A comparison of apoptosis and necrosis induced by ent-kaurene-type diterpenoids in HL-60 cells

We previously reported that ent-11alpha-hydroxy-16-kauren-15-one (KD) induces apoptosis through a caspase-dependent pathway and the induction of apoptosis is dependent on its enone group in human leukemia cells. Here we investigated the abilities of some KD-related compounds with enone group (Fig. 1) to induce apoptosis and to activate some caspases. The IC50 values of ent-kaurene-related compounds possessing the enone group, ent-1beta-hydroxy-9(11),16-kauradien-15-one (1), ent-9(11),16-kauradiene-12,15-dione (2) and the rearranged ent-kaurane-type diterpene (3), against HL-60 cells after 12 h of treatment were 40 microM, 1.8 microM and 5.5 microM, respectively. Although treatment with 3 induced apoptosis, DNA ladder formation was not observed after treatment with 1 or 2. Induction of necrosis, as assayed by trypan blue staining, was observed after treatment with 1 or 2. Treatment with compound 1, 2 or 3 induced proteolysis of poly(ADP-ribose) polymerase (PARP), a substrate of caspase-3, and processing of caspase-3. Activation of caspase-8 and processing of Bid, a typical substrate of caspase-8, were also observed on treatment with these compounds. Pretreatment with a broad-spectrum inhibitor of caspases attenuated apoptosis induced by 3 but not necrosis induced by 1 and 2. In summary, KD-related compounds are a unique family of diterpenes that cause either caspase-dependent apoptotic or necrotic cell death.     

Inhibition by vitamin C of apoptosis induced by N-nitrosamines in HepG2 and HL-60 cells

The aim of this study was to evaluate the effect of vitamin C towards N-nitrosopyrrolidine (NPYR)- and N-nitrosodimethylamine (NDMA)-induced apoptosis in human hepatoma (HepG2) and leukemia (HL-60) cell lines using flow cytometry analysis and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay (TUNEL). None of the vitamin C concentrations tested (1-100 microM) caused cytotoxicity in HepG2 cells. However, there were significant losses of HL-60 cells viability, measured by MTT assay, 72 h after treatment with 50 and 100 microM vitamin C (29 and 46%, respectively). Moreover, an increase of lactate dehydrogenase release was significant with 50 microM at 72 h (28%) and with 100 microM of vitamin C at 48 and 72 h (27 and 36%, respectively). Also, the percentage of apoptotic HL-60 cells found in TUNEL assay increased to 21% when they were treated with 100 microM vitamin C for 72 h. Thus, in subsequent simultaneous treatments with NPYR (30 and 50 mM) or NDMA (27 and 68 mM) and vitamin C, concentrations of 5-50 microM vitamin C were used. Our results revealed that vitamin C, at all concentrations and times tested, reduced the apoptosis induced by NPYR and NDMA in both cell lines, showing a similar effect in HepG2 and HL-60 cells towards NPYR (50 mM)--65 and 63% of reduction, respectively--whereas towards NDMA (27 mM) the inhibition was higher in HL-60 than in HepG2 cells--75 and 57%, respectively. Therefore, our findings suggest that inhibition of apoptosis may be one of the mechanisms by which vitamin C exerts its protective effect.     

沉默MALAT1基因对蜂毒素诱导HepG2细胞增殖和凋亡的影响

目的探讨沉默HepG2细胞株中MALAT1基因对蜂毒素诱导的细胞增殖抑制和凋亡的影响。方法采用MTT法检测蜂毒素对HepG2细胞的增殖抑制作用;流式细胞仪检测细胞凋亡率;qPCR法检测HepG2细胞中MALAT1基因的表达;采用特异性siRNA对HepG2细胞的MALAT1基因进行沉默;比较单独用蜂毒素处理和给予蜂毒素同时沉默MALAT1的细胞增殖抑制率和凋亡率变化。结果蜂毒素明显抑制HepG2细胞的增殖并促进细胞凋亡,呈浓度依赖性;和正常肝细胞株L0-2相比,MALAT1 mRNA在HepG2细胞中存在高表达(P<0.05);蜂毒素可下调细胞中MALAT1的表达,并随着浓度的增加抑制率升高;给予蜂毒素同时沉默MALAT1的研究组中,细胞增殖抑制和凋亡率都明显高于单独给予蜂毒素组(P<0.05)。结论蜂毒素可下调HepG2细胞株中MALAT1的表达,且沉默MALAT1可促进蜂毒素诱导的HepG2细胞增殖抑制和凋亡。     

