Background AG8 is a triterpenoid saponin isolated from Ardisia gigantifolia stapf.,we previously found that AG8 demonstrated excellent antitumor activity against triple negative breast cancers(TNBCs)through reactive o...Background AG8 is a triterpenoid saponin isolated from Ardisia gigantifolia stapf.,we previously found that AG8 demonstrated excellent antitumor activity against triple negative breast cancers(TNBCs)through reactive oxygen species(ROS)generation and mitochondrial apoptotic pathways triggering.Intracellular ROS plays a vital role in cell survival,it can activate both apoptotic and ferroptosis signaling pathways.However,the effects of AG8 on ferroptosis of TNBCs have not been investigated.Objective In this study,we selected basal-like 1(BL1)and luminal androgen receptor(LAR)subtypes of TNBC to further investigate the effects of AG8 on ferroptosis and apoptosis.Methods The cell viabilities were tested using MTT.Glutathione/oxidized glutathione(GSH/GSSG)ratio,malondialdehyde(MDA)and superoxide dismutase(SOD)levels were measured by ELISA kits.C11-BODIPY 581/591and flow cytometry were used to test lipid ROS.Ferrous ion was analyzed using FeRhoNox-1.Molecular docking was used to test the interactions of AG8 with caspase 3,solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4).The influences of AG8 on caspase 3,cleaved-caspase 3,SLC7A11 and GPX4 protein levels were measured using western blotting.In vivo antitumor effect of AG8 was tested by HCC-1937 tumor bearing nude mice.Results AG8 significantly suppressed cell proliferation and colony-formation in HCC-1937(BL1)and MDA-MB-453(LAR)cells.Interestingly,Z-VAD-FMK(an apoptosis inhibitor)and Ferrostatin-1(Fer-1,ferroptosis inhibitor)both rescued AG8-induced cell death,but MDA-MB-453 was more sensitive to Fer-1.AG8 induced ferroptosis of TNBC cells by the accumulation of ROS,increasing ferrous ion and MDA levels,and decreasing GSH/GSSG ratio and SOD levels.AG8 reduced caspase 3,SLC7A11 and GPX4 protein levels,but induced cleaved caspase 3 level in TNBC cells.The results of molecular docking showed that AG8 bound effectively to caspase 3,SLC7A11 and GPX4.Moreover,AG8 treatment significantly inhibited the tumor volumes in HCC-1937 tumor bearing nude mice.Conclusion Above all,AG8 induced apoptosis and ferroptosis through regulating caspase 3 and SLC7A11/GPX4 signaling pathway.Ferroptosis of LAR subtypes are easily to be induced by AG8,the underlying mechanism need to be further investigated.展开更多
The gene, SLC7All, which encodes the solute carrier family 7 member 11 （anionic amino acid transporter light chain, xCT）, has been reported to be implicated in multiple processes such as in pheomelanin production, c...The gene, SLC7All, which encodes the solute carrier family 7 member 11 （anionic amino acid transporter light chain, xCT）, has been reported to be implicated in multiple processes such as in pheomelanin production, cell proliferation and migration, Kaposi＇s sarcoma herpesvirus （KSHV） entry into the host cells, learning and memory. Its involvement in cancer cell proliferation and metastasis has been widely studied. Its role in pheomelanogenesis is likely conserved in sheep. The full-length cDNA of sheep SLC7A11 was cloned from sheep skin fibroblasts for evaluating its role in regulating sheep coat color. The complete open reading frame of sheep xCT （sxCT） is 1512 bp in length, encoding a 503 amino acid polypeptide. We explored its function on pheomelanogenesis in vitro and in vivo. In the melan-a non-agouti mouse melanocytes that mainly produce eumelanin, overexpressed sxCT reduced the content of eumelanin. Using a testicular injection transgenic method, sxCT-transgenic sheep were generated and exhibited patches of brown/yellow coat, suggesting that sxCT can be selectively expressed to increase the pheomelanin production in wool. Our studies suggest that testicular injection of transgene can be used to genetically modify sheep coat color.展开更多
