Clarithromycin effectively enhances doxorubicin-induced cytotoxicity and apoptosis in MCF7 cells through dysregulation of autophagy

PurposeUse of autophagy inhibitors in combination with chemotherapy has become a novel chemotherapeutic strategy. In this study, we aimed to determine whether the effectiveness of doxorubicin (DOX) is augmented by clarithromycin (CAM) in MCF7 cells and the molecular mechanisms involved.Materials and methodsCombined cytotoxicity of CAM and DOX was assessed by MTT assay and was analyzed using the Chou-Talalay's method. To clarify the underlying mechanisms, several factors, including apoptosis (Annexin V/propidium iodide staining), intracellular level of DOX (spectrofluorimetry) and P-glycoprotein activity (Rhodamin 123 efflux assay) were measured. In addition, autophagy was evaluated by intracellular labeling with anti-LC3II and LysoTrackerGreen (LTG) staining and analyzed by flowcytometry.ResultsThe anti-proliferation effect of DOX was synergistically enhanced by CAM in MCF7 cells and was associated with an increase in the apoptotic cell death. However, the intracellular level of DOX remained unchanged in the presence of CAM. Based on the findings, 100 μM of CAM did not exhibit any inhibitory effects on P-glycoprotein activity. Flow cytometric analysis indicated that DOX at IC₂₀ concentration induced the autophagy flux, as confirmed by the increased level of LC3II and LTG signals. Moreover, combined treatment with DOX and CAM resulted in more pronounced LTG signals, but no change in LC3II. These results indicate that CAM blocks the autophagy flux induced by DOX.ConclusionsThese findings suggest that suppression of autophagy by CAM may promote chemotherapeutic outcome in breast cancer. However, further investigations are needed to evaluate the application of CAM in adjuvant breast cancer therapy.     

沉默GRP94 基因对乳腺癌MCF7 细胞增殖和凋亡的影响

目的： 研究沉默葡萄糖调节蛋白GRP94 (Glucose regulated protein,GRP94)对乳腺癌MCF7细胞增殖、凋亡的影响及潜在机制。方法：设计并化学合成靶向沉默GRP94基因的小干扰RNA,通过脂质体转染入MCF7细胞中,采用qRT-PCR和Western blot分别检测GRP94、cyclinD1、Bax和Bcl-2 mRNA和蛋白的表达水平;通过流式细胞术检测细胞凋亡比例变化,Hoechst 33258染色检测凋亡细胞核变化、CCK8实验检测细胞增殖能力的变化。结果： GRP94 siRNA组GRP94基因的表达水平被有效抑制;与对照组相比,GRP94-siRNA转染组的细胞凋亡比例明显增加;凋亡细胞核形态发生变化;增殖能力明显下降;mRNA及蛋白水平cyclinD1、Bcl-2表达明显下调,Bax表达增加。结论：沉默GRP94基因可明显抑制乳腺癌MCF7细胞增殖能力,促进细胞凋亡的发生,且其可能通过下调cyclinD1、Bcl-2和上调Bax表达参与其中。     

Cyclin E overexpression and amplification in human tumours

Cyclin E amplification and overexpression have recently been described in several tumour types. However, many tumour entities have never been examined for cyclin E alterations. Numerous and time-consuming experiments were previously required to determine the significance of potential oncogenes across different tumour types. To overcome this problem, tissue microarrays (TMAs) consisting of 3670 primary tumours from 128 different tumour types, 709 metastases, and 354 normal tissues were generated. Cyclin E alterations were then analysed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Cyclin E gene amplification was observed in 15 different tumour types and subtypes, ie rhabdomyosarcoma, urinary bladder cancer (three subtypes), ovarian cancer (two subtypes), malignant fibrous histiocytoma, adenocarcinoma of the small intestine, medullary breast cancer, gall bladder adenocarcinoma, phaeochromocytoma, gastric adenocarcinoma, squamous cell carcinoma of the uterine cervix, colonic adenocarcinoma, and endometrial carcinoma. Cyclin E protein accumulation was found in 48 different tumour types. The use of TMA technology has enabled us to expand considerably our knowledge of cyclin E alterations in human tumours. The occurrence of amplification and overexpression in many different tumour types suggests that cyclin E plays an important role in tumour biology.     

