Involvement of bax/bcl-2 in wogonin-induced apoptosis of human hepatoma cell line SMMC-7721

The molecular mechanisms of wogonin-induced apoptosis of human hepatoma SMMC-7721 cells are reported. Wogonin treatment resulted in significant inhibition of SMMC-7721 cells in a time-dependent and concentration-dependent manner. Typical morphological changes and apoptotic blebbing in SMMC-7721 cells were observed after treatment with 1x10(-4) mol/l wogonin for a period of 0-48 h. Flow cytometry and Annexin-V/propidium iodide double-staining experiments revealed a dramatic increase in the number of apoptotic and G0/G1 phase cells after wogonin treatment. The proapoptotic activity of wogonin is attributed to its ability to modulate the expression of bcl-2 and bax proteins. It is observed that the expression of bax protein is dramatically increased whereas the synthesis of bc1-2 protein is significantly decreased when cells are treated with wogonin. The results presented in this paper suggested an important relationship between gene regulation and wogonin-induced apoptosis, and indicated the possibility of developing naturally occurring monoflavonoids as novel anticancer agents for better management of human cancers.     

新型金属铜络合物对SMMC-7721细胞增殖与凋亡的影响

目的研究新型金属铜络合物(N-Cu)在体外对人肝癌SMMC-7721细胞增殖与凋亡的影响及其作用机制。方法将不同浓度的N-Cu(0.3～24μmol.L-1)作用于体外培养的SMMC-7721细胞,应用MTT法检测细胞生长抑制率,FCM法检测细胞周期及凋亡率,RT-PCR和Western blot法检测细胞中Bcl-2、Bax、Caspase-3 mRNA和蛋白表达的变化。结果 N-Cu可明显抑制SMMC-7721细胞的增殖,呈明显的量效与时效关系。随着药物浓度的增加,G0/G1期的细胞比率上升,G2/M和S期细胞比率下降,并促进凋亡率增加。N-Cu可上调细胞中Bax、Caspase-3基因及蛋白的表达,抑制Bcl-2基因及蛋白的表达,且均呈剂量依赖性。结论一定浓度的N-Cu可抑制SMMC-7721细胞的增殖并诱导其凋亡,阻滞细胞周期于G0/G1期。上调Bax、Caspase-3基因及蛋白的表达,降低Bcl-2/Bax比值,可能是其诱导细胞凋亡的重要机制。     

General gambogic acids inhibited growth of human hepatoma SMMC-7721 cells in vitro and in nude mice

AIM: To study the inhibitory effect of general gambogic acids (GGA) on transplantation tumor SMMC-7721 in experimental animal model and SMMC-7721 cells in vitro. METHODS: Anti-tumor activity of GGA in the experimental transplantation tumor SMMC-7721 was evaluated by relative tumor growth ratio. Cell morphology was observed with inverted microscope and electron microscope. Cell proliferation was measured by MTT assay and the telomerase activity was determined by PCR. RESULTS: In vivo study indicated that GGA (2, 4, and 8 mg/kg, iv, 3 times per week for 3 weeks) displayed an inhibitory effect on the growth of transplantation tumor SMMC-7721 in nude mice compared with the normal saline group (P<0.01). At the concentrations of 0.625-5.0 mg/L, GGA remarkably inhibited the proliferation of SMMC-7721 cells in vitro. GGA 2 mg/L dramatically changed morphology of SMMC-7721 cells and inhibited the telomerase activity in SMMC-7721 cells. CONCLUSION: GGA had inhibitory effect on the growth of SMMC-7721, which might be related to its inhibition of telomerase activity.     

