雷公藤甲素诱导人肝细胞凋亡的机制

目的探讨雷公藤甲素诱导人肝细胞L-02凋亡的机制。方法将L-02细胞分为对照组和雷公藤甲素50nM实验组,处理48h后,采用MTT法检测细胞存活率,流式细胞术检测细胞凋亡率、活性氧(ROS)的荧光强度,试剂盒检测细胞色素C的浓度以及半胱天冬氨酸蛋白酶9(Caspase-9)和Caspase-3的活性,Western blot检测细胞中B细胞淋巴瘤-白血病2(Bcl-2)和Bcl相关X蛋白(Bax)的表达。结果与对照组相比,实验组细胞存活率下降,细胞凋亡率上升,Bcl-2表达下调,Bax表达、细胞色素C浓度、ROS荧光强度、Caspase-9和Caspase-3活性均明显上调(P<0.05或P<0.01)。结论雷公藤甲素可以抑制L-02细胞的存活,通过激活线粒体凋亡途径促进L-02细胞凋亡,下调Bcl-2与Bax比率,促进细胞色素C释放,诱导ROS生成,进一步破坏线粒体膜,增加细胞色素C的释放,激活Caspase-9和Caspase-3介导的细胞凋亡通路,最终诱导细胞凋亡。

Casticin-induced apoptosis involves death receptor 5 upregulation in hepatocellular carcinoma cells

AIM:To investigate the apoptotic activities of casticin in hepatocellular carcinoma(HCC) cells and its molecular mechanisms.METHODS:PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of casticin on the growth of cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolim bromide(MTT) assay.The apoptotic cell death was examined using the cell apoptosis enzyme linked immunosorbent assay(ELISA) detection kit,flow cytometry(FCM) after propidium iodide(PI) staining and DNA agarose gel electrophoresis.The caspase activities were measured using ELISA.Reactive oxygen species(ROS) production was evaluated by FCM after dichlorodihydrofluorescein diacetate(DCFH-DA) probe labeling.Intracellular glutathione(GSH) content was measured using a glutathione assay kit.The expression of death receptor(DR)4 and DR5 proteins was analyzed by Western blotting and FCM.RESULTS:Casticin significantly inhibited the growth of human HCC(PLC/PRF/5 and Hep G2) cells in a dosedependent manner(P < 0.05).Casticin increased the percentage of the sub-G1 population in HCC cells in a concentration-dependent manner.The potency of casticin to PLC/PRF/5 cells was higher than that of 5-flurouracil(26.8% ± 4.8% vs 17.4% ± 5.1%) at 10 μmol/L for 24 h.Casticin increased the levels of Histone/DNA fragmentation and the levels of active caspase-3,-8 and-9 in a concentration-dependent manner(P < 0.05).Treatment with 30 μmol/L casticin for 24 h resulted in the formation of a DNA ladder.Casticin reduced the GSH content(P < 0.05),but did not affect the level of intracellular ROS in PLC/PRF/5 and Hep G2 cells.The thiol antioxidants,acetylcysteine(NAC) and GSH restored GSH content and attenuated casticin-induced apoptosis.In contrast,the nonthiol antioxidants,butylated hydroxyanisole and mannitol failed to do so.In the HCC cells treated with casticin for 24 h,DR5 protein level was increased.The expression of DR5 protein induced by casticin was inhibited by NAC.Pretreatment with DR5/Fc chimera protein,a blocking antibody,effectively attenuated the induction of apoptosis by casticin.CONCLUSION:Casticin-induced apoptosis of HCC cells is involved in GSH depletion and DR5 upregulation.

Expression of Bcl-2 inhibited Fas-mediated apoptosis in human hepatocellular carcinoma BEL-7404 cells

Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and "Death factor" family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of "Death factor" family, the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl-2. The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis. Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.

Fascaplysin sensitizes cells to TRAIL-induced apoptosis through upregulating DR5 expression

This study investigated the molecular mechanism of anti-tumor effect of fascaplysin, a nitrogenous red pigment firstly isolated from a marine sponge. Microarray analysis show that the TNF and TNF receptor superfamily in human umbilical vein endothelial cells (HUVEC) and human hepatocarcinoma cells (BEL-7402) were significantly regulated by fascaplysin. Western Blot results reveal that fascaplysin increased the expression of cleaved caspase-9, active caspase-3, and decreased the level of procaspase-8 and Bid. Flow cytometry and cytotoxicity tests indicate that fascaplysin sensitized cells to tumor necrosis-related apoptosis-inducing ligand-(TRAIL) induced apoptosis, which was markedly blocked by TRAIL R2/Fc chimera, a dominant negative form of TRAIL receptor DR5. Therefore, our results demonstrate that fascaplysin promotes apoptosis through the activation of TRAIL signaling pathway by upregulating DR5 expression.

