Enhancing cisplatin sensitivity in MCF-7 human breast cancer cells by down-regulation of Bcl-2 and cyclin D1

MCF-7 human breast cancer cells are relatively resistant to cisplatin treatment compared to other breast cancer cell lines. In order to identify possible targets for sensitizing the breast cancer cells to cisplatin treatment protein expression levels and the phosphorylation status of 27 different signaling proteins were examined. MCF-7 cells expressed high levels of anti-apoptotic Bcl-2 protein relative to more cisplatin sensitive breast cancer cells. After cisplatin treatment a decrease in cyclin D1 was seen in all the breast cancer cells studied. Therefore, Bcl-2 and cyclin D1 were chosen as putative targets for increasing cell death and growth arrest induced by cisplatin, thereby enhancing the drug sensitivity in MCF-7. RNA interference, using Bcl-2- and cyclin D1- siRNAs sensitized MCF-7 cells to cisplatin treatment and by simultaneous knockdown of both Bcl-2 and cyclin D1 further sensitization was seen. This shows the potential of targeting both apoptotic- and cell cycle-regulating pathways to enhance the effect of chemotherapy.     

HuR contributes to cyclin E1 deregulation in MCF-7 breast cancer cells

Many cancers overexpress cyclin E1 and its tumor-specific low molecular weight (LMW) isoforms. However, the mechanism of cyclin E1 deregulation in cancers is still not well understood. We show here that the mRNA-binding protein HuR increases cyclin E1 mRNA stability in MCF-7 breast carcinoma cells. Thus, mRNA stabilization may be a key event in the deregulation of cyclin E1 in MCF-7 cells. Compared with MCF10A immortalized breast epithelial cells, MCF-7 cells overexpress full-length cyclin E1 and its LMW isoforms and exhibit increased cyclin E1 mRNA stability. Increased mRNA stability is associated with a stable adenylation state and an increased ratio of cytoplasmic versus nuclear HuR. UV cross-link competition and UV cross-link immunoprecipitation assays verified that HuR specifically bound to the cyclin E1 3'-untranslated region. Knockdown of HuR with small interfering RNA (siRNA) in MCF-7 cells decreased cyclin E1 mRNA half-life (t(1/2)) and its protein level: a 22% decrease for the full-length isoforms and 80% decrease for the LMW isoforms. HuR siRNA also delayed G(1)-S phase transition and inhibited MCF-7 cell proliferation, which was partially recovered by overexpression of a LMW isoform of cyclin E1. Overexpression of HuR in MCF10A cells increased cyclin E1 mRNA t(1/2) and its protein level. Taken together, our data show that HuR critically contributes to cyclin E1 overexpression and its growth-promoting function, at least in part by increasing cyclin E1 mRNA stability, which provides a new mechanism of cyclin E1 deregulation in breast cancer.     

Efficacy of sulforaphane is mediated by p38 MAP kinase and caspase-7 activations in ER-positive and COX-2-expressed human breast cancer cells.

Sulforaphane is an antioxidant and a potent stimulator of natural detoxifying enzyme and associated with lowered risk of cancer that is associated with the consumption of cruciferous vegetables. The chemopreventive effects of SFN was investigated using the MCF-7 human breast cancer cells and the M13SV1-immortalized human breast luminal epithelial cells. Sulforaphane reduced proliferation in MCF-7 cells and inhibited cyclooxygenase-2 expression in M13SV1 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). The chemopreventive effects of sulforaphane were associated with p38 mitogen-activated protein kinase suggest its important role in cell survival/apoptosis regulation and stabilization of cyclooxygenase-2. Sulforaphane upregulates p38 in MCF-7 cells and prevented TPA-reduced phosphorylation of p38 in M13SV1 cells, but activated caspase-7 associated with apoptosis in MCF-7 cells. These results suggest that sulforaphane may be an alternative candidate for targeted prevention of ER-positive and cyclooxygenase-2-induced phenotypes and breast cancer.     

Antiestrogen regulation of cell cycle progression and cyclin D1 gene expression in MCF-7 human breast cancer cells

