5,7,4⿲-Trihydroxy-6,8-diprenylisoflavone and lupalbigenin,active components of Derris scandens,induce cell death on breast cancer cell lines

BackgroundNatural products are a potential source for cancer chemotherapeutic development. This current study was performed to investigate the anti-tumor potential of 5,7,4⿲-trihydroxy-6,8-diprenylisoflavone (TD) and lupalbigenin (LB), plant flavonoids found in Derris scandens Benth (family: Leguminosae), in cancer and normal cell lines.MethodsThe human breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468, the human colon cancer cell line SW-620, and the mouse fibroblast cell line L-929 were used to test their anti-cancer activity. Apoptotic cell levels were measured by staining with annexin-V and propidium iodide and Western blot analysis was performed to confirm the apoptotic mechanism.ResultsThe results revealed that TD and LB showed specific cytotoxicity against MDA-MB-231 and MCF-7 cells. To elucidate mode of cell death via cytotoxic activities, breast cancer cell lines were treated. TD and LB induced MDA-MB-231 and MCF-7 cells to apoptosis, with the highest number of apoptotic cells at 24 and 72 h, respectively. Furthermore, TD and LB inhibited cell cycle progression via up-regulation of p21. Both compounds stimulated apoptosis through down-regulation of bcl-2, up-regulation of bax and releasing of cytochrome C proteins.ConclusionsTD and LB have significant anti-cancer effects against human breast cancer cells via cell cycle arrest and the induction of apoptosis through mitochondria signaling pathways, and may be potential anti-cancer agents for the treatment of breast cancer.     

Caprin-1 is a novel microRNA-223 target for regulating the proliferation and invasion of human breast cancer cells

MicroRNAs (miRNAs) are 21–22 nucleotides regulatory small non-coding RNAs that inhibit gene expression by binding to complementary sequences especially the 3’ untranslated region (3’UTR) of mRNA. One miRNA can target many messenger RNAs, leading to a complex metabolic network. Previous studies have shown that miRNA-223 regulates migration and invasion of tumor cells and targets cytoplasmic activation/proliferation-associated protein-1 (Caprin-1). In the present study, we detected the expression of miRNA-223 and Caprin-1 in MCF-7, T-47D and MDA-MB-231 cancer cell lines, and MCF-10A normal breast cell line, and analyzed the role of miRNA-223 in Caprin-1-induced proliferation and invasion of human breast cancer cells. We found that miRNA-223 expression levels are significantly lower in MCF-7, T-47D and MDA-MB-231 cancer cells than in MCF-10A normal breast cells, while Caprin-1 expression is higher in cancer cells than in normal breast cells. The most malignant cancer cell line MDA-MB-231 has the lowest expression of miR-223, but the highest expression of Caprin-1. Further, we found that miR-223 targets the 3’UTR of Caprin-1 miRNA and down-regulates the expression of Caprin-1. We also found that over-expression of Caprin-1 can promote the proliferation and the invasion of breast cancer cells, but miRNA-223 can inhibit the proliferation and the invasion. miRNA-223-induced inhibition can be reversed by ectopic over-expression of Caprin-1. These findings suggest that miR-223 may suppress the proliferation and invasion of cancer cells by directly targeting Caprin-1. Our study also indicates that expression levels of miR-223 and Caprin-1 can be used to predict the state of cancer in breast cancer patient.     

印记基因SLC22A18的表达与乳腺癌侵袭能力的关系

目的:研究印记基因SLC22A18(solute carrier family 22,member 18)在乳腺癌中的表达情况及其与乳腺癌侵袭能力的关系。方法:采用Transwell方法评估2种不同恶性程度的乳腺癌细胞株MDA-MB-231(恶性程度高)和MCF-7(恶性程度低)的侵袭转移能力。分别采用实时荧光定量逆转录聚合酶链反应(RT-PCR)和蛋白质印迹法检测SLC22A18的mRNA和蛋白在这2种乳腺癌细胞株中的表达情况。结果:MDA-MB-231细胞株恶性程度高,穿过膜的细胞多,侵袭能力强;MCF-7细胞株恶性程度低,穿过膜的细胞少,侵袭能力弱;SLC22A18在MCF-7中的mRNA和蛋白表达水平高于MDA-MB-231;差异有统计学意义(P0.01)。结论:印记基因SLC22A18的表达与乳腺癌细胞的侵袭能力相关,该基因有望作为一个抑癌基因抑制乳腺癌的转移。     

