基于微流控芯片的姜黄素诱导MCF-7细胞凋亡的研究

目的探讨应用微流控芯片实现高内涵药物筛选（high content screening,HCS）的可行性。方法本文将微流控芯片技术与HCS技术相结合,通过自行设计、制作聚二甲基硅氧烷（polydimelhylsiloxane,PDMS）-玻璃微流控芯片,并在芯片上实现人乳腺癌MCF-7细胞培养、脂质体转染、药物姜黄素刺激等操作,最后通过显微成像技术进行检测。结果姜黄素可以诱导MCF-7细胞凋亡,并呈浓度依赖性,同时获得了细胞在凋亡过程中一些生物信息的改变：随着姜黄素浓度的增加,细胞凋亡比例、EndoG—GFP重定位比例增大,膜通透性增加,细胞核固缩变小。结论上述微流控芯片可以为HCS技术提供良好的研究平台。     

全反式维甲酸对人乳腺癌MCF-7细胞凋亡的影响

目的:探讨全反式维甲酸(ATRA)对人乳腺癌MCF-7细胞凋亡的影响及作用途径.方法:在人乳腺癌细胞株MCF-7培养基中加入一定浓度ATRA和PKC-δ的专一抑制剂rottlerin(RO)并分组,通过SubG1assay by FACS、琼脂糖凝胶电泳检测基因组DNA ladder来观察ATRA对MCF-7的影响.结果:在浓度为5μM的ATRA作用下,MCF-7的凋亡率显著高于其它各组(P＜0.01),并可观察到明显梯状DNA.结论:ATRA能够诱导乳腺癌MCF-7细胞凋亡,但受PKC-δ的专一抑制剂RO的抑制.     

DMSO诱导MCF-7细胞凋亡的研究

目的研究二甲基亚砜(DMSO)对MCF-7细胞凋亡的诱导作用。方法用不同浓度DMSO处理体外培养的MCF-7细胞,应用倒置光显微镜观察细胞形态学变化,用MTT比色法检测细胞存活率;Hoechst33258/PI荧光染色,用荧光显微镜分析凋亡细胞比率;琼脂糖凝胶电泳检测DNA梯状条带。结果在倒置光学显微镜下观察1%DMSO处理细胞12h后细胞形态发生变化。约有50%以上的细胞变圆,细胞内有多泡小体形成。随DMSO浓度增加和作用时间的延长,细胞存活率明显下降,经MTT检测其IC_(50)值为1%;荧光显微镜下可见60%以上细胞核染色质凝集,核碎裂等凋亡细胞的形态学变化;琼脂糖凝胶电泳呈现梯状条带(DNA ladder)。结论适当浓度的DMSO能够抑制乳腺癌细胞增殖并诱导其凋亡。     

The Effects of X-Ray Irradiation on the Proliferation and Apoptosis of MCF-7 Breast Cancer Cells

Abstract

To investigate the effects of X-ray irradiation on the proliferation and apoptosis of MCF-7 breast cancer cells; MCF-7 breast cancer cells were irradiated with X-ray. After irradiation, morphological changes and growth inhibition rate of the irradiated cells were observed under an inverted microscope. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess the proliferation of the irradiated MCF-7 cells. Transmission electron microscope was used to observe the morphology and ultrastructure of the irradiated MCF-7 cells. Western blotting was used to analyze the expression level of apoptosis-related protein caspase-3. Our results showed, at 48?h after the irradiation (0?Gy and 8?Gy), cells oval in shape, cell shrinkage or swelling and partial formation of debris under inverted microscope; as well as cytoplasmic vacuolization or inspissation, increased electron density of cytoplasm, structural damage of organelles, blurred mitochondrial cristae and chromatin margination under transmission electron microscopy; the survival rate of MCF-7 cells in X-ray group was 17.3% lower than that in control group (0?Gy) (p?<?0.001); while caspase-3 expression increased evidently in X-ray group compared with control group (0?Gy) (p?<?0.05). In conclusion, X-ray irradiation can inhibit the proliferation of MCF-7 cells and induce apoptosis through increasing caspase-3 expression.     

