PROM2通过β-catenin/HER2通路调控乳腺癌细胞SK-BR-3的迁移、侵袭、增殖和凋亡

乳腺癌是当今威胁女性健康最主要的恶性肿瘤之一。虽然当前在乳腺癌的诊治方面获得了一定的进展,但其发病率和死亡率仍然较高,寻找有效的乳腺癌预后标记和治疗靶点是当前研究的热点。本研究通过对高通量基因表达数据库(Gene Expression Omnibus,GEO)分析发现,Prominin 2(PROM2)在诱导、增强和增殖成瘤能力的3组MCF7乳腺癌细胞中的表达,高于3组对照组MCF7细胞。且在诱导具有侵袭性的MCF10A细胞中的表达,高于未处理非侵袭性MCF10A细胞。研究选择了人乳腺癌细胞系SK-BR-3,成功地将过表达或敲减PROM2质粒转染到细胞中,并检测PROM2对SK-BR-3细胞的迁移、侵袭和增殖和凋亡的作用。划痕结果显示,敲减PROM2的SKBR-3细胞愈合面积更小(P<0.01),过表达PROM2的SK-BR-3细胞愈合面积更大(P<0.01);Transwell的结果显示,敲减PROM2的SK-BR-3细胞侵袭数量更少(P<0.01),过表达PROM2的SK-BR-3细胞侵袭数量更多(P<0.01);流式细胞术的结果显示,敲减PROM2的SK-BR-3细胞增殖能力减弱且凋亡比率增高(2.50%vs.0.93%),过表达PROM2的SK-BR-3细胞增殖能力增强且凋亡比率降低(0.43%vs.0.72%)。然后,探讨PROM2作用的可能机制。通过基因表达谱交互分析(gene expression profiling interactive analysis,GEPIA),对肿瘤基因图谱数据库(The Cancer Genome Atlas,TCGA)中的乳腺癌数据分析发现,PROM2与β-联蛋白(β-catenin)、人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)之间表达正相关。通过定量实时聚合酶链反应、免疫荧光和蛋白质印迹也发现,PROM2促进了β-联蛋白和HER2的表达。证明PROM2可能通过激活β-catenin/HER2通路,促进乳腺癌细胞SK-BR-3的迁移、侵袭和增殖,并抑制其凋亡。     

小干扰RNA抑制LRP16基因表达限制了MCF-7乳腺癌细胞增殖

雌激素雌二醇上调人乳腺癌细胞MCF 7中LRP16基因表达 ,该基因过表达促进MCF 7细胞增殖 .为进一步探讨LRP16基因不同表达水平对MCF 7细胞增殖的影响以及对雌激素的反应性增殖能力 ,采用针对LRP16基因特异的小干扰RNA策略 ,通过逆转录病毒介导及抗性筛选构建了LRP16基因被稳定抑制的 2个MCF 7细胞系 ,针对绿色荧光蛋白的干扰序列作为阴性对照 .Northern印迹实验检测了LRP16基因在各个细胞株中mNRA的水平 ,与对照组细胞比较 ,针对LRP16基因不同位置的 2个小干扰RNA可分别将该基因抑制 90 %和 6 0 % .细胞增殖试验结果显示 ,MCF 7细胞中LRP16基因表达抑制率越高 ,细胞增殖速率减慢越显著 (P <0 0 5 ) ;软琼脂集落形成试验结果显示 ,抑制LRP16基因在MCF 7细胞中表达 ,限制了细胞锚定非依赖性生长 ;细胞周期分析结果表明 ,LRP16基因抑表达使MCF 7细胞G1 S周期转换受抑 ;Western印迹结果表明 ,LRP16基因表达抑制的细胞中细胞周期蛋白E及细胞周期蛋白D1蛋白水平显著下调 ,但未检测到P5 3及Rb蛋白表达水平的影响 .雌二醇刺激的增殖实验结果显示 ,抑制LRP16基因表达没有消除MCF 7细胞的反应性增殖特征 .上述结果表明 ,LRP16基因表达量与MCF 7细胞增殖能力密切相关 ,抑制其表达可有效限制MCF 7细胞的增殖能力 ,提     

Real-Time Motion Analysis Reveals Cell Directionality as an Indicator of Breast Cancer Progression