表没食子儿茶素没食子酸酯对乳腺癌细胞生长及迁移的影响

目的：探讨表没食子儿茶素没食子酸酯（EGCG）对乳腺癌细胞MCF-7生长的影响及对乳腺癌细胞MDA-MB-231迁移的影响。方法：MCF-7细胞培养贴壁之后,加入EGCG处理,2d后收集蛋白,采用Western Blot检测磷酸化p38丝裂原活化蛋白激酶（phospho-p38MAPK）的表达;同样处理后收集活细胞,用细胞计数法检测细胞的存活;取对数生长期的MDA-MB-231细胞,分至6孔板培养,使用EGCG处理后,采用细胞划线法探测乳腺癌细胞的迁移。结果：使用EGCG处理乳腺癌细胞后,phospho-p38MAPK的表达降低,EGCG处理乳腺癌细胞4d后其增殖率降低50%,迁移活性降低。结论：EGCG处理乳腺癌细胞能抑制肿瘤细胞的生长以及迁移,这与p38 MAPK信号通路相关。     

Stathmin表达水平与乳腺癌细胞侵袭能力相关性研究

目的 探讨乳腺癌细胞MDA-MB-231和MCF-7中Stathmin基因表达水平与细胞生长、黏附、侵袭等生物学行为之间的关系,为进一步研究乳腺癌转移机制奠定实验基础。方法 应用RT-PCR和Western Blot方法检测MDA-MB-231和MCF-7细胞中Stathmin基因的表达水平,同时利用细胞增殖试验、细胞黏附试验和细胞侵袭试验检测MDA-MB-231和MCF-7细胞的生长、黏附、侵袭能力,分析Stathmin表达与细胞的生长、黏附、侵袭能力之间的关系。结果 RT-PCR和Western Blot检测结果显示,Stathmin基因在MDA-MB-231和MCF-7细胞中表达均高于正常对照细胞(F=10.173,P<0.05),且MDA-MB-231细胞中的表达水平明显高于MCF-7细胞中的表达水平(t=4.562,P<0.05)。而MDA-MB-231细胞在生长、黏附和侵袭能力方面均强于MCF-7细胞(P<0.05)。结论 Stathmin表达水平高的乳腺癌细胞相应的生长、黏附、侵袭能力较强,Stathmin表达水平与细胞侵袭能力密切相关。     

Recombinant epoetins do not stimulate tumor growth in erythropoietin receptor-positive breast carcinoma models

We investigated the significance of erythropoietin receptor (EPOR) expression following treatment with recombinant human erythropoietin (rHuEPO; epoetin alpha) and the effect of recombinant epoetins (epoetin alpha, epoetin beta, and darbepoetin alpha) alone or in combination with anticancer therapy on tumor growth in two well-established preclinical models of breast carcinoma (MDA-MB-231 and MCF-7 cell lines). Expression and localization of EPOR under hypoxic and normoxic conditions in MDA-MB-231 and MCF-7 cells were evaluated by immunoblotting, flow cytometry, and immunohistochemistry. EPOR binding was evaluated using [125I]rHuEPO. Proliferation, migration, and signaling in MDA-MB-231 and MCF-7 cells following treatment with rHuEPO were evaluated. Tumor growth was assessed following administration of recombinant epoetins alone and in combination with paclitaxel (anticancer therapy) in orthotopically implanted MDA-MB-231 and MCF-7 breast carcinoma xenograft models in athymic mice. EPOR expression was detected in both tumor cell lines. EPOR localization was found to be exclusively cytosolic and no specific [125I]rHuEPO binding was observed. There was no stimulated migration, proliferation, or activation of mitogen-activated protein kinase and AKT following rHuEPO treatment. In mice, treatment with recombinant epoetins alone and in combination with paclitaxel resulted in equivalent tumor burdens compared with vehicle-treated controls. Results from our study suggest that although EPOR expression was observed in two well-established breast carcinoma cell lines, it was localized to a cytosolic distribution and did not transduce a signaling cascade in tumors that leads to tumor growth. The addition of recombinant epoetins to paclitaxel did not affect the outcome of paclitaxel therapy in breast carcinoma xenograft models. These results show that recombinant epoetins do not evoke a physiologic response on EPOR-bearing tumor cells as assessed by numerous variables, including growth, migration, and cytotoxic challenge in preclinical in vivo tumor models.     