蝎毒联合ADM对人乳腺癌MCF-7/细胞的凋亡诱导作用及其耐药逆转的研究

目的探讨蝎毒(BMK)联合阿霉素(ADM)对人乳腺癌MCF-7/ADM细胞的凋亡诱导及其耐药逆转作用。方法MTT法、荧光分光光度法、流式细胞术、紫外分光光度术和免疫细胞化学法。结果①非细胞毒性剂量(3.0μg/m l)蝎毒能显著降低MCF-7/ADM的IC50(P<0.01),增加MCF-7/ADM细胞内ADM的药物积累(P<0.01)。②蝎毒(3.0μg/m l)联合ADM后增强了对耐药细胞的凋亡诱导作用,凋亡率由8.9±0.01%上升为12.6±0.21%(P<0.01)。③蝎毒(3.0μg/m l)能够显著降低耐药细胞内谷胱甘肽S转移酶(GSTs)酶的活性(P<0.01)。结论蝎毒能够部分逆转人乳腺癌MCF-7/ADM细胞对阿霉素的耐药性,其逆转机制与抑制耐药细胞GSTs活性有关。     

Apoptosis Inducing Effect of Andrographolide on TD-47 Human Breast Cancer Cell Line

Andrographolide isolated from Andrographis paniculata Ness (Acanthaceae) at 0.35 mM, 0.70 mM and 1.40 mM induced DNA fragmentation and increased the percentage of apoptotic cells when TD-47 human breast cancer cell line was treated for 24, 48 and 72 h. The results demonstrated that andrographolide can induce apoptosis in TD-47 human breast cancer cell line in a time and concentration-dependent manner by increase expression of p53, bax, caspase-3 and decrease expression of bcl-2 determined by immunohistochemical analysis.     

Molecular Mechanism of SAHA on Regulation of Autophagic Cell Death in Tamoxifen-Resistant MCF-7 Breast Cancer Cells

Objective: Tamoxifen is currently used for the treatment of estrogen receptor-positive breast cancer patients, but acquired resistance to tamoxifen is a critical problem in breast cancer therapy. Suberoylanilide hydroxamic acid (SAHA) is a prototype of the newly developed HDAC inhibitor. The aim of this study is to investigate the anticancer effects of SAHA in tamoxifen-resistant MCF-7 (TAMR/MCF-7) cells.Methods: Cytotoxicity, apoptosis and autophagic cell death induced by SAHA were studied. A TAMR/MCF-7 cells xenograft model was established to investigate the inhibitory effect of SAHA on tumor growth in vivo.Results: SAHA inhibited the proliferation of TAMR/MCF-7 cells in a dose-dependent manner. SAHA significantly reduced the expression of HDAC1, 2, 3, 4 and 7 and increased acetylated histone H3 and H4. Although SAHA induced G2/M phase arrest of cell cycle, apoptotic cell death was very low, which is correlated with the slight change in the activation of caspases and PARP cleavage. Interestingly, expression of the autophagic cell death markers, LC3-II and beclin-1, was significantly increased in TAMR/MCF-7 cells treated with SAHA. Autophagic cell death induced by SAHA was confirmed by acridine orange staining and transmission electron microscopy (TEM) in TAMR/MCF-7 cells. In mice bearing the TAMR/MCF-7 cell xenografts, SAHA significantly reduced the tumor growth and weight, without apparent side effects.Conclusion: These results suggest that SAHA can induce caspase-independent autophagic cell death rather than apoptotic cell death in TAMR/MCF-7 cells. SAHA-mediated autophagic cell death is a promising new strategy to treatment of tamoxifen-resistant human breast cancer.     

硼替佐米和3-甲基腺嘌呤对人乳腺癌细胞株MCF-7的抑瘤效应

目的:研究蛋白酶体活性抑制剂硼替佐米(Bortezomib)对人乳腺癌细胞株MCF-7的自噬诱导和增殖抑制作用,以及自噬抑制剂3-甲基腺嘌呤(3-MA)与Bortezomib联合应用对MCF-7细胞自噬及增殖的影响。方法:采用MTT法检测细胞增殖,蛋白质印迹法检测蛋白酶体活性及自噬相关蛋白LC3的表达。结果:Bortezomib能明显抑制MCF-7细胞增殖并诱导其自噬,3-MA与Bortezomib联合应用,可逆转Bortezomib诱导的细胞自噬,并增强Bortezomib对MCF-7细胞增殖的抑制(P<0.05)。结论:联合应用Bortezomib和3-MA可能对乳腺癌细胞自噬和增殖具有协同抑制作用。     

