MiaPaCa-2 and macrophages were assessed by flow cytometry. We recorded the effect of co-culture on migration rate of pancreatic cancer cells MiaPaCa-2. RESULTS: Macrophages increased the migration rate of pancreatic cancer cells. Co-culture activated STAT1, STAT3, STAT5, AKT, and NFkB in macrophages and STAT3 in MiaPaCa-2 cells. IL-12 polarized macrophages towards M1 and decreased the migration rate of pancreatic cancer cells in co-cultures as well as P6. IL-10 skewed macrophage polarization towards M2 and induced increase of pancreatic cancer cells in co-cultures. CONCLUSION: Co-culture with macrophages increased pancreatic cancer cell migration and activated STAT3. It is possible to activate and deactivate migration of pancreatic cancer cells trough macrophage polarization.
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BACKGROUND: We have found that the expression of the novel focal adhesion protein kindlin-2 had a significant positive correlation with poor survival in gastric cancer. However, the mechanism by which kindlin-2 acts in gastric cancer warrants further evaluation. METHODS: Kindlin-2 mRNA expression in gastric cancer cell lines was measured by realtime RT-PCR under normal and hypoxic conditions. Cell proliferation, apoptosis, cell cycle, tumor adhesion, cell invasion ability, and phosphorylation of integrin ß1 and ß3 proteins were measured to assess the influence of kindlin-2 on the malignant behavior of gastric cancer cells. RESULTS: Kindlin-2 mRNA expression was highest in the distant metastasis gastric cancer cell line Hs-746T. Cell proliferation, adhesion with endothelium and collagen IV, invasion rate, and angiogenesis genes expression, as well as phosphorylation of integrin ß1 and ß3 in Hs-746T, were decreased significantly after kindlin-2 downregulation, but there was no change in apoptosis and cell cycle. CONCLUSIONS: Kindlin-2 might promote the invasion of gastric cancer cells through enhancing proliferation and adhesion by the phosphorylation of integrin ß1 and ß3.
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Biomarcadores de Tumor/metabolismo , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Western Blotting , Adhesión Celular , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de la Membrana/genética , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Fosforilación , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologíaRESUMEN
OBJECTIVE: Transforming growth factor ß (TGF-ß) superfamily plays an important role in regulating gastric cancer progression. As previously demonstrated, tumor-associated macrophages (TAMs) promoted the invasion of gastric cancer cells in Matrigel. However, the role of TGF-ß superfamily signaling between TAMs and gastric cancer remains unclear. MATERIAL AND METHODS: Three-dimensional dynamic migration imaging system was used to detect gastric cancer invasion rate cocultured with macrophages in Matrigel before or after TGF-ß receptor 1 or bone morphogenic protein (BMP) receptor 1A and 1B inhibition; real-time RT-PCR was used to quantitatively investigate gene expression (TGF-ß1, TGF-ß2, BMP4, and BMP7, ADAM9, MMP9, TIMP3, VEGF-A, and VEGF-C). RESULTS: TGF-ß1, TGF-ß2, BMP4, and BMP7 expressions were increased significantly in macrophages grown with cancer cells as compared to macrophages grown alone. The invasion rate and invasion-related genes expressions of both AGS and Hs-746T gastric cancer cell lines were upregulated by macrophages, although the expression profile was different. Invasion rate and invasion-related genes' expressions of AGS cells cocultured with macrophages were downregulated significantly after TGF-ßR1 and BMPR1 inhibition. CONCLUSIONS: Macrophages associated with tumor might promote gastric cancer cells invasion though enhancing TGF-ß/BMPs signal pathway. Inhibiting TGF-ß/BMPs signal between TAMs and gastric cancer cells might provide a new therapeutic method of gastric cancer.
