The effects of retinoids on proliferative capacities and macromolecular synthesis in human breast cancer MCF-7 cells

The effects of various retinoids on the proliferative capacities and on the synthesis of DNA, RNA, and protein have been investigated in MCF-7 mammary carcinoma cells in culture. Of the various retinoids tested, retinoic acid revealed maximum activity in inhibiting cell proliferation and thymidine incorporation. The degree of inhibition of cell proliferation by the various retinoids paralleled their capacity to inhibit thymidine incorporation, suggesting suppression of DNA synthesis as a primary cause of restriction of cell growth by these compounds. Two nonepithelial human cell lines were tested for sensitivity to retinoids, and showed diminished responses compared with MCF-7 cells. This suggests a correlation between the ability of retinoids to exert control of differentiation and cell proliferation for a given cell type. Reversibility of the effect of retinoid treatment, high cell viability, and lack of retinoid-induced lysosomal enzyme release, as shown in our studies, indicate that cytotoxicity may be excluded as a cause of decreased cell proliferation and inhibition of thymidine incorporation by retinoids.     

转铁蛋白和RGD肽共修饰脂质体的制备及其肺癌组织细胞的靶向性

目的:构建转铁蛋白(transferrin,TF)与整合素受体结合肽(RGD)共修饰脂质体(TF/RGD-LP),从体内外评价TF/RGD-LP的肺癌组织细胞靶向性。方法:薄膜分散法制备整合素受体结合肽RGD修饰的脂质体(RGD-LP),采用后插入法制备TF与RGD共修饰脂质体TF/RGD-LP,分析脂质体的理化性质。将肺癌A549细胞分为TF/RGD-LP、TF-LP、RGD-LP和LP组,每组设5个复孔。细胞摄取实验和肿瘤细胞球穿透实验研究TF/RGD-LP与肺癌A549细胞的亲和力和肿瘤组织的穿透能力,通过荷瘤裸鼠活体成像实验检测TF/RGD-LP的肺癌组织细胞靶向性。结果:所制备TF/RGD-LP粒径为(122.8±11.5)nm,电位为(6.4±3.85)m V。体外细胞摄取实验表明,肺癌A549细胞对TF/RGD-LP的摄取效率分别是TFLP、RGD-LP和LP的2.8、2.2和3.9倍,TF-LP(P=0.006)、RGD-LP(P=0.007)和LP(P=0.001)的主效应有统计学意义,三者间有交互效应(P=0.006);肿瘤细胞球摄取实验以及裸鼠肿瘤组织活体成像实验表明,TF/RGD-LP具有良好的肺癌组织靶向性。结论:TF/RGD-LP具有良好的肺癌组织细胞靶向性,是一种潜在的肺癌组织细胞靶向给药系统。     

三氯生对离体鹅尾臀腺和人乳腺癌细胞脂肪酸合成酶的抑制作用

背景与目的;研究表明,人乳腺癌早期和病程中癌细胞脂肪酸合成酶（fatty acid synthase,FAS)呈高效表达,抑制FAS可引起癌细胞凋亡。本研究采用酶促反应动力学实验观察三氯生对离体鹅尾臀腺FAS的影响,建立实验方法,研究三氯生对人乳腺癌SKBr3细胞FASS的抑制作用。方法：运用超速离心和Superdex PG200层析技术分离纯化鹅尾臀腺FAS,用超速离心技术部分纯化人乳腺癌SKBr3细胞FAS,不同浓度三氯生与FAS相互作用不同时间后,加入底物,用分光光度法观察三氯生对FAS的抑制作用。结果：在鹅尾臀腺组,FAS被纯化为SDS-PAGE电泳单条区带（分子量250kDa)。三种浓度三氯生（12.5,25.0,100.0μmol/L）与FAS在催化作用前相互作用0,5和10min,对FAS的抑制率分别为26.40%,28.30%和43.93%(12.5μmol/L）;46.22%,50.28%和97.05%(25μmol/L）;98.11%,97.75%和97.37%(100μmol/L）。在人乳腺癌SKBr3细胞组FAS被部分纯化,25,50,100和200μmol/L三氯生分别与FAS在催化前相互作用5min,对FAS活性的抑制率分别为20.00%,26.67%,60.00%和100.00%。结论：三氯生对离体鹅尾臀腺FAS和人乳腺癌SKBr3细胞FAS均具有抑制作用;作用强度既依赖于抑制剂浓度,又依赖于抑制剂与酶相互作用时间。     

