Anti-proliferative Activities of Metallic Nanoparticles in an in Vitro Breast Cancer Model

Aims: To investigate effect of metallic nanoparticles, silver (AgNPs) and gold nanoparticles (AuNPs) asantitumor treatment in vitro against human breast cancer cells (MCF-7) and their associated mechanisms. Thiscould provide new class of engineered nanoparticles with desired physicochemical properties and may presentnewer approaches for therapeutic modalities to breast cancer in women. Materials and Methods: A humanbreast cancer cell line (MCF-7) was used as a model of cells. Metallic nanoparticles were characterized usingUV-visible spectra and transmission electron microscopy (TEM). Cytotoxic effects of metallic nanoparticles onMCF-7 cells were followed by colorimetric SRB cell viability assays, microscopy, and cellular uptake. Natureof cell death was further investigated by DNA analysis and flow cytometry. Results: Treatment of MCF-7 withdifferent concentrations of 5-10nm diameter of AgNPs inhibited cell viability in a dose-dependent manner, withIC50 value of 6.28μM, whereas treatment of MCF-7 with different concentrations of 13-15nm diameter of AuNPsinhibited cell viability in a dose-dependent manner, with IC50 value of 14.48μM. Treatment of cells with a IC50concentration of AgNPs generated progressive accumulation of cells in the S phase of the cell cycle and preventedentry into the M phase. The treatment of cells with IC50 concentrations of AuNPs similarly generated progressiveaccumulation of cells in sub-G1 and S phase, and inhibited the entrance of cells into the M phase of the cell cycle.DNA fragmentation, as demonstrated by electrophoresis, indicated induction of apoptosis. Conclusions: Ourengineered silver nanoparticles effectively inhibit the proliferation of human breast carcinoma cell line MCF-7in vitro at high concentration (1000 μM) through apoptotic mechanisms, and may be a beneficial agent againsthuman carcinoma but further detailed study is still needed.     

Bactericidal Application and Cytotoxic Activity of Biosynthesized Silver Nanoparticles with an Extract of the Red Seaweed Pterocladiella capillacea on the HepG2 Cell Line

Background: Nano-biotechnology is recognized as offering revolutionary changes in various fields of medicine.Biologically synthesized silver nanoparticles have a wide range of applications. Materials and Methods: Silvernanoparticles (AgNPs) were biosynthesized with an aqueous extract of Pterocladiella (Pterocladia) capillacea,used as a reducing and stabilizing agent, and characterized using UV-VIS spectroscopy, Fourier Transform Infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM) and energy dispersive analysis (EDX). Thebiosynthesized AgNPs were tested for cytotoxic activity in a human hepatocellular carcinoma (HepG2) cell linecultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% antibiotic andantimycotic solution and 2 mM glutamine. Bacterial susceptibility to AgNPs was assessed with Staphylococcusaureus, Bacillus subtilis [Gram+ve] and Pseudomonas aeruginosa and Escherichia coli [Gram-ve]. The agar welldiffusion technique was adopted to evaluate the bactericidal activity of the biosynthesized AgNPs using Ampicillinand Gentamicin as gram+ve and gram-ve antibacterial standard drugs, respectively. Results: The biosynthesizedAgNPs were 11.4±3.52 nm in diameter. FT-IR analysis showed that carbonyl groups from the amino acid residuesand proteins could assist in formation and stabilization of AgNPs. The AgNPs showed potent cytotoxic activityagainst the human hepatocellular carcinoma (HepG2) cell line at higher concentrations. The results also showedthat the biosynthesized AgNPs inhibited the entire panel of tested bacteria with a marked specificity towardsBacillus subtillus. Conclusions: Cytotoxic activity of the biosynthesized AgNPs may be due to the presence ofalkaloids present in the algal extract. Our AgNPs appear more bactericidal against gram-positive bacteria (B.subtillus).     