野苋菜提取物抗肿瘤作用及诱导人肝癌HepG2细胞凋亡的分子机制

目的研究野苋菜提取物的抗肿瘤作用及诱导人肝癌HepG2细胞凋亡的可能机制。方法采用Alamar blue法检测野苋菜提取物对肿瘤细胞的抑制作用;光学显微镜和Hoechst 33258荧光染色观察HepG2细胞的形态变化;流式细胞术检测该提取物对HepG2细胞凋亡的影响;Western blot和caspase-3活性检测试剂盒分析野苋菜提取物诱导HepG2细胞凋亡的作用机制;caspase-9、caspase-3抑制剂(Z-LEHDFMK和Ac-DEVD-CHO)验证相关的调控信号转导通路。结果野苋菜提取物对多种肿瘤细胞均有抑制作用,其中HepG2细胞最敏感;经提取物处理过的HepG2细胞出现明显的凋亡特征,流式细胞术进一步证实野苋菜提取物能够诱导HepG2细胞的凋亡;野苋菜提取物处理48h后的HepG2细胞中Bcl-2、survivin表达下调,Bax、PARP、Apaf-1、caspase-9表达量增加,并且caspase-3、caspase-9酶活性明显提高;caspase-9和caspase-3的抑制剂能够逆转野苋菜提取物对HepG2细胞的抑制活性。结论野苋菜提取物具有抗肿瘤作用,并且能够激活caspase-9内源性凋亡信号通路,发挥其促进HepG2细胞凋亡的作用。     

Ghrelin对肿瘤坏死因子-α诱导HepG_2细胞纤溶酶原激活物抑制剂-1分泌的干预作用

目的探讨ghrelin对肿瘤坏死因子-α(TNF-α)诱导的HepG2细胞纤溶酶原激活物抑制剂-1(PAI-1)和组织型纤溶酶原激活物(tPA)分泌的影响。方法HepG2细胞培养,加入不同浓度和时间TNF-α,采用酶联免疫吸附试验法测定细胞上清、PAI-1、tPA含量,采用ghrelin预处理1h后,加TNF-α检测二者变化。结果TNF-α(0.1、1、10μg/L)浓度、时间(2、6、24h)依赖地增高PAI-1,tPA轻度增加,tPA/PAI-1下降;ghrelin抑制单独作用和TNF-α诱导的PAI-1分泌,增加tPA,提高tPA/PAI-1。结论Ghrelin可抑制基础的和TNF-α诱导的PAI-1分泌。     

Cytotoxicity and apoptosis induced by silver nanoparticles in human liver HepG2 cells in different dispersion media

Silver nanoparticles (Ag NPs) have been widely used in medical and healthcare products owing to their unique antibacterial activities. However, their safety for humans and the environment has not yet been established. This study evaluated the cellular proliferation and apoptosis of Ag NPs suspended in different solvents using human liver HepG2 cells. The ionization of Ag NPs in different dispersion media [deionized water, phosphate‐buffered saline (PBS), saline and cell culture] was measured using an Ag ion selective electrode. The MTT assay was used to examine the cell proliferation activities. The effects of Ag NPs on cell cycle, induction of apoptosis, production of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were analyzed using flow cytometry. The degree of Ag NPs ionization differed with dispersion media, with the concentrations of silver ions in deionized water being the highest in all suspensions. Ag NPs could inhibit the viability of HepG2 cells in a time‐ and concentration‐dependent manner. Ag NPs (40, 80 and 160 µg ml^?1) exposure could cause cell‐cycle arrest in the G2/M phase, significantly increasing the apoptosis rate and ROS generation, and decreasing the MMP in HepG2 cells more sensitive to deionized water than in cell culture. These results suggested that the cellular toxicological mechanism of Ag NPs might be related to the oxidative stress of cells by the generation of ROS, leading to mitochondria injury and induction of apoptosis. It also implies that it is important to assess the physicochemical properties of NPs in the media where the biological toxicity tests are performed. Copyright © 2015 John Wiley & Sons, Ltd.     