Cancer cells alter their migratory properties during tumor progression to invade surrounding tissues and metastasize to distant sites. However, it remains unclear how migratory behaviors differ between tumor cells of different malignancy and whether these migratory behaviors can be utilized to assess the malignant potential of tumor cells. Here, we analyzed the migratory behaviors of cell lines representing different stages of breast cancer progression using conventional migration assays or time-lapse imaging and particle image velocimetry (PIV) to capture migration dynamics. We find that the number of migrating cells in transwell assays, and the distance and speed of migration in unconstrained 2D assays, show no correlation with malignant potential. However, the directionality of cell motion during 2D migration nicely distinguishes benign and tumorigenic cell lines, with tumorigenic cell lines harboring less directed, more random motion. Furthermore, the migratory behaviors of epithelial sheets observed under basal conditions and in response to stimulation with epidermal growth factor (EGF) or lysophosphatitic acid (LPA) are distinct for each cell line with regard to cell speed, directionality, and spatiotemporal motion patterns. Surprisingly, treatment with LPA promotes a more cohesive, directional sheet movement in lung colony forming MCF10CA1a cells compared to basal conditions or EGF stimulation, implying that the LPA signaling pathway may alter the invasive potential of MCF10CA1a cells. Together, our findings identify cell directionality as a promising indicator for assessing the tumorigenic potential of breast cancer cell lines and show that LPA induces more cohesive motility in a subset of metastatic breast cancer cells.     

METCAM/MUC18 augments migration, invasion, and tumorigenicity of human breast cancer SK-BR-3 cells

Previous research has identified METCAM/MUC18, an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family, as a promoter or a suppressor in the development of human breast cancer by MCF7, MDA-MB-231, and MDA-MB-468. To resolve these conflicting results we have investigated the role of this CAM in the progression of the three aforementioned cell lines plus one additional human breast cancer cell line, SK-BR-3. We transfected the SK-BR-3 cells with human METCAM/MUC18 cDNA to obtain G418-resistant clones, which expressed different levels of the protein and which were used to test the effect of human METCAM/MUC18 expression on in vitro motility, invasiveness, anchorage-independent colony formation in soft agar, disorganized growth in a 3D basement membrane culture assay, and in vivo tumorigenesis in athymic nude mice. Enforced METCAM/MUC18 expression increased in vitro motility, invasiveness, and anchorage-independent colony formation of SK-BR-3 cells and favored disorganized growth of the cells in 3D basement membrane culture. Enforced expression also increased tumorigenicity and final tumor weights of SK-BR-3 clones/cells after subcutaneous injection of the cells under the left third nipple of female athymic nude mice. To understand the mechanisms, we also determined the expression of several downstream key effectors in the tumors. Tumor cells from METCAM/MUC18 expressing clones exhibited elevated expression of an anti-apoptotic and survival index (Bcl2), an aerobic glycolysis index (LDH-A), and pro-angiogenesis indexes (VEGF and VAGFR2). We concluded that human METCAM/MUC18 promotes the development of breast cancer cells by increasing an anti-apoptosis and survival pathway and augmenting aerobic glycolysis and angiogenesis.     

Transforming growth factors type beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell lines

At least one member of the TGF-beta family, TGF-beta 1, has been previously shown to inhibit the anchorage-independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF-beta family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF-beta 1 and TGF-beta 2 on the anchorage-independent growth of the oestrogen receptor-negative cell lines MDA-MB-231, SK-BR-3, Hs578T, MDA-MB-468, and MDA-MB-468-S4 (an MDA-MB-468 clone not growth inhibited by EGF) and the estrogen receptor-positive cell lines MCF7, ZR-75-1, T-47D. TGF-beta 1 and TGF-beta 2 caused a 75-90% growth inhibition of MDA-MB-231, SK-BR-3, Hs578T, and MDA-MB-468 cells and a 50% growth inhibition of ZR-75-1 and early passage (less than 100) MCF7 cells. T-47D cells responded to TGF-beta only in serum-free conditions in the presence of IGF-1 or EGF. The growth of MDA-MB-468-S4 cells and late passage (greater than 500) MCF7 cells was not inhibited by TGF-beta 1 or TGF-beta 2. TGF-beta-sensitive MCF7 and MDA-MB-231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF-beta-related polypeptide. TGF-beta 1 or TGF-beta 2 were mutually competitive for receptor binding with a similar affinity (Kd 25-130 pM, 1,000-13,000 sites per cell). To determine the time course of the TGF-beta effect, an anchorage-dependent growth assay was carried out using MDA-MB-231 cells. Growth inhibition occurred at 6 days, and cell-cycle changes were seen 12 hr after the addition of TGF-beta. Cells accumulated in the G1 phase and were thus inhibited from entering the S-phase. These data indicate that TGF-beta is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF-beta effects in vivo.     