The molecular mechanisms by which antiestrogens inhibit breast cancer cell proliferation are not well understood. Using cultured breast cancer cell lines, we studied the effects of antiestrogens on proliferation and cell cycle progression and used this information to select candidate cell cycle regulatory genes that are potential targets for antiestrogens. Under estrogen- and serum-free conditions antiestrogens inhibited proliferation of MCF-7 cells stimulated with insulin. Cells were blocked at a point in G₁ phase. These effects are comparable with those in serum- and estrogen-containing medium and were also seen to a lesser degree in nude mice bearing MCF-7 tumors. Similar observations with other peptide mitogens suggest that the process inhibited by antiestrogens is common to estrogen and growth factor activated pathways. Other studies have identified G₁ cyclins as potential targets for growth factor and steroid hormone/steroid antagonist regulation of breast epithelial cell proliferation. In MCF-7 cells growing in the presence of fetal calf serum, cyclin D1 mRNA was rapidly down-regulated by steroidal and nonsteroidal antiestrogens by an apparently estrogen receptor mediated mechanism. Cyclin D1 gene expression was maximally inhibited before effects on entry into S phase and inhibition was therefore not merely a consequence of changes in cell cycle progression. Together with data on the effects of antiestrogens in serum-free conditions [1], these results suggest down-regulation of cyclin D1 by antiestrogens may be a general phenomenon in estrogen receptor-positive breast cancer cells, independent of culture conditions and class of antiestrogen. These observations are compatible with the hypothesis that reductions in cyclin D1 levels may mediate in part the action of antiestrogens in blocking entry of cells into S phase.     

Indomethacin-induced radiosensitization and inhibition of ionizing radiation-induced NF-kappaB activation in HeLa cells occur via a mechanism involving p38 MAP kinase

Although ionizing radiation (IR) activates multiple cellular factors that vary depending on dose and tissue specificity, the activation of NF-kappaB appears to be a well-conserved response in tumor cells exposed to IR. Recently, it also has been demonstrated that nonsteroidal anti-inflammatory agents inhibit tumor necrosis factor and interleukin-1-induced NF-kappaB activation and act as radiosensitizing agents. These observations reinforce the growing notion that NF-kappaB may be a protective cellular factor responding to the cytotoxicity of IR and other damaging stimuli. As such, we addressed the idea and mechanism that NF-kappaB is a downstream target of the nonsteroidal anti-inflammatory agent indomethacin and is involved in the process of radiosensitization. In this study, we report that indomethacin inhibited IR-induced activation of NF-kappaB and sensitized HeLa cells to IR-induced cytotoxicity at similar concentrations. Pretreatment of HeLa cells with SB 203580, a pyridinyl imidazole compound that specifically inhibits p38 mitogen-activated protein kinase (MAPK), abrogated the ability of indomethacin to inhibit IR-induced activation of NF-kappaB and diminished the indomethacin radiosensitizing effect. In addition, the transient genetic activation of p38(MAPK) inhibited IR induction of NF-kappaB gene expression in the absence of indomethacin. Finally, permanently transfected cell lines genetically unable to activate NF-kappaB, because of expression of a dominant negative I-kappaBalpha gene, demonstrated increased sensitivity to IR-induced cytotoxicity. Taken together, these results suggest that p38 MAPK is a target involved in indomethacin-induced radiosensitization and that NF-kappaB may be one downstream target in this process.     

EMT经p38-MAPK调节乳腺癌MCF-7细胞P-gp介导的多药耐药

摘 要　目的：探讨乳腺癌细胞中上皮间质转化（epithelial-mesenchymal transition, EMT）与P-糖蛋白（P-glucoprotein, P-gp）介导的多药耐药（multidrug resistance, MDR）的关系及其可能的机制。方法：将携Snail基因的真核表达载体pcDNA-Snail转染人乳腺癌细胞MCF-7,用多柔比星（doxorubicin, DOX）诱导各组细胞耐药。免疫细胞荧光检测上皮标志物E-钙黏素（E-cadherin）、间质标志物波形蛋白（vimentin）以及P-gp的表达,MTT检测耐药细胞的增殖,RT-PCR检测Snail、MDR1、p38-MAPK mRNA的表达。结果：细胞免疫荧光显示,转染pcDNA-Snail载体后,MCF-7细胞发生EMT,E-cadherin表达显著降低,vimentin表达显著升高;P-gp在发生EMT的MCF-7细胞中表达显著升高。经DOX诱导,MCF-7/Snail细胞的耐药能力较MCF-7/DOX细胞显著增强（P<0.05）。RT-PCR显示,MCF-7细胞发生EMT后,p38-MAPK表达显著升高（P<0.05）,MDR1表达较亲本细胞明显升高（P<0.01）。结论：MCF-7细胞发生EMT后,可能通过p38-MAPK引发P-gp介导的MDR。     

Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells: Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression

Summary We have examined the role of cyclin D1 and cyclin-dependent kinase-4 (CDK4) in the cell cycle progression and proliferation of MCF-7 breast cancer cells. Forced expression of cyclin D1 using a tetracycline-regulated expression system, and suppression of endogenous cyclin D1 and CDK4 using small interfering RNA (siRNA) were used to validate this protein complex as a drug target in cancer drug discovery. Overexpression of cyclin D1 increased both phosphorylation of the retinoblastoma gene product (RB) and passage through the G1–S phase transition, resulting in increased proliferation of cells. When cyclin D1 expression was shut off, growth rates fell below those seen in control cell lines transfected with the vector, indicating an increased dependence on this protein for proliferation. Inhibition of endogenous cyclin D1 or CDK4 expression by RNA interference resulted in hypophosphorylation of RB and accumulation of cells in G1. These results support the prevailing view that pharmacological inhibition of cyclin D1/CDK4 complexes is a useful strategy to inhibit the growth of tumors. Furthermore, since MCF-7 cells appear to be dependent on this pathway for their continued proliferation, it is a suitable cell line to test novel cyclin D1/CDK4 inhibitors.     