硒多糖对MCF-7乳腺癌小鼠的抗肿瘤作用研究

目的 探究硒多糖对MCF-7乳腺癌小鼠脾指数、胸腺指数、脾淋巴细胞转化率及SBP-1表达的影响,分析硒多糖对乳腺癌小鼠的抑瘤效果及作用机制。方法 将40只小鼠随机分为4组,每组10只,其中1组作为正常组,其余3组将MCF-7乳腺癌细胞悬液注射于小鼠左侧膈腧穴处建立乳腺癌模型,证明成瘤后予以给药,模型组（予等剂量的生理盐水）,硒多糖用药组（400 mg/kg硒多糖）、联合用药组（50 mg/kg环磷酰胺＋400 mg/kg硒多糖）,连续给药14 d。脱颈处死小鼠,测胸腺指数、脾指数,无菌条件下取脾脏研磨进行体外淋巴细胞培养,检测PHA诱导下的小鼠脾淋巴细胞转化率并应用免疫组化技术检测肿瘤组织中SBP-1的表达情况。结果 与正常组和模型组相比较硒多糖用药组、联合用药组的胸腺指数、脾指数及淋巴细胞转化率明显提高,差异有统计学意义（P＜0.05）;与硒多糖用药组比较联合用药组的胸腺指数、脾指数及淋巴细胞转化率均有所提高,差异有统计学意义（P＜0.05）;与模型组相比硒多糖用药组及联合用药组的SBP-1蛋白表达均有所提高,差异有统计学意义（P＜0.05）;与硒多糖用药组相比联合用药组的SBP-1蛋白表达有所增强,差异有统计学意义（P＜0.05）。结论 硒多糖可以通过改善MCF-7乳腺癌小鼠的免疫功能及增强SBP-1的表达来起到抗肿瘤作用,与环磷酰胺联合用药作用更显著。     

Nutrient Starvation Sensitizes Human Ovarian Cancer SKOV3 Cells to BH3 Mimetic via Modulation of Mitochondrial Dynamics

The aberrant proliferation of tumor cells necessitates compensatory changes in tumor metabolic processes. Previous studies on tumor growth and metabolism have established a relationship between nutrient stress and Bcl‐2 anti‐apoptotic proteins, although the mechanisms connecting these processes remain unclear. We induced nutrient deprivation in human ovarian cancer SKOV3 cells by culturing cells in Earle's balanced salt solution (EBSS) as a starvation model. We used EBSS treatment with the BH3 domain of Bcl‐2 family proteins (BH3) mimetic ABT737, which targets Bcl‐2/Bcl‐xL, to examine mitochondrial dynamics and the interactive regulatory mechanisms between nutrition and Bcl‐2 proteins. We found that EBSS combined with ABT737 can promote SKOV3 cells to undergo apoptosis and convert tubular mitochondria into small, fragmented morphologies. Bcl‐2 family proteins participated in the regulation of mitochondrial fusion and fission through apoptosis, and the decrease of Mcl‐1 expression was the key to ABT737 sensitization. Our findings showed that nutrient stress could sensitize SKOV3 cells to ABT737 via regulation of the mitochondrial dynamic balance and interaction of Bcl‐2 family proteins. Our data suggest that nutrient starvation combined with the BH3 mimetic ABT737 could reduce the required effective dose of ABT737, and that inhibition of Bcl‐2 and Mcl‐1 together with nutrient starvation could serve as an effective strategy for the treatment of human ovarian cancer. Anat Rec, 300:326–339, 2017. © 2016 Wiley Periodicals, Inc.     

Apoptosis Induction of Centella Asiatica on Human Breast Cancer Cells

The present study evaluated the ability of methanolic extract of Centella asiatica (Linn) Urban (Umbelliferae) to induce apoptosis in different cancer cell lines. MCF-7 cells emerged as the most sensitive cell line for in vitro growth inhibitory activity. C. asiatica extract induced apoptosis in MCF-7 cells as indicated by nuclear condensation, increased annexin staining, loss of mitochondrial membrane potential and induction of DNA breaks identified by TUNEL reactivity. It is possible that the use of C. asiatica extract as a component in herbal medicines could be justifiable.     

The Ultrastructure of MCF-7 Breast Cancer Cells after Vasodilator-Stimulated Phosphoprotein Knockdown

Inhibition of vasodilator-stimulated phosphoprotein (VASP) expression could modulate the adhesion and proliferation of breast cancer cells. However, the underlying mechanisms are not well defined. Here, we show that knockdown of the VASP changes the ultrastructure of human MCF-7 breast cancer cells. Transfection of VASP shRNA significantly lowered the expression of VASP protein in MCF-7 cells. In the shRNA-VASP group, immunofluorescence showed diminished presence of F-actin, and it was lower in the nucleus than in the cytoplasm. After VASP was inhibited, the MCF-7 cells were oval in shape with blunt lamellipodium, disappearance of the cristae of mitochondria, decreased microvilli and more vacuoles. Collectively, our findings elucidated the morphological mechanism that knockdown of the VASP changed the ultrastructure of MCF-7 cells.     

