AIM To investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives on human colorectal cancer cell lines.METHODS Changes in cell proliferation and the cytotoxic effect of the ...AIM To investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives on human colorectal cancer cell lines.METHODS Changes in cell proliferation and the cytotoxic effect of the tested compounds on human colorectal cancer cell lines and human fibroblasts were evaluated using MTS and Brd U assay,allowing us to choose the most potent substance.Cell cycle alterations were analyzed using flow cytometric analysis.The apoptosis-inducing effect of compound K-453 on the HCT116 cell line was examined with annexin V/PI double staining using flow cytometry,as well as acridine orange/propidium iodide(AO/PI)staining.The flow cytometry method also allowed us to measure changes in levels or activation states of other factors associated with apoptosis,such as poly(ADP-ribose)polymerase(PARP),caspase-3 and-9,cytochrome c,Bcl-2 family proteins,and also the integrity of the mitochondrial membrane.To evaluate activity of the transcription factors and proteins involved in signaling pathways we used Western blot analysis together with flow cytometry.RESULTS Among the ten tested compounds,compound K-453{(±)-trans-1,2-dimethoxy-2’-(3,5-bis-trifluoromethylphenylamino)spiro{indoline-3,5’[4’,5’]dihydrothiazol}exhibited the most potent activity with IC50=32.22±1.14μmol/L in human colorectal HCT116 cells and was thus selected for further studies.Flow cytometric analysis revealed a K-453-induced increase in the population of cells with sub-G1 DNA content,which is considered as a marker of apoptotic cell death.The apoptosis-inducing effect of compound K453 was also confirmed by annexin V/PI double staining and AO/PI staining.The apoptosis was associated with the loss of mitochondrial membrane potential,PARP cleavage,caspase-3 and caspase-9 activation,release of cytochrome c,as well as changes in the levels of Bcl-2family members.Moreover,flow cytometry showed that compound K-453 stimulates phosphorylation of p38MAPK but decreases phosphorylation of Akt and Erk1/2.Activation of p38 MAPK was also confirmed using Western blot analysis.This analysis also revealed downregulation of NF-κB1(p50)and Rel A(p65)proteins and the loss of their anti-apoptotic activity.CONCLUSION In our study compound K-453 exhibited an antiproliferative effect by induction of intrinsic apoptosis as well as modulation of several signaling pathways.展开更多
The phytoalexin elicitor β-(1→3)-branched β-( 1→6)-linked glucohexatose has been regio- and stereospecifically synthesized by coupling of the 3, 6-branched gluco-trisaccharide Schmidt reagent 10 with a mixture of ...The phytoalexin elicitor β-(1→3)-branched β-( 1→6)-linked glucohexatose has been regio- and stereospecifically synthesized by coupling of the 3, 6-branched gluco-trisaccharide Schmidt reagent 10 with a mixture of multiol 3,6-branched gluco-trisaccharides 13 which consists of free 5,6'-OH trisaccharide, free 5,2' ,6'-OH trisaccharide, free 5,3' ,6'-OH trisaccharide and so on. The compounds 10 and 13 were prepared from 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose , 2, 3, 4, 6-tetra-O-ben-zoyi-a-D-glucopyranosyl trichioroacetimldate, and 2,3,4, 6-tetra-O-acetyl-α-D-glucopyranosyl trichloreacetimidate through regio- and stereoselective manners.展开更多
文摘AIM To investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives on human colorectal cancer cell lines.METHODS Changes in cell proliferation and the cytotoxic effect of the tested compounds on human colorectal cancer cell lines and human fibroblasts were evaluated using MTS and Brd U assay,allowing us to choose the most potent substance.Cell cycle alterations were analyzed using flow cytometric analysis.The apoptosis-inducing effect of compound K-453 on the HCT116 cell line was examined with annexin V/PI double staining using flow cytometry,as well as acridine orange/propidium iodide(AO/PI)staining.The flow cytometry method also allowed us to measure changes in levels or activation states of other factors associated with apoptosis,such as poly(ADP-ribose)polymerase(PARP),caspase-3 and-9,cytochrome c,Bcl-2 family proteins,and also the integrity of the mitochondrial membrane.To evaluate activity of the transcription factors and proteins involved in signaling pathways we used Western blot analysis together with flow cytometry.RESULTS Among the ten tested compounds,compound K-453{(±)-trans-1,2-dimethoxy-2’-(3,5-bis-trifluoromethylphenylamino)spiro{indoline-3,5’[4’,5’]dihydrothiazol}exhibited the most potent activity with IC50=32.22±1.14μmol/L in human colorectal HCT116 cells and was thus selected for further studies.Flow cytometric analysis revealed a K-453-induced increase in the population of cells with sub-G1 DNA content,which is considered as a marker of apoptotic cell death.The apoptosis-inducing effect of compound K453 was also confirmed by annexin V/PI double staining and AO/PI staining.The apoptosis was associated with the loss of mitochondrial membrane potential,PARP cleavage,caspase-3 and caspase-9 activation,release of cytochrome c,as well as changes in the levels of Bcl-2family members.Moreover,flow cytometry showed that compound K-453 stimulates phosphorylation of p38MAPK but decreases phosphorylation of Akt and Erk1/2.Activation of p38 MAPK was also confirmed using Western blot analysis.This analysis also revealed downregulation of NF-κB1(p50)and Rel A(p65)proteins and the loss of their anti-apoptotic activity.CONCLUSION In our study compound K-453 exhibited an antiproliferative effect by induction of intrinsic apoptosis as well as modulation of several signaling pathways.
基金Project supported by the Beijing Natural Science Foundation(No.6021004)and the Ministry of Science and Technology(No.2001AA246014).
文摘The phytoalexin elicitor β-(1→3)-branched β-( 1→6)-linked glucohexatose has been regio- and stereospecifically synthesized by coupling of the 3, 6-branched gluco-trisaccharide Schmidt reagent 10 with a mixture of multiol 3,6-branched gluco-trisaccharides 13 which consists of free 5,6'-OH trisaccharide, free 5,2' ,6'-OH trisaccharide, free 5,3' ,6'-OH trisaccharide and so on. The compounds 10 and 13 were prepared from 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose , 2, 3, 4, 6-tetra-O-ben-zoyi-a-D-glucopyranosyl trichioroacetimldate, and 2,3,4, 6-tetra-O-acetyl-α-D-glucopyranosyl trichloreacetimidate through regio- and stereoselective manners.