灵芝多糖诱导人肝癌细胞HepG2凋亡的研究

目的：研究灵芝多糖（GLPS）与化疗药氟尿嘧啶联合使用诱导人肝癌细胞HepG2凋亡作用。方法：MTT法测定细胞毒作用,流式细胞术检测细胞凋亡率,比色法测定Caspase-3、Caspase-8酶活性,Western—Blotting法检测细胞色素C、Caspase-3、Caspase-8蛋白表达情况。结果：灵芝多糖与氟尿嘧啶联合使用具有显著的细胞毒作用;流式细胞术检测,随着GLPS浓度增加,细胞凋亡率升高,Caspase-3、Caspase-8酶活性亦增强;Western-Blotting检测发现GLPS作用后细胞色素C、Caspase-3、Caspase-8蛋白表达不同程度增加。结论：灵芝多糖与氟尿嘧啶联合使用可通过引起细胞色素C的释放,活化Caspase诱导肝癌细胞凋亡。     

Role of reactive oxygen species in apoptosis induced by N-ethylmaleimide in HepG2 human hepatoblastoma cells.

We have previously reported that N-ethylmaleimide induces apoptosis through activation of K(+), Cl(-)-cotransport in HepG2 human hepatoblastoma cells. In this study, we investigated the role for reactive oxygen species as a mediator of the apoptosis induced by N-ethylmaleimide. N-ethylmaleimide induced a significant elevation of intracellular level of reactive oxygen species. Treatment with antioxidants (N-acetyl cysteine, N,N'-diphenyl-p-phenylenediamine) which markedly suppressed generation of reactive oxygen species, significantly inhibited the N-ethylmaleimide-induced activation of K(+), Cl(-)-cotransport and apoptosis. Inhibitors of NADPH oxidase (diphenylene iodonium, apocynin, D-(+)-neopterine) also significantly blunted the generation of reactive oxygen species, activation of K(+), Cl(-)-cotransport and apoptosis induced by N-ethylmaleimide. These results suggest that reactive oxygen species generated through activation of NADPH oxidase may play a role in the N-ethylmaleimide-induced stimulation of K(+), Cl(-)-cotransport and apoptosis in HepG2 cells.     

地塞米松对顺铂诱导的HepG2细胞凋亡及Bcl-2和Caspase-3蛋白表达的影响

目的:探讨地塞米松对顺铂诱导HepG2细胞凋亡的影响,并观察Bcl-2和Caspase-3蛋白的表达。方法:HepG2细胞与顺铂及不同浓度的地塞米松共培养,RT-PCR检测Bcl-2、Caspase-3的mRNA表达,Western blot检测Bcl-2、Caspase-3蛋白表达,流式细胞术(FACS)检测各组HepG2细胞的凋亡情况。结果:随着地塞米松浓度的增加,HepG2细胞中Bcl-2蛋白表达量升高,Caspase-3蛋白表达量下降。同时细胞凋亡率降低。结论:地塞米松部分通过Bcl-2途径抑制肝癌细胞的凋亡,而Caspase-3又在调控细胞凋亡中起重要作用。     