慢性缺氧应激可能通过EMT增强乳腺癌MCF-7细胞恶性生物学行为

目的:探讨慢性缺氧应激对人乳腺癌MCF-7细胞恶性生物学行为的影响及可能机制。方法:将人乳腺癌MCF-7细胞分为缺氧组(1%O_2、5%CO_2和94%N_2)和正常对照组(常氧)进行培养。利用MTT法、CCK-8实验、细胞直接计数法及细胞侵袭和迁移实验对MCF-7细胞活力、增殖及侵袭和迁移能力进行检测;用软琼脂集落形成实验及Matrigel 3D培养技术检测MCF-7细胞非锚定生长能力及极性改变情况;利用MCF-7细胞构建裸鼠皮下种植瘤模型,检测慢性缺氧应激对体内肿瘤生长及肺转移的影响;利用倒置显微镜观察MCF-7细胞形态改变;Western blot检测低氧诱导因子1(hypoxia-inducible factor-1,HIF-1)和磷酸化的糖原合成酶激酶3β(glycogen synthase kinase-3β,GSK-3β)在缺氧环境下表达水平的改变,以及E-钙黏连蛋白(E-cadherin)、N-钙黏连蛋白(Ncadherin)、波形蛋白(vimentin)、基质金属蛋白酶3(matrix metalloproteinase-3,MMP-3)、MMP-9等上皮-间充质转化(epithelial-mesenchymal transition,EMT)相关蛋白的表达水平。结果:与正常对照组相比较,慢性缺氧组MCF-7细胞活力、增殖能力及侵袭迁移能力增强,细胞非锚定生长能力提高且在3D培养系统更容易发生极性改变,呈现侵袭样生长,体内生长及转移能力增强;除了HIF-1被缺氧诱导表达升高外,GSK-3β呈现活化趋势,且上皮样标志物E-cadherin蛋白表达水平明显下降,而间充质样标志物N-cadherin、vimentin、MMP-3和MMP-9蛋白表达水平明显升高。结论:慢性缺氧应激促进了乳腺癌细胞恶性生物学行为,且其机制可能与EMT有关。     

Sodium butyrate-induced apoptosis and ultrastructural changes in MCF-7 breast cancer cells

This study investigated the effects of sodium butyrate (NaB) on Michigan Cancer Foundation-7 (MCF-7) breast cancer cells and analyzed the relevant mechanism. Here, we demonstrated that a certain concentration of NaB effectively induced MCF-7 cell apoptosis. Cell counting kit-8 (CCK-8) assay was used to detect cell viability and the apoptosis rate. Western blotting was used to detect changes in the Bcl-2 expression level. We observed cell shape changes with microscopy. Immunofluorescence revealed some apoptotic nuclei. Electron microscopy revealed thick nucleoli, chromatin margination, reduced mitochondria, and dramatic vacuoles. Collectively, our findings elucidated the morphological mechanism by which NaB changed the ultrastructure of MCF-7 cells.     

PUMA增强依托泊苷诱导宫颈癌细胞凋亡

目的探讨PUMA蛋白在依托泊苷诱导的宫颈癌细胞凋亡中的作用。方法采用MTT方法检测依托泊苷对宫颈癌细胞系SiHa细胞活力的影响。通过Annexin-v／PI双染检测和光学显微镜形态观察检测依托泊苷对宫颈癌SiHa细胞凋亡的影响。通过转染siRNA干扰PUMA的表达,检测低表达PUMA后,宫颈癌细胞对依托泊苷的敏感性。通过免疫印迹方法检测PUMA干扰后PUMA的表达水平。结果依托泊苷显著抑制宫颈癌细胞的活力和诱导其凋亡,且随着浓度和时间的增加而细胞活力依次减少而凋亡率依次升高,且光学显微镜下有明显的细胞皱缩等凋亡形态。当转染PUMA的siRNA后．依托泊苷诱导的细胞凋亡显著减少。结论PUMA在依托泊苷诱导宫颈癌细胞凋亡中起了重要作用,它能够增强宫颈癌细胞对依托泊苷的敏感性。     

An ethanol extract of Iris nertschinskia induces p53-dependent apoptosis in the MCF7 human breast cancer cell line

Iris nertschinskia, an ornamental plant, is utilized in traditional East Asian medicine for the treatment of skin diseases. However, the biological activity underlying its therapeutic effects remains to be established. In this study, we investigated the anti-tumor effect of the plant extract on MCF7 human breast cancer cells. An ethanol extract of Iris nertschinskia triggered cell death in a dose-dependent manner. Moreover, treatment with the extract promoted p53 phosphorylation in MCF7 cells. Increased phosphorylation of p53, in turn, led to induction of Bax protein, a key regulator of p53-dependent apoptotic cell death, as well as of caspase-7 cleavage in MCF7 cells. Consistently, cells treated with p53-specific siRNA or the caspase inhibitor, Z-VAD, resisted apoptotic cell death induced by the Iris nertschinskia extract. Our results suggest that p53 sensitizes tumor cells to the ethanol extract of Iris nertschinskia by Bax protein induction and caspase-dependent apoptosis.     

