| The macrophages in tumor microenvironment macrophages?Tumor-associated macrophages TAM?play a catalytic role in malignant tumor progression [3,4,5,52],but the chemotherapy can kill TAM from incidental secondary effect.5-FU is a commonly used cancer chemotherapy drugs for malignant entity tumors such as melanoma,colon cancer,head and neck tumors [1,2],but its killing effect on tumor-associated macrophage remains unclear.Tumor-associated macrophages?TAM?sensitivity to chemotherapy,for exploring the mechanism of resistance to chemotherapy in tumors has important significance.In preliminary experiments,we found that lipid catabolism in colon cancer TAM significantly increased,suggesting that the survival and functional activity of TAM depends on strong energy metabolism [54],so intervention TAM material and energy metabolism which may affect the sensitivity of 5-FU anti-macrophages [54],to make the killing effect of 5-FU and other chemotherapy drugs on TAM is more obvious.In preliminary experiments,we also found that: CGI-58 of macrophages can promote lipolysis and mitochondrial oxygen and suppress ROS generation,suggesting that CGI-58 may affect the effect of 5-FU anti-macrophage [54].Previous studies have shown that the key genes CGI-58 of fat catabolism in colon TAM was significantly increased [6];and CGI-58 is the important factors regulating mitochondrial function [7],prompting TAM may resist killing effect of 5-FU,leading to poor effect of chemotherapy.We respectively in vitro used CCK-8 assay to detect the cell proliferation of 5-FU treated ordinary macrophages?PLKO?and CGI-58 specific knockdown macrophages?CGI-58-KD?,used flow cytometry?FCM?to test the influence of the cell cycle and apoptosis of PLKO and CGI-58 KD which induced by 5-FU,employed western blotting to inspect 5-FU induced PLKO and CGI-58 KD of apoptosis protein?Cleaved Caspase-3?expression level,applied seahorse assays for testing the mitochondrial function of the PLKO and CGI-58 KD,and with an active oxygen blocker NAC carried on a reply experiments and found that CGI-58 deletion will promote inhibitory effects of 5-FU on the proliferation activity of macrophage,mainly by promoting macrophage apoptosis induced by 5-FU and its cycle arrest had no significant effect,and will damage the macrophage mitochondrial function,leading to ROS accumulation,but after clearing ROS CGI-58 deletion promoting effect of 5-FU on macrophage killing activity disappears.Based on studies which found out in vitro that we simultaneously further verified these studies in vivo.We using a melanoma xenograft model of CGI-58 overexpressed transgenic mouse which successfully constructed in vivo to examined the effect of 5-FU on tumor growth and prognosis of transgenic mice(TGCGI-58)and wild type mice?WT?,and after used the macrophages removed agent and reactive oxygen species ROS inhibitor NAC to intervene,we examined the effect of 5-FU on tumor growth and prognosis of transgenic mice(TGCGI-58)and wild type mice?WT?,and found TAM can resist to 5-FU chemotherapy and make poor prognosis,and CGI-58 overexpression of macrophages resist to anti-tumor effect of 5-FU,and the effect of CGI-58 overexpression resistance to 5-FU anti-tumor increases after cleared ROS.These results suggest that CGI-58 deletions may promote the 5-FU cytotoxicity of macrophages,and CGI-58 overexpression inhibits 5-FU cytotoxicity of macrophages;the effect of CGI-58 to 5-FU-induced killing TAM sensitivity,mainly through affect apoptosis rather than cycle arrest;Effect of CGI-58 on 5-FU to cytotoxicity of macrophages by produce macrophages ROS achieved;CGI-58 by influencing TAM mitochondrial function,inhibiting ROS generation,promoting the role of melanoma TAM resistance to 5-FU,thereby weakening the effect of 5-FU chemotherapy for tumors,leading to tumor malignant progression and poor prognosis.Therefore,CGI-58 as the starting point from the perspective of TAM lipid decomposition metabolism to elucidate the effecs and the regulative role of CGI-58 on 5-FU induced TAM apoptosis and its mechanism,the results for the exploration of improving chemotherapy