MicroR-760 suppresses cancer stem cell subpopulation and breast cancer cell proliferation and metastasis: By down-regulating NANOG

Background and objectiveEmerging evidences suggest that cancer stem cells are responsible for tumor aggressive, metastasis and therapeutic resistance. To data, the mechanism underlying breast cancer stem cell (BCSC) population within tumor metastasis remains to be fully elucidated. The current study was to investigate the potential role of microRNA-760 (miR-760) and its associated target gene in population and metastasis of BCSC.MethodsCharacteristic BCSCs surface markers (CD44⁺/CD24^−/low) were determined by flow cytometry in breast cancer MCF-7 and BT-549 cells. Quantitative RT-PCR was used to evaluate miR-760 and NANOG mRNA expression. Expression of NANOG protein was determined using western blot. Cell proliferation was determined by MTT assay. The model of breast cancer cell xenograft was used to evaluate the effect of miR-760 on tumor growth.ResultsBT-549 cell has substantially more CD44⁺/CD24^−/low subpopulation than MCF-7 cell. Moreover, BT-549 cell expressed lower level of miR-760 and higher level of NANOG than MCF-7cell. By result from cellular miR-760 modulation, we found that miR-760 overexpression suppressed CD44⁺/CD24^−/low population as well as inhibited cell proliferation and migration of BT-549. On the contrary, knockdown of miR-760 promoted CD44⁺/CD24^−/low population and migration of MCF-7 cells. By luciferase reporter assay, miR-760 was proved to be functional associated with NANOG via regulating its expression. This functional interaction was showed to be involved in controlling proliferation and migration of MCF-7 and BT-549 cell.ConclusionThese data suggest that the target of miR-760/NANOG axis may represent a new therapeutic approach to suppress breast cancer stem cell subpopulation thereby prevent cancer metastasis.     

巨噬细胞迁移抑制因子受体CD74与乳腺癌转移及预后的相关性

目的：研究巨噬细胞迁移抑制因子受体CD74在乳腺癌细胞株MCF-7和MDAMB-231以及人乳腺癌组织中的表达及其与乳腺癌患者预后的关系。方法：应用荧光定量聚合酶链式反应（fluorescentquantitativepo[ymerasechainreaction,FQ-PCR）和蛋白质印迹（Westernblotting）方法分别检测不同转移潜能乳腺癌细胞株MCF-7和MDA—MB-231CD74的mRNA和蛋白表达量,并比较CD74在两者间的表达差异;应用免疫组织化学检测89例乳腺癌组织中CD74的表达情况,分析CD74与乳腺癌患者总生存率和无瘤生存率的关系。结果：高转移潜能乳腺癌细胞株MDA—MB-231中CD74表达显著高于低转移潜能细胞株MCF-7（P〈0．001）。CD74表达水平是乳腺癌总生存率（P=0．001）和无瘤生存率（P〈0．001）的独立预测因素。结论：CD74的表达水平与乳腺癌患者预后呈负相关。     

Effects of decitabine on the expression of selected endogenous control genes in human breast cancer cells

