AIM: To study the effect of hydroxyapatite (HAP) nanoparticleson human hepatoma cell line BEL-7402 in vitro.METHODS: The human hepatoma cell line BEL-7402 wascultured and treated with HAP nanoparticles at variousconce...AIM: To study the effect of hydroxyapatite (HAP) nanoparticleson human hepatoma cell line BEL-7402 in vitro.METHODS: The human hepatoma cell line BEL-7402 wascultured and treated with HAP nanoparticles at variousconcentrations. Growth suppression was detected with MTTcolorimetric assay, cell apoptotic alterations were evaluatedby cytochemical staining (Hoechst 33258), transmissionelectron microscopy (TEM), and flow cytometry (FCM).RESULTS: HAP nanoparticles inhibited the growth ofhepatoma cells in a dose-dependent manner, with IC50 valuesof 29.30 mg/L. Treated with 50-200 mg/L HAP nanoparticlesfor 48 h, BEL-7402 cells apoptosis with nuclear chromatincondensation and fragmentation as well as cell shrinkageand the formation of apoptotic bodies were observed undercytochemical staining and transmission electron microscopy.FCM analysis showed hypodiploid peaks on histogram, theapoptotic rates at the concentrations of 50, 75, 100, 150and 200 mg/L of HAP nanoparticles were 20.35±2.23%,25.35±1.92%, 29.34±4.61%, 44.92±3.78 % and53.64±3.49%, respectively, which were all significantlyhigher than that of control group 2.23±0.14%. There wasa significant correlation between HAP nanoparticleconcentration and apoptotic rate (r=0.994, P＜0.01).CONCLUSION: HAP nanoparticles not only inhibitproliferation but also induce apoptosis of human hepatoma cell line BEL-7402 in vitro.展开更多
AIM: To investigate the characteristics of PPAR gamma ligands induced apoptosis in liver cancer cells.METHODS: The effects of ligands for each of the PPAR gamma ligands on DNA synthesis and cell viability were examine...AIM: To investigate the characteristics of PPAR gamma ligands induced apoptosis in liver cancer cells.METHODS: The effects of ligands for each of the PPAR gamma ligands on DNA synthesis and cell viability were examined in BEL-7402 liver cancer cells. Apoptosis was characterized by Hochest33258 staining, DNA fragmentation,TUNEL and ElISA, and cell cycle kinetics by FACS. Modulation of apoptosis related caspases expression by PPAR gamma ligands was examined by Western blot.RESULTS: PPARgamma ligands, 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ), suppressed DNA synthesis of BEL-7402 cells. Both 15d-PGJ2 and TGZ induced BEL-7402 cell death in a dose dependent manner, which was associated with an increase in fragmented DNA and TUNEL-positive cells. At concentrations of 10 and 30 μM,15d-PGJ2 or troglitazone increased the proportion of cells with G0/G1 phase DNA content and decreased those with S phase DNA content. There was no significant change in the proportion of cells with G2/M DNA content. The activities of Caspases-3, -6, -7 and -9 were increased by 15d-PGJ2and TGZ treatment, while the activity of Caspase 8 had not significantly changed.CONCLUSION: The present results suggest the potential usefulness of PPAR gamma ligands for chemoprevention and treatment of liver cancers.展开更多
AIM: Mitotic cell death has been focused on in tumor therapy.However, the precise mechanisms underlying it remain unclear. We have reported previously that enediyne antibiotic lidamycin induces mitotic cell death at l...AIM: Mitotic cell death has been focused on in tumor therapy.However, the precise mechanisms underlying it remain unclear. We have reported previously that enediyne antibiotic lidamycin induces mitotic cell death at low concentrations in human epithelial tumor cells. The aim of this study was to investigate the possible link between centrosome dynamics and lidamycin-induced mitotic cell death in human hepatoma BEL-7402 cells.METHODS: Growth curve was established by Ml-l assay.Cell multinucleation was detected by staining with Hoechst 33342. Flow cytometry was used to analyze cell cycle.Aberrant centrosomes were detected by indirect immunofluorescence. Western blot and senescenceassociated β-galactosidase (SA-β-gal) staining were used to analyze protein expression and senescence-like phenotype,respectively.RESULTS: Exposure of BEL-7402 cells to a low concentration of lidamycin resulted in an increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death and activation of SA-β-gal in some cells, accompanied by the changes of protein expression for the regulation of proliferation and apoptosis. The mitochondrial signaling pathway, one of the major