The p21-dependent radiosensitization of human breast cancer cells by MLN4924, an investigational inhibitor of NEDD8 activating enzyme

Radiotherapy is a treatment choice for local control of breast cancer. However, intrinsic radioresistance of cancer cells limits therapeutic efficacy. We have recently validated that SCF (SKP1, Cullins, and F-box protein) E3 ubiquitin ligase is an attractive radiosensitizing target. Here we tested our hypothesis that MLN4924, a newly discovered investigational small molecule inhibitor of NAE (NEDD8 Activating Enzyme) that inactivates SCF E3 ligase, could act as a novel radiosensitizing agent in breast cancer cells. Indeed, we found that MLN4924 effectively inhibited cullin neddylation, and sensitized breast cancer cells to radiation with a sensitivity enhancement ratio (SER) of 1.75 for SK-BR-3 cells and 1.32 for MCF7 cells, respectively. Mechanistically, MLN4924 significantly enhanced radiation-induced G2/M arrest in SK-BR-3 cells, but not in MCF7 cells at early time point, and enhanced radiation-induced apoptosis in both lines at later time point. However, blockage of apoptosis by Z-VAD failed to abrogate MLN4924 radiosensitization, suggesting that apoptosis was not causally related. We further showed that MLN4924 failed to enhance radiation-induced DNA damage response, but did cause minor delay in DNA damage repair. Among a number of tested SCF E3 substrates known to regulate growth arrest, apoptosis and DNA damage response, p21 was the only one showing an enhanced accumulation in MLN4924-radiation combination group, as compared to the single treatment groups. Importantly, p21 knockdown via siRNA partialy inhibited MLN4924-induced G2/M arrest and radiosensitization, indicating a causal role played by p21. Our study suggested that MLN4924 could be further developed as a novel class of radiosensitizer for the treatment of breast cancer.     

Lin28 Mediates Radiation Resistance of Breast Cancer Cells via Regulation of Caspase,H2A.X and Let-7 Signaling

Resistance to radiation therapy is a major obstacle for the effective treatment of cancers. Lin28 has been shown to contribute to breast tumorigenesis; however, the relationship between Lin28 and radioresistance remains unknown. In this study, we investigated the association of Lin28 with radiation resistance and identified the underlying mechanisms of action of Lin28 in human breast cancer cell lines. The results showed that the expression level of Lin28 was closely associated with resistance to radiation treatment. The T47D cancer cell line, which highly expresses Lin28, is more resistant to radiation than MCF7, Bcap-37 or SK-BR-3 cancer cell lines, which have low-level Lin28 expression. Transfection with Lin28 siRNA significantly led to an increase of sensitivity to radiation. By contrast, stable expression of Lin28 in breast cancer cells effectively attenuated the sensitivity to radiation treatment. Stable expression of Lin28 also significantly inhibited radiation-induced apoptosis. Moreover, further studies have shown that caspases, H2A.X and Let-7 miRNA were the molecular targets of Lin28. Stable expression of Lin28 and treatment with radiation induced H2AX expression, while inhibited p21 and γ-H2A.X. Overexpression of Let-7 enhanced the sensitivities to radiation in breast cancer cells. Taken together, these results indicate that Lin28 might be one mechanism underlying radiation resistance, and Lin28 could be a potential target for overcoming radiation resistance in breast cancer.     

Identification of Cytoskeleton-Associated Proteins Essential for Lysosomal Stability and Survival of Human Cancer Cells

Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 siRNAs was preceded by lysosomal membrane permeabilization, and all identified siRNAs induced several changes in the endo-lysosomal compartment, i.e. increased lysosomal volume (KIF11, KIF20A, KIF25, MYO1G, MYH1), increased cysteine cathepsin activity (KIF20A, KIF25), altered lysosomal localization (KIF25, MYH1, TPM2), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide or cisplatin. Similarly to KIF11 siRNA, the KIF11 inhibitor monastrol induced lysosomal membrane permeabilization and sensitized several cancer cell lines to siramesine. While KIF11 inhibitors are under clinical development as mitotic blockers, our data reveal a new function for KIF11 in controlling lysosomal stability and introduce six other molecular motors as putative cancer drug targets.     

Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.     

