Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocreba...Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocrebanine on microtubule network homeostasis at both molecular and cellular levels.Methods:the EBI site competition method and molecular docking method were used to determine the occupation of the microtubule site of oxocrebanine.Western Blot was used to detect the effect of oxocrebanine on microtubule-associated proteins including STAT3,PAK1,CAMK4,and PKA.Results:The results of EBI site competition assay showed that the binding of EBI toβ-Tubulin covalent fusions produced adducts that appeared in regions of lower molecular weight thanβ-tubulin(ctrl 2).Molecular docking results showed that oxocrebanine could occupy the colchicine site of microtubule proteins.As revealed by Western Blot,the expression of STAT3 protein was decreased after MCF-7 cells have been treated with low,medium,and high concentration of oxocrebanine or the positive drug taxol for 48 h(P<0.01).The expression levels of PAK1 and Camk4 proteins aslo showed significant reductions(P<0.05,or P<0.01).Oxocrebanine also decreased the PKA protein in MCF-7 cells compared to the control group(P<0.01).Conclusions:Oxocrebanine,a ligand that binds at the colchicine site of tubulin,perturbs tubulin polymerization and causes mitosis in MCF-7 cells,thus leading to MCF-7 cell death.Oxocrebanine may promote microtubule dynamics through stathmin by inhibiting the expression levels of STAT3,PAK1,Camk4,and PKA proteins in MCF-7 cells.Oxocrebanine interfers with spindle formation,and ultimately causes mitotic catastrophe in MCF-7 cells.展开更多
BACKGROUND Finding active lead anti-hepatocellular carcinoma compounds from traditional Chinese medicine has important research value.AIM To assess the detailed mechanism of oxocrebanine,a compound separated from the ...BACKGROUND Finding active lead anti-hepatocellular carcinoma compounds from traditional Chinese medicine has important research value.AIM To assess the detailed mechanism of oxocrebanine,a compound separated from the traditional Chinese medicinal plant Stephania hainanensis H.S.Lo et Y.Tsoong,and to evaluate its inhibition of the proliferation of human hepatocellular carcinoma cells via apoptosis and autophagy.METHODS MTT,BrdU labeling,and colony formation assays were used to assess the inhibitory effect of oxocrebanine on the growth and proliferation of human hepatocellular carcinoma Hep3B2.1-7 cells.Flow cytometry was used to detect the effect of oxocrebanine on the apoptosis of Hep3B2.1-7 cells.Western blotting was used to assess the expression of apoptosis-related proteins in Hep3B2.1-7 cells.The aforementioned methods were also used to evaluate the effects of oxocrebanine on cell proliferation,autophagy markers,and autophagy-related protein expression levels after adding autophagy inhibitor 3-mA.Furthermore,to verify the anti-hepatocellular carcinoma effect of oxocrebanine in vivo,a nude mouse model was used to investigate the inhibitory effect of oxocrebanine treatment and its mechanism.Apoptosis was detected using a TUNEL assay and the expression of microtubule-associated protein 1 LC3 in tumor specimens was assessed using immunohistochemistry.RESULTS Oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells,whilst upregulating the protein expression of cleaved caspase-3,downregulating poly(ADP-ribose)polymerase 1 protein expression,increasing the levels of Bax and Bcl-2 antagonist/killer 1 protein expression,and decreasing the levels of Bcl-2 and myeloid cell leukemia 1 protein expression,which could promote apoptosis in Hep3B2.1-7 cells.Oxocrebanine promoted the transformation of LC3-I to LC3-II in Hep3B2.1-7 cells,suggesting the occurrence of autophagy,whilst the autophagy inhibitor 3-MA could reverse this process.Oxocrebanine was also shown to reduce the phosphorylation levels of the eukaryotic translation initiation factor 4EBP1 and ribosomal protein S6 kinase B1(P70S6K),two downstream effector molecules in the PI3K/Akt/mTOR pathway,inducing autophagy in Hep3B2.1-7 cells.Moreover,the tumor-bearing nude mouse experiment indicated that oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells in vivo.The results of the TUNEL assay and immunohistochemistry also revealed that oxocrebanine induced apoptosis in vivo and increased the expression level of LC3,an autophagy marker.CONCLUSION Oxocrebanine can inhibit the proliferation of human hepatocellular carcinoma cells by promoting apoptosis and inducing autophagy in vitro and in vivo.展开更多
基金Natural Science Foundation of Hainan Province(No.820RC776)。