一氧化氮供体PABA/NO经线粒体途径诱导HepG2细胞凋亡

目的研究新型一氧化氮(NO)供体,O~2-{2,4-二硝基-5-[4-(N-甲基氨基)苯甲酰氧基]苯基}-1-(N,N-二甲基氨基)偶氮-1-鎓-1,2-二醇(PABA/NO)对人肝癌细胞凋亡的影响。方法 PABA/NO 7.5,15.0和30.0μmol·L~(-1)处理细胞24 h后,CCK-8法检测细胞存活率,倒置显微镜下检测细胞形态,DAF-FM DA荧光染色法检测细胞内NO水平,膜联蛋白(Annexin)Ⅴ-FITC/PI双染法检测细胞凋亡率,罗丹明123(Rh123)染色法检测细胞线粒体膜电位,Western蛋白质印迹法检测Bcl-2、Bax、活化胱天蛋白酶3、细胞色素c(Cyt c)和凋亡诱导因子(AIF)蛋白表达。结果与细胞对照组比较,PABA/NO可显著抑制HepG2细胞存活,24 h时IC50值为(10.8±0.6)μmol·L~(-1);形态上出现胞膜皱缩、细胞扁圆等变化;细胞内NO水平提高,荧光强度分别为121±9(P<0.05),174±31(P<0.05)和230±43(P<0.01);凋亡率由原来的(2.9±0.5)%增加至(17.0±4.5)%,(39.8±5.4)%和(74.3±45.2)%(P<0.01);细胞内Rh123的荧光强度由原来的668±69下降到605±73,420±65(P<0.05)和242±47(P<0.01);PABA/NO引起Bcl-2表达下调(P<0.01)、Bax及活化胱天蛋白酶3表达上调,胞浆中Cyt c的表达由原来的0.15±0.04升高到0.27±0.06(P<0.05),0.38±0.07(P<0.01)和0.82±0.16(P<0.01)。胞核中AIF的表达由原来0.183±0.032升高至0.231±0.011,0.682±0.020(P<0.01)和0.966±0.090(P<0.01)。加入NO清除剂羧基(carboxy)-PTIO后,可逆转PABA/NO引起的Bcl-2表达下调,对Bax、活化胱天蛋白酶3、胞浆中Cyt c和胞核中AIF蛋白表达的上调也有一定的逆转作用(P<0.01)。结论 PABA/NO可能经线粒体途径诱导HepG2细胞凋亡。     

血根碱通过诱导活性氧促进HepG2细胞凋亡的机制研究

目的研究血根碱对Hep G2细胞中活性氧(ROS)调控细胞凋亡途径的影响。方法采用血根碱干预Hep G2细胞,MTT法检测血根碱对细胞活性的影响;DCFH-DA和DHE染色观察血根碱作用Hep G2细胞12 h后细胞内ROS的变化;Hoechst 33342和Annexin V/PI染色检测细胞凋亡;Rho123染色检测细胞线粒体膜电位;Western blot检测凋亡相关蛋白的表达。结果随着血根碱药物浓度的增加,Hep G2细胞活力明显被抑制;血根碱可以明显提高Hep G2细胞内的ROS含量,并且降低线粒体膜电位,促进细胞凋亡;Western blot检测发现血根碱促进Bax、cleaved-caspase-3和细胞质Cyt-C的表达,抑制Bcl-2蛋白的表达。结论血根碱通过升高细胞内ROS的含量,激活线粒体凋亡途径,进而促进Hep G2细胞发生凋亡。     