The chlorophyllin-induced cell cycle arrest and apoptosis in human breast cancer MCF-7 cells is associated with ERK deactivation and Cyclin D1 depletion

Targeting the mitogen-activated protein kinases (MAPKs) has been suggested as a novel strategy to treat cancer. Chlorophyllin (CHL) is the sodium-copper salt of chlorophyll derivative and is a commonly used food dye for green coloration; CHL was found previously to retard growth of the human breast carcinoma MCF-7 cells. Extracellular signal-regulated kinases (ERKs) constitute a subfamily of MAPKs, participating in cell survival, proliferation and differentiation. We report here the first evidence that CHL deactivates ERKs to inhibit the breast cancer cell proliferation. The results from flow cytometry showed that 200 microg/ml CHL reduced the phosphorylated and activated ERK-positive cells in different cell cycle phases from the control of >96 to <38% at 24 h of incubation; the ERK deactivations occurred in both dose- and time-dependent manner, so that nearly all ERKs were de-activated by 400 microg/ml CHL at 72 h of treatment. Immunoblot studies, however, illustrated that the levels of total ERKs were not significantly affected by the CHL treatments, suggesting that the phytochemical retards the enzyme activation rather than its expression. Cyclin D1, but not its enzyme Cdk6, was also depleted after the CHL treatments; the depletions were associated with elevations of G0/G1 cells. Apoptosis occurred time-dependently with the ERK deactivations by 400 microg/ml CHL; the apoptotic cells elevated from 2.7-fold of the control level at 24 h, to 4.7-fold at 48 h and to 16.6-fold at 72 h of treatment. Bcl-2 was also depleted at 72 h when there was the most prominent elevation of the apoptotic cells, suggesting that it participates during the exacerbation rather than the initiation phases of the CHL-induced apoptosis. Results from this study support further research on CHL for preventing and treating those tumors with deregulated ERK activations.     

阿霉素诱导人乳腺癌细胞系耐药后的细胞行为变化

目的研究和完善乳腺癌细胞多药耐药机制及多药耐药后肿瘤细胞的性质、行为改变。方法分别对野生组(MCF-7)、阿霉素诱导的耐药组(MCF-7/ADM)、撤药60d组(MCF-7/撤药60d)进行细胞学分析,观察细胞增殖速度及群体增殖时间,SP免疫组化法检测细胞表型变化。结果MCF-7/ADM与MCF-7细胞增殖速度无明显差异,撤药组随着撤药时间的延长,细胞的增殖速度加快,群体倍增时间缩短;MCF-7/ADM、MCF-7/撤药60d细胞的分化程度低于MCF-7组;MCF-7/ADM的耐药相关标记蛋白Pgp、LRP、GST-π、HER-2表达较MCF-7明显增加,而雌激素受体(ER)、TOPO-Ⅱ转为阴性表达,孕激素受体(PR)随撤药时间的延长表达逐步减低。结论应用阿霉素诱导的MCF-7具有明显的耐药细胞性状,耐药细胞具有去分化的能力;耐药细胞的遗传和生化特性发生了变化,可用于肿瘤多药耐药机制的研究。     

黄芪多糖抑制人乳腺癌MCF－7细胞增殖和诱导凋亡

目的探讨中药黄芪多糖的体外抗人乳腺癌MCF．7细胞活性。方法实验分为空白对照组、黄芪多糖组和阳性对照组,黄芪多糖组MCF．7细胞给予不同浓度（2．5、5、10、20mg／L）的黄芪多糖,阳性对照组给予10gmol／L顺铂,空白对照组给予等体积培养基。48h后应用四甲基偶氮唑蓝（MTT）法测黄芪多糖对MCF-7细胞增殖抑制率,计算IC_50;吖啶橙（AO）,溴化乙啶（EB）荧光染色法测定黄芪多糖对MCF-7细胞诱导凋亡作用;应用流式细胞仪分析黄芪多糖对MCF-7细胞凋亡和细胞周期的影响。结果在给予2．5、5、10、20mg／L黄芪多糖48h后,黄芪多糖呈浓度依赖性抑制MCF．7细胞的增殖（r=0．985,P〈0．05）,抑制率分别为（4．14±2．96）％、（7．14±2．10）％、（20．13±2．33）％、（64．66±5．15）％,高于空白对照组0％,但4个不同浓度组的抑制率均低于阳性对照组（90．31±4．92）％。黄芪多糖48h的IC_50=16．83mg／L。随着黄芪多糖浓度的逐渐增高,MCF-7细胞中代表凋亡的玛瑙色逐渐增多,细胞核骤缩、核分裂,细胞形态呈现典型的凋亡特征。2．5、5、10、20mg／L黄芪多糖的凋亡率分别为（2．37±0．98）％、（6．76±1．31）％、（11．65±1．46）％、（20．75±2．68）％,高于空白对照组（1．14±1．25）％（均P〈0．05）,但均低于阳性对照组（35．09±2．88）％（均P〈0．05）。与空白对照组比较,黄芪多糖以浓度依赖性诱导MCF-7细胞凋亡（r=0．991,P〈0．05）,随浓度的增加,细胞凋亡率升高,但均低于阳性对照组。黄芪多糖随浓度的增加促使s期细胞比例逐渐升高,但低于空白和阳性对照组（均P〈0．05）,使处于G_0-G_1期细胞的比例逐渐减少,仍高于空白和阳性对照组（均P〈0．05）。结论黄芪多糖抑制人乳腺癌MCF-7细胞增殖并诱导其凋亡,使MCF-7细胞生长增殖停滞在S期?     