To quantify gene expression levels, appropriate controls have to be used to adjust for experimental variation. Endogenous control genes are widely used as they are stably expressed independent of cell cycle and experimental conditions, however, they can be altered upon drug treatment. DNA methylation is widely studied in chemotherapy drug resistance and the DNA methylation inhibitor decitabine showed promising results reversing drug resistance in cancer. We aimed to investigate the effect of different decitabine concentrations on the expression of selected endogenous control genes (GAPDH, 18S rRNA, PPIA, RPL13A, OAZ1) in two docetaxel-resistant human breast cancer cell lines (MCF-7 and MDA-MB-231) compared to untreated cells. In MCF-7 cells, 18S rRNA remained stable, however, GAPDH, PPIA and OAZ1 gene expression was increased after treatment. RPL13A was stably expressed at 8 μM decitabine but was increased at lower drug concentrations. In MDA-MB-231 cells, GAPDH levels remained relatively stable following decitabine treatment and so was PPIA expression at low decitabine concentrations. Decitabine increased 18S rRNA, RPL13A and OAZ1 gene expression. In this study, we observed cell line specific effects of decitabine and suggest that 18S rRNA is most suitable to use in MCF-7 cells, while GAPDH is recommended to use in MDA-MB-231 cells during decitabine treatment.     

Rab27A在4种不同人乳癌细胞株中的表达

目的检测Rab27A在4种人乳癌细胞中的定位、表达情况,初步研究Rab27A表达对乳癌细胞生物学特性的影响。方法采用RT-PCR技术,检测Rab27A mRNA在4种人乳癌细胞MCF-7、MDA-MB-231、MDA-MB-435和MDA-MB-435HM中的表达并进行半定量分析。结果Rab27A mRNA在人乳癌细胞MDA-MB-231、MDA-MB-435及MDA-MB-435HM中的表达水平分别是MCF-7中的2.1、3.4和6.9倍,差异有显著意义（F=17.74～136.23,P〈0.05、0.01）;Rab27A蛋白在人乳癌细胞MDA-MB-231、MDA-MB-435及MDA-MB-435HM中的表达分别是MCF-7中的2.8、4.9和9.2倍,差异有显著性（F=37.74～154.29,P〈0.05、0.01）。Rab27A蛋白弥散性分布于MCF-7、MDA-MB-231和MDA-MB-435细胞浆中,MDA-MB-231和MDA-MB-435中又可见Rab27A蛋白在核周凝聚。结论Rab27A表达可能与乳癌细胞侵袭转移能力有关。     

Expression and silencing of the microtubule-associated protein Tau in breast cancer cells

The microtubule-associated protein Tau has been reported to be a predictive factor for clinical response to taxanes in metastatic breast cancer. We generated a panel of eight taxane-resistant variants from four human breast cancer cell lines (MCF-7, T-47D, MDA-MB-231, and BT-549). Four variants had higher levels of Tau compared with their T-47D and MDA-MB-231 parental cells. Using isoform-specific primers, we found that Tau 0N, 1N, 2N, 3R, and 4R isoforms are overexpressed in the resistant variants, as is Tau exon 6 but not exons 4A or 8. To determine whether Tau overexpression produces resistance to taxanes, we derived three independent T-47D clones stably overexpressing Tau 3R and 4R isoforms. Tau overexpression did not result in taxane resistance compared with parental cells transfected with vector alone. We then knocked down Tau expression in three cell lines that expressed Tau constitutively (MCF-7 and ZR-75-1 breast cancer cells, and OVCAR-3 ovarian cancer cells). Lentivirus-mediated silencing of Tau expression in MCF-7 and OVCAR-3 cells did not result in increased taxane sensitivity compared with luciferase short hairpin RNA-infected cells and uninfected parental cells. Transient silencing using Tau-specific small interfering RNAs also did not alter taxane sensitivity relative to nontargeting controls in both MCF-7 and ZR-75-1 cells. These results show that neither overexpression nor depletion of Tau modulates cellular sensitivity to taxanes. Although Tau overexpression has been reported to be a predictive marker of taxane resistance, it is not likely to be a direct mechanism of taxane resistance in breast cancer.     