apoptotic pathways, was not activated during mitotic cell death. The aberrant centrosomes contributed to the multipolar mitotic spindles formation, which might lead to an unbalanced division of chromosomes and mitotic cell death characterized by the manifestation of multi- or micronucleated giant cells.Cell cycle analysis revealed that the lidamycin treatment provoked the retardation at G2/M phase, which might be involved in the centrosome overduplication.CONCLUSION: Mitotic cell death and senescence can be induced by treatment of BEL-7402 cells with a low concentration of lidamycin. Centrosome dysregulation may play a critical role in mitotic failure and ultimate cell death following exposure to intermediate dose of lidamycin.展开更多
AIM:To evaluate the effects of 3,3′-diethyl-9-methylthia-carbocyanine iodide (DMTCCI) on DNA primase activity and on apoptosis of human hepatocellular carcinoma BEL-7402 cells.METHODS: DNA primase assay was used to i...AIM:To evaluate the effects of 3,3′-diethyl-9-methylthia-carbocyanine iodide (DMTCCI) on DNA primase activity and on apoptosis of human hepatocellular carcinoma BEL-7402 cells.METHODS: DNA primase assay was used to investigate DNA primase activity. MTT assay was applied to determine cell proliferation. Flow cytometric analysis, transmission electron microscopy, DNA fragmentation assay were performed to detect DMTCCI-induced apoptosis. Expression levels of p53, Bcl-2, Bcl-xL, Bad, Bax, survivin, Caspase-3 and poly (ADP-ribose) polymerase (PARP) were evaluated by immunoblot analysis. Caspase-3 activity was assessed with ApoAlert Caspase-3 colorimetric assay kit.RESULTS:DMTCCI had inhibitory effects on eukaryotic DNA primase activity with IC50 value of 162.2 nmol/L. It also inhibited proliferation of human hepatocellular carcinoma BEL-7402 cells with IC50 value of 2.09μmol/L. Furthermore,DMTCCI-induced BEL-7402 cell apoptosis was confirmed by DNA fragmentation (DNA ladders and sub-G1 formation) and transmission electron microscopy (apoptotic bodies formation). During the induction of apoptosis, expression of Bcl-2, Bcl-xL and survivin was decreased, and that of p53,Bad and Bax was increased. Caspase-3 was activated and poly (ADP-ribose) polymerase (PARP) was cleaved in BEL-7402 cells treated with DMTCCI.CONCLUSION: The present data suggest that DMTCCI has inhibitory effects on eukaryotic DNA primase and can induce apoptosis of BEL-7402 cells. The modulation of expression of p53 and Bcl-2 family proteins, and activation of Caspase-3 might be involved in the induction of apoptosis.展开更多
This study aimed to observe the effects of tyroserleutide (tyrosyl-seryl-leucine, YSL) on the growth of human hepatocarcinoma BEL-7402 that was transplanted into nude mice, and explore its anti-tumor mechanism prelimi...This study aimed to observe the effects of tyroserleutide (tyrosyl-seryl-leucine, YSL) on the growth of human hepatocarcinoma BEL-7402 that was transplanted into nude mice, and explore its anti-tumor mechanism preliminarily. YSL, at doses of 80 μg·kg?1·d?1, 160 μg·kg?1·d?1 and 320 μg·kg?1·d?1 significantly inhibited the growth of the human hepatocarcinoma BEL-7402 tumor in nude mice, producing inhibition of 21.66%, 41.34%, and 34.78%, respectively. Ultra structure of BEL-7402 tumor in nude mice showed that YSL could induce tumor cells apoptosis and necrosis, cell organelle mitochondria and endoplasmic reticulum damage, and calcium over-load. By confocal laser scanning microscopy and flow cytometry, we found that 10 μg/mL YSL rapidly induced an increase of the concentration of cytoplasmic free calcium in BEL-7402 cells in vitro, and maintained high concentrations of cytoplasmic free calcium for 1 h. Then the calcium concentration began to decrease after 2 h, and was lower than that of the control group at 4 h and 24 h (p<0.05). YSL also decreased the mitochondrial transmembrane potential of BEL-7402 cells in vitro, but had no effect on the calcium homeostasis or mitochondrial transmembrane potential of Chang liver hepatocytes. So affecting calcium homeostasis, then inducing apoptosis and necrosis may be a mechanism by which YSL inhibits the tumor growth in animal model.展开更多