乙肝病毒x蛋白结合蛋白抑制阿霉素诱导HepG2细胞凋亡的机制研究

目的探讨乙型肝炎病毒x蛋白结合蛋白(hepatitis Bvirus x-interacting protein,HBXIP)抑制阿霉素(doxorubicinhydrochloride,DOX,adriamycin,ADM)诱导HepG2肝癌细胞凋亡的作用及可能的分子机制,为研究肝细胞癌的临床耐药性奠定基础。方法以建立的稳定高表达HBXIP基因的HepG2细胞系为研究对象,分别用不同浓度的DOX处理高表达HBXIP组及对照组细胞,MTT法检测细胞的生存率,DAPI染色及Annexin V-FITC/PI染色检测细胞凋亡,免疫印迹法检测蛋白表达水平。结果 MTT结果表明,DOX能够抑制HepG2细胞的存活,但HBXIP对DOX诱导的HepG2细胞凋亡作用具有明显的拮抗效应,并抑制DOX诱导的caspase-3、caspase-9及其底物PARP的活化,同时促进Bcl-2蛋白的表达。结论 HBXIP蛋白能够抑制化疗药物DOX诱导的HepG2细胞凋亡,其机制可能与调节胱天蛋白酶家族关键因子的活性相关。     

Bcl-2 siRNA induced apoptosis and increased sensitivity to 5-fluorouracil and HCPT in HepG2 cells

To investigate the changes in drug sensitivity of Bcl-2 siRNA transfected HepG2 cells. Bcl-2 siRNA and negative siRNA expression vector were constructed and stably transfected into HepG2 cells. RT-PCR and Immunofluorescence were used to detect the target gene expression. Western Blotting was used to detect Bcl-2, Bax and caspase-3 protein expressiom. Drug sensitivity of the cells to 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) were analyzed with MTT and flow cytometry. Results were following: (1) the mRNA and protein expression level of Bcl-2 in Bcl-2 siRNA stable transfectants were reduced compared with negative siRNA transfected or untreated cells. Accordingly, Bax protein expression had no change and caspase-3 protein expression showed significantly be up regulated; (2) MTT results showed that Bcl-2 siRNA transfectants had higher cell inhibitory rates after treated with 5-FU or HCPT; (3) flow cytometry results demonstrated that sub G1 population increased in Bcl-2 siRNA transfected cells compared with negative siRNA or untreated cells. After addition 5-FU (1300 mg/l) and HCPT (0.72 mg/l), Bcl-2 siRNA cells showed higher sub G1 population than negative siRNA or untreated cells. siRNA targeting Bcl-2 gene can specifically down-regulate Bcl-2 expression, increased Bax/Bcl-2 ratio expression and caspase-3 activity in HepG2 cells, which lead to increase cells spontaneous apoptosis and sensitize cells to 5-FU or HCPT. Bcl-2 siRNA may be a potential therapy agent against human hepatoblastoma.     

沉默Cdk7导致pRb和Cdk2磷酸化水平下降并诱导HepG2细胞凋亡

目的　研究转染反义寡脱氧核苷酸 (antisenseoligode oxynucleotides, ASODN)对Cdk7特异性的沉默作用及其对体外培养人肝细胞癌HepG2细胞pRb和Cdk2磷酸化水平以及细胞周期进展的影响,确定Cdk7作为抗肿瘤药物研发靶点的可行性。方法　以免疫印迹检测转染ASODN对Cdk7的特异性沉默作用及其对pRb和Cdk2磷酸化水平的影响;以流式细胞术测定转染ASODN后细胞DNA含量,确定细胞周期进展和凋亡情况,透射电子显微镜观察细胞凋亡形态。结果　转染ASODN 72h后,Cdk7蛋白水平显著下降,有明显剂量 -效应关系,最高可降至对照组的 0 12±0 05;在ASODN浓度>100nmol L-1时,pRb和Cdk2磷酸化水平显著下降,两种蛋白的磷酸化水平与给药剂量有明显依赖关系。随转染ASODN浓度上升及转染时间的延长,G0 /G1 期细胞、凋亡细胞比例迅速升高,与对照组比较,出现明显的G0 /G1 期阻滞(P<0 01)和细胞程序性死亡(P<0 01);透射电子显微镜观察显示>50nmol·L-1各组细胞具特征性凋亡形态。结论　用ASODN沉默Cdk7可降低pRb以及Cdk2的磷酸化水平,使其生物活性下降,对体外培养HepG2细胞可产生显著的G0 /G1 期阻滞,引起细胞周期延长和细胞凋亡,产生抗增殖作用,可以Cdk7作为抗肿瘤药物研发的新靶点。     