表阿霉素诱导人肝癌SMMC-7721细胞凋亡的实验研究

目的 进一步探讨表阿霉素治疗原发性肝癌的作用机理。方法 应用DNA电泳、电镜、流式细胞仪分析技术，观察表阿霉素诱导人肝癌SMMC－7721细胞凋亡模式。结果 0.1μg/ml表阿霉素作用细胞12、24、36、48小时及0.1、1.0、10.0、100μg/ml表阿霉素作用细胞24小时均可出现细胞凋亡现象，其诱导凋亡效率与剂量、时间呈正相关。结论 表阿霉素可诱导人肝癌SMMC－7721细胞凋亡而发挥抗肿瘤作用。     

Mechanism of apoptotosis induced by ortho-topolin riboside in human hepatoma cell line SMMC-7721

The naturally occurring cytokinin, ortho-topolin riboside (oTR), has been recently reported to have a strong anticancer effect. However, the molecular mechanism has not been elucidated. From our research we found that oTR strongly inhibited the proliferation of SMMC-7721 cells inducing apoptosis. After oTR treatment, up-regulation of the protein levels of pro-apoptotic Bax and the down-regulation of the anti-apoptotic proteins, Bcl-2 and Bcl-xL was observed, leading to the loss of mitochondrial membrane potential, the release of cytochrome c from the mitochondria into the cytosol, the downstream activation of caspase-9 and caspase-3, as well as the cleavage of poly ADP-ribose-polymerase (PARP), the effect of apoptosis could be blocked by the pan-specific caspase inhibitor z-VAD-fmk and caspase-9-specific inhibitor z-LEHD-fmk. Moreover, oTR was shown to inhibit the activation of the extracellular signal-regulated kinase-1/2 (ERK_1/2) as well as the Akt pathway. These results suggest that oTR interferes with the mitogen-activated protein kinase (MAPK) and Akt pathways and induces the apoptosis of human SMMC-7721 cells through the activation of intrinsic mitochondria-mediated pathways. However, the apoptosis was completely prevented when cells were treated with A-134974, an inhibitor of adenosine kinase, it indicated that the intracellular phosphorylation of oTR is necessary for its cytotoxic effects to SMMC-7721 cells.     

没食子酸诱导人肝癌细胞SMMC-7721凋亡机制的探讨

目的探讨没食子酸(gallic acid,GA)抑制人肝癌细胞SMMC-7721增殖的作用,揭示其促凋亡的相关分子机制。方法体外培养人肝癌细胞SMMC-7721,MTT法观察细胞在GA作用24、48、72 h后的增殖情况;用倒置显微镜观察细胞形态学的变化;透视电镜观察细胞内部结构变化;Annexin V-FITC/PI检测细胞凋亡;采用RT-PCR技术研究p53 mRNA的变化;应用Western blot法检测p53蛋白水平的表达。探讨GA对人肝癌细胞SMMC-7721的诱导凋亡作用及机制。结果剂量为6.25⁵0μmol·L-1GA作用SMMC-7721细胞48 h有明显的增殖抑制活性,引起核固缩、凝聚、碎裂,诱导细胞凋亡,并呈剂量依赖性;RT-PCR和Western blot结果显示,GA能升高p53 mRNA水平和p53蛋白表达。结论 GA可抑制人肝癌细胞SMMC-7721的增殖,诱导凋亡,可能与其上调肿瘤相关抑癌基因p53有关。     

姜黄素具有抑制血管生成与诱导肿瘤细胞凋亡双重作用

目的　探讨姜黄素的抗肿瘤机制。方法　采用鸡胚绒毛尿囊膜模型观察姜黄素对血管生成的影响 ;利用培养的肿瘤细胞SMMC 772 1,采用电子显微镜及流式细胞仪观察姜黄素诱导SMMC 772 1细胞凋亡的作用。结果　姜黄素能明显抑制鸡胚绒毛尿囊膜内的血管生成 ,2 0 μmol·L-1的姜黄素即可诱导SMMC 772 1细胞凋亡。结论　姜黄素的抗肿瘤机制是多方面的 ,既可抑制血管生成又可诱导肿瘤细胞凋亡     

蟾蜍灵对肝癌细胞SMMC 7721的细胞毒作用及生长相关基因表达的影响

为探讨蟾蜍灵的抗癌作用机理 ,以肝癌SMMC772 1细胞为靶细胞 ,应用噻唑蓝还原法检测细胞毒作用 ,双荧光染色法和DNA电泳技术检测细胞凋亡与坏死 ;免疫组织化学方法检测细胞生长相关基因p2 1waf1/cip1和增殖细胞核抗原 (PCNA)蛋白表达 .结果表明 ,0 .0 1μmol·L- 1及以上浓度蟾蜍灵对SMMC772 1细胞具有显著细胞毒作用 ,形态学和DNA片段化检测证实蟾蜍灵诱导的细胞死亡以凋亡为主 ;p2 1waf1/cip1在蟾蜍灵诱导下表达上调 ,同时PCNA的表达下降 ,两者呈负相关 (P <0 .0 1) .提示蟾蜍灵通过上调p2 1waf1/cip1表达 ,下调PCNA表达 ,从而抑制肝癌细胞生长     