Berbamine induces apoptosis in human hepatoma cell line SMMC7721 by loss in mitochondrial transmembrane potential and caspase activation

Objective： To investigate the effect ofberbamine on human hepatoma cell line SMMC7721. Methods： The effects of 24 h and 48 h incubation with different concentrations （0-64 μg/ml） of the berbamine on SMMC7721 cells were evaluated using 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide （MTT） assay. Hoechst 33258 staining was conducted to distinguish the apoptotic cell, and the appearance of sub-G1 stage was determined by PI （propidium iodide） staining, the percentage of apoptotic cell was determined by flow cytometry following annexin V/PI staining. Flow cytometry was performed to analyze the cell cycle distribution and the mitochondrial membrane potential （△ψm）, the expression of activated caspase3 and caspase9 was analyzed by Western-blot. Results： The proliferation of SMMC7721 was decreased after treatment with berbamine in a dose- and time-dependent manner. Berbamine could induce apoptosis in SMMC7721 cells and could cause cell cycle arrest in G0/G1 phase, to induce loss of mitochondrial membrane potential （AVm） and activate caspase3 and caspase9. Berbamine-induced apoptosis could be blocked by the broad caspase inhibitor z-VAD-fmk. Conclusion： Berbamine exerts antiproliferative effects on human hepatocellular carcinoma SMMC7721 cells. The anticancer activity of berbamine could be attributed partly to its inhibition of cell proliferation and induction of apoptosis in cancer cells through loss in mitochondrial transmembrane potential and caspase activation.

Secretory Transactivating Transcription-apoptin fusion protein induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM:To determine whether SP-TAT-apoptin induces apoptosis and also maintains its tumor cell specificity. METHODS:In this study,we designed a secretory protein by adding a secretory signal peptide(SP) to the N terminus of Transactivating Transcription(TAT)-apoptin(SP-TAT-apoptin),to test the hypothesis that it gains an additive bystander effect as an anti-cancer therapy. We used an artificial human secretory SP whose amino acid sequence and corresponding cDNA sequence were generated by the SP hidden Markov model. RESULTS:In human liver carcinoma HepG2 cells,SP-TAT-apoptin expression showed a diffuse pattern in the early phase after transfection. After 48 h,however,it translocated into the nuclear compartment and caused massive apoptotic cell death,as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and annexin-V binding assay. SP-TAT-apoptin did not,however,cause any cell death in non-malignant human umbilical vein endothelial cells(HUVECs). Most importantly,the conditioned medium from Chinese hamster ovary(CHO) cells transfected with SP-TAT-apoptin also induced significant cell deathin HepG2 cells,but not in HUVECs. CONCLUSION:The data demonstrated that SP-TAT-apoptin induces apoptosis only in malignant cells,and its secretory property might greatly increase its potency once it is delivered in vivo for cancer therapy.

Antisense EGFR sequence enhances apoptosis in a human hepatoma cell line BEL-7404

Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells. In the cells of JX-1, a sub clone of BEL-7404 stably transfected with antisense EGFR vector (Cell Research, 3:75, 1993), an enhanced rate (9.5%) of spontaneous apoptosis was detected by flow cytometry, whereas the rates of spontaneous apoptosis in JX-0 cells, a sub-clone of BEL-7404 transfected by control vector, and the parellt BEL-7404 cells were almost equal and about 1.7%. Serum-starvation for 72 h increased the rate of apoptosis of JX-1cells up to 33.7%, while JX-0 and BEL-7404 cells, under the same condition, produced less than 5% of apoptotic cells. Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies of ten occurred in JX-1 cells, especially during serumstarvation. These results, combined with the data of DNA fragmentation Elisa test, suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.Comparison of intracellular Ca2+ level and the responsiveness of JX-1 cells to the induced action of EGF and tharpsigargin (TG) treatment with that of control JX-0cells indicated that antisense EGFR might interrupt the EGF/EGFR signaling pathway resulting in the decreass of intracellular Ca2+ pool content as well as the responsiveness of these cells to the extracellular signals. These findings suggest that antisense EGFR either directly or indirectly reglllates Ca2+ storage in endoplasmic reticulum,thereby enhances apoptosis in the hnman hepatoma cells.