Inhibition of cell growth and telomerase activity of breast cancer cells in vitro by retinoic acids

The effects of retinoic acid (RA) and its analogs, all-trans RA, 9-cis RA and 13-cis RA, were investigated in human breast cancer MCF-7 cells and immortalized breast epithelial cell line MCF-10A. RA inhibited the telomerase activity of MCF-7 cells in a wide range of concentrations. RA at 10 microM also inhibited the growth of MCF-7 cells in a time-dependent manner. However, no significant growth inhibition was found between untreated control and RA-treated MCF-10A cells. Moreover, a marked inhibition of telomerase activity by RA was detected early in MCF-7 cells (after 24 h of RA treatment), which was preceded by a reduction of hTERT mRNA expression (after 12 h of RA treatment). However, MCF-10A cells showed a reduction of telomerase activity and down-regulation of hTERT after 4 days of RA treatment. Simultaneous changes in hTERT mRNA expression and telomerase activity were found for MCF-10A cells. The expressions of hTR and hTEP1 telomerase component genes were not changed after RA treatment. These results indicate that the anti-breast cancer activity of RA could be mediated by its ability to down-regulate the expression of hTERT telomerase gene.     

Inhibition of growth of breast cancer cells in vitro by the ribosome-inactivating protein saporin 6

The antiproliferative activity of Saporin 6, a Ribosome-inactivating protein purified from the seeds of Saponaria officinalis has been tested on human breast cancer cells in vitro by the analysis (a) of colony formation in cells from surgical specimens from 27 patients bearing primary breast cancer and (b) of protein synthesis inhibition in the MCF/7 cell line. Results indicate a very high sensitivity of breast cancer cells from most patients to a short-term treatment with Saporin 6 at concentrations (10(-9) M), until now found effective only in acellular systems or after conjugation with monoclonal antibodies. On the contrary, the treatment of the human cell line MCF/7 indicate a very reduced sensitivity compared to fresh human neoplastic cells, with the necessity of a long lag for the effect to begin.     

Inhibition by retinoids of in vitro invasive ability of human lung carcinoma cells

The effect of retinoid treatment of A549 human lung carcinoma cells on in vitro cell invasion using the human amnion basement membrane (BM) was investigated. A 2-day retinoid pretreatment of the cells resulted in a significant reduction in their invasive ability. The most effective retinoid, retinol acetate, inhibited cell migration through the BM and degradation of [3H] proline labeled BM components by 50% at noncytotoxic concentrations of 0.09, and 3 micrograms/ml, respectively. Inhibition by retinol acetate of A549 cell invasive potential was accompanied by a significant decrease in type IV collagenase activity and no change in transglutaminase activity.     