微小RNA-375调控自噬抑制肝癌细胞SMMC-7721增殖和转移

目的 探讨微小 RNA-375（miR-375）通过调控自噬相关蛋白（Atg）14 介导的自噬对肝癌细胞（SMMC-7721）增殖和转移能力的影响。方法 将SMMC-7721细胞分别用miR-375模拟物、抑制物以及Atg14的干扰RNA处理,并建立缺氧1 h复氧6 h的模型,分为miR-375 NC组、miR-375 mimics组、miR-375 inhibitor组以及siRNA NC组、Atg14 siRNA 组、miR-375+Atg14 siRNA 组。TargetScan 预测 miR-375 与 Atg14 基因相关性。荧光实时定量 PCR（qRT-PCR）检测miR-375 NC组、miR-375 mimics组、miR-375 inhibitor组miR-375、Atg14 mRNA相对表达量。免疫细胞化学染色检测3组细胞内N-钙黏素（N-Cadherin）和β-连环蛋白（β-catenin）表达分布情况。绿色荧光蛋白-红 色荧光蛋白-轻链（mGFP-RFP-LC3）自噬双标腺病毒转染3组细胞,荧光显微镜下观察自噬体形成情况。平板克隆检测肝癌细胞增殖情况。Western blot 检测Atg14、泛素结合蛋白（P62）、轻链3Ⅰ（LC3Ⅰ）、轻链3Ⅱ（LC3Ⅱ）、酵母Atg6同源物（Beclin1）、N-Cadherin、β-catenin和波形蛋白（Vimentin）表达情况。结果 miR-375与Atg14基因相关性较高,qRT-PCR显示过表达miR-375能抑制Atg14 mRNA表达;反之能促进Atg14 mRNA表达。过表达miR-375能抑制肝癌细胞内N-Cadherin表达、提高β-catenin表达水平,抑制肝癌细胞增殖能力（P＜0.01）;而抑制miR-375表达能促进N-Cadherin表达、降低β-catenin表达水平,提高肝癌细胞增殖能力（P＜0.01）。过表达miR-375能抑制Atg14、LC3Ⅱ、Beclin1表达,促进P62表达（P＜0.01）;反之能促进Atg14、LC3Ⅱ、Beclin1表达,抑制P62表达（P＜0.01）。荧 光显微镜下观察到过表达 miR-375 可抑制自噬体形成,而抑制 miR-375 表达能促进自噬体形成（P＜0.01）。干扰Atg14表达后可增强miR-375对肝癌细胞增殖、转移能力的抑制作用（P＜0.01）。结论 激活miR-375能抑制肝癌细胞增殖和转移,而抑制miR-375能促进肝癌细胞增殖和转移,其机制可能与miR-375通过靶向调控Atg14抑制自噬,从而抑制SMMC-7721肝癌细胞增殖和转移有关。     

黄芪总黄酮对K562细胞凋亡及细胞周期的影响

<正>白血病是严重危害人类健康的恶性肿瘤,儿童尤其多见。黄芪总黄酮(total flavonoids of Astragalus,TFA)是从蒙古黄芪中分离的抗氧化、清除自由基的主要活性成分[1,2],     