BCL—2基因表达与乳腺癌抗凋亡及免疫耐受的关系

目的 观察乳腺癌细胞株ＢＣＬ－２基因表达与其耐受肿瘤浸润淋巴细胞（ＴＩＬ）免疫攻击的关系,探索应用ＢＣＬ－２硫代反义寡核苷酸（ＢＣＬ－２ＳＯＮ）治疗乳腺癌的途径。方法 ＢＣＬ－２硫代反义寡核苷酸处理乳腺癌细胞株ＭＣＦ－７,Ｗｅｓｔｅｒｎ印迹法观察其对ＢＣＬ－２表达的抑制;并将ＭＣＦ－７细胞与乳腺标本分离出来的ＴＩＬ共同培养,通过ＪＡＭ试验观察ＴＩＬ诱导ＭＣＦ－７细胞凋亡的情况。结果 ＢＣＬ－２ＳＯＮ处理的ＭＣＦ－７细胞无ＢＣＬ－２表达,并在ＪＡＭ试验中随着ＴＩＬ浓度增加,凋亡细胞也增加,未经处理的对照组可见ＢＣＬ－２表达,ＪＡＭ试验中随着ＴＩＬ浓度增加,凋亡细胞的量无明显变化。结论 ＢＣＬ－２表达的乳腺癌细胞可对抗肿瘤浸润淋巴细胞的免疫攻击,ＢＣＬ－２ＳＯＮ可作为治疗乳腺癌的新途径。     

BCL-2基因表达与乳腺癌抗凋亡及免疫耐受的关系

目的观察乳腺癌细胞株BCL-2基因表达与其耐受肿瘤浸润淋巴细胞(TIL)免疫攻击的关系,探索应用BCL-2硫代反义寡核苷酸(BCL-2SON)治疗乳腺癌的途径.方法BCL-2硫代反义寡核苷酸处理乳腺癌细胞株MCF-7,Western印迹法观察其对BCL-2表达的抑制;并将MCF-7细胞与乳腺癌标本分离出来的TIL共同培养,通过JAM试验观察TIL诱导MCF-7细胞凋亡的情况.结果BCL-2SON处理的MCF-7细胞无BCL-2表达,并在JAM试验中随着TIL浓度增加,凋亡细胞也增加;未经处理的对照组可见BCL-2表达,JAM试验中随着TIL浓度增加,凋亡细胞的量无明显变化.结论BCL-2表达的乳腺癌细胞可对抗肿瘤浸润淋巴细胞的免疫攻击,BCL-2SON可作为治疗乳腺癌的新途径.     

雄黄诱导MCF-7/ADM细胞凋亡及逆转其耐药性的研究

目的探讨雄黄诱导人乳腺癌MCF-7／ADM细胞凋亡及逆转其耐药性的调节机制。方法经MTT法探讨雄黄对人乳腺癌MCF-7／ADM细胞药物敏感性的影响;用透射电镜观察凋亡细胞形态改变;应用流式细胞术,探讨凋亡抑制基因bcl-2的编码蛋白Bcl-2的改变及凋亡百分率的改变;通过荧光分光光度法观察雄黄对细胞内化疗药物阿霉素积累的影响。结果雄黄对MCF-7／ADM的耐药性有明显的逆转作用,无毒剂量（15mg,L）及低毒剂量（25mg,L）雄黄作用后,逆转倍数分别为2．3倍及2．8倍;出现凋亡细胞,凋亡百分率由0．6％分别增至2．0％（P〈0．05）及3．4％（P〈0．01）;Bcl-2表达由90．2％分别降至63．6％（P〈0．01）及52．7％（P〈0．01）;MCF-7／ADM的细胞内阿霉素浓度明显增加（P〈0．01）。结论雄黄通过降低Bcl-2表达,促进细胞凋亡,增加耐药细胞内阿霉素积累量等耐药机制的调节,部分逆转了MCF-7／ADM细胞对阿霉素的耐药性。     

Inhibition of JNK3 Promotes Apoptosis Induced by BH3 Mimetic S1 in Chemoresistant Human Ovarian Cancer Cells