舒林酸抗胃癌细胞体外生长的实验研究

目的观察舒林酸对胃癌BGC-823细胞增殖、凋亡的影响,探讨其作用机制。方法将不同浓度的舒林酸体外作用于人胃癌BGC-823细胞,采用倒置显微镜观察BGC-823细胞形态改变,四甲基偶氮唑蓝（MTT）比色法检测胃癌细胞增殖,流式细胞仪检测胃癌细胞周期分布及凋亡,透射电镜观察凋亡,免疫组化检测ki-67、bcl-2及环氧合酶-2（COX-2）蛋白的表达。结果舒林酸作用后,细胞伪足回缩,变小、变圆,排列松散或聚集成团,出现悬浮现象,瘤巨细胞减少或不见瘤巨细胞。舒林酸可抑制BGC-823细胞增殖,使G0／G1期细胞比例增高,S期细胞比例降低。1．2mmol／L舒林酸作用48h,G0／G1期细胞增至93．8％,S期细胞比例降至3．4％。舒林酸作用后,细胞凋亡率显著上升,1．2mmol／L舒林酸作用48h,细胞凋亡率升至54．9％,而ki-67、bcl-2及COX-2蛋白表达阳性率显著降低;透射电镜可观察到细胞凋亡的形态特征及凋亡小体。上述作用均呈时间和剂量依赖性。结论舒林酸可抑制胃癌BGC--823细胞体外生长,其机制涉及影响细胞周期分布、诱导细胞凋亡及抑制ki-67、bcl-2及COX-2蛋白的表达。     

Inhibition of MCL-1 in breast cancer cells promotes cell death in vitro and in vivo

The present studies have examined approaches to suppress MCL-1 function in breast cancer cells, as a means to promote tumor cell death. Treatment of breast cancer cells with CDK inhibitors (flavopiridol; roscovitine) enhanced the lethality of the ERBB1 inhibitor lapatinib in a synergistic fashion. CDK inhibitors interacted with lapatinib to reduce MCL-1 expression and overexpression of MCL-1 or knock down of BAX and BAK suppressed drug combination lethality. Lapatinib-mediated inhibition of ERK1/2 and to a lesser extent AKT facilitated CDK inhibitor-induced suppression of MCL-1 levels. Treatment of cells with the BH3 domain/MCL-1 inhibitor obatoclax enhanced the lethality of lapatinib in a synergistic fashion. Knock out of MCL-1 and BCL-X_L enhanced lapatinib toxicity to a similar extent as obatoclax and suppressed the ability of obatoclax to promote lapatinib lethality. Pre-treatment of cells with lapatinib or with obatoclax enhanced basal levels of BAX and BAK activity and further enhanced drug combination toxicity. In vivo tumor growth data in xenograft and syngeneic model systems confirmed our in vitro findings. Treatment of cells with CDK inhibitors enhanced the lethality of obatoclax in a synergistic fashion. Overexpression of MCL-1 or knock down of BAX and BAK suppressed the toxic interaction between CDK inhibitors and obatoclax. Obatoclax and lapatinib treatment or obatoclax and CDK inhibitor treatment or lapatinib and CDK inhibitor treatment radiosensitized breast cancer cells. Lapatinib and obatoclax interacted to suppress mammary tumor growth in vivo. Collectively our data demonstrate that manipulation of MCL-1 protein expression by CDK inhibition or inhibition of MCL-1 sequestering function by Obatoclax renders breast cancer cells more susceptible to BAX/BAK-dependent mitochondrial dysfunction and tumor cell death.Key words: MCL-1, Lapatinib, Obatoclax, Flavopiridol, Roscovitine, CDK inhibitor, RTK inhibitor, BCL-2 inhibitor, BAK     

Expression profiling of breast cancer cells by differential peptide display

Expression profiling of RNAs or proteins has become a promising means to investigate the heterogeneity of histopathologically defined classes of cancer. Peptides, representing degradation as well as processing products of proteins offer an even closer insight into cell physiology. Peptides are related to the turnover of cellular proteins and are capable to reflect disease-related changes in homoeostasis of the human body. Furthermore, peptides derived from tumor cells are potentially useful markers in the early detection of cancer.In this study, we introduced a method called differential peptide display (DPD) for separating, detecting, and identifying native peptides derived from whole cell extracts. This method is a highly standardized procedure, combining the power of reversed-phase chromatography with mass spectrometry. This technology is suitable to analyze cell lines, various tissue types and human body fluids. Peptide-based profiling of normal human mammary epithelial cells (HMEC) and the breast cancer cell line MCF-7 revealed complex peptide patterns comprising of up to 2300 peptides. Most of these peptides were common to both cell lines whereas about 8% differed in their abundance. Several of the differentially expressed peptides were identified as fragments of known proteins such as intermediate filament proteins, thymosins or Cathepsin D. Comparing cell lines with native tumors, overlapping peptide patterns were found between HMEC and a phylloides tumor (CP) on the one hand and MCF-7 cells and tissue from a invasive ductal carcinoma (DC) on the other hand.     