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Proteínas Morfogenéticas Óseas/genética , Macrófagos/metabolismo , Neoplasias Gástricas/genética , Factor de Crecimiento Transformador beta/genética , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 7/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Línea Celular Tumoral , Técnicas de Cocultivo/métodos , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Macrófagos/patología , Invasividad Neoplásica , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta2/genéticaRESUMEN
BACKGROUND: As previously demonstrated, tumor associated macrophages (TAMs) infiltration is associated with some cancers invasion and metastasis. However, the role of TAMs in the gastric cancer remains unclear. METHODS: Three- dimensional dynamic migration imaging system and real time RT-PCR were used to quantitatively investigate the effect of macrophages on the cancer cell mobility and gene expression related to cancer invasion and metastasis, including ADAM8, ADAM9, MMP9, TIMP3, VEGF-A and IL8 genes, in AGS, HGC-27, Hs-746T and NCI-N87 gastric cancer cell lines under normal or hypoxic conditions. RESULTS: Under normal conditions, the cancer cell invasion rate was increased significantly and all six gene expressions were upregulated in all four cancer cell lines by macrophages. Under hypoxia the changes in the cancer cell invasion rate induced by macrophages was negatively correlated to the TIMP3 expression. In non- metastatic cell line AGS, the increase in migration rate induced by macrophages was further elevated under hypoxia with increased ADAM8 and ADAM9 expression and decreased MMP9 and TIMP3 expressions. Under hypoxia, the induction by macrophages for IL-8 expression was increased significantly in distant metastatic cell lines NCI-N87 and HS-746T, VEGF-A was increased in HGC-27 cell line. CONCLUSIONS: Both macrophages and hypoxia play an indispensable role in regulating the invasion of gastric cancer cells in vitro; ADAMs, MMP9 and TIMP3 might be involved in TAM induced invasive power of gastric cancer cells.
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Adenocarcinoma/patología , Macrófagos/fisiología , Neoplasias Gástricas/patología , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Hipoxia de la Célula/genética , Hipoxia de la Célula/fisiología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Macrófagos/metabolismo , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Invasividad Neoplásica , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Oxígeno/farmacología , Oxígeno/fisiología , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/genética , Inhibidor Tisular de Metaloproteinasa-3/metabolismoRESUMEN
BACKGROUND: Recent evidence shows that chronic inflammation mediated by tumor-associated macrophage (TAM) play an important role in malignant tumor formation and progression. Interleukins expressed in TAMs regulate this progress. Hypoxia is a salient feature of solid tumors and has a profound influence on the biology of TAMs However, the role of interleukins in the gastric cancer progression under hypoxia is not clear. METHODS: Realtime RT-PCR was used to quantitatively investigate the IL10, IL11 and IL18 expression in CD14(-) normal macrophages and CD14(+) TAMs co-cultured with four gastric cancer cell lines including non-metastatic cell line AGS and metastatic cell lines HGC-27, Hs-746T and NCI-N87 under normal or hypoxic conditions. In addition, the correlation between IL10, IL11, IL18 expression in TAMs under hypoxia and mobility of gastric cancer cells were analyzed. RESULTS: Under normal conditions, the IL10 and IL18 expressions were significantly higher in CD14(+) TAMs co-cultured with non-metastatic cell line than with metastatic cell lines. IL11 expression was significantly higher in CD14(+) TAMs co-cultured with distant metastasis cell lines. Hypoxia induced IL10, IL11 and IL18 expression up regulated significantly in TAMs co-cultured with AGS, Hs-746T and NCI-N87 cell line. There was a significant negative correlation between IL11 expression in CD14(+) TAMs and gastric cancer cell invasion speed under hypoxic conditions (r=0.861, P<0.001). CONCLUSION: The up-regulation of IL10, IL11 and IL18 expression in TAMs by hypoxia differed in gastric cancer cell lines. IL11 expression in TAMs might play more important role than IL10 and IL18 expression in regulating the invasion of gastric cancer cells under hypoxia.