Inhibition of apoptosis facilitates necrosis induced by cisplatin in gastric cancer cells

Although cisplatin has been shown to induce both apoptosis and necrosis in cancer cells, the potential interconnections between these modes of cell death induced by the drug remain unknown. We studied this phenomenon in gastric cancer cell lines and identified one cell line (SGC-7901) that underwent apoptosis, and another cell line (BGC-823) that primarily underwent nonapoptotic cell death, in response to cisplatin. Apoptosis in cisplatin-treated SGC-7901 cells seemed to be caspase dependent and was mediated, at least in part, by the BH3-only protein, Noxa. This was evidenced by the rapid upregulation of Noxa and inhibition of apoptosis by small interfering RNA knockdown of Noxa. Nonapoptotic cell death induced by cisplatin in BGC-823 cells was characterized by lack of DNA fragmentation, delayed externalization of phosphatidylserine, caspase independence, plasma membrane disruption, and intracellular vacuole formation, indicative of necrosis. Surprisingly, blockage of apoptosis induction by a general caspase inhibitor or by Noxa small interfering RNA in SGC-7901 failed to protect against cisplatin-induced cell death. Under such conditions, SGC-7901 cells displayed cellular features associated with necrosis. Cisplatin-induced apoptosis, thus, seems to precede necrosis when the apoptotic machinery is operative. When the apoptosis program is defective, necrotic cell death takes place as an alternative pathway leading to cell demise. Induction of different modes of cell death that are interrelated in the same cells by cisplatin has the potential to be exploited in formulating new adjuvant cancer therapies.     

硫芥诱导HeLa细胞坏死和凋亡的超微结构变化

硫芥是一种细胞毒药物，能烃化生物体内一系列大分子物质，其中核酸是硫芥作用最敏感的靶分子．它还是首次证实有致突变的化学物质［１］．硫芥损伤的短期效应是引起ＤＮＡ断裂，细胞死亡；长期效应是致畸，致癌，致突变．硫芥对人体各种组织都有损伤作用，对皮肤，眼睛，...     

Bcl-2 siRNA induced apoptosis and increased sensitivity to 5-fluorouracil and HCPT in HepG2 cells

To investigate the changes in drug sensitivity of Bcl-2 siRNA transfected HepG2 cells. Bcl-2 siRNA and negative siRNA expression vector were constructed and stably transfected into HepG2 cells. RT-PCR and Immunofluorescence were used to detect the target gene expression. Western Blotting was used to detect Bcl-2, Bax and caspase-3 protein expressiom. Drug sensitivity of the cells to 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) were analyzed with MTT and flow cytometry. Results were following: (1) the mRNA and protein expression level of Bcl-2 in Bcl-2 siRNA stable transfectants were reduced compared with negative siRNA transfected or untreated cells. Accordingly, Bax protein expression had no change and caspase-3 protein expression showed significantly be up regulated; (2) MTT results showed that Bcl-2 siRNA transfectants had higher cell inhibitory rates after treated with 5-FU or HCPT; (3) flow cytometry results demonstrated that sub G1 population increased in Bcl-2 siRNA transfected cells compared with negative siRNA or untreated cells. After addition 5-FU (1300 mg/l) and HCPT (0.72 mg/l), Bcl-2 siRNA cells showed higher sub G1 population than negative siRNA or untreated cells. siRNA targeting Bcl-2 gene can specifically down-regulate Bcl-2 expression, increased Bax/Bcl-2 ratio expression and caspase-3 activity in HepG2 cells, which lead to increase cells spontaneous apoptosis and sensitize cells to 5-FU or HCPT. Bcl-2 siRNA may be a potential therapy agent against human hepatoblastoma.     

