利用献血者捐献血液中白细胞制备的异体CIK细胞与肿瘤靶细胞体外杀伤试验研究

目的:探讨利用献血者捐献血液中的白细胞制备的异体细胞因子诱导的杀伤(CIK)细胞与肿瘤靶细胞体外杀伤试验情况。方法:重悬献血者CIK细胞与肝癌细胞、人乳腺癌细胞、白血病细胞、B淋巴瘤细胞浓度,以CIK细胞为效应细胞,肿瘤细胞为靶细胞,使效靶比为32∶1、16∶1、8∶1、4∶1、2∶1、1∶1时共培养48h后检测450nm下的吸收度A值。结果:献血者异体CIK细胞对肝癌、乳腺癌、白血病、B淋巴瘤细胞4种肿瘤靶细胞的杀伤率随着效应细胞数的增加而增大,当效靶比为32∶1时,献血者异体CIK细胞的杀伤率分别为86.1%、84.3%、62.5%、87.9%;献血者异体CIK细胞对肝癌、乳腺癌、B淋巴瘤细胞杀伤活性明显强于白血病细胞,同一效靶比时,CIK细胞对肝癌细胞杀伤活性较高,对白血病细胞杀伤活性较差。结论:献血者CIK细胞对肝癌、人乳腺癌、白血病、B淋巴瘤细胞等肿瘤细胞的生长均有明显的抑制作用。4种肿瘤靶细胞的杀伤率随着效应细胞数的增加而增大,同一效靶比时,CIK细胞对肝癌细胞杀伤活性较高,对白血病细胞杀伤活性较差。     

白藜芦醇对人乳腺癌骨高转移细胞株MDA-MB-231BO生长抑制和凋亡的影响

目的观察白藜芦醇（Res）对人乳腺癌骨高转移细胞株MDA-MB-231BO生长抑制和凋亡的影响,探讨其在抑制乳腺癌骨转移中的应用价值。方法将0、12.5、50、100、200μmo/L的Res分别与MDA-MB-231BO细胞作用24、48、72 h;用CCK-8法检测MDA-MB-231BO细胞的生长抑制率,流式细胞仪分析细胞周期并检测细胞凋亡率,荧光显微镜观察细胞形态。结果随Res浓度的增加及作用时间的延长,MDA-MB-231BO细胞的生长抑制率逐渐升高,S期细胞逐渐增多。Res浓度为200μmol/L、作用48 h,MDA-MB-231BO生长抑制率为82.17%±5.37%、早期凋亡率为4.48%±1.23%、晚期凋亡率为9.48%±2.30%,荧光显微镜下观察用药组随着Res浓度的增加,凋亡细胞逐渐增多。结论Res能抑制人乳腺癌骨高转移细胞株MDA-MB-231BO细胞增殖,使MDA-MB-231BO细胞阻滞于S期,并可诱导该细胞凋亡。     

脂筏结构蛋白1基因在乳腺癌细胞中的表达及靶向抑制其表达对癌细胞凋亡的诱导

目的探讨脂筏结构蛋白(FLOT)1基因在乳腺癌细胞中的表达及靶向抑制其表达对癌细胞凋亡的影响及机制。方法 Western印迹法检测正常乳腺上皮细胞MCF-10A及MCF7、MDA-MB-231和HCC1569乳腺癌细胞FLOT1的蛋白表达。将设计合成的针对FLOT1的特异性siRNA序列(si-FLOT1组)及阴性对照siRNA序列(NC组)转染至MDA-MB-231细胞,仅仅加入脂质体的为空白对照组,AG490作为信号转导与转录因子(STAT)3信号通路抑制剂,转染48 h, Western印迹法检测FLOT1、磷酸化蛋白酪氨酸激酶(p-JAK)2、p-STAT3、细胞增殖核抗原(PCNA)和B细胞淋巴瘤(Bcl)-2的蛋白表达。流式细胞术检测细胞凋亡率。结果 FLOT1在MCF7、MDA-MB-231和HCC1569乳腺癌细胞中的蛋白表达均显著高于正常乳腺上皮细胞MCF-10A(P0.05)。与空白对照组比较,si-FLOT1组FLOT1表达受到抑制(P0.05);与NC组比较,si-FLOT1组细胞凋亡率显著升高(P0.05),p-JAK2、p-STAT3、PCNA和Bcl-2的蛋白表达显著下调(P0.05)。与si-FLOT1组比较,si-FLOT+AG490组细胞凋亡率显著升高(P0.05)。结论 FLOT1在乳腺癌细胞中呈高表达,通过RNA干扰抑制其表达可诱导癌细胞凋亡,机制与下调STAT3信号通路有关。     