Differential anti-tumor activity of coriolus versicolor (Yunzhi) extract through p53- and/or Bcl-2-dependent apoptotic pathway in human breast cancer cells

Coriolus versicolor (CV), also called Yunzhi, has been demonstrated to exert anti-tumor effects on various types of cancer cells, but the underlying mechanism has not been fully elucidated. The present study aimed to evaluate the in vitro anti-tumor activity of a standardized aqueous ethanol extract prepared from CV on four breast cancer cell lines using MTT assay, and test whether the mechanism involves apoptosis induction and modulation of p53 and Bcl-2 protein expressions using cell death detection ELISA, p53 and Bcl-2 ELISAs respectively. Our results demonstrated that the CV extract dose-dependently suppressed the proliferation of three breast tumor cell lines, with ascending order of IC50 values: T-47D, MCF-7, MDA-MB-231, while BT-20 cells were not significantly affected. Tumoricidal activity of the CV extract was found to be comparable to a chemotherapeutic anti-cancer drug, mitomycin C. Nucleosome productions in apoptotic MDA-MB-231, MCF-7 and T-47D cells were significantly augmented in a time-dependent manner and paralleled the anti-proliferative activity of CV extract. Expression of p53 protein was significantly upregulated only in T-47D cells treated with the CV extract in a dose- and time-dependent fashion, but not in MCF-7 (except at 400 mug/ml after 16 h) and MDA-MB-231 cells. The CV extract significantly induced a dose-dependent downregulation of Bcl-2 protein expression in MCF-7 and T-47D cells, but not in MDA-MB-231 cells. These results suggested that apoptosis induction, differentially dependent of p53 and Bcl-2 expressions, might be the possible mechanism of CV extract-mediated cytotoxicity in human breast cancer cells in vitro.     

积雪草甙诱导肿瘤细胞凋亡及增强长春新碱的抗肿瘤作用

背景与目的:中药积雪草的三萜类成分积雪草甙(asiaticoside,ATS)具有促进胶原蛋白合成作用,对创伤愈合有较好的疗效。近年有文献报道积雪草甙有一定的抗肿瘤活性。本文研究积雪草甙诱导肿瘤细胞凋亡及其与长春新碱(vincristine,VCR)的协同作用。方法:采用MTT法检测积雪草甙单用或合用VCR对KB、KBv200、MCF-7和MCF-7/ADM细胞的增殖抑制作用,流式细胞术分析KB细胞周期和凋亡率的变化,琼脂糖凝胶电泳、荧光显微镜和Westernblot方法观察KB细胞凋亡和细胞周期相关性质改变。结果:积雪草甙对KB、KBv200、MCF-7和MCF-7/ADM细胞的IC50分别为(1.11±0.13)、(1.82±0.08)、(1.58±0.15)mg/ml和(3.25±0.46)mg/ml,且KBv200和MCF-7/ADM细胞对积雪草甙表现出与相应的亲本细胞相近的药物敏感性。经积雪草甙处理后的KB细胞表现出凋亡细胞特征性的改变。积雪草甙与VCR合用对多种肿瘤细胞均有显著协同作用,合并用药组KB细胞凋亡率、Bcl-2蛋白磷酸化水平明显高于单用组,且仅有合用组检测到活化后的caspase-3蛋白。积雪草甙与VCR合用组KB细胞阻滞于S-G2/M期的比率和CyclinB1蛋白的表达水平高于单用组,P34cdc2蛋白表达略有降低。结论:积雪草甙可诱导肿瘤细胞凋亡并与长春新碱显示协同作用,有可能作为生化调节剂应用于肿瘤化     

维生素D3对人乳腺癌细胞株表皮生长因子受体mRNA表达的影响

目的 探讨维生素Ｄ３对人乳腺癌细胞株细胞增殖等生物学行为的影响。方法 选用两种人类乳腺癌细胞株ＭＣＦ－７及ＺＲ－７５－１,应用细胞培养及核酸分子杂交方法,研究使用维生素Ｄ３以及并用三苯氧胺后,上述细胞株生长及表皮生长因子受体（ＥＧＦＲ）ｍＲＮＡ表达的改变。结果 （１０＾－１０～１０＾－７）ｍｏｌ／Ｌ维生素Ｄ３对ＭＣＦ－７及ＺＲ－７５－１细胞呈现明显的抑制生长作用,并能抑制（１０＾－９～１０＾－７）     

Bioactive Compounds in the Ethanol Extract of Marine Sponge Stylissa carteri Demonstrates Potential Anti-Cancer Activity in Breast Cancer Cells