efficacy of 5-FU and other chemotherapy drugs for tumor has important guiding significance.Objective:To investigate the effecs and the regulative role of CGI-58 on 5-fluorouracil?5-FU?induced tumor associated macrophages apoptosis and its mechanism.Methods:Part One To invest the effect of CGI-58 to 5-FU resistant macrophage cell killing and its mechanism in vitro1.Effect of CGI-58 deficiency on 5-FU-induced macrophage proliferationTo study the effect of CGI-58 on cytotoxicity of 5-FU-induced TAM,in vitro we examined the cell proliferation of 5-FU treated PLKO and CGI-58-KD by CCK-8 assay.2.Effect of CGI-58 deficiency on cell cycle of 5-FU-induced macrophageTo study the mechanism of CGI-58 to the cytotoxicity of 5-FU-induced TAM,in vitro we first examined the cell cycle of 5-FU treated PLKO and CGI-58-KD by flow cytometry,then observed the proportion of cells in the G1 phase under different process conditions.3.Effect of CGI-58 deficiency on apoptosis of 5-FU-induced macrophageTo research the effect of CGI-58 deficiency on apoptosis 5-FU induced TAM,in vitro we examined the apoptosis of 5-FU treated PLKO and CGI-58-KD by flow cytometry and using Western Blot detect the apoptosis protein?Cleaved Caspase-3?expression levels.4.Effect of CGI-58 deficiency on mitochondrial function of macrophageTo further investigate the mechanism of CGI-58 to the cytotoxicity of 5-FU-induced macrophage,in vitro we again investigated the mitochondrial function of PLKO and CGI-58-KD: used Seahorse to detect their foundation oxygen consumption rate?OCR?,employed bioluminescent method to test their ATP production volume,applied fluorescence method to assay the generation of reactive oxygen species ROS.5.Effect of CGI-58 deficiency on mitochondrial function of macrophages and 5-FU-induced macrophage proliferation and apoptosis after clearing ROSIn order to verify the above effacy and mechanism we use active oxygen inhibitors NAC make responded experiments,in the case of NAC blocking in vitro we applied fluorescence method to detect the generation of reactive oxygen species ROS of PLKO and CGI-58-KD,used CCK-8 assay to test the cell proliferation of 5-FU treated PLKO and CGI-58-KD and using Western Blot detect the apoptosis protein?Cleaved Caspase-3?expression levels.Part Two To invest the effect of tumor-associated macrophages CGI-58 to resist 5-FU chemotherapy in vivo1.Construction of the CGI-58 transgenic mice(TGCGI-58)We use the Western Blot identified the expression levels of the CGI-58 in CGI-58 over-expressing transgenic mice(TGCGI-58)and wild-type mice?WT?primary peritoneal macrophages and other tissues and organs.2.Effect of 5-FU on prognosis of transgenic mice(TGCGI-58)and wild type mice?WT?after remove macrophages in tumor?melanoma?typeIn vivo subcutaneously transplanted melanoma to CGI-58 high expression of transgenic(TGCGI-58)mice and wild-type?WT?mice,then dynamically monitored body weight and survival of mouse after administration of 5-FU chemotherapy and clodronate liposomes.3.Effect of 5-FU on prognosis of transgenic mice(TGCGI-58)and wild type mice?WT?in tumor?melanoma?typeIn vivo subcutaneously transplanted melanoma to CGI-58 high expression of transgenic(TGCGI-58)mice and wild-type?WT?mice,then dynamically monitored body weight,tumor growth and survival of mouse after administration of 5-FU chemotherapy.4.Effect of 5-FU on prognosis of transgenic mice(TGCGI-58)and wild type mice?WT?after clear ROS in tumor?melanoma?typeIn vivo subcutaneously transplanted melanoma to CGI-58 high expression of transgenic(TGCGI-58)mice and wild-type?WT?mice,then dynamically monitored body weight,tumor growth and survival of mouse after administration of 5-FU chemotherapy and active oxygen inhibitors NAC.Results:Part One To invest the effect of CGI-58 to 5-FU resistant macrophage cell killing and its mechanism in vitro1.Effect of CGI-58 deficiency on 5-FU-induced macrophage proliferationAfter 5-FU treatment,the cell proliferation of CGI-58-KD was significantly lower than that of PLKO,showing that CGI-58 deletion will promote inhibitory effects of 5-FU on the proliferation activity of