文摘AIM: To study the effect of hydroxyapatite (HAP) nanoparticleson human hepatoma cell line BEL-7402 in vitro.METHODS: The human hepatoma cell line BEL-7402 wascultured and treated with HAP nanoparticles at variousconcentrations. Growth suppression was detected with MTTcolorimetric assay, cell apoptotic alterations were evaluatedby cytochemical staining (Hoechst 33258), transmissionelectron microscopy (TEM), and flow cytometry (FCM).RESULTS: HAP nanoparticles inhibited the growth ofhepatoma cells in a dose-dependent manner, with IC50 valuesof 29.30 mg/L. Treated with 50-200 mg/L HAP nanoparticlesfor 48 h, BEL-7402 cells apoptosis with nuclear chromatincondensation and fragmentation as well as cell shrinkageand the formation of apoptotic bodies were observed undercytochemical staining and transmission electron microscopy.FCM analysis showed hypodiploid peaks on histogram, theapoptotic rates at the concentrations of 50, 75, 100, 150and 200 mg/L of HAP nanoparticles were 20.35±2.23%,25.35±1.92%, 29.34±4.61%, 44.92±3.78 % and53.64±3.49%, respectively, which were all significantlyhigher than that of control group 2.23±0.14%. There wasa significant correlation between HAP nanoparticleconcentration and apoptotic rate (r=0.994, P＜0.01).CONCLUSION: HAP nanoparticles not only inhibitproliferation but also induce apoptosis of human hepatoma cell line BEL-7402 in vitro.
文摘AIM: To investigate the characteristics of PPAR gamma ligands induced apoptosis in liver cancer cells.METHODS: The effects of ligands for each of the PPAR gamma ligands on DNA synthesis and cell viability were examined in BEL-7402 liver cancer cells. Apoptosis was characterized by Hochest33258 staining, DNA fragmentation,TUNEL and ElISA, and cell cycle kinetics by FACS. Modulation of apoptosis related caspases expression by PPAR gamma ligands was examined by Western blot.RESULTS: PPARgamma ligands, 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ), suppressed DNA synthesis of BEL-7402 cells. Both 15d-PGJ2 and TGZ induced BEL-7402 cell death in a dose dependent manner, which was associated with an increase in fragmented DNA and TUNEL-positive cells. At concentrations of 10 and 30 μM,15d-PGJ2 or troglitazone increased the proportion of cells with G0/G1 phase DNA content and decreased those with S phase DNA content. There was no significant change in the proportion of cells with G2/M DNA content. The activities of Caspases-3, -6, -7 and -9 were increased by 15d-PGJ2and TGZ treatment, while the activity of Caspase 8 had not significantly changed.CONCLUSION: The present results suggest the potential usefulness of PPAR gamma ligands for chemoprevention and treatment of liver cancers.
基金Supported by Grants from the Major State Basic Research Development Program Foundation of China,No.G2000057010 and G 1999053901 and Grants from the National Natural Science Foundation of China,No.30271438 and 30300424 and Grant from the Key Laborator
文摘AIM: Mitotic cell death has been focused on in tumor therapy.However, the precise mechanisms underlying it remain unclear. We have reported previously that enediyne antibiotic lidamycin induces mitotic cell death at low concentrations in human epithelial tumor cells. The aim of this study was to investigate the possible link between centrosome dynamics and lidamycin-induced mitotic cell death in human hepatoma BEL-7402 cells.METHODS: Growth curve was established by Ml-l assay.Cell multinucleation was detected by staining with Hoechst 33342. Flow cytometry was used to analyze cell cycle.Aberrant centrosomes were detected by indirect immunofluorescence. Western blot and senescenceassociated β-galactosidase (SA-β-gal) staining were used to analyze protein expression and senescence-like phenotype,respectively.RESULTS: Exposure of BEL-7402 cells to a low concentration of lidamycin resulted in an increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death and activation of SA-β-gal in some cells, accompanied by the changes of protein expression for the regulation of proliferation and apoptosis. The mitochondrial signaling pathway, one of the major apoptotic pathways, was not activated during mitotic cell death. The aberrant centrosomes contributed to the multipolar mitotic spindles formation, which might lead to an unbalanced division of chromosomes and mitotic cell death characterized by the manifestation of multi- or micronucleated giant cells.Cell cycle analysis revealed that the lidamycin treatment provoked the retardation at G2/M phase, which might be involved in the centrosome overduplication.CONCLUSION: Mitotic cell death and senescence can be induced by treatment of BEL-7402 cells with a low concentration of lidamycin. Centrosome dysregulation may play a critical role in mitotic failure and ultimate cell death following exposure to intermediate dose of lidamycin.