6-姜辣素对人乳腺癌细胞增殖、凋亡的影响及机制

目的探究6-姜辣素对人乳腺癌MCF-7和MDA-MB-231细胞增殖、凋亡的影响,并探讨其分子机制。方法不同浓度6-姜辣素处理人乳腺癌MCF-7和MDA-MB-231细胞,使用CCK-8法检测6-姜辣素对乳腺癌细胞增殖能力的影响;应用流式细胞仪检测6-姜辣素对乳腺癌凋亡的影响;6-姜辣素处理人乳腺癌细胞,应用Western印迹法检测促凋亡蛋白(Bad)、免抗人单克隆抗体(Bax)、B淋巴细胞瘤(Bcl)-2、磷酸化腺苷酸活化蛋白激酶(p-AMPK)和磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)蛋白水平。结果 6-姜辣素抑制乳腺癌MCF-7和MDA-MB-231细胞增殖能力,并呈浓度依赖性和时间依赖性;流式细胞仪检测细胞凋亡率,提示6-姜辣素促进乳腺癌细胞凋亡发生;6-姜辣素处理乳腺癌细胞,促进Bad和Bax表达,而抑制Bcl-2表达;同时,6-姜辣素促进p-AMPK的表达,抑制p-mTOR的表达。结论 6-姜辣素通过激活AMPK/mTOR信号通路,抑制乳腺癌细胞增殖并促进其凋亡,发挥抗肿瘤功能。     

CIK联合紫杉醇对胃癌细胞株SGC-7901的杀伤作用观察

目的观察由细胞因子诱导的杀伤细胞（CIK）联合紫杉醇对胃癌细胞株SGC-7901的杀伤作用。方法将健康人外周血单核细胞（PBMC）在体外诱导形成CIK。取SGC-7901,分别加入CIK、紫杉醇、CIK＋紫杉醇培养,采用MTT法分别检测各组细胞的杀伤率。结果5μg/ml的紫杉醇作用于SGC-7901细胞4 h后,分别加入效靶比为10∶1和20∶1的CIK细胞作用24 h,SGC-7901杀伤效率明显高于单用紫杉醇及单用CIK时的杀伤率,P均〈0.01。结论CIK联合紫杉醇对胃癌细胞株SGC-7901的杀伤作用明显增强。     

过表达miR-769-5p对乳腺癌细胞增殖、凋亡、侵袭及迁移能力的影响和机制

目的 探讨乳腺癌细胞中miR-769-5p的表达变化及其过表达对乳腺癌细胞增殖、凋亡、侵袭能力、迁移能力的影响及机制.方法 通过qRT-PCR法检测乳腺癌细胞MCF-7、MDA-MB-231、SK-BR-3、HCC1806和正常乳腺上皮细胞HBL-100中miR-769-5p的表达.通过脂质体转染技术对HCC1806转...     

中药扶正祛瘤对乳腺癌干细胞MDA-MB-231增殖、凋亡的影响及其机制的研究

目的探究中药扶正祛瘤对乳腺癌干细胞MDA-MB-231抑制增殖、凋亡的影响。方法体外富集培养乳腺癌干细胞MDA-MB-231,饲养小鼠并灌输中药获得含药血清;实验分为对照组、等药量组、1倍药量组、2倍药量组,MTT法检测每组乳腺癌干细胞MDA-MB-231细胞抑制率;在最适药物浓度条件下连续培养MDA-MB-23细胞5 d,MTT法测定每天细胞的抑制率,绘制乳腺癌干细胞MDA-MB-231抑制增长曲线;在最佳药物浓度及培养时间下培养细胞,用流式细胞仪检测乳腺癌干细胞MDA-MB-231的凋亡情况;q RT-PCR法检测中药扶正祛瘤处理后细胞MDA-MB-231中mi R-34a及其靶基因SIRT1的基因表达。结果含药血清对乳腺癌干细胞MDA-MB-231有不同程度的抑制,抑制率:2倍药量组1倍药量组等量药量组对照组;采用2倍药量的小鼠血清连续培养细胞,1～4 d内随处理时间增长细胞抑制率增加,第5天有下降趋势;含药血清处理后的细胞内mi R-34a基因表达量高于对照组,SIRT1基因表达量低于对照组;2倍药量的中药扶正祛瘤血清作用乳腺癌干细胞MDA-MB-231 4 d后与对照组相比出现大量的凋亡。结论中药扶正祛瘤能够效抑制乳腺癌干细胞MDA-MB-231的增殖,可能通过调控mi R-34a靶基因SIRT1的表达而抑制癌细胞的增殖凋亡。     