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Interleucinas/genética , Receptores de Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Hipoxia de la Célula/genética , Línea Celular Tumoral , Movimiento Celular , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-11/genética , Interleucina-11/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucinas/metabolismo , Macrófagos/patología , Invasividad Neoplásica , Neoplasias Gástricas/inmunologíaRESUMEN
Recent studies suggest that pro-inflammatory type M1 macrophages inhibit tumor progression and that anti-inflammatory M2 macrophages enhance it. The aim of this study was to examine the interaction of type M1 and M2 macrophages with pancreatic cancer cells. We studied the migration rate of fluorescein stained pancreatic cancer cells on Matrigel cultured alone or with Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) differentiated macrophages or with Macrophage Colony Stimulating Factor (M-CSF) differentiated macrophages, skewing the phenotype towards pro- and anti-inflammatory direction, respectively. Macrophage differentiation was assessed with flow cytometry and the cytokine secretion in cell cultures with cytokine array. Both GM-CSF and M-CSF differentiated macrophages increased the migration rate of primary pancreatic adenocarcinoma cell line (MiaPaCa-2) and metastatic cell line (HPAF-II). Stimulation with IL6 or IL4+LPS reversed the macrophages' increasing effect on the migration rate of MiaPaCa-2 completely and partly of HPAF-II. Co-culture with MiaPaCa-2 reduced the inflammatory cytokine secretion of GM-CSF differentiated macrophages. Co-culture of macrophages with pancreatic cancer cells seem to change the inflammatory cytokine profile of GM-CSF differentiated macrophages and this might explain why also GM-CSF differentiated macrophages promoted the invasion. Adding IL6 or IL4+LPS to the cell culture with MiaPaCa-2 and GM-CSF or M-CSF differentiated macrophages increased the secretion of inflammatory cytokines and this could contribute to the reversion of the macrophage induced increase of cancer cell migration rate.
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Patients with chronic pancreatitis with local inflammation have high risk for pancreatic cancer. The aim of this study was to examine the role of the inflammatory cells in the invasion of pancreatic cancer cells, focusing on the involvement of a disintegrin and metalloproteinase 8 (ADAM8) and matrix metalloproteinase 9 (MMP9) proteins. ADAM8 expression is associated with worse survival of pancreatic cancer patients. Monocytes from healthy donors were differentiated into macrophages. Pancreatic adenocarcinoma cells were cultured either alone or with differentiated macrophages. The cancer cell migration rate in Matrigel was measured by imaging fluorescently stained cells for 24 h. After invasion, cells were sorted into CD14 positive/negative macrophages and cancer cells with magnetic separation. The expression of ADAM8 and MMP9 was measured by the real-time PCR. Protein-level expression of ADAM8 and MMP9 was analyzed by Western blotting. In two series, siRNA technique was used to reduce either ADAM8 or MMP9 expression in the cancer cells. The coculture with macrophages increased cancer cell migration rate in Matrigel, and increased ADAM8 and MMP9 mRNA expression and protein level in the cancer cells. Reduction of ADAM8 expression with siRNA in the cancer cells decreased macrophage-induced migration rate of the cancer cells from 11.7±0.3 µm/h to 9.0±0.2 µm/h (p<0.01), and reduction of MMP9 expression decreased the migration rate to 10.1±0.2 µm/h (p<0.01). Anti-inflammatory macrophages increase pancreatic cancer cell migration rate in basement membrane matrix by inducing ADAM8 and MMP9 expression in cancer cells, thereby possibly enhancing the invasiveness of cancer.