基金Supported by the National High Technology Research and Development Program of China(863 Program),No.2002AA2Z341C and the National Natural Science Foundation of China,No.39870886
文摘AIM:To evaluate the effects of 3,3′-diethyl-9-methylthia-carbocyanine iodide (DMTCCI) on DNA primase activity and on apoptosis of human hepatocellular carcinoma BEL-7402 cells.METHODS: DNA primase assay was used to investigate DNA primase activity. MTT assay was applied to determine cell proliferation. Flow cytometric analysis, transmission electron microscopy, DNA fragmentation assay were performed to detect DMTCCI-induced apoptosis. Expression levels of p53, Bcl-2, Bcl-xL, Bad, Bax, survivin, Caspase-3 and poly (ADP-ribose) polymerase (PARP) were evaluated by immunoblot analysis. Caspase-3 activity was assessed with ApoAlert Caspase-3 colorimetric assay kit.RESULTS:DMTCCI had inhibitory effects on eukaryotic DNA primase activity with IC50 value of 162.2 nmol/L. It also inhibited proliferation of human hepatocellular carcinoma BEL-7402 cells with IC50 value of 2.09μmol/L. Furthermore,DMTCCI-induced BEL-7402 cell apoptosis was confirmed by DNA fragmentation (DNA ladders and sub-G1 formation) and transmission electron microscopy (apoptotic bodies formation). During the induction of apoptosis, expression of Bcl-2, Bcl-xL and survivin was decreased, and that of p53,Bad and Bax was increased. Caspase-3 was activated and poly (ADP-ribose) polymerase (PARP) was cleaved in BEL-7402 cells treated with DMTCCI.CONCLUSION: The present data suggest that DMTCCI has inhibitory effects on eukaryotic DNA primase and can induce apoptosis of BEL-7402 cells. The modulation of expression of p53 and Bcl-2 family proteins, and activation of Caspase-3 might be involved in the induction of apoptosis.
基金This study was supported by the“863”Project(Grant No.2004AA2Z3170)from the Nat ional Important Project Grant(Grant No.03007)from the Ministry of Education of ChinaPre-Explore of Key Basic Science Program of Ministry of Science and Technology ofChina(Grant No.2003CCA04300).
文摘This study aimed to observe the effects of tyroserleutide (tyrosyl-seryl-leucine, YSL) on the growth of human hepatocarcinoma BEL-7402 that was transplanted into nude mice, and explore its anti-tumor mechanism preliminarily. YSL, at doses of 80 μg·kg?1·d?1, 160 μg·kg?1·d?1 and 320 μg·kg?1·d?1 significantly inhibited the growth of the human hepatocarcinoma BEL-7402 tumor in nude mice, producing inhibition of 21.66%, 41.34%, and 34.78%, respectively. Ultra structure of BEL-7402 tumor in nude mice showed that YSL could induce tumor cells apoptosis and necrosis, cell organelle mitochondria and endoplasmic reticulum damage, and calcium over-load. By confocal laser scanning microscopy and flow cytometry, we found that 10 μg/mL YSL rapidly induced an increase of the concentration of cytoplasmic free calcium in BEL-7402 cells in vitro, and maintained high concentrations of cytoplasmic free calcium for 1 h. Then the calcium concentration began to decrease after 2 h, and was lower than that of the control group at 4 h and 24 h (p<0.05). YSL also decreased the mitochondrial transmembrane potential of BEL-7402 cells in vitro, but had no effect on the calcium homeostasis or mitochondrial transmembrane potential of Chang liver hepatocytes. So affecting calcium homeostasis, then inducing apoptosis and necrosis may be a mechanism by which YSL inhibits the tumor growth in animal model.