Objective: Despite advanced treatment options available, drug resistance develops in breast cancer (BC) patientsrequiring novel effective drugs. Stylissa carteri, a marine sponge predominantly living in Indonesia territories, hasnot been extensively studied as anti-cancer. Therefore, this study targeted to assess the anti-tumor activity of theethanol extract of S. carteri in BC cells. Methods: S. carteri was collected from Pramuka Island, at Kepulauan SeribuNational Park, Jakarta, Indonesia and extracted using ethanol. Different BC cells including MDA MB 231, MDAMB 468, SKBR3, HCC-1954 and MCF-7 cells were treated with this extract for cytotoxic analysis using MTT assay.Spheroid growth assay and apoptosis assay were conducted in HCC-1954 cells. In addition, cell migration analysis andsynergistic activity with doxorubicin or paclitaxel were conducted in MDA MB 231 cells. This extract was subjectedalso for GC-MS analysis. Results: The results show that ethanol extract of S. carteri demonstrated a cytotoxic activityin BC cells. The IC50 of this extract was lower 15 μg/ml in MDA MB 231, MDA MB 468, SKBR3, and HCC-1954cells. Moreover, this extract inhibited spheroids growth and induced apoptosis in HCC-1954 cells. It inhibited cellmigration and demonstrated a synergistic activity with doxorubicin or paclitaxel on triggering cell death in MDA MB231 cells. Furthermore, GC-MS analysis indicated that this extract contained 1,2-Benzenediol, Dibutyl phthalate and9,12-Octadecadienoic acid, ethyl ester. Conclusion: Our preliminary data indicate a potential anti-tumor activity ofethanol extract of S. carteri in breast cancer cells.     

Anti-cancer effect of metformin by suppressing signaling pathway of HER2 and HER3 in tamoxifen-resistant breast cancer cells

Development of new therapeutic strategies is becoming increasingly important to overcome tamoxifen resistance. Recently, much interest has been focused on anti-tumor effects of metformin commonly used to treat type II diabetes. Increased protein expression and signaling of epidermal growth factor receptor (EGFR) family is a possible mechanism involved in tamoxifen resistance. Since HER2/HER3 heterodimers are able to induce strong downstream signaling and activate various biological responses such as cellular proliferation and growth, we investigated the anti-cancer effect of metformin by inhibition of signaling pathway via downregulation of HER2 and HER3 using tamoxifen-resistant MCF-7 (TR MCF-7) cells. Compared to MCF-7 cells, TR MCF-7 cells showed increased expression of EGFR, HER2, and HER3, and metformin inhibited the expression of these proteins in a dose- and time-dependent manner. Metformin inhibited activation of HER2 (Tyr1248)/HER3 (Tyr1289)/Akt (Ser473) as well as cell proliferation and colony formation by estrogenic promotion in MCF-7 and TR MCF-7 cells. Known as a HER3 ligand, heregulin (HRG)-β1-induced phosphorylation of HER2, HER3 and Akt, and protein interaction of HER2/HER3 and colony formation were inhibited by metformin in both cells. Consistent with the results in the two cell lines, we identified that metformin inhibited HER2/HER3/Akt signaling axis activated by HRG-β1 using the HER2 and HER3-overexpressing breast cancer cell line SK-BR-3. Lastly, lapatinib-induced HER3 upregulation was significantly inhibited by treatment of metformin in HER3 siRNA-transfected TR MCF-7 cells. These data suggest that metformin might overcome tamoxifen resistance through the inhibition of expression and signaling of receptor tyrosine kinase HER2 and HER3.     

乳腺癌中COX-2表达及其抑制剂塞来昔布对乳腺癌细胞系增生和凋亡的影响

目的为进一步观察COX-2对乳腺癌的作用,我们检测了乳腺癌组织、乳腺癌细胞株MCF-7和B37中COX-2的表达,并观察选择性COX-2抑制剂塞来昔布对乳腺癌细胞株生长和凋亡的影响。方法应用免疫组织化学染色的方法检测132例乳腺癌组织标本中COX-2蛋白的表达;应用免疫细胞化学染色、原位杂交、逆转录多聚酶联反应（RT-PCR）的方法,分别检测COX-2蛋白和mRNA在乳腺癌细胞株MCF-7、B37中的表达;采用MTT法、AO/EB双荧光染色法和流式细胞术,研究塞来昔布对乳腺癌细胞增殖和凋亡的影响。结果52.3%(69/132)的乳腺癌组织标本表达COX-2蛋白;在乳腺癌细胞株MCF-7和B37中均有COX-2的表达,25μmol/L塞来昔布作用72h后,COX-2表达明显减少;25、50、100μmol/L的塞来昔布与MCF-7细胞作用72h,生长抑制率分别为36.3%、57.7%、74.5%。100μmol/L塞来昔布作用72h后,MCF-7细胞凋亡率为38.5％。结论乳腺癌组织和乳腺癌细胞株MCF-7、B37中存在COX-2的表达;塞来昔布可抑制乳腺癌细胞增殖,并促进细胞凋亡,塞来昔布可能作为新的药物治疗乳腺癌。     