hTERT反义寡核苷酸对肝癌SMMC-7721细胞株细胞增殖和端粒酶活性的影响

目的研究hTERT基因反义寡核苷酸（ASPSODN）对SMMC-7721细胞端粒酶活性及细胞增殖的影响。方法不同浓度的ASPSODN转染到SMMC-7721细胞株24h和48h后分别采用MTT法、TRAP-ELISA法检测SMMC-7721细胞的增殖活性及端粒酶活性。结果各浓度组的ASPSODN均能降低端粒酶活性,作用24h时,不同浓度均明显受抑,其中0.8μmol/L ASPSODN较0.2μmol/L和0.4μmol/L组抑制作用更明显（P〈0.05）,但再增大浓度抑制作用并不随着增加。作用48h后,抑制作用减弱,低浓度组（0.2、0.4μmol/L）端粒酶活性恢复到正常水平,而高浓度组（0.8、1.0、1.2μmol/L）抑制作用依然明显,端粒酶活性低于60%。MTT法检测显示,不同浓度ASPSODN组对SMMC-7721细胞的生长均有抑制作用,随着浓度的增加,抑制作用明显,但随着时间的延长,抑制作用增加不明显。结论 ASPSODN靶向hTERT能特异性抑制SMMC-7721细胞增殖,明显下调端粒酶活性,hTERT可能成为肝癌治疗的一个靶点。     

短发夹RNA沉默YAP表达对肝癌细胞SMMC-7721增殖和凋亡的影响

目的本实验通过短发夹RNA(shRNA)抑制Yes相关蛋白(YAP)基因的表达,并观察其对肝癌细胞SMMC-7721的增殖、凋亡和周期影响。方法以人YAP mRNA编码区中的4条序列作为RNA干扰靶点,分别构建4个真核表达载体质粒YAP-shRNA-1、YAP-shRNA-2、YAP-shRNA-3、YAP-shRNA-4,用阳离子聚合物转染法瞬时转染,筛选出2个干扰效果较好的质粒,将它们瞬时转染肝癌细胞SMMC-7721,应用实时荧光定量聚合酶链反应(PCR)和蛋白印迹法检测SMMC-7721细胞中YAP在mRNA和蛋白水平表达的变化。噻唑蓝(MTT)法检测转染后细胞增殖能力,流式细胞术检测细胞凋亡和周期的变化。结果转染后293T细胞中shRNA-YAP-2组与shRNA-YAP-4组YAP mRNA表达明显降低,在SMMC-7721中shRNA-YAP-2组与shRNAYAP-4组的YAP mRNA及蛋白表达水平分别为(0.871±0.081,0.106±0.013)和(1.010±0.097,0.192±0.013),与未转染组(2.399±0.148,0.372±0.007)比较差异有统计学意义(P<0.01)。MTT显示,shRNA-YAP-2、shRNA-YAP-4组细胞生长明显抑制,但未出现阻滞;流式细胞仪分析显示,shRNA-YAP-2组和shRNA-YAP-4组细胞凋亡率分别为(12.0±0.81)%和(5.03±1.01)%,明显高于空质粒组的(2.89±0.08)%(P<0.01)。细胞被阻滞在G2期,G0/G1期出现DNA亚二倍体峰。结论运用shRNA干扰技术,可以有效地干扰肝癌SMMC-7721细胞YAP的表达并能降低YAP的生成,抑制SMMC-7721细胞的增殖和促进细胞的凋亡。     

杨梅素对肝癌SMMC-7721细胞迁移及侵袭影响的研究

目的探讨杨梅素对肝癌SMMC-7721细胞迁移和侵袭的影响。方法以不同浓度杨梅素处理的肝癌SMMC-7721细胞为研究对象,分别采用Wound healing实验、Transwell细胞迁移和侵袭实验检测细胞的迁移和侵袭过程,用RT-q PCR和Western blot检测E-cadherin和N-cadherin的表达。结果杨梅素呈浓度依赖性(10~40μ mol · L~(-1))抑制肝癌SMMC-7721细胞活力;杨梅素还可以抑制SMMC-7721细胞迁移和侵袭过程,并且随着杨梅素浓度的增加,细胞中丝状、片状伪足逐渐减少,细胞排列越来越紧密;RT-qPCR和Western blot结果均显示,杨梅素可以上调SMMC-7721细胞的E-cadherin表达,下调N-cadherin表达。结论杨梅素能抑制肝癌SMMC-7721细胞的迁移和侵袭过程,此作用的发生可能是通过上调E-cadherin、下调N-cadherin等EMT相关的信号通路以及肌动蛋白细胞骨架的重排实现的。     

Piperonal ciprofloxacin hydrazone induces growth arrest and apoptosis of human hepatocarcinoma SMMC-7721 cells

Aim:

To investigate the cytotoxic effects of piperonal ciprofloxacin hydrazone (QNT4), a novel antibacterial fluoroquinolone derivative, against human hepatocarcinoma SMMC-7721 cells.