macrophage.2.Effect of CGI-58 deficiency on cell cycle of 5-FU-induced macrophageNo significant differences in cell proportion of CGI-58-KD and PLKO in G1 phase after 5-FU treatment,indicated that lack of CGI-58 had no significant effect on cell cycle arrest of 5-FU-induced macrophage.3.Effect of CGI-58 deficiency on apoptosis of 5-FU-induced macrophageAfter 5-FU treatment,CGI-58-KD apoptosis was significantly increased comparing with PLKO,and the expression levels of Caspase-3 in CGI-58-KD was lower than in PLKO,suggesting that CGI-58 deletion can promote apoptosis of 5-FU-induced macrophage.4.Effect of CGI-58 deficiency on mitochondrial function of macrophageCompared with PLKO,the OCR reduced,ATP reduced and ROS increased in CGI-58-KD cells,revealing that the lack of CGI-58 can damage mitochondrial function of TAM.5.Effect of CGI-58 deficiency on mitochondrial function of macrophages and 5-FU-induced macrophage proliferation and apoptosis after clearing ROSAfter ROS inhibitors NAC blocked,ROS generation of CGI-58-KD and PLKO cells with 5-FU treatment was basically the same,no significant difference in cell proliferation and apoptosis,indicating that under conditions of NAC blocking to remove reactive oxygen species?ROS?,these phenomena can be eliminated which the proliferation reduction,apoptosis increasion and mitochondrial function damage of 5-FU-induced TAM due to CGI-58 deletion,revealing us that after clearing ROS CGI-58 deletion promoting effect of 5-FU on macrophage killing activity disappears.Part Two To invest the effect of tumor-associated macrophages CGI-58 to resist 5-FU chemotherapy in vivo1.Construction of the CGI-58 transgenic mice(TGCGI-58)The CGI-58 expression level of transgenic mice(TGCGI-58)was significantly higher than that of wild-type mice?WT?only in primary peritoneal macrophages,described transgenic mice(TGCGI-58)was successfully constructed.2.Effect of 5-FU on prognosis of transgenic mice(TGCGI-58)and wild type mice?WT?after remove macrophages in tumor?melanoma?typeAfter remove macrophages mice body weight and survival of transgenic(TGCGI-58)mice and wild-type?WT?mice had no significant difference,indicating that after 5-FU chemotherapy,transgenic mice(TGCGI-58)compared to wild-type mice?WT?,the prognosis was poor,but to remove the macrophages can eliminate this difference,and the prognosis was getting better,revealing us that TAM can resistance to 5-FU chemotherapy and prognosis.3.Effect of 5-FU on prognosis of transgenic mice(TGCGI-58)and wild type mice?WT?in tumor?melanoma?typeAfter 5-FU treatment,the body weight of transgenic mice(TGCGI-58)was lighter than that of WT mice,the tumor growth of transgenic mice(TGCGI-58)was faster than that of WT mice,and the survival of TGCGI-58 mice was shorter than that of wild-type?WT?mice,and indicating that CGI-58 overexpression of macrophages resist to anti-tumor effect of 5-FU and make the prognosis get poorer.4.Effect of 5-FU on prognosis of transgenic mice(TGCGI-58)and wild type mice?WT?after clear ROS in tumor?melanoma?typeAfter ROS inhibitors NAC blocked mice body weight,tumor volume and survival of transgenic(TGCGI-58)mice and wild-type?WT?mice had no significant difference,indicating that after 5-FU chemotherapy,transgenic mice(TGCGI-58)compared to wild-type mice?WT?,the prognosis was poor,but to remove the ROS can eliminate this difference,and the effect of CGI-58 overexpression resistance to 5-FU anti-tumor increases.Conclusion:1.CGI-58 deletions may promote the killing effect of 5-FU on macrophages,and CGI-58 overexpression inhibits cytotoxicity of 5-FU to macrophages.2.Anti-sensitivity of CGI-58 to 5-FU-induced macrophages mainly depend on the influence of 5-FU on TAM apoptosis and had no effect on the cycle block of macrophages.3.Effect of CGI-58 on 5-FU to cytotoxicity of macrophages was by produce macrophages ROS achieved.4.CGI-58 by influencing TAM mitochondrial function,inhibiting ROS generation,promoting the role of melanoma TAM resistance to 5-FU,thereby weakening the effect of 5-FU chemotherapy for tumors,leading to tumor malignant progression and poor prognosis. |
PDF Full Text Request