Scutellaria baicalensis, a herbal medicine: anti-proliferative and apoptotic activity against acute lymphocytic leukemia, lymphoma and myeloma cell lines

Scutellaria baicalensis (S.B.) is a widely used Chinese herbal medicine. We initially investigated its in vitro anti-tumor activities. S.B inhibited the growth of ALL, lymphoma and myeloma cell lines by inducing apoptosis and cell cycle arrest at clinically achievable concentrations. The anti-proliferative effect was associated with mitochondrial damage, modulation of the Bcl family of genes, increased level of the CDK inhibitor p27(KIP1) and decreased level of c-myc oncogene. HPLC analysis of S.B. showed it contains 21% baicalin and further studies confirmed it was the major anti-cancer component of S.B. Thus, Scutellaria baicalensis should be tested in clinical trials for these hematopoietic malignancies.     

Progressive resistance to apoptosis in a cell lineage model of human proliferative breast disease

BACKGROUND: Proliferative breast disease (PBD) may increase a woman's risk of developing breast cancer, perhaps by decreasing cellular sensitivity to apoptosis. To determine whether resistance to apoptosis develops during PBD, we investigated apoptosis initiated through the Fas pathway in a series of cell lines that recapitulates the morphologic changes of PBD in nude/beige mice. METHODS: The series of cell lines used was MCF-10A cells (parental preneoplastic human breast epithelial cells), MCF-10AT cells (transformed with T(24) Ha-ras), and MCF-10ATG3B cells (derivative cells that progress to carcinoma). Fas-mediated apoptosis, induced when a Fas monoclonal antibody bound to and activated the Fas receptor on these cells, was assessed morphologically and by flow cytometry. Levels of proteins involved in Fas-mediated apoptosis and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP), an end product of caspase activation, were determined by immunoblotting. Bcl-2 and Bax heterodimerization was examined by coimmunoprecipitation. All statistical tests were two-sided. RESULTS: Sensitivity to Fas-mediated apoptosis decreased with the tumorigenic potential of cells: MCF-10A cells were extremely susceptible, MCF-10AT cells were less susceptible, and MCF-10ATG3B cells were resistant. The percentage of apoptotic cells declined, from 24% to 8% to 6%, respectively. All lines produced Fas ligand (FasL) and had comparable levels of Fas receptor, FasL, Fas-associated death-domain protein, and caspases 3 and 6. Levels of caspase 8 were similar in MCF-10A and MCF-10AT cells but about 30% lower in MCF-10ATG3B cells (P>.01 but <.05). Levels of caspase 10 were about 20% lower in MCF-10AT cells (P>.005 but <.01) and about 59% lower in MCF-10ATG3B cells than in MCF-10A cells (P>.01 but <.05). PARP cleavage was detected in MCF-10A and MCF-10AT cells but not in MCF-10ATG3B cells. Levels of Bax, Bid, and Bak proteins were similar in all lines, but levels of Bcl-2 were lower in MCF-10AT and MCF-10ATG3B cells than in MCF-A cells, and Bcl-2-Bax heterodimerization progressively declined in the series. CONCLUSION: Resistance to Fas-mediated apoptosis appears to develop progressively in the MCF-10AT cell series.     

