BACKGROUND Hepatocellular carcinoma(HCC)is one of the most aggressive tumors worldwide.Chronic inflammation contributes to tumor evolution,and the infiltration of tumor-associated macrophages(TAMs),also known as M2-li...BACKGROUND Hepatocellular carcinoma(HCC)is one of the most aggressive tumors worldwide.Chronic inflammation contributes to tumor evolution,and the infiltration of tumor-associated macrophages(TAMs),also known as M2-like macrophages,is associated with the most aggressive behavior.Therefore,these macrophages provide the primary growth and migratory factors to the tumor cells,including those of HCC.Current therapies are not well optimized for eliminating trans-formed cells or neutralizing the tumor immune microenvironment leukocytes,such as TAMs.Growth differentiation factor 11(GDF11)may represent a promi-sing dual therapeutic target due to its reported anti-tumorigenic and immuno-modulatory properties.AIM To characterize the effects of GDF11 in M2-like macrophages and the HCC cell interaction using a functional in vitro model.METHODS This research used THP-1 and Huh7 cell lines.We applied recombinant GDF11(50 ng/mL)every 24 hours on THP-1 differentiated macrophages with M2-like polarization using interleukin-4 and interleukin-13.Firstly,the GDF11 effects on signaling,viability,proliferation,metabolism,and redox state in macrophages were charac-terized.Subsequently,we extracted conditioned media(CM)from macrophages and performed indirect co-cultures with Huh7 cells.The functional parameters were proliferation and migration assays.Finally,we charac-terized secretion in the CM using the cytokine array membrane assay.RESULTS The present study demonstrated that GDF11 activates the canonical pathway Smad2/3 without cytotoxic or prolif-erative effects.We provide evidence that GDF11 also diminishes the pro-tumoral properties of M2-like macrophages.GDF11 promoted the reduction of the M2-like macrophage marker,cluster of differentiation 206,indicating a loss of pro-tumoral properties in these leukocytes.Furthermore,this molecule induced changes in metabolism and an increase in reactive oxygen species.Using CM derived from GDF11-treated M2-like macrophages,we observed a reduction in the proliferation and migratory capacity of liver cancer cells.Moreover,the cytokine profile was affected by GDF11 stimulus,demonstrating that this molecule alters the pro-tumoral properties of TAMs,which in turn impact the behavior of HCC-derived cells.CONCLUSION This in vitro study suggests that mitigating tumor-promoting or M2-like macrophages with GDF11 may be an effective strategy for controlling the aggressiveness of HCC.展开更多
AIM： To investigate the association of cyclooxygenase-2 （COX-2） expression with angiogenesis and the number and type of inflammatory cells （macrophages/Kupffer cells; mast cells） within primary hepatocellular car...AIM： To investigate the association of cyclooxygenase-2 （COX-2） expression with angiogenesis and the number and type of inflammatory cells （macrophages/Kupffer cells; mast cells） within primary hepatocellular carcinoma （HCC） tissues and adjacent non-tumorous （NT） tissues. METHODS： Immunohistochemistry for COX-2, CD34, CD68 and mast cell tryptase （MCT） was performed on 14 well-characterized series of liver-cirrhosis-associated HCC patients. COX-2 expression and the number of inflammatory cells in tumor lesions and surrounding liver tissues of each specimen were compared. Moreover, COX-2, CD34 staining and the number of inflammatory cells in areas with different histological degrees within each tumor sample were comparatively analyzed. RESULTS： The percentage of COX-2 positive cells was significantly higher in NT tissues than in tumors. COX-2 expression was higher in well-differentiated HCC than in poorly-differentiated tissues. Few mast cells were observed within the tumor mass, whereas a higher number was observed in the surrounding tissue, especially in peri-portal spaces of NT tissues. Abundant macrophages/ Kupffer cells were observed in NT tissues, whereas the number of cells was significantly lower in the tumor mass. However, a higher cell number was observed in the welldifferentiated tumor and progressively decreased in relation to the differentiation grade. Within the tumor, a positive correlation was found between COX-2 expression and the number of macrophages/Kupffer cells and mastcells. Moreover, there was a positive correlation between CD34 and COX-2 expression in tumor tissues. Comparison between well- and poorly-differentiated HCC showed that the number of CD34-positive cells decreased with dedifferentiation. However, COX-2 was the only independent variable showing a positive correlation with CD34 in a multivariate analysis. CONCLUSION： The presence of inflammatory cells and COX-2 expression in liver tumor suggests a possible relationship with tumor angiogenesis. COX-2 expressing cells and the number of macrophages/Kupffer cells and mast cells decrease with progression of the disease.展开更多