TNF-α可诱导乳腺癌细胞凋亡

目的研究TNF-α对乳腺癌的影响。方法采用RT-PCR和WesternBlotting分析30例乳腺浸润性导管癌及癌旁正常乳腺组织,乳腺正常上皮细胞系及乳腺癌细胞系中TNF-α的表达情况;采用流式细胞术观察TNF-α对乳腺癌细胞凋亡的影响。结果RT-PCR和Western Blotting结果碌示,TNF-αmRNA和蛋白在乳腺癌组织中表达都明显低于配对的癌旁正常组织（P〈0．05）,在乳腺上皮细胞系中表达均高于乳腺癌三利一细胞系,流式细胞术检测结果显示与未处理组桐比．经TNF-α处理的MDA-MB-435S（16．7±0．31）和MCF7（18．6±0．42）细胞的凋亡率明显增加,差异均有统计学意义（P〈0．05）。结论TNF-α可促进乳腺癌细胞的凋亡,TNF-α可为乳腺癌的治疗提供新靶点。     

Tamoxifen-resistant human breast cancer cell growth: inhibition by thioridazine, pimozide and the calmodulin antagonist, W-13.

Estrogen receptor (ER)-negative human breast cancer cell lines (MDA-MB-231 and MDA-MB-435) and ER-positive derivatives of the MCF-7 cell line selected for growth in the presence of antiestrogens (LY2 and RR) were used as in vitro models of tamoxifen-resistant human breast cancer in this study. The sensitivity of the tamoxifen-sensitive (MCF-7) and tamoxifen-resistant human breast cancer cell growth to two noncytotoxic neuroleptic drugs, pimozide and thioridazine, and the anticalmodulin agent, W-13, were compared. Inhibition of cell growth was measured as a decrease in cell number following a 72-h incubation with drug. Growth of the ER-negative cell lines MDA-MB-231 and MDA-MB-435 was inhibited by all three drugs. The average Ki values in these two lines were 6.3 and 3.8 microM for pimozide and 4.1 and 15 microM for thioridazine, respectively. Both ER-negative cell lines were more sensitive than MCF-7 cells to growth inhibition by W-13. MCF-7 cells selected for antiestrogen resistance were sensitive to growth inhibition by W-13 and thioridazine (LY2, average Ki = 10.4 microM; RR, average Ki = 5.2 microM). LY2 and RR cells were resistant to pimozide except when treated with estradiol (Ki = 4.6 and 7.9 microM, respectively). Pimozide, thioridazine and W-13 all exerted different effects on the distribution of human breast cancer cells within the cell cycle, suggesting that each drug may utilize a distinct pathway for inhibition of cell growth. We conclude that all three drugs are potential noncytotoxic alternatives to tamoxifen for the treatment of tamoxifen-resistant human breast cancer.     

A novel all-trans retinoid acid derivatives inhibits the migration of breast cancer cell lines MDA-MB-231 via myosin light chain kinase involving p38-MAPK pathway

Objective

To explore the effect and its probable mechanism of a synthetic retinoid 4-amino-2-tri-fluoromethyl-phenyl ester (ATPR) on the migration of human breast cancer MDA-MB-231 cells.