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Proteínas ADAM/metabolismo , Adenocarcinoma/patología , Carcinoma Ductal Pancreático/patología , Regulación Neoplásica de la Expresión Génica , Macrófagos/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias Pancreáticas/patología , Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/genética , Adenocarcinoma/inmunología , Adenocarcinoma/metabolismo , Western Blotting , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/metabolismo , Estudios de Casos y Controles , Movimiento Celular , Células Cultivadas , Técnicas de Cocultivo , Humanos , Hipoxia , Macrófagos/inmunología , Metaloproteinasa 9 de la Matriz/química , Metaloproteinasa 9 de la Matriz/genética , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Invasividad Neoplásica , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Kindlin-2 is a novel focal adhesion gene mediating the cell-extracellular matrix (ECM) adhesion. Tumor-associated macrophages (TAMs) play an important role in linking chronic inflammation to cancer progression. Both kindlin-2 and TAMs have been found to promote the invasion of gastric cancer cells in our previous studies. However, the correlation between kindlin-2 and TAMs remains unclear. Real-time RT-PCR was used to investigate kindlin-2 expression in the AGS, NCI and Hs-746T gastric cancer cell lines co-cultured with TAMs under normal or hypoxic conditions. IL8, IL10, IL11, IL17b, IL18, IL22 and IL24 expressions were measured by real-time RT-PCR in the gastric cancer lines with varying levels of kindlin-2 expression, as well as after downregulation of kindlin-2 mRNA expression by the siRNA method. We found that kindlin-2 was upregulated in all three gastric cancer cell lines when co-cultured with TAMs under normal conditions. Under hypoxic conditions, the induction of kindlin-2 expression induced by macrophages was significantly downregulated in the Hs-746T cell line. IL8, IL11, IL17b, IL22 and IL24 expression was significantly higher in gastric cell lines with high kindlin-2 expression. Downregulation of kindlin-2 mRNA decreased IL10, IL11, IL17b, IL22 and IL24 expression but IL8 and IL18 expression was upregulated. Therefore, the novel focal adhesion gene kindlin-2 may play an important role in promoting the invasion of gastric cancer cells mediated by TAMs through regulating interleukin expression.
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Regulación Neoplásica de la Expresión Génica , Interleucinas/genética , Macrófagos/metabolismo , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular , Técnicas de Cocultivo , Regulación hacia Abajo/genética , Matriz Extracelular/fisiología , Expresión Génica , Humanos , Hipoxia/metabolismo , Invasividad Neoplásica , Interferencia de ARN , ARN Mensajero/metabolismo , Neoplasias Gástricas/metabolismoRESUMEN
Vasohibin-1 has been detected in endothelial cells as an intrinsic angiogenesis inhibitor. Both tumor-associated macrophages (TAMs) and transforming growth factor-ß (TGF-ß)/bone morphogenic protein (BMP) signaling have been reported to promote angiogenesis in cancer. However, whether vasohibin-1 expression is regulated by TGF-ß/BMP signaling between TAMs and cancer cells remains unclear. The expression of TGF-ß1, TGF-ß2, BMP-4, and BMP-7 in TAMs and the expression of vasohibin-1, vascular endothelial growth factor-A (VEGF-A), and VEGF-C in two pancreatic cancer cell lines (a nonmetastatic cell line Panc-1 and a distant metastatic cell line HPAF-II) were measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). The TGF-ß receptor 1 and BMP receptor 1 were inhibited by the inhibitor SB-431542 and LDN193189, respectively. Thereafter, vasohibin-1, VEGF-A, and VEGF-C expression was detected by real-time RT-PCR. We found that the expression of TGF-ß1, TGF-ß2, BMP-4, and BMP-7 was upregulated in TAMs cocultured with pancreatic cancer cells. Vasohibin-1, VEGF-A, and VEGF-C mRNA expression in pancreatic cancer cells was upregulated by TAMs. Vasohibin-1 expression in pancreatic cancer cells cocultured with TAMs was upregulated significantly when TGF-ß receptors or BMP receptors were inhibited, but VEGF-C expression was downregulated. Therefore, Vasohibin-1 expression is regulated by the TGF-ß/BMP signaling between TAMs and pancreatic cancer cells. These results might shed a new light on the antiangiogenesis therapy in the pancreatic cancer.