纳米银对人肺癌和肝癌细胞毒性的差异

目的:研究纳米银对人肺癌(A549)和人肝癌(HepG2)细胞系增殖和凋亡的影响,并探讨纳米银对两种细胞系毒效应的差异及其机制。方法:用20、40、80、160、320、640 μg/mL的纳米银分别染毒A549和HepG2细胞,染毒时长均为24和48 h,以细胞培养液为对照,采用MTT法检测各组细胞的存活率;谷胱甘肽(GSH)和超氧化物歧化酶(SOD)试剂盒检测细胞内GSH含量和SOD活力。除上述各染毒组,两种细胞均另设置N-乙酰半胱氨酸(NAC)预处理后160 μg/mL纳米银染毒组,采用流式细胞术检测各组细胞凋亡率。结果:与对照组比较,染毒24和48 h后, ≥40 μg/mL剂量组A549细胞和所有剂量组HepG2细胞存活率均降低(P<0.05或P<0.01);与相同染毒条件下的A549细胞比较,40~640 μg/mL剂量组HepG2细胞的存活率较高(P<0.05或P<0.01)。SOD活力和GSH含量检测发现,纳米银染毒A549细胞24 h后,40 μg/mL剂量组SOD活力与对照组比较显著降低(P<0.05),而GSH含量上升(P<0.05);染毒48 h后,SOD活力和GSH含量在40~160 μg/mL剂量组下降,其中80 μg/mL剂量组GSH含量与对照组间的差异显著(P<0.05)。纳米银染毒HepG2细胞24、48 h后,SOD活力和GSH含量都表现为上升,其中40、80 μg/mL组SOD活力和20 μg/mL组GSH含量与对照组间的差异显著(P<0.05)。细胞凋亡率检测发现,染毒24 h时,各剂量组A549细胞凋亡率与对照组比较差异无统计学意义(P>0.05),而各剂量组HepG2细胞凋亡率均显著增加(P<0.05或P<0.01);染毒48 h时, ≥40 μg/mL剂量组A549细胞和160 μg/mL剂量组HepG2细胞凋亡率均高于对照组(P<0.05或P<0.01)。使用NAC预处理细胞后,160 μg/mL剂量组两种细胞系凋亡率均显著下降(P<0.01)。结论:纳米银可以引起A549和HepG2细胞表现不同程度的增殖抑制和凋亡,而细胞内不同的SOD和GSH改变可能是纳米银引起两种细胞系不同毒作用的原因之一。     

Effect of landomycin E on expression of mRNA coding for transforming growth factor beta ligands and specific receptors in MCF-7 cells

The aim of the present study was to investigate regulation of mRNA, coding for TGFbeta ligands and specific receptors, by novel antitumor antibiotic landomycin E (LE) in human breast adenocarcinoma cells of MCF-7 line, sensitive and resistant to anti-cancer agent doxorubicin. METHODS: Semi-quantitative analysis of mRNA expression was estimated using RT-PCR and DNA gel-electrophoresis methods. For comparison, another anti-cancer drug doxorubicin (adriamycin) was utilized. THE RESULTS obtained indicate that LE induces up-regulation of mRNA coding for TGFbeta receptors type I and type II in MCF-7 cells, as well as in their doxorubicin-resistant sub-line. CONCLUSION: Anti-tumor action of LE may be mediated by TGFbeta, and a signaling pathway of this cytokine is not affected by the development of tumor cell resistance to doxorubicin.     

FG020327逆转肿瘤多药耐药性的作用及其机制

背景与目的肿瘤细胞过量表达P鄄糖蛋白穴P-glycoprotein.P-gp雪导致多药耐药穴multidrug resistance,MDR雪是目前肿瘤化疗的一大障碍,使用多药耐药逆转剂与抗癌药物联合化疗是克服临床多药耐药的重要方法。本研究对一种新的多芳基取代咪唑化合物FG020327的体外逆转活性及其逆转机制进行了探讨。方法以MTT法检测FG020327对多药耐药肿瘤细胞MCF-7/ADR及KBv200的耐药逆转活性;以荧光分光光度计法检测FG020327对MCF-7/ADR细胞内抗癌药物阿霉素累积的影响;以罗丹明蓄积实验检测该化合物对P-gp功能的影响。结果FG020327在体外具有较强的逆转活性,在5μmol/L浓度下使多药耐药细胞KBv200对长春新碱的敏感性增加44.9倍,逆转活性是公认的强逆转剂维拉帕米的3倍熏但它对敏感株对抗癌药物的敏感性基本无影响。2.5、5和10μmol/L的FG020327使MCF-7/ADR细胞中阿霉素的累积分别增加2.3、2.7和3.7倍,但是在敏感株MCF鄄7细胞却观察不到阿霉素累积的增加。FG020327浓度依赖性增加KBv200细胞内的罗丹明蓄积,但对敏感株KB细胞内的罗丹明蓄积无影响。结论FG020327具有较强的体外逆转MDR的活性,它可能通过抑制P鄄gp功能及增加MDR细胞内抗癌药物的累积逆转MDR。     

Loss of epithelial markers and acquisition of vimentin expression in adriamycin- and vinblastine-resistant human breast cancer cell lines.