Inhibition of breast cancer metastasis via PITPNM3 by pachymic acid

Breast cancer metastasis is the most common cause of cancer-related death in women. Thus, seeking targets of breast tumor cells is an attractive goal towards improving clinical treatment. The present study showed that CCL18 from tumor-associated macrophages could promote breast cancer metastasis via PITPNM3. In addition, we found that pachymic acid (PA) could dose-dependently inhibit migration and invasion of MDA-MB-231 cells, with or without rCCL18 stimulation. Furthermore, evidence was obtained that PA could suppress the phosphorylation of PITPNM3 and the combination of CCL18 and PITPNM3. Therefore, we speculate that PA could inhibit breast cancer metastasis via PITPNM3.     

Inhibition of hepatocyte growth factor-induced motility and in vitro invasion of human colon cancer cells by gamma-linolenic acid.

In this study we have determined the effects of the n-6 essential fatty acid gamma-linolenic acid (GLA) on the motility and invasive/metastatic nature of the human colon cancer cell lines HT115, HT29 and HRT18. Cell motility was induced by hepatocyte growth factor/scatter factor (HGF/SF) and measured by both colony scattering and dissociation from carrier beads. Invasiveness was measured in vitro by cellular invasion into extracellular matrix. At concentrations up to 100 microM (which had no effect on cell growth over the duration of the experiments) both cell motility and invasion induced by HGF/SF were markedly reduced by GLA and its lithium salt. The attachment of these cells to the extracellular matrix components (Matrigel and fibronectin) was also inhibited. There were also changes in the cell-surface E-cadherin, but not fibronectin receptor at similar concentrations. It is concluded that n-6 essential fatty acids have the ability to inhibit both motility and invasiveness of human colon cancer cells, perhaps by modifying cell-surface adhesion molecules.     

乳腺癌细胞体外摄取2-脱氧葡萄糖荧光类似物

目的 观察2-脱氧葡萄糖(2-DG)荧光类似物2-N[7-硝基苯-2-乙二酸,34羟氨基]-2-脱氧葡萄糖(2-NBDG)被高表达葡萄糖转运蛋白1(GLUT-1)的乳腺癌细胞靶向摄取的情况.方法 应用逆转录聚合酶链反应(RT-PCR)法和免疫组化法检测乳腺癌MDA-MB-231细胞GLUT-1 mRNA和蛋白的表达,采用Western blot法比较乳腺癌MDA-MB-231细胞和MCF-7细胞GLUT-1的蛋白表达量.应用2-NBDG孵育人乳腺癌MDA-MB-231细胞,采用荧光显微镜及流式细胞仪观察、分析对2-NBDG的摄取情况,比较MDA-MB-231和MCF-7细胞吸收2-NBDG量的差异.结果 RT-PCR和免疫组化检测结果显示,MDA-MB-231细胞高表达GLUT-1;Western blot检测结果进一步显示,MDA-MB-231细胞的GLUT-1表达(0.946 4±0.007)高于MCF-7(0.833±0.010).荧光成像及流式细胞仪分析结果显示,MDA-MB-231细胞能快速摄取2-NBDG,且加入50 mmol/L D-葡萄糖后,荧光强度降低了46.0%.2-NBDG孵育乳腺癌细胞20 min后,MDA-MB-231细胞荧光强度(25.10±0.57)明显高于MCF-7细胞(10.12±0.62).结论 2-NBDG能迅速被高表达GLUT-1的乳腺癌MDA-MB-231细胞靶向吸收.     

Inhibition of breast cancer cell growth in vitro by a tyrosine kinase inhibitor.