树突状细胞与细胞因子诱导的杀伤细胞共培养对白血病耐药细胞杀伤作用的实验研究

目的探讨负载P-糖蛋白(P-gp)高表达的多药耐药(MDR)白血病K562/A02细胞冻融抗原的树突状细胞(DC)与同源细胞因子诱导的杀伤细胞(CIK)共培养对MDRK562/A02杀伤作用的影响。方法提取健康人骨髓单个核细胞,常规诱导出DC及CIK,将K562/A02细胞冻融物作为抗原冲击的DC,与CIK共培养作为实验组,抗原不冲击的DC与CIK共培养作为对照组,以CIK及DC单独培养分别作为空白对照组1和空白对照组2。光镜下观察细胞形态,流式细胞术分析细胞表型,MTT法检测杀伤活性。结果实验组、对照组细胞增殖活性均大于CIK组(P<0.05)。实验组对K562/A02、K562的杀伤活性在效靶比5∶1、10∶1、20∶1时分别为(42.90±0.67)%、(49.85±0.28)%、(63.36±0.46)%和(23.56±0.43)%、(26.11±0.34)%、(34.46±0.35)%,均高于对照组及空白对照组1(P<0.05);实验组对K562/A02的杀伤活性高于K562和MCF7(P<0.05)。结论DC与CIK共培养物是一种增殖活性和细胞毒活性高于CIK的免疫活性细胞,而经冻融抗原冲击的DC与CIK共培养能明显提高对MDRK562/A02的杀伤活性。     

骨髓瘤独特型抗原负载树突细胞介导的抗肿瘤效应研究

目的:研究骨髓瘤独特型抗原(Idiotype,Id)负载树突细胞(DC)对同源细胞因子诱导的杀伤细胞(CIK)体外抗瘤活性的影响。方法:采集健康供者外周血单个核细胞(PBMNC)用常规方法诱导DC和CIK细胞,将骨髓瘤OPM-2细胞培养上清提取的Id冲击或未冲击的DC与CIK细胞共培养(CIK、DC加CIK、Id-DC加CIK),用流式细胞术分析细胞表型,MTT法检测体外效应细胞杀伤活性。结果:在(5～20):1效靶比范围内, CIK细胞对OPM-2和K562细胞的杀伤率分别为(24．47±3．00)％～(40．64±1．62)％和(23．36±1．51)％～(42．52±2．06)％。DC加CIK及Id—DC加CIK对OPM-2和K562细胞的杀伤活性均高于CIK组,差异有统计学意义(P<0．05);而在相同效靶比之下,Id-DC加CIK对OPM-2细胞的杀伤活性最强,差异有统计学意义(P <0．05)。结论:CIK细胞对骨髓瘤细胞有强的杀伤活性,经Id负载的DC与CIK细胞共培养能进一步增强其特异性杀伤活性,对骨髓瘤可能有免疫治疗作用。     

CIK细胞对结肠癌SW620和LOVO细胞株的杀伤作用观察

目的观察细胞因子诱导的杀伤细胞（C IK细胞）对结肠癌SW 620和LOVO细胞株的杀伤作用。方法无菌采集健康人和结肠癌患者外周血,诱导制备C IK细胞。采用流式细胞术检测两者细胞表型。将健康人、结肠癌患者来源的C IK细胞分别以20∶1、10∶1的效靶比作用于结肠癌SW 620、LOVO细胞株,测算杀伤活性。结果用健康人来源的C IK细胞处理SW 620细胞,效靶比为20∶1时的杀伤活性为80.86%±6.08%,10∶1时为78.00%±7.63%;处理LOVO细胞,效靶比为20∶1时的杀伤活性为86.13%±6.97%,10∶1时为82.15%±6.60%。用结肠癌患者来源的C IK细胞处理SW 620细胞,效靶比为20∶1时的杀伤活性为63.36%±5.26%,10∶1时为65.35%±6.28%;处理LOVO细胞,效靶比为20∶1时的杀伤活性为60.33%±4.09%,10∶1时为55.16%±5.82%。相同靶细胞和相同效靶比下健康人来源的C IK细胞的杀伤活性明显高于结肠癌来源的C IK细胞（P均〈0.05）。结论健康人、结肠癌患者来源的C IK细胞对结肠癌SW 620、LOVO细胞均有杀伤作用。健康人来源的C IK细胞对结肠癌SW 620、LOVO细胞株的杀伤作用更强。     