Methods:

Human hepatocarcinoma cells (SMMC-7721), human breast adenocarcinoma cells (MCF-7) and human colon adenocarcinoma cells (HCT-8) were tested. The effects of QNT4 on cell proliferation were examined using MTT assay. Cell apoptosis was determined using Hoechst 33258 fluorescence staining, TUNEL assay and agarose gel electrophoresis. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. Mitochondrial membrane potential (Δψm) was measured using a high content screening imaging system. Protein expression of caspase-9, caspase-8, caspase-3, p53, Bcl-2, Bax, and cytochrome c was detected with Western blot analysis.

Results:

Treatment with QNT4 (0.625–10 μmol/L) potently inhibited the proliferation of the cancer cells in time- and dose-dependent manners (the IC₅₀ value at 24 h in SMMC-7721 cells, MCF-7 cells and HCT-8 cells was 2.956±0.024, 3.710±0.027, and 3.694±0.030 μmol/L, respectively). Treatment of SMMC-7721 cells with QNT4 (0.2146, 2.964, and 4.600 μmol/L) for 24 h dose-dependently increased the percentage of apoptotic cells, elicited characteristic DNA “ladder” bands, and decreased the mitochondrial membrane potential. QNT4 dose-dependently increased topoisomerase II-mediated DNA breaks while inhibiting DNA relegation, thus keeping the DNA in fragments. Treatment of SMMC-7721 cells with QNT4 significantly increased cytochrome c in the cytosol, and decreased cytochrome c in the mitochondrial compartment. QNT4 (3–7.39 μmol/L) significantly increased the protein expression of p53, Bax, caspase-9, caspase-3, and the cleaved activated forms of caspase-9 and caspase-3 in SMMC-7721 cells. In contrast, the expression of Bcl-2 was decreased, while caspase-8 had no significant change.

Conclusion:

QNT4 induced the apoptosis of SMMC-7721 cells via inhibiting topoisomerase II activity and modulating mitochondrial-dependent pathways.     

荷人膀胱癌原位移植瘤Balb／c裸小鼠动物模型的建立

目的建立一种简便、可靠的荷人膀胱癌原位移植瘤动物模型,核磁共振成像(MRI)评价成瘤过程。方法常规培养人膀胱癌E-J细胞株细胞,Balbc裸小鼠膀胱穿刺后腔炮灌注E-J细胞悬液,建立荷人膀胱癌原位移植瘤动物模型,核磁共振成像(MRI)监测肿瘤的生长,45～50天处死动物,标本常规行HE染色,并使用抗人膀胱癌单克隆抗体BDI-1通过免疫组化鉴定移植瘤。结果20只动物有19只荷瘤,成瘤率为95%;接种后10～14天MRI检查可发现明显的充盈缺损。肿瘤病理分级为Ⅰ～Ⅱ级,T1期65%(13只),T2期20%(4只),T3期10%(2只),T4期5%(1只),所有动物无远处转移。HE染色示移行细胞癌,免疫组化(S-P法)染色检测瘤细胞表面抗原的表达呈阳性,与体外培养的E-J细胞免疫组化染色结果一致。结论膀胱穿刺腔内灌注法建立荷人膀胱癌原位移植瘤裸小鼠动物模型十分简便,成瘤率高,有助于研究膀胱癌的生物学行为和腔内灌注治疗。     

Effect of honey bee venom on proliferation of K1735M2 mouse melanoma cells in-vitro and growth of murine B16 melanomas in-vivo