Methods

MTT assay was performed to measure the proliferation of MDA-MB-231 cells treated with different concentrations of all-trans retinoic acid (ATRA) and ATPR. The effect of ATPR and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, an inhibitor of p38, on the migration of MDA-MB-231 cells were analyzed by wound healing assay. The expression of MLCK and phosphorylation of myosin light chain (MLC), ERK, JNK, p38 proteins were detected by western blot

Results

After the cells were treated by ATRA and ATPR, the proliferation and migration of breast cancer MDA-MB-231 cells were inhibited significantly. The IC 50 of ATRA and ATPR is 34.08 μmol/l and 18.06 μmol/l respectively. The relative migration rate of MDA-MB-231 cells treated with ATPR reached 50% at 48 h while the ATRA group is over 90%. The relative migration rate of ML-7 group and SB group had significant decrease compared with control group. The expression level of MLCK and phosphorylation of MLC of breast cancer cells was reduced when the cells were treated by ATPR with 48 h, the phosphorylation of ERK, JNK and p38 in breast cancer also reduced when cells were treated by ATPR with 2 h. In addition, ML-7 (50 μmol/l) could inhibit the phosphorylation of p38 and SB (50 μmol/l) could inhibit the expression of MLCK and phosphorylation of MLC.

Conclusions

ATPR had a better inhibition on the proliferation and the migration of breast cancer MDA-MB-231 cells than ATRA, and its probable mechanism was associated with the down regulation of expression of MLCK and phosphorylation of MLC protein involving p38-MAPK pathway.     

厚朴酚抑制体外乳腺癌细胞株生物活性的实验研究

[目的]探讨中药厚朴提取物厚朴酚(Magnolol,Mag)抑制不同乳腺癌细胞株生物活性的作用.[方法]选取不同乳腺癌高转移细胞株(MCF-7、MDA-MB-231),在培养液中分别加入不同浓度的Mag溶液,观察处理后细胞的凋亡、侵袭、转移和黏附能力,判断Mag对乳腺癌细胞株生物活性的影响.[结果]Mag可以抑制细胞的抗凋亡、侵袭、转移和黏附能力(P<0.05),随着Mag浓度的增大,抑制生物活性的作用逐渐增强(P<0.05).[结论]Mag可以通过抑制不同受体状态乳腺癌细胞株的抗凋亡率、侵袭能力、黏附能力和迁移能力,从而抑制其生物活性,在乳腺癌的治疗及预防乳腺癌复发、转移中具有重要的临床应用价值.     

乳腺癌细胞膜蛋白的SELDI-TOF-MS分析

目的比较乳腺癌细胞株和正常乳腺上皮细胞膜蛋白指纹图谱,为乳腺癌亚细胞蛋白组学的研究奠定基础。方法试剂盒提取两株乳腺癌细胞株MDA-MB-231,MCF-7及一株乳腺正常上皮细胞HBL-100的膜蛋白,SEL-DI-TOF-MS分析蛋白指纹图谱,寻找差异蛋白。结果在分子量2kD-100kD范围内3株细胞均可检测到近100个蛋白点,MDA-MB-231和MCF-7与HBL-100相比,膜蛋白差异点分别为32个和34个(P<0.05),其中7.8kD,8.3kD和8.8kD的蛋白在两株癌细胞中表达均降低,9.6kD的蛋白在两株癌细胞中表达均增高。结论 SELDI-TOF-MS可以快速灵敏地检测到乳腺癌亚细胞蛋白水平的差异,为乳腺癌早期诊断提供依据。     

表柔比星对乳腺癌细胞c-FLIP表达的影响

目的:探讨表柔比星对乳腺痛细胞中c-FLIP表达的影响.方法:以MCF-7及MDA-MB-231乳腺癌细胞株为对象分两组:处理组中加入2 mg/L的表柔比星分别作用24 h、48 h和72 h,对照组加入等量生理盐水.采用RT-PCR技术检测两组细胞株中c-FLIP的表达,漉式细胞仪检测细胞凋亡百分率.结果:MCF-7及MDA-MB-231乳腺癌细胞中有c-FLIP的表达;与对照组相比,表柔比星作用24 h,乳腺癌细胞中c-FLIP的表达明显下降,48 h时c-FLIP表达有所升高,72 h时表达最低.随着表柔比星作用时间延长,细胞凋亡率逐渐增加,72 h达到最高.结论:表柔比星通过抑制c-FLIP的表达促进乳腺癌细胞的凋亡.