文摘Background AG8 is a triterpenoid saponin isolated from Ardisia gigantifolia stapf.,we previously found that AG8 demonstrated excellent antitumor activity against triple negative breast cancers(TNBCs)through reactive oxygen species(ROS)generation and mitochondrial apoptotic pathways triggering.Intracellular ROS plays a vital role in cell survival,it can activate both apoptotic and ferroptosis signaling pathways.However,the effects of AG8 on ferroptosis of TNBCs have not been investigated.Objective In this study,we selected basal-like 1(BL1)and luminal androgen receptor(LAR)subtypes of TNBC to further investigate the effects of AG8 on ferroptosis and apoptosis.Methods The cell viabilities were tested using MTT.Glutathione/oxidized glutathione(GSH/GSSG)ratio,malondialdehyde(MDA)and superoxide dismutase(SOD)levels were measured by ELISA kits.C11-BODIPY 581/591and flow cytometry were used to test lipid ROS.Ferrous ion was analyzed using FeRhoNox-1.Molecular docking was used to test the interactions of AG8 with caspase 3,solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4).The influences of AG8 on caspase 3,cleaved-caspase 3,SLC7A11 and GPX4 protein levels were measured using western blotting.In vivo antitumor effect of AG8 was tested by HCC-1937 tumor bearing nude mice.Results AG8 significantly suppressed cell proliferation and colony-formation in HCC-1937(BL1)and MDA-MB-453(LAR)cells.Interestingly,Z-VAD-FMK(an apoptosis inhibitor)and Ferrostatin-1(Fer-1,ferroptosis inhibitor)both rescued AG8-induced cell death,but MDA-MB-453 was more sensitive to Fer-1.AG8 induced ferroptosis of TNBC cells by the accumulation of ROS,increasing ferrous ion and MDA levels,and decreasing GSH/GSSG ratio and SOD levels.AG8 reduced caspase 3,SLC7A11 and GPX4 protein levels,but induced cleaved caspase 3 level in TNBC cells.The results of molecular docking showed that AG8 bound effectively to caspase 3,SLC7A11 and GPX4.Moreover,AG8 treatment significantly inhibited the tumor volumes in HCC-1937 tumor bearing nude mice.Conclusion Above all,AG8 induced apoptosis and ferroptosis through regulating caspase 3 and SLC7A11/GPX4 signaling pathway.Ferroptosis of LAR subtypes are easily to be induced by AG8,the underlying mechanism need to be further investigated.
基金supported by the grants from the Ministry of Agriculture of the People's Republic of China(No. 2009ZX08009-158B)the National Natural Science Foundation of China(No.31071252)
文摘The gene, SLC7All, which encodes the solute carrier family 7 member 11 （anionic amino acid transporter light chain, xCT）, has been reported to be implicated in multiple processes such as in pheomelanin production, cell proliferation and migration, Kaposi＇s sarcoma herpesvirus （KSHV） entry into the host cells, learning and memory. Its involvement in cancer cell proliferation and metastasis has been widely studied. Its role in pheomelanogenesis is likely conserved in sheep. The full-length cDNA of sheep SLC7A11 was cloned from sheep skin fibroblasts for evaluating its role in regulating sheep coat color. The complete open reading frame of sheep xCT （sxCT） is 1512 bp in length, encoding a 503 amino acid polypeptide. We explored its function on pheomelanogenesis in vitro and in vivo. In the melan-a non-agouti mouse melanocytes that mainly produce eumelanin, overexpressed sxCT reduced the content of eumelanin. Using a testicular injection transgenic method, sxCT-transgenic sheep were generated and exhibited patches of brown/yellow coat, suggesting that sxCT can be selectively expressed to increase the pheomelanin production in wool. Our studies suggest that testicular injection of transgene can be used to genetically modify sheep coat color.