Peroxisome proliferator-activated receptor gamma agonists induce proteasome-dependent degradation of cyclin D1 and estrogen receptor alpha in MCF-7 breast cancer cells

Treatment of MCF-7 cells with the peroxisome proliferator-activated receptor (PPAR) gamma agonists ciglitazone or 15-deoxy-Delta 12,14-prostaglandin J2 resulted in a concentration- and time-dependent decrease of cyclin D1 and estrogen receptor (ER) alpha proteins, and this was accompanied by decreased cell proliferation and G(1)-G(0)-->S-phase progression. Down-regulation of cyclin D1 and ER alpha by PPARgamma agonists was inhibited in cells cotreated with the proteasome inhibitors MG132 and PSII, but not in cells cotreated with the protease inhibitors calpain II and calpeptin. Moreover, after treatment of MCF-7 cells with 15-deoxy-Delta 12,14-prostaglandin J2 and immunoprecipitation with cyclin D1 or ER alpha antibodies, there was enhanced formation of ubiquitinated cyclin D1 and ER alpha bands. Thus, PPARgamma-induced inhibition of breast cancer cell growth is due, in part, to proteasome-dependent degradation of cyclin D1 (and ER alpha), and this pathway may be important for other cancer cell lines.     

Estrogenic regulation of a novel 34 kDa protein associated with cyclin B1 in MCF-7 breast cancer cells

Recent studies have revealed altered regulation of cyclins in breast cancer cells. To understand the role of aberrant cyclin B1 expression in the proliferation of breast cancer cells, we examined cyclin B1-associated proteins in estrogen-responsive MCF-7 cells in a cell cycle-dependent manner. Immunoprecipitation of cell lysate with a monoclonal anti-human cyclin B1 antibody, followed by Western blot probing with an anti-human cdc2 (PSTAIR) antibody revealed the presence of a 34 kDa protein in estradiol-treated cells at 16 h after initiation of cell cycle progression. Flow cytometry and [H-3]-thymidine (Thd) incorporation experiments showed a dramatic increase in the percentage of S phase cells at this time point. This protein was suppressed by an antiestrogen, 4-hydroxytamoxifen. It was not found in MCF-1OA, a normal breast epithelial cell line. The 34 kDa protein was not reactive with antibodies raised against other cyclin dependent kinases (CDKs), including p34(cdc2(Carboxy terminal)). This protein was functionally active as determined by histone H1 kinase activity. These data suggest that the induction of a cyclin B1-associated 34 kDa protein during the G1 --> S transition might be a positive regulator of cell cycle progression in estrogen-responsive breast cancer cells.     

Gene delivery of cyclin-dependent kinase inhibitors p21 Waf1 and p27 Kip1 suppresses proliferation of MCF-7 breast cancer cells in vitro

Background

Because tumorigenesis depends on a variety of oncogenes, symphyseal study of combined genes may lead to more significant knowledge about tumorigenesis and progression. Combined deficiency of p21 and p27 proteins in mice is linked to more aggressive spontaneous tumorigenesis. We investigated the effect of the transfected p21 ^Waf1 -p27 ^Kip1 gene on centrosome duplication, cell proliferation, and apoptosis of MCF-7, a breast cancer cell line.

Methods

The pIRES-p21 ^Waf1 , pIRES-p27 ^Kip1 , and pIRES-p21 ^Waf1 -p27 ^Kip1 genes were transfected into MCF-7 cells by lipofection. The effect on proliferation was evaluated by MTT assay and clone-formation assay. Cell cycle and apoptosis were analyzed by flow cytometry. Apoptosis was tested by flow cytometry and TUNEL assay. Centrosome duplication was detected by use of indirect immunofluorescence microscopy.

Results

The results showed that the pIRES-p21 ^Waf1 , pIRES-p27 ^Kip1 , and pIRES-p21 ^Waf1 -p27 ^Kip1 significantly inhibited proliferation of MCF-7 cells, followed by accumulation of MCF-7 cells in cycle G₁, induced apoptosis, and a decrease in the proportion of MCF-7 cells which contained abnormal centrosomes. Compared with p21 ^Waf1 or p27 ^Kip1 alone, combination of p21 ^Waf1 and p27 ^Kip1 had a much more significant effect (P < 0.05).