Human breast cancer cell proliferation is regulated by growth factors that bind to receptors with intrinsic tyrosine kinase (TK) activity, including the epidermal growth factor (EGF) receptor. To determine whether inhibition of receptor TK activity inhibits tumor growth, we studied the effects of a tyrosine kinase inhibitor, RG-13022, on cultured human breast cancer cells. RG-13022 represents a class of compounds which have been shown to inhibit preferentially the TK activity of the EGF receptor in a cell-free system and also to inhibit EGF-stimulated growth of cultured cells. RG-13022 significantly inhibited EGF-stimulated autophosphorylation of its receptor in two breast cancer cell lines that have abundant, although not amplified, EGF receptor content (MDA-231 and T47D). RG-13022 also inhibited EGF-stimulated DNA synthesis and proliferation of T47D and MCF-7 breast cancer cells in a reversible and dose-dependent manner. Inhibition was observed at 0.1 microM, and it was maximal at 10 microM. The effect was rapid (within 3 h), persisted for 18 h, and was partially reversed by 24 h at 1 microM. At 5 microM, inhibition persisted for more than 50 h. Inhibitory effects were also observed in a panel of estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines. RG-13022 inhibited not only EGF-induced growth but also growth stimulated by insulin, insulin-like growth factor I, insulin-like growth factor II, or transforming growth factor alpha. RG-13022 also totally blocked estrogen-stimulated phosphorylation of the EGF receptor, as well as estrogen-induced cell proliferation, suggesting that functioning TK pathways are required for estrogen action. The TK inhibitor RG-13022 is a potent inhibitor of hormonally regulated growth of human breast cancer. Tyrosine kinase inhibitors have the potential of providing a new strategy for the "endocrine therapy" of breast cancer.     

Functional characterization of E- and P-cadherin in invasive breast cancer cells

Background  

Alterations in the cadherin-catenin adhesion complexes are involved in tumor initiation, progression and metastasis. However, the functional implication of distinct cadherin types in breast cancer biology is still poorly understood.     

In vitro synergistic cytoreductive effects of zoledronic acid and radiation on breast cancer cells

Introduction  

Bisphosphonates are mostly used in the treatment of bone metastases. They have been shown to act synergistically with other chemotherapeutic agents. It is not known, however, whether similar synergistic effects exist with radiation on breast cancer cells.     

Membrane disrupting lytic peptide conjugates destroy hormone dependent and independent breast cancer cells in vitro and in vivo

We have prepared conjugates of a membrane disrupting lytic peptide (hecate) and a 15-amino acid segment of the -chain of CG and hecate and the decapeptide, luteinizing hormone releasing hormone (LHRH). We have tested the concept that these conjugates will target breast cancer cells expressing LH/CG or LHRH receptors. In previous studies, we were able to destroy prostate cancers in vitro and in vivo with lytic peptide conjugates [1]. Hecate, hecate–CG and LHRH–hecate were added to cultures of the human breast cancer cell lines MCF-7 and MDA-MB-435S. Hecate and its conjugates showed concentration dependent toxicity to both cell lines. The lytic peptide alone showed similar EC₅₀ values for both cell lines; however, there was a significant difference between the EC₅₀ values when the conjugates were tested. The hormone dependent MCF-7 cell line was less sensitive to the CG conjugate than to the LHRH conjugate; the reverse was found for the hormone independent MDA-MB-435S cells. Removal of steroids decreased the sensitivity of MCF-7 cells to both lytic peptide conjugates and this sensitivity could be restored by adding estradiol. Activation of protein kinase C further increased the sensitivity to the drug. MDA-MB-435S xenografts were established in intact female athymic nude mice, which were treated once a week for 3 weeks with hecate–CG via the lateral tail vein. The ability of hecate–CG to destroy xenografts of human breast cancer cells (MDA-MB-435S) in nude mice was demonstrated for the first time. We conclude that hecate–CG and LHRH–hecate conjugates could serve as useful drugs for the treatment of breast cancer.