RPN2 gene confers docetaxel resistance in breast cancer

Drug resistance acquired by cancer cells has led to treatment failure. To understand the regulatory network underlying docetaxel resistance in breast cancer cells and to identify molecular targets for therapy, we tested small interfering RNAs (siRNAs) against 36 genes whose expression was elevated in human nonresponders to docetaxel for the ability to promote apoptosis of docetaxel-resistant human breast cancer cells (MCF7-ADR cells). The results indicate that the downregulation of the gene encoding ribophorin [corrected] II (RPN2), which is part of an N-oligosaccharyl transferase complex, most efficiently induces apoptosis of MCF7-ADR cells in the presence of docetaxel. RPN2 silencing induced reduced glycosylation of the P-glycoprotein, as well as decreased membrane localization, thereby sensitizing MCF7-ADR cells to docetaxel. Moreover, in vivo delivery of siRNA specific for RPN2 markedly reduced tumor growth in two types of models for drug resistance. Thus, RPN2 silencing makes cancer cells hypersensitive response to docetaxel, and RPN2 might be a new target for RNA interference-based therapeutics against drug resistance.     

RNAi-mediated silencing of insulin receptor substrate 1 (IRS-1) enhances tamoxifen-induced cell death in MCF-7 breast cancer cells

Insulin receptor substrate 1 (IRS-1) is a major downstream signaling protein for insulin and insulin-like growth factor I (IGF-I) receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In breast cancer, IRS-1 overexpression has been associated with tumor development, hormone-independence and antiestrogen-resistance. In part, these effects are related to potentiation of IRS-1/PI-3K/Akt signaling. In estrogen sensitive breast cancer cell lines, tamoxifen treatment reduces IRS-1 expression and function; consequently, inhibiting IRS-1/PI-3K signaling. We tested whether anti-IRS1 siRNA could inhibit growth and survival of estrogen-sensitive MCF-7 breast cancer cells, when used alone or in combination with TAM. Our results indicated: (a) out of four tested anti-IRS1 siRNAs, two siRNAs reduced IRS-1 protein by approximately three-fold in both growing and IGF-I-stimulated cells without affecting a closely related protein, IRS-2; (b) these effects paralleled IRS1 mRNA downregulation by approximately three-fold, measured by quantitative real time-polymerase chain reaction; (c) action of anti-IRS1 siRNAs induced the apoptotic response, observed by altered mitochondrial membrane potential coupled with downregulation of NF-kappaB target Bcl-xL and reduced cell viability; (d) anti-IRS1 siRNA treatment enhanced the cytotoxic effects of TAM by approximately 20%. In summary, anti-IRS1 RNAi strategy could become a potent tool to induce breast cancer cell death, especially if combined with standard TAM therapy.     

Insulin receptor substrate 1 knockdown in human MCF7 ER+ breast cancer cells by nuclease-resistant IRS1 siRNA conjugated to a disulfide-bridged D-peptide analogue of insulin-like growth factor 1

IRS-1 overexpression has been associated with breast cancer development, hormone independence and antiestrogen resistance. IRS-1 is a major downstream signaling protein for insulin and IGF1 receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In estrogen-sensitive breast cancer cell lines, the widely used antiestrogen tamoxifen treatment reduces IRS-1 expression and function, thereby inhibiting IRS-1/PI-3K signaling. IRS-1 may serve as an alternative target to overexpressed IGF1R in breast cancer. While siRNA technology has become commonplace in many laboratories for in vitro gene knockdown studies, and in vivo stability issues are largely solved, its use in vivo is limited by an inability to efficiently and specifically deliver it to the intended site of action. We previously reported reduced survival of human MCF7 estrogen receptor positive breast cancer cells treated with a normal IRS1 siRNA delivered by a cationic lipid, plus an additive effect in combination with tamoxifen. We now report enhanced cellular uptake, relative to the unconjugated serum-stabilized IRS1 siRNA, of a serum-stabilized IRS1 siRNA conjugated with our previously characterized peptide mimetic of IGF1, D-(Cys-Ser-Lys-Cys), without the use of cationic lipids or electroporation, in MCF7 cells that overexpress IGF1R. Excess native IGF1 blocked uptake. An IRS1 siRNA cholesterol conjugate, targeted universally to cell membranes, was taken up by MCF7 cells as much as the peptide mimetic conjugate. IRS1 mRNA knockdown and IRS-1 protein knockdown were comparable for the IGF1 peptide and cholesterol conjugates. The unconjugated serum-stabilized IRS1 siRNA control showed negligible effects. Viability assays showed additive effects of siRNA treatment in combination with tamoxifen. In summary, we have taken the first step in converting an siRNA into a pharmacologically active agent for breast cancer.     