Combretastatin A-1 phosphate （CA1P） is a tubulin polymerization inhibitor that binds to the colchicine- binding site of tubulin and shows potential anti-tumor activity to acute myelocytic leukemia as reported. We de...Combretastatin A-1 phosphate （CA1P） is a tubulin polymerization inhibitor that binds to the colchicine- binding site of tubulin and shows potential anti-tumor activity to acute myelocytic leukemia as reported. We demon- strated that CA1P also showed an outstanding anti-cancer effect on hepatocellular carcinoma （HCC） in vivo and in vitro. As determined by DCFH-DA dye and Western blot, CA1P induced ROS accumulation and apoptosis in HepG2 cells with the down-regulation of Mcl-1. Additonal western blot and immunofluorescence assays further indi- cated that CA1P inhibited Wnt/β-catenin pathway through GSK-3β activition with an increasing of Mcl phosphoryl- ation and subsequent degradation mediated by tubulin-dynactin p l50-AKT signaling pathway axis. Apoptosis of HepG2 cells induced by CA1P was reversed by the GSK-3β inhibitor （ CHIR-99021 ）. Furthermore, determined by immunohistochemistry of an orthotopic HCC tumor model, CA1P showed a significantly effect on tumor associated macrophage （TAM） apoptosis in vitro and eliminated TAM in tumor microenviroment in vivo, while the infiltration of Treg cells and expression of TGF-β were also altered. Adoptive transfer of macrophages reinstated tumor growth treated by CA1P. These results indicated that CA1P presented potent potential on the regulation of hepatocellular carcinoma cells and TAMs, and also revealed a novel anti-HCC mechanism of CA1P, which acted on both cancer cells and tumor microenvironment. The findings would be beneficial for exploring new application of anti-microtubu- lar drugs on oncotherapy.展开更多
文摘BACKGROUND Hepatocellular carcinoma(HCC)is one of the most aggressive tumors worldwide.Chronic inflammation contributes to tumor evolution,and the infiltration of tumor-associated macrophages(TAMs),also known as M2-like macrophages,is associated with the most aggressive behavior.Therefore,these macrophages provide the primary growth and migratory factors to the tumor cells,including those of HCC.Current therapies are not well optimized for eliminating trans-formed cells or neutralizing the tumor immune microenvironment leukocytes,such as TAMs.Growth differentiation factor 11(GDF11)may represent a promi-sing dual therapeutic target due to its reported anti-tumorigenic and immuno-modulatory properties.AIM To characterize the effects of GDF11 in M2-like macrophages and the HCC cell interaction using a functional in vitro model.METHODS This research used THP-1 and Huh7 cell lines.We applied recombinant GDF11(50 ng/mL)every 24 hours on THP-1 differentiated macrophages with M2-like polarization using interleukin-4 and interleukin-13.Firstly,the GDF11 effects on signaling,viability,proliferation,metabolism,and redox state in macrophages were charac-terized.Subsequently,we extracted conditioned media(CM)from macrophages and performed indirect co-cultures with Huh7 cells.The functional parameters were proliferation and migration assays.Finally,we charac-terized secretion in the CM using the cytokine array membrane assay.RESULTS The present study demonstrated that GDF11 activates the canonical pathway Smad2/3 without cytotoxic or prolif-erative effects.We provide evidence that GDF11 also diminishes the pro-tumoral properties of M2-like macrophages.GDF11 promoted the reduction of the M2-like macrophage marker,cluster of differentiation 206,indicating a loss of pro-tumoral properties in these leukocytes.Furthermore,this molecule induced changes in metabolism and an increase in reactive oxygen species.Using CM derived from GDF11-treated M2-like macrophages,we observed a reduction in the proliferation and migratory capacity of liver cancer cells.Moreover,the cytokine profile was affected by GDF11 stimulus,demonstrating that this molecule alters the pro-tumoral properties of TAMs,which in turn impact the behavior of HCC-derived cells.CONCLUSION This in vitro study suggests that mitigating tumor-promoting or M2-like macrophages with GDF11 may be an effective strategy for controlling the aggressiveness of HCC.