Structural Consideration in Designing Organotin Polyethers to Arrest the Growth of Breast Cancer Cells In Vitro

The ability to inhibit cancer is inherent in organotin materials yet the structural relationships that regulate/direct this activity remains unknown. We measured antitumor activity using a matched pair of cell lines MDA-MB-231 cells that are estrogen-independent, estrogen receptor negative and MCF-7 cells, a cell line that is estrogen receptor (ER) positive. Those polyethers that contained a O-phenyl unit were able to significantly inhibit the non-estrogen sensitive cell line but were much less effective against the estrogen sensitive cell line; that is, the human breast cancer cell line MDA-MB-231 showed better test results for polymers derived from diols containing the O-phenyl moiety than the breast cancer cell line MCF-7, a well-characterized estrogen receptor positive control cell line. Those polyethers that did not contain the O-phenyl unit inhibited both cell lines approximately the same. The differential activity of the O-phenyl-containing polyethers is likely due to the estrogen-sensitive cells combining with some of the organotin polyethers minimizing their ability to inhibit cell growth.     

Effect of brefeldin A on membrane localization of MUC1 mucin and adhesive properties of cancer cells

Transmembrane glycoproteins play a significant role in cancer cells adhesion and metastatic process, just for that reason the glycosylation inhibitors are used to change the glycan structure and in this way the membrane expression of glycoproteins. The inhibitory effect of brefeldin A (BFA) on the expression of some glycoproteins: MUC1 mucin and alpha2beta1 integrin on cell surface of breast (MCF-7 and MDA-MB-231 lines) and endometrial (Ishikawa line) cancer cells was evaluated in our study. In MCF-7 and MDA-MB-231 cells, a decrease in MUC1 expression depended on brefeldin A concentration and equaled about 40% in cells treated with 1mg% of drug. In Ishikawa cells, a decrease in MUC1 expression was lower and amounted to about 25%. The expression of alpha2beta1 integrin was greatly inhibited in brefeldin-treated MCF-7 and Ishikawa cells, though it was unchanged in MDA-MB-231 cells. A decrease in MUC1 mucin and alpha2beta1 integrin level reduced the adhesive properties of BFA-treated cells. Adhesion to type I collagen was greatly diminished in BFA-treated MCF-7 and Ishikawa cells (above 70%), and to a lesser degree in MDA-MB-231 cells (about 50%); which was mainly caused by the inhibited integrin expression. These findings have proved that brefeldin A, by changing the surface glycoproteins level, can alter carcinoma cells adhesion to extracellular matrix proteins.     

Comparison of mammosphere formation from breast cancer cell lines and primary breast tumors

Background

Breast cancer stem cells (BCSCs) can be enriched by culturing of cells in non-adherent non-differentiating conditions. However, culturing mammospheres from primary breast tumors are costly and difficult to control. In order to overcome problems associated with using primary human tissues, continuous breast cancer cell lines have been developed from various sources.

Methods

In this study, a luminal subtype breast cancer cell line MCF-7 and a basal subtype cell line MDA-MB-231 were chosen. We explored the optimal culturing system for BCSCs from the two cell lines and primary breast tumors. Then, mammosphere formation efficiency (MFE), CD44⁺/CD24^–/lowESA⁺Lin^– cell proportion in mammospheres, and tumorigenecity of mammospheres generated from the two breast cancer cell lines and primary breast tumors were compared.

Results

Enzymatic digestion of 60 mins and the addition of B27 to the culture medium were optimal for mammosphere culturing. Mammospheres could be formed in all the three cells, in which MCF-7 had the highest MFE. After 3 weeks culture, CD44⁺/CD24^–/lowESA⁺Lin^– cell proportion in mammospheres from MCF-7, MDA-MB-231 cells and primary breast tumors was 95.0%±2.5%, 82%±22% and 21.5%±1.0%, respectively. A total of 1,000 cells from MCF-7, MDA-MB-231 mammospheres but not primary mammospheres were tumorigenic.

Conclusions

This study validates the use of breast cancer cell lines as models to elucidate the nature of BCSCs.     