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Proteínas Morfogenéticas Óseas/genética , Proteínas de Ciclo Celular/genética , Expresión Génica , Macrófagos/metabolismo , Factor de Crecimiento Transformador beta/genética , Benzamidas/farmacología , Western Blotting , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Proteína Morfogenética Ósea 7/genética , Proteína Morfogenética Ósea 7/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Dioxoles/farmacología , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor C de Crecimiento Endotelial Vascular/genética , Factor C de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Kindlin-2 has been confirmed as an essential element of bidirectional integrin signaling. In recent years, the relationship between Kindlin-2 expression and cancers has been a focus of interest. However, the relationship between Kindlin-2 expression in gastric cancer and tumor invasion, metastasis, and the outcome of patients have not been studied. METHODS: Kindlin-2 expression at protein and RNA levels were detected by Western blot and real-time polymerase chain reaction in 40 pairs of gastric cancer samples. In addition, the correlations between Kindlin-2 expression and clinicopathologic factors as well as the prognosis of the patients were analyzed. Multivariate Cox regression was used to study the effect of Kindlin-2 expression on overall and progression-free survival. RESULTS: We found that Kindlin-2 was up-regulated both at RNA (P = .027) and protein levels (P = .014) in gastric cancer tissues. Tumor samples with high Kindlin-2 expression (Kindlin-2/ß-actin:tumor tissue/paraneoplastic tissue, ≥2) was observed in 55% of the patients. Moreover, Kindlin-2 expression had a significant positive correlation with tumor stromal invasion (P = .014), lymph node metastasis (P = .007), and TNM stage (P = .014). Patients with high Kindlin-2 expression had significantly poorer overall survival (P = .012) and progression-free survival (P = .012). High Kindlin-2 expression was an independent risk factor of progression-free survival (hazard ratio, 5.2; 95% confidence interval, 1.1-3.3; P = .032). CONCLUSIONS: Kindlin-2 may play an important role in the development of gastric cancer and it is a potential factor that could be used to evaluate the outcome of gastric cancer. Kindlin-2 may shed new light on evaluating the prognosis and targeted therapy of gastric cancer.
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Biomarcadores de Tumor/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/metabolismo , Anciano , Anciano de 80 o más Años , Western Blotting , Femenino , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Reacción en Cadena en Tiempo Real de la Polimerasa , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Neoplasias Gástricas/terapia , Análisis de SupervivenciaRESUMEN
Accumulating evidence suggests that TAMs contribute to tumor progression. Recently, vasohibin-1 and vasohibin-2 were detected in endothelial cells and considered as intrinsic angiogenesis inhibitors. However, it is not known whether they are also expressed in cancer cells or tumor-associated macrophages (TAMs). Realtime RT-PCR was used to investigate the vasohibin-1 and vasohibin-2 expression in four gastric cancer cell lines, including a non-metastatic cell line AGS, and metastatic cell lines HGC-27, Hs-746T and NCI-N87, co-cultured with or without TAMs. The effect of hypoxic conditions on vasohibin expression was evaluated as well, and the correlation between vasohibin-1, vasohibin-2 and VEGF-A expression under different culture conditions was analyzed. We found that both vasohibin-1 and vasohibin-2 were expressed in the four gastric cancer cell lines and in TAMs. Under normal conditions, vasohibin-1 and vasohibin-2 expressions were significantly upregulated by TAMs in all the gastric cancer cell lines. Under hypoxia, both vasohibin-1 and vasohibin-2 expressions were significantly decreased in the distant metastasis cancer cell line Hs-746T, cultured with or without TAMs (P<0.001). After induction by TAMs or hypoxia, the vasohibin-1 and vasohibin-2 expressions correlated with that of VEGF-A. In addition, TAMs, when co-cultured with the metastatic cancer cell lines, showed hypoxia-induced vasohibin-1 upregulation (P<0.05). In conclusion, both vasohibin-1 and vasohibin-2 mRNA are expressed in gastric cancer cells and in TAMs, and their expressions are altered by hypoxia.