Previous studies have suggested that the novel BH3 mimetic S1 could induce apoptosis in diverse tumor cell lines through endoplasmic reticulum (ER) stress or mitochondrial cell death pathways. The activation of c‐Jun N‐terminal kinase (JNK) through inositol requiring enzyme‐1 (IRE1) is closely connected to ER stress‐induced apoptosis. However, the role of JNK is complex, as there are different JNK subtypes and the function of each subtype is still not entirely clear. Here we found that the mRNA expression of JNK3 was continuously high in S1‐treated human ovarian cancer SKOV3/DDP cells using a human unfolded protein response (UPR) pathway PCR array. Pharmacological inhibition of JNK3 increased cell sensitivity to apoptosis induced by S1. Furthermore, inhibition of JNK3 induced accumulation of both acidic compartment and p62, and upregulated ROS production. Our results suggest that JNK3 plays a pro‐survival role during ER stress through preventing the block of autophagic flux and reducing oxidative stress in SKOV3/DDP cells. Inhibition of JNK3 may be a potential method to enhance the killing effect of the Bcl‐2 inhibitor S1. Anat Rec, 298:386–395, 2015. © 2014 Wiley Periodicals, Inc.     

A Study on the Inhibitory Effect of Polysaccharides from Radix Ranunculus Ternati on Human Breast Cancer MCF-7 Cell Lines

The objective of this paper was to study the in vitro anti-breast cancer activity of polysaccharides from Radix ranunculus ternati. Different concentrations of polysaccharide extracts were selected, and MTT assay and flow cytometry (FCM) were used to investigate their growth-inhibitory and apoptosis-inducing effects on human breast cancer MCF-7 cell lines. Radix ranunculus ternati polysaccharides had varying degrees of effects on the growth of human breast cancer MCF-7 cell lines, and the differences were significant compared with the blank control group. FCM showed that the polysaccharides can induce apoptosis. In addition, it can also enhance NK cell activity. Radix ranunculus ternati polysaccharides have a relatively good in-vitro anti-breast cancer activity.     

Effect of Norcantharidin on the Human Breast Cancer Bcap-37 Cells

Norcantharidin (NCTD), a chemically modified form of cantharidin, is a potential anticancer drug. In this study, the effects of NCTD on the cellular viability, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and DNA damage in the human breast cancer cell line Bcap-37 were investigated with confocal and fluorescence microscopy. The cell cycle was further analyzed using the CellQuest software of a Becton-Dickinson FACS flow cytometer. The results indicated that the cellular viability was decreased with the growing concentrations of NCTD and time exposure. Moreover, the fluorescence intensity of ROS was increased, whereas the MMP was decreased in Bcap-37 cells with the growing concentrations of NCTD. NCTD induced a dose-dependent DNA damage and reduced the G1 peak in Bcap-37 cells. The G2/M peak of Bcap-37 was also decreased by the higher concentration of NCTD.     

GM-CSF对TNF-α诱导白血病细胞凋亡的抑制作用

通过TNFα以及TNFα+GM-CSF对18例白血病患者髓性白血病细胞凋亡的影响研究,发现加TNFα组凋亡细胞数明显高于TNFα+GM-CSF组,两者比较有显著性差异（P<0.01）。本结果提示TNFα有捉进肿瘤细胞凋亡作用,而 GM-CSF则可抑制这种作用。     

Inhibition Effects of Scorpion Venom Extracts (Buthus Matensii Karsch) on the Growth of Human Breast Cancer MCF-7 Cells

Background

To observe the inhibition effects of the Buthus matensii Karsch (BmK) scorpion venom extracts on the growth of human breast cancer MCF-7 cells, and to explore its mechanisms.

Methods

Two common tumor cells (SMMC7721, MCF-7) were examined for the one which wasmore sensitivity to scorpion venom by MTT method. Cell cycle was determined by flow cytometry. Immunocytochemistry was applied to detect apoptosis-related protein Caspase-3 and Bcl-2 levels, while the expression of cell cycle-related protein Cyclin D1 was shown by Western blotting.

Results

Our data indicated that MCF-7 was the more sensitive cell line to scorpion venom. The extracts of scorpion venom could inhibit the growth and proliferation of MCF-7 cells. Furthermore, the extract of scorpion venom induced apoptosis through Caspase-3 up-regulation while Bcl-2 down-regulation in MCF-7 cells. In addition, the extracts of scorpion venom blocked the cells from G₀/G₁ phase to S phase and decreased cell cycle-related protein Cyclin D1 level after drug intervention compared with the negative control group.

Conclusions

These results showed that the BmK scorpion venom extracts could inhibit the growth of MCF-7 cells by inducing apoptosis and blocking cell cycle in G₀/G₁ phase. The BmK scorpion venom extracts will be very valuable for the treatment of breast cancer.