Thapsigargin increases apoptotic cell death in human hepatoma BEL-7404 cells

Effects of thapsigargin, an inhibitor of Ca2+-ATPase in surface of endoplasmic reticulum, on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron mi-croscopy Propidium iodide staining and flow cytome- try revealed that in the serum-free condition, thapsigar-gin increased the rate of apoptosis of BEL- 7404 cells in a dose-dependent manner. Prolongation of the period of serum-free condition enhanced the apoptosis induced by thapsigargin treatment. Morphological observation with electron microscope further demonstrated that chromatin condensation and fragmentation, apoptotic bodies existed in TG-treated cells, supporting that thapsigargin is a po-tent activator of apoptosis in the cells.

Celastrus orbiculatus extract induces mitochondrial-mediated apoptosis in human hepatocellular carcinoma cells

OBJECTIVE： To investigate the apoptotic effects and underlying molecular mechanisms of Celastrus orbiculatus （C. orbiculatus） extract in human hepa- tocellular carcinoma cells. METHODS： Human hepatocellular carcinoma cells （HCCLM6） were treated with C. orbiculatus extract （COE） at different nontoxic concentrations （10, 20, 40, 80, and 160 IJg/mL）. The effect of COE on HC-CLM6 viability was examined using 3-（4,5-dimethyl- thiazol-2-yl）-2,5-diphenyl tetrazolium bromide （MTT） assays. Cellular apoptosis following COE treatment was assessed by flow cytometry and western blot analysis. RESULTS： COE significantly inhibited cell viability and induced apoptosis of HCCLM6 cells in a dose-dependent manner. Apoptosis was accompa- nied by increased Bax expression and decreased Bcl-2 expression. In addition, COE treatment led to the release of cytochrome c, activation of cas- pase-3, and cleavage of poly （ADP-ribose） poly- merase （PARP）. Furthermore, activation of extracel- lular signal-regulated kinase （ERK）, p38 kinase, and c-Jun N-terminal kinase （JNK） phosphorylation, and down-regulation of Akt phosphorylation was ob- served. CONCLUSION： COE induces mitochondrial-mediat- ed, caspase-dependent apoptosis in HCCLM6 cells, which might be attributed to the activation of mito- gen-activated protein kinase （MAPK） and inhibition of Akt signaling pathways. These data suggest that COE may be a potential treatment for human hepa- tocellular carcinoma.

The pharmacological mechanism underlying the apoptosis of human hepatic stellate cells LX-2 induced by NF-κB inhibitor PDTC

In the present study,we aimed to confirm whether NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC)could induce apoptosis of human hepatic stellate cells(HSCs)LX-2 and explore the potential pharmacological mechanism underlying these effects.In this study,LX-2 cells were cultured in vitro,and the experiment was divided into two groups,including the control and PDTC groups.The viability of LX-2 cells was measured by CCK8 assay after the cells were exposed to PDTC.The anti-apoptotic effect of PDTC was detected by AO/EB double assay staining kit.Additionally,the activities of NF-κB,Fas/FasL,apoptosis-related proteins,as well as the cellular localization of AIF,were determined by Western blotting analysis and immunofluorescence staining respectively.After PDTC treatment for 12 and 24 h,AO/EB dual staining showed typical apoptotic changes,such as cell volume reduction,cell shrinkage,nuclear fragmentation,and so on.PDTC at 60μmol/L significantly increased the proliferation inhibition rate and decreased the secretion of collagen I,collagen III,andα-SMA in LX-2 cells.The Western blotting analysis and RT-PCR showed no significant difference in the expression of AIF between the control group and PDTC group,and the expressions of Fas and FasL were not observed in all groups(P>0.05).Further results showed that PDTC could promote the displacement of AIF from mitochondria to the nucleus,activate the apoptotic signaling in the cell nucleus,and possibly participate in the apoptosis process of LX-2 cells.In conclusion,the pharmacological mechanism of PDTC against hepatic fibrosis might be to promote the displacement of AIF from mitochondria to the nucleus,then activate the apoptotic signaling in the cell nucleus,and finally induce the apoptosis of LX-2 cells.Meanwhile,these results also revealed that the Fas/FasL-mediated apoptosis pathway was not involved in the PDTC-induced apoptosis process of LX-2 cells.