Bee venom has been reported to exhibit antitumour activity in-vitro and in-vivo. Apoptosis, necrosis and lysis of tumour cells were suggested as possible mechanisms by which bee venom inhibited tumour growth. The aim of this study was to investigate potential mechanisms by which bee venom inhibits K1735M2 mouse melanoma cells in-vitro and B16 melanoma, a transplantable solid melanoma in C57BL/6 mice, in-vivo. The proliferation of K1735M2 cells in-vitro was inhibited by bee venom in a concentration- and time-dependent manner. The inhibition was indicated by the arrest of the cell cycle at the G1 stage, as detected by flow cytometric measurements. The bee venom induced apoptosis-like cell death as identified by histological observations and by DNA fragmentation. In the in-vivo experiments, the bee venom (1.0, 3.0, 9.0 mg kg-1 of body weight, on days 1-12) was injected intraperitoneally into mice 24 h after the mice were inoculated with B16 cells. Inhibition of the solid tumour was observed. Apoptosis of the K1735M2 cells was suggested as the possible mechanism by which bee venom inhibited cell proliferation and induced K1735M2 cell differentiation in-vitro. The in-vivo experiment indicated that bee venom could be used as a chemotherapeutic agent against malignant tumours.     

丹参酮衍生物对K562细胞的生长抑制及诱导凋亡作用研究

目的研究丹参酮衍生物对人红白血病细胞株K562的生长抑制以及诱导凋亡作用。方法MTT比色法检测不同浓度丹参酮1、丹参酮2和丹参酮B对K562细胞的增殖抑制作用;PI单染法检测3者对K562细胞周期的影响;An-nexin V/PI双染法检测3者对K562细胞诱导凋亡的作用。结果丹参酮1和丹参酮B能够抑制K562细胞增殖,IC50分别为5.22和15.11μmol·L-1,且分别在2.5~10μmol·L-1和10~40μmol·L-1剂量范围内,G0/G1期细胞比例的增加及早期凋亡细胞百分率的提高均呈剂量相关性;丹参酮2在100μmol·L-1的剂量下,对K562细胞的抑制率仅为27.8%,无诱导其凋亡作用,但可以使G0/G1期细胞增多。结论丹参酮1和丹参酮B抑制K562细胞增殖,阻滞其于G0/G1期并诱导细胞凋亡;丹参酮2对K562细胞没有明显生长抑制及诱导凋亡作用,但可将其阻滞于G0/G1期。     

紫杉醇对人肝癌细胞SMMC-7721黏附及侵袭转移潜能的影响

目的探讨紫杉醇对人肝癌细胞株SMMC-7721的黏附及侵袭转移潜能的影响及机制。方法应用体外细胞．基质黏附实验检测不同浓度紫杉醇对SMMC-7721与Matrigel胶（人工基底膜胶）黏附性的影响：Transwell小室模型测定紫杉醇对细胞侵袭和迁移能力的影响;蛋白免疫印迹检测紫杉醇对细胞内黏着斑激酶（FAK）蛋白表达量的影响。结果不同浓度紫衫醇（10、20、40nmol／L）作用SMMC-7721细胞60min后,细胞与Matrigel胶黏附性下降;紫杉醇干预24h后,细胞的侵袭能力及迁移能力随紫杉醇浓度提高而明显减弱;细胞内FAK蛋白的表达量也随紫杉醇浓度增高而明显下降。结论紫杉醇能抑制肝癌细胞SMMC-7721的黏附及侵袭转移,其机制可能与其抑制肝癌细胞内FAK的表达相关。     

Induction of apoptosis by chelerythrine chloride through mitochondrial pathway and Bcl-2 family proteins in human hepatoma SMMC-7721 Cell

The objective of this study was to evaluate the antitumor activity of chelerythrine chloride (CHE) and investigate its potential apoptotic induction mechanism in SMMC-7721 cells. Our results suggested that the proliferation of SMMC-7721 cells was inhibited by CHE in a time and dose dependent manner, with a significant accumulation in S phase, and the cells exhibited typical apoptotic features. Moreover, CHE remarkably induced apoptosis by disruption of the mitochondrial membrane potential, release of Cyt-c, activation of caspase-3, and cleavage of poly-ADP-ribose polymerase in a dose dependent manner. Furthermore, the expression of Bcl-xl was downregulated while Bax and Bid expression was upregulated, and no variation was found for Bcl-2. These results indicated that CHE may play an important role in suppression of tumor growth by inducing apoptosis in human hepatoma cells via the activation of a mitochondrial pathway and regulating the expression of Bcl-2 family proteins.