基金Supported by the MIUR and Progetto Strategico Oncologia "Terapia Preclinica Moleculare Oncologia" MIUR-CNR
文摘AIM： To investigate the association of cyclooxygenase-2 （COX-2） expression with angiogenesis and the number and type of inflammatory cells （macrophages/Kupffer cells; mast cells） within primary hepatocellular carcinoma （HCC） tissues and adjacent non-tumorous （NT） tissues. METHODS： Immunohistochemistry for COX-2, CD34, CD68 and mast cell tryptase （MCT） was performed on 14 well-characterized series of liver-cirrhosis-associated HCC patients. COX-2 expression and the number of inflammatory cells in tumor lesions and surrounding liver tissues of each specimen were compared. Moreover, COX-2, CD34 staining and the number of inflammatory cells in areas with different histological degrees within each tumor sample were comparatively analyzed. RESULTS： The percentage of COX-2 positive cells was significantly higher in NT tissues than in tumors. COX-2 expression was higher in well-differentiated HCC than in poorly-differentiated tissues. Few mast cells were observed within the tumor mass, whereas a higher number was observed in the surrounding tissue, especially in peri-portal spaces of NT tissues. Abundant macrophages/ Kupffer cells were observed in NT tissues, whereas the number of cells was significantly lower in the tumor mass. However, a higher cell number was observed in the welldifferentiated tumor and progressively decreased in relation to the differentiation grade. Within the tumor, a positive correlation was found between COX-2 expression and the number of macrophages/Kupffer cells and mastcells. Moreover, there was a positive correlation between CD34 and COX-2 expression in tumor tissues. Comparison between well- and poorly-differentiated HCC showed that the number of CD34-positive cells decreased with dedifferentiation. However, COX-2 was the only independent variable showing a positive correlation with CD34 in a multivariate analysis. CONCLUSION： The presence of inflammatory cells and COX-2 expression in liver tumor suggests a possible relationship with tumor angiogenesis. COX-2 expressing cells and the number of macrophages/Kupffer cells and mast cells decrease with progression of the disease.
文摘Combretastatin A-1 phosphate （CA1P） is a tubulin polymerization inhibitor that binds to the colchicine- binding site of tubulin and shows potential anti-tumor activity to acute myelocytic leukemia as reported. We demon- strated that CA1P also showed an outstanding anti-cancer effect on hepatocellular carcinoma （HCC） in vivo and in vitro. As determined by DCFH-DA dye and Western blot, CA1P induced ROS accumulation and apoptosis in HepG2 cells with the down-regulation of Mcl-1. Additonal western blot and immunofluorescence assays further indi- cated that CA1P inhibited Wnt/β-catenin pathway through GSK-3β activition with an increasing of Mcl phosphoryl- ation and subsequent degradation mediated by tubulin-dynactin p l50-AKT signaling pathway axis. Apoptosis of HepG2 cells induced by CA1P was reversed by the GSK-3β inhibitor （ CHIR-99021 ）. Furthermore, determined by immunohistochemistry of an orthotopic HCC tumor model, CA1P showed a significantly effect on tumor associated macrophage （TAM） apoptosis in vitro and eliminated TAM in tumor microenviroment in vivo, while the infiltration of Treg cells and expression of TGF-β were also altered. Adoptive transfer of macrophages reinstated tumor growth treated by CA1P. These results indicated that CA1P presented potent potential on the regulation of hepatocellular carcinoma cells and TAMs, and also revealed a novel anti-HCC mechanism of CA1P, which acted on both cancer cells and tumor microenvironment. The findings would be beneficial for exploring new application of anti-microtubu- lar drugs on oncotherapy.