NS-398诱导肝癌细胞凋亡的实验研究

目的 :研究选择性环氧化酶 2 (COX 2 )抑制剂NS 398对人肝癌细胞HepG2 凋亡的影响 ,及其相关蛋白Bcl 2在细胞凋亡中的作用。方法 :通过体外细胞培养 ,应用荧光显微镜、透射电镜、流式细胞仪观察NS 398对HepG2 的凋亡诱导作用 ,应用免疫细胞化学法观察Bcl 2在人肝癌细胞HepG2 凋亡中的表达。结果 :NS 398处理HepG2 细胞后 ,电镜下可见到细胞核固缩、染色质凝集成新月型紧靠核膜周边 ,核碎裂、染色质片断化等典型的细胞凋亡形态变化。荧光显微镜及流式细胞检测则未见凋亡征象。免疫细胞化学分析显示 ,NS 398处理后的HepG2 细胞 ,其Bcl 2表达较对照组明显下降。结论 :COX 2选择性抑制剂NS 398对人肝癌HepG2 细胞有显著的凋亡诱导作用 ;抗凋亡基因Bcl 2在NS 398诱导的HepG2 细胞凋亡中有重要的调控作用。     

Inhibition of autophagy promotes caspase-mediated apoptosis by tunicamycin in HepG2 cells

Tunicamycin (TM) causes accumulation of unfolded protein in endoplasmic reticulum (ER) lumen and introduces from elsewhere ER stress. This study was to assess the apoptosis and autophagy effect induced by TM on HepG2 cells and the role of autophagy in the system. The viability of HepG2 cells was significantly inhibited by TM in a dose-dependent manner detected by MTT assay. Then, the apoptotic morphology change, increasing apoptotic cell rate suggested that apoptosis was induced by TM in a time- and dose-dependent manner. To further determine the involvement of caspase-dependent pathway in TM-induced apoptosis, we discover that the activity of caspase-3/7, 8, 9 and cleavage of PARP markedly increased after TM treatment and the apoptosis was effectively attenuated by using caspase-9 and pan caspase inhibitor. Moreover, provided the rising stained acidic vacuoles and an increased level of LC3II and activation of Beclin1, we concluded that autophagy could be triggered by TM in a time- and dose-dependent manner. In addition, the inhibition of autophagy efficiently promoted TM-induced cell death identified by MTT assay. Meanwhile, the apoptotic cell rate and caspase-3 activation increased significantly after autophagy blockage. In conclusion, we found that TM initiated apoptosis and autophagy both in a time- and dose-dependent manner in HepG2 cells; and inhibition of autophagy may promote TM-induced cell death through enhancing apoptosis.     

三萜皂苷Saxifragifolin D抑制人肝癌耐药细胞HepG2/ADM生长并诱导细胞凋亡作用研究

目的研究Saxifragifolin D(SD)对人肝癌耐药细胞HepG2/ADM的生长抑制及诱导凋亡作用。方法采用MTT法观察SD对HepG2/ADM细胞的增殖抑制作用,应用流式细胞仪分析SD对细胞周期的影响,AnnexinⅤ-FITC/PI双染检测凋亡细胞比率,JC-1染色观察SD对细胞内线粒体膜电位的影响,Western blot检测凋亡相关蛋白caspase-9,caspase-3和PARP的激活及c-Raf,MEK和ERK蛋白的表达和磷酸化水平。结果 SD可以明显抑制人肝癌耐药细胞HepG2/ADM的增殖。细胞周期检测发现SD诱导细胞产生亚二倍体凋亡峰,同时细胞凋亡率也由对照组的5.3%增加到34.8%和47.8%。线粒体膜电位检测结果显示SD导致细胞内线粒体膜电位的明显降低。Western blot检测结果表明caspase-9,caspase-3被激活,PARP被剪切活化,cytochrome C由线粒体释放至胞质,c-Raf、MEK和ERK蛋白的磷酸化水平降低。结论 SD可以抑制人肝癌耐药细胞HepG2/ADM增殖并诱导其凋亡,作用机制可能与线粒体功能障碍及抑制c-Raf/MEK/ERK通路的活化有关。