Differential impact of Cetuximab, Pertuzumab and Trastuzumab on BT474 and SK-BR-3 breast cancer cell proliferation

OBJECTIVES: The potential of epidermal growth factor receptor (EGFR)- and Her2-targeted antibodies Cetuximab, Pertuzumab and Trastuzumab, used in combination to inhibit cell proliferation of breast cancer cells in vitro, has not been extensively investigated. It is anticipated that there would be differences between specific erbB receptor co-expression profiles that would affect tumour cell growth. MATERIALS AND METHODS: We have examined the effects of Cetuximab, Pertuzumab and Trastuzumab, applied separately or in combination, on cell proliferation of BT474 and SK-BR-3 breast cancer cell lines. Cell cycle progression of BT474 and SK-BR-3 cells was statically and dynamically assessed using flow cytometry. In order to discover a potential influence of differential EGFR co-expression on sensitivity to antibody treatment, EGFR was down-regulated by siRNA in SK-BR-3. An annexinV/propidium iodide assay was used to identify potential induction of apoptosis. RESULTS: Treatment with Pertuzumab and Trastuzumab, both targeted to Her2, resulted in a reduced fraction of proliferating cells, prolongation of G(1) phase and a great increase in quiescent BT474 cells. Cetuximab had no additional contribution to the effect of either Pertuzumab or Trastuzumab when administered simultaneously. Treatment with the antibodies did not induce an appreciable amount of apoptosis in either BT474 or SK-BR-3 cells. In contrast to SK-BR-3, the BT474 cell line appears to be more sensitive to antibody treatment due to low EGFR content besides Her2 overexpression. CONCLUSION: The extent of decelerated or blocked cell proliferation after antibody treatment that is targeted to EGFR and to Her2 depends both on EGFR and Her2 co-expression and on antibody combination used in the treatment setting. Cetuximab did not enhance any inhibitory effect of Trastuzumab or Pertuzumab, most probably due to the dominant overexpression of Her2. Cell susceptibility to Trastuzumab/Pertuzumab, both targeted to Her2, was defined by the ratio of EGFR/Her2 co-expression.     

曲妥珠单抗联合NSC 74859 对曲妥珠耐药的乳腺癌细胞的抑制作用研究

目的:观察曲妥珠单抗(Trastuzumab)与转录信号转导子与激活子3蛋白(STAT3)抑制剂NSC 74859联用对曲妥珠耐药细胞株SK-BR-3R的生长抑制作用及机理研究。方法:采用四甲基偶氮唑蓝(MTT)法鉴定曲妥珠耐药的SK-BR-3R细胞株并检测曲妥珠单药处理、NSC 74859单药处理以及两药联用处理对SK-BR-3R细胞的生长抑制程度。建立SK-BR-3R的皮下肿瘤模型,观察两药联用对肿瘤生长的抑制效果;通过免疫印迹(Western Blot)实验检测SK-BR-3R细胞中磷酸化HER2(p-HER2),磷酸化STAT3(p-STAT3)及磷酸化AKT(p-AKT)的水平。结果:当曲妥珠浓度在50 nmol/L及NSC 74859的浓度在50μmol/L联用时,较之两药单用显示了显著的抑制效果,其差异具有统计学意义;进一步在建立的SK-BR-3R小鼠肿瘤模型中观察到了曲妥珠联合NSC74859治疗组显示了比曲妥珠或NSC 74859单独使用时更显著的抑瘤效果。最后,免疫印迹实验显示了曲妥珠和NSC74859联合处理显著降低了SK-BR-3R细胞的HER2,STAT3及AKT的磷酸化水平。结论:曲妥珠单抗联合NSC 74859使用可显著抑制曲妥珠耐药的乳腺癌细胞SK-BR-3R的生长,其机制可能是药物协同抑制了对肿瘤生长重要的PI3K/AKT信号通路。本研究可为临床上治疗曲妥珠耐药的乳腺癌提供参考。     