文摘Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocrebanine on microtubule network homeostasis at both molecular and cellular levels.Methods:the EBI site competition method and molecular docking method were used to determine the occupation of the microtubule site of oxocrebanine.Western Blot was used to detect the effect of oxocrebanine on microtubule-associated proteins including STAT3,PAK1,CAMK4,and PKA.Results:The results of EBI site competition assay showed that the binding of EBI toβ-Tubulin covalent fusions produced adducts that appeared in regions of lower molecular weight thanβ-tubulin(ctrl 2).Molecular docking results showed that oxocrebanine could occupy the colchicine site of microtubule proteins.As revealed by Western Blot,the expression of STAT3 protein was decreased after MCF-7 cells have been treated with low,medium,and high concentration of oxocrebanine or the positive drug taxol for 48 h(P<0.01).The expression levels of PAK1 and Camk4 proteins aslo showed significant reductions(P<0.05,or P<0.01).Oxocrebanine also decreased the PKA protein in MCF-7 cells compared to the control group(P<0.01).Conclusions:Oxocrebanine,a ligand that binds at the colchicine site of tubulin,perturbs tubulin polymerization and causes mitosis in MCF-7 cells,thus leading to MCF-7 cell death.Oxocrebanine may promote microtubule dynamics through stathmin by inhibiting the expression levels of STAT3,PAK1,Camk4,and PKA proteins in MCF-7 cells.Oxocrebanine interfers with spindle formation,and ultimately causes mitotic catastrophe in MCF-7 cells.
基金Supported by National Natural Science Foundation of China,No.82060778the Hainan Provincial Natural Science Foundation of China,No.820RC776+1 种基金the Hainan Province Health Science and Technology Innovation Joint Project,No.WSJK2024MS162the Heilongjiang Province Scientific Research Project of Traditional Chinese Medicine,No.ZHY2024-098.
文摘BACKGROUND Finding active lead anti-hepatocellular carcinoma compounds from traditional Chinese medicine has important research value.AIM To assess the detailed mechanism of oxocrebanine,a compound separated from the traditional Chinese medicinal plant Stephania hainanensis H.S.Lo et Y.Tsoong,and to evaluate its inhibition of the proliferation of human hepatocellular carcinoma cells via apoptosis and autophagy.METHODS MTT,BrdU labeling,and colony formation assays were used to assess the inhibitory effect of oxocrebanine on the growth and proliferation of human hepatocellular carcinoma Hep3B2.1-7 cells.Flow cytometry was used to detect the effect of oxocrebanine on the apoptosis of Hep3B2.1-7 cells.Western blotting was used to assess the expression of apoptosis-related proteins in Hep3B2.1-7 cells.The aforementioned methods were also used to evaluate the effects of oxocrebanine on cell proliferation,autophagy markers,and autophagy-related protein expression levels after adding autophagy inhibitor 3-mA.Furthermore,to verify the anti-hepatocellular carcinoma effect of oxocrebanine in vivo,a nude mouse model was used to investigate the inhibitory effect of oxocrebanine treatment and its mechanism.Apoptosis was detected using a TUNEL assay and the expression of microtubule-associated protein 1 LC3 in tumor specimens was assessed using immunohistochemistry.RESULTS Oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells,whilst upregulating the protein expression of cleaved caspase-3,downregulating poly(ADP-ribose)polymerase 1 protein expression,increasing the levels of Bax and Bcl-2 antagonist/killer 1 protein expression,and decreasing the levels of Bcl-2 and myeloid cell leukemia 1 protein expression,which could promote apoptosis in Hep3B2.1-7 cells.Oxocrebanine promoted the transformation of LC3-I to LC3-II in Hep3B2.1-7 cells,suggesting the occurrence of autophagy,whilst the autophagy inhibitor 3-MA could reverse this process.Oxocrebanine was also shown to reduce the phosphorylation levels of the eukaryotic translation initiation factor 4EBP1 and ribosomal protein S6 kinase B1(P70S6K),two downstream effector molecules in the PI3K/Akt/mTOR pathway,inducing autophagy in Hep3B2.1-7 cells.Moreover,the tumor-bearing nude mouse experiment indicated that oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells in vivo.The results of the TUNEL assay and immunohistochemistry also revealed that oxocrebanine induced apoptosis in vivo and increased the expression level of LC3,an autophagy marker.CONCLUSION Oxocrebanine can inhibit the proliferation of human hepatocellular carcinoma cells by promoting apoptosis and inducing autophagy in vitro and in vivo.