Conclusion

Altogether, these results indicate that the p21 ^Waf1 -p27 ^Kip1 gene combination has a more obvious antitumor effect than p21 ^Waf1 or p27 ^Kip1 alone. This study provides preclinical evidence that combination of p21 ^Waf1 and p27 ^Kip1 could be a novel and promising therapeutic approach to treatment of breast cancer with suppressed p21 ^Waf1 and p27 ^Kip1 expression.     

AEG-1基因促进乳腺癌细胞株MCF-7转移

目的 观察AEG-1基因在细胞水平对乳腺癌细胞MCF-7转移的影响。方法 通过将siRNA转染进MCF-7细胞,沉默细胞中AEG-1表达量,以转染阴性siRNA作为对照组。分别采用Transwell小室检测细胞迁移侵袭能力、CCK8实验检测细胞增殖能力。同时通过检测细胞中VEGF的变化及HUVEC细胞体外管腔形成实验考察AEG-1对于血管新生的影响。结果 沉默AEG-1,MCF-7细胞的迁移能力、侵袭能力和增殖能力明显受到抑制。沉默AEG-1,MCF-7细胞的VEGF表达明显降低。上清处理HUVEC细胞,沉默AEG-1组的血管新生能力明显受到抑制。结论 沉默AEG-1基因能显著抑制MCF-7细胞转移的多个层面,包括细胞迁移、侵袭、增殖以及血管新生。表明AEG-1基因在乳腺癌转移过程中起着重要作用,也为将来乳腺癌治疗开拓了新思路。     

p70/p85 核糖体蛋白S6激酶1重组真核表达载体的构建及其在人乳腺癌MCF-7细胞内功能的初步鉴定

目的：构建p70 核糖体蛋白S6激酶1（p70 ribosomal protein S6 kinase 1,p70 S6K1）及p85 S6K1基因的真核表达载体pcDNA3.1(-)-flag-p70 S6K1和pcDNA3.1(-)-flag-p85 S6K1,并鉴定其在人乳腺癌MCF-7细胞内的表达及功能。 方法： 以pRK7-HA-S6K1为模板,采用PCR扩增出目的基因片段p70 S6K1、p85 S6K1,克隆入真核表达载体pcDNA3.1(-)-flag构建重组表达载体pcDNA3.1(-)-flag-p70 S6K1和pcDNA3.1(-)-flag-p85 S6K1,采用PCR、双酶切和DNA测序鉴定。将重组载体转染MCF-7细胞,24 h后采用Western blotting方法检测细胞内p70 S6K1、p85 S6K1蛋白的表达;同时向转染细胞内加入1 mmol/L H2O2处理36 h,观察p70 S6K1、p85 S6K1蛋白对H2O2诱导的细胞死亡的影响。 结果： 成功扩增得到p70 S6K1、p85 S6K1基因片段并构建重组真核表达载体pcDNA3.1(-)-flag-p70 S6K1和pcDNA3.1(-)-flag-p85 S6K1,重组载体经PCR、双酶切鉴定均出现p70 S6K1和p85 S6K1预期条带,DNA测序结果显示其全长基因阅读框完整、正确。重组载体在MCF-7细胞中高效表达flag-p70 S6K1和flag-p85 S6K1,且p85 S6K1能增强H2O2诱导的细胞死亡。 结论：成功构建重组真核表达载体pcDNA3.1(-)-flag-p70 S6K1和pcDNA3.1(-)-flag-p85 S6K1,均能在MCF-7细胞中高效表达,且p85 S6K1能够增强H2O2诱导的细胞死亡。     

Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells

To investigate the mechanisms underlying apoptosis in breast cancer cells, staurosporine was used as an apoptotic stimulus in the human breast cancer cell lines MCF-7 and T47D. Staurosporine induced dose and time dependent increases in DNA fragmentation which was abrogated by z-VAD-fmk. MCF-7 cells did not express caspase-3, suggesting that DNA fragmentation occurred in the absence of caspase-3 and that other caspases may be involved. Staurosporine induced DEVDase activity in T47D cells suggesting the involvement of caspase-3 and/or caspase-7, yet there was no DEVDase activity in MCF-7 cells, probably ruling out the involvement caspase-7. However, staurosporine induced the cleavage of pro-caspase-6 in MCF-7 cells, but not in T47D cells. Caspase dependent PARP cleavage was detected in MCF-7 cells at 3 h, whereas only partial PARP cleavage was detected in T47D cells and then only after 24 h. Moreover, staurosporine led to cytochrome c release at 2 h in MCF-7 cells and 6 h in T47D cells. In addition, a time dependent and caspase-independent reduction of the mitochondrial transmembrane potential was observed; which appeared to occur after the release of cytochrome c. Translocation of Bax from the cytosol to mitochondria was observed in both cell types, and this preceded cytochrome c release in both T47D and MCF-7 cells. Apoptotic events in both cell types differ temporally, involving activation of different caspases and mitochondrial changes.