迁移诱导蛋白7促进胃癌血管生成拟态

目的:体外观察胃癌细胞株形成血管生成拟态(VM)的能力,并通过Mig-7-siRNA转染SGC7901细胞,观察其对VM形成的影响,初步探讨其可能机制.方法:采用体外三维培养技术,在光镜及扫描电镜下观察不同分化程度的胃癌细胞形成VM的能力,并检测迁移诱导蛋白7(Mig-7)表达;通过Mig-7-siRNA转染SGC7901细胞,观察其对VM、侵袭及迁移能力的影响;Western blot检测SGC7901细胞中Mig-7、磷酸化细胞外调节蛋白激酶1、2(p-ERK1/2)及基质金属蛋白酶2(MMP-2)表达.结果:体外实验显示MKN45和SGC7901能形成VM,表达Mig-7;而MKN28和GES-1无形成VM能力,Mig-7表达阴性;Mig-7-siRNA转染SGC7901细胞后可干扰VM形成、侵袭及迁移能力;下调p-ERK1/2和MMP-2蛋白表达,可干扰VM形成.结论:能形成VM的细胞,可表达Mig-7;干扰Mig-7的表达,抑制VM的形成,可能与下调p-ERK1/2和MMP-2蛋白表达有关.     

miR-145-5p的过表达对乳腺癌细胞凋亡作用的研究

目的 探讨miR-145-5p靶向调控激活转录因子1(ATF1)对人乳腺癌MCF7细胞凋亡的影响.方法 运用TargetScan在线分析miR-145-5p与ATF1的相关性;将ATF1的3'UTR构建进PMIR-RB-REPORT质粒,利用荧光素酶报告实验检测miR-145-5p是否靶向调控ATF1;用HiPerFe...     

乙型肝炎病毒X基因促TRAIL诱导Huh7细胞凋亡的机制

目的探讨乙型肝炎病毒(hepatitis B virus,HBV)X基因(HBX)促肿瘤坏死因子相关凋亡诱导配体(TNF relatedapoptosis inducing ligand,TRAIL)蛋白诱导人肝癌细胞Huh7凋亡的机制。方法应用脂质体转染法将pcDNA3.1-HBX、pcDNA3.1分别转染Huh7细胞,建立稳定表达HBX的细胞模型Huh7-HBX和空载体对照细胞模型Huh7-3.1;运用TRAIL蛋白分别作用Huh7-HBX细胞、Huh7-3.1及Huh7细胞后,采用CCK8、流式细胞术检测细胞增殖和凋亡情况;Western blot检测死亡受体4(deathreceptor 4,DR4)和死亡受体5(death receptor 5,DR5)的表达情况;分光光度法检测细胞caspase8和caspase3活性。结果 RT-PCR及Western blot证实成功构建细胞株Huh7-HBX;CCK8和流式细胞术检测结果均显示Huh7-HBX细胞的凋亡率明显高于Huh7-3.1及Huh7(P<0.05);Western blot显示Huh7-HBX细胞的DR4、DR5的表达量明显高于Huh7-3.1及Huh7细胞(P<0.05);caspase活性检测结果显示,Huh7-HBX细胞caspase8及caspase3的活性明显高于Huh7-3.1及Huh7(P<0.05)。结论 HBX能够通过上调Huh-7细胞表面死亡受体DR4、DR5的表达,进而促进TRAIL蛋白诱导的细胞凋亡,为进一步研究HBX的促凋亡机制提供实验依据。