端粒酶反义cDNA对乳腺癌细胞恶性表型的影响

 为了进一步探讨端粒酶在肿瘤发生中的作用 ,实验应用反义核酸技术研究端粒酶反义cDNA对乳腺癌细胞MCF 7恶性表型的影响 采用的方法包括 :基因重组、脂质体共转染法获得端粒酶反义重组病毒 ,病毒感染MCF 7后 ,检测细胞的生长曲线、细胞周期及集落形成能力 结果表明 ,与对照组细胞相比 ,反义病毒感染后的MCF 7细胞恶性表型明显降低 提示端粒酶RNA的反义cDNA的导入 ,可以显著抑制乳腺癌细胞恶性表型     

siRNA对乳腺癌细胞Cyclin E表达和生长抑制作用

研究siRNA对乳腺癌MCF-7细胞株cyclin E表达的抑制及对细胞生长的影响。化学合成针对cyclin E基因的小干扰RNA（siRNA）,转染MCF-7细胞株;分别应用荧光定量PCR和免疫印迹测定cyclin E mRNA和蛋白质的表达,CCK-8测定细胞的增殖活性,流式细胞仪检测细胞周期,软琼脂培养检测细胞克隆形成能力。10、50、100nmol／L siRNA-cyclin E分别使MCF-7细胞cyclin E基因表达降低了24．7％、62．5％和71．0％,蛋白质表达降低了40．8％、66．5％和71．3％。转染siRNA-cyclin E后,G1期细胞增多,S期减少,增殖受到抑制,软琼脂克隆形成率降低。结果提示,在MCF-7细胞株中,导入针对cyclin E的siRNA,可有效抑制cyclin E的表达,进而使细胞增殖减缓,逆转其恶性表型。     

BMP9 Inhibits Proliferation and Metastasis of HER2-Positive SK-BR-3 Breast Cancer Cells through ERK1/2 and PI3K/AKT Pathways

Bone morphogenetic protein 9 (BMP9), a member of TGF-β superfamily, is reported to inhibit the growth and migration of prostate cancer, osteosarcoma and triple-negative MDA-MB-231 breast cancer cells. However, little is known about the effect of on the biological behaviors of HER2-positive SK-BR-3 breast cancer cells and the underlying mechanisms. This study aimed to investigate the effects of BMP9 on the proliferation and metastasis of SK-BR-3 cells with BMP9 over-expression or BMP9 down-regulated expression. Results indicated that exogenously expressed BMP9 inhibited the proliferation and metastasis of SK-BR-3 cells while decreased endogenous BMP9 expression in SK-BR-3 cells promoted the proliferation and migration of breast cancer cells in vitro and in vivo. In SK-BR-3 cells with BMP9 over-expression, the phosphorylation of HER2, ERK1/2 and AKT was markedly suppressed and the HER2 expression decreased at both mRNA and protein levels, while opposite results were observed in SK-BR-3 cells with BMP9 knock down. When the phosphorylation of ERK1/2 and PI3K/AKT was inhibited by PD98059 and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, respectively, the decreased proliferation and invasion induced by BMP9 knock down were eliminated. These findings suggest that BMP9 can inhibit the proliferation and metastasis of SK-BR-3 cells via inactivating ERK1/2 and PI3K/AKT signaling pathways. Thus, BMP9 may serve as a useful agent in the treatment of HER-2 positive breast cancer.     

3-O-methylfunicone, from Penicillium pinophilum, is a selective inhibitor of breast cancer stem cells

Objectives: Cancer stem cells make up a subpopulation of cells within tumours that drive tumour initiation, growth and recurrence. They are resistant to many current types of cancer treatment, causing failure of such therapeutic approaches, including chemotherapy and radiotherapy. In the study described here, anti‐proliferative effects of 3‐O‐methylfunicone (OMF), a metabolite from Penicillium pinophilum, were investigated on human breast cancer MCF‐7 cells and cancer stem cells selected as mammospheres derived from MCF‐7s. Materials and methods: Stemness markers were analysed on isolated mammospheres showing positive expression of CD24, CD29, CD44, CD133, CD184 and CD338. Cell proliferation and apoptosis were analysed by flow cytometry and RT‐PCR. Cell colony formation assays were performed to evaluate colony formation of mammospheres. Results and conclusion: OMF treatment affected both MCF‐7 and mammosphere growth, inducing apoptosis. In addition, OMF strongly reduced stemness markers and survivin, hTERT and Nanog‐1 gene expression. Growth of colonies in soft‐agar was significantly affected by OMF treatment, too. Lastly, we tested ability of MCF‐7 cells to form mammospheres after treatment with OMF or cisplatin, demonstrating that OMF treatment resulted in drastic reduction in number of mammospheres. These results introduce OMF as an effective molecule in suppressing breast cancer stem cells.