P2Y2 receptor-mediated modulation of estrogen-induced proliferation of breast cancer cells

It is known that estrogen promotes the proliferation of breast cancer cells. Agonists to P2Y(2) receptors promote or suppress proliferation in different cancers. In the present study, the methods of methylthiazoltetrazolium (MTT) assay, real-time RT-PCR, Western blot and fluorescent calcium imaging analysis were used to investigate whether P2Y(2) receptors play a role in the effects of estrogen on the breast cancer cell lines, MCF-7 and MDA-MB-231. We found that P2Y(2) receptors were expressed in both the estrogen receptor alpha (ER(α))-positive breast cancer cell line MCF-7 and the ER(α)-negative breast cancer cell line MDA-MB-231. 17β-Estradiol (17β-E(2)) (1 pM to 1000 nM) promoted proliferation of MCF-7 cells, which was blocked by the ER antagonist ICI 182,780 (1 μM) and the ER(α) antagonist methyl-piperidino-pyrazole (MPP, 50 μM), but not by the ER(β) antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 50 μM) or ER(β) small interfering RNA. The P2Y(2) and P2Y(4) receptor agonist UTP (10-100 μM) suppressed the viability of breast cancer cells in both MCF-7 and MDA-MB-231 cells. The effect was blocked by suramin (10-100 μM), known to be an effective antagonist against P2Y(2), but not P2Y(4), receptor-mediated responses. 17β-E(2) played a more positive role in promoting proliferation in MCF-7 cells when suramin blocked the functional P2Y(2) receptors. 17β-E(2) (0.1-1000 nM) downregulated the expression of P2Y(2) receptors in terms of both mRNA and protein levels in MCF-7 cells. The effect was blocked by ICI 182,780 and MPP, but not PHTPP or ER(β) small interfering RNA. 17β-E(2) did not affect the expression of P2Y(2) receptors in MDA-MB-231. UTP (10-100 μM) led to a sharp increase in intracellular Ca(2+) in MCF-7 cells. Pre-incubation with 17β-E(2) (0.1 μM) attenuated UTP-induced [Ca(2+)](i), which was blocked by ICI182,780 and MPP, but not PHTPP. It is suggested that estrogen, via ER(α) receptors, promotes proliferation of breast cancer cells by down-regulating P2Y(2) receptor expression and attenuating P2Y(2)-induced increase of [Ca(2+)](i).     

MUC1 expression in human breast cancer cells is altered by the factors affecting cell proliferation

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. In the present study, we have investigated if the factors affecting cells proliferation could influence MUC1 mucin biosynthesis and shedding from cell surface into the culture medium in two human breast cancer cell lines: MCF-7 (ER+) and MDA-MB-231 (ER-). Using MCF-7 line we found that estradiol at a concentration of 10(-7) M increased [3H]glucosamine incorporation into mucin in cell lysate approximately twofold in comparison with control cultures, and a similar increase was observed in the culture medium. The selective estrogen receptor modulator, tamoxifen (at concentrations of 10(-6) M and 10(-5) M) had a little inhibitory effect. MDA-MB-231 cells in culture were stimulated with phorbol ester PMA, the protein kinase C activator. We noted that PMA greatly stimulated MUC1 synthesis and its shedding to culture medium and that this effect was abolished by protein kinase C specific inhibitor--bisindolylmaleimide.     

Effects of genistein and synergistic action in combination with eicosapentaenoic acid on the growth of breast cancer cell lines

Genistein, a prominent isoflavone in soy products, produced dose- and time-dependent in vitro growth inhibition at high concentrations (at least 185 microM) with an IC50 of 7.0-274.2 microM after 72 h incubation in four breast cancer cell lines (DD-762, Sm-MT, MCF-7 and MDA-MB-231) and one breast epithelial cell line (HBL- 100) of human and animal origin; it stimulated estrogen-receptor-positive MCF-7 cells at low concentrations (3.7 nM-37 microM). Genistein-exposed cells underwent apoptosis, confirmed by G2/M arrest followed by the appearance of a sub-G1 fraction in cell-cycle progression, and by a characteristic cell ultrastructure. The apoptosis cascade was due to up-regulation of Bax protein, down-regulation of Bcl-XL protein, and activation of caspase-3. Genistein acted in synergism with eicosapentaenoic acid (EPA), a fish oil component, on human breast cancer MCF-7 cells (genistein > 93.2 microM and EPA > 210.9 microM) and on MDA-MB-231 cells (genistein > 176.1 microM and EPA > 609.3 microM). Dietary intake of genistein in combination with EPA may be beneficial for breast cancer control.