We have previously observed that breast cancer cell lines could exhibit either epithelial or fibroblastic phenotypes as reflected by their morphologies and intermediate filament protein expression (C. L. Sommers, D. Walker-Jones, S. E. Heckford, P. Worland, E. Valverius, R. Clark, M. Stampfer, and E. P. Gelmann, Cancer Res., 49:4258-4263, 1989). Fibroblastoid, vimentin-expressing breast cancer cell lines are more invasive in vitro and in vivo (E. W. Thompson, S. Paik, N. Brunner, C. L. Sommers, G. Zugmaier, R. Clarke, T. B. Shima, J. Torri, S. Donahue, M. E. Lippman, G. R. Martin, and R. B. Dickson, J. Cell. Physiol., 150: 534-544, 1992). We hypothesized that a breast cancer cell with an epithelial phenotype could undergo a transition to a fibroblastic phenotype, possibly resulting in more invasive capacity. We now show that two Adriamycin-resistant MCF-7 cell lines and a vinblastine-resistant ZR-75-B cell line have undergone such a transition. Adriamycin-resistant MCF-7 cells express vimentin, have diminished keratin 19 expression, have lost cell adhesion molecule uvomorulin expression, and have reduced formation of desmosomes and tight junctions as determined by reduced immunodetection of their components desmoplakins I and II and zonula occludens (ZO)-1. Other MCF-7 cell lines selected for resistance to vinblastine and to Adriamycin and verapamil did not have these characteristics, indicating that drug selection does not invariably cause these phenotypic changes. In addition, to determine if vimentin expression in MCF-7 cells alone could manifest a fibroblastic phenotype, we transfected the full-length human vimentin complementary DNA into MCF-7 cells. Although vimentin expression was achieved in MCF-7 cells, it did not affect the phenotype of the cells in terms of the distribution of keratins, desmoplakins I and II, ZO-1, or uvomorulin or in terms of in vitro invasiveness. We conclude that vimentin expression is a marker for a fibroblastic and invasive phenotype in breast cancer cells but does not by itself give rise to this phenotype.     

Effects of Rapamycin on Cell Apoptosis in MCF-7 Human Breast Cancer Cells

Background: Rapamycin is an effective anti-angiogenic drug. However, the mode of its action remainsunclear. Therefore, in this study, we aimed to elucidate the antitumor mechanism of rapamycin, hypotheticallyvia apoptotic promotion, using MCF-7 breast cancer cells. Materials and Methods: MCF-7 cells were platedat a density of 15105 cells/well in 6-well plates. After 24h, cells were treated with a series of concentrations ofrapamycin while only adding DMEM medium with PEG for the control regiment and grown at 37oC, 5% CO2and 95% air for 72h. Trypan blue was used to determine the cell viability and proliferation. Untreated andrapamycin-treated MCF-7 cells were also examined for morphological changes with an inverted-phase contrastmicroscope. Alteration in cell morphology was ascertained, along with a stage in the cell cycle and proliferation.In addition, cytotoxicity testing was performed using normal mouse breast mammary pads. Results: Our resultsclearly showed that rapamycin exhibited inhibitory activity on MCF-7 cell lines. The IC50 value of rapamycin onthe MCF-7 cells was determined as 0.4μg/ml (p<0.05). Direct observation by inverted microscopy demonstratedthat the MCF-7 cells treated with rapamycin showed characteristic features of apoptosis including cell shrinkage,vascularization and autophagy. Cells underwent early apoptosis up to 24% after 72h. Analysis of the cell cycleshowed an increase in the G0G1 phase cell population and a corresponding decrease in the S and G2M phasepopulations, from 81.5% to 91.3% and 17.3% to 7.9%, respectively. Conclusions: This study demonstrated thatrapamycin may potentially act as an anti-cancer agent via the inhibition of growth with some morphologicalchanges of the MCF-7 cancer cells, arrest cell cycle progression at G0/G1 phase and induction of apoptosis inlate stage of apoptosis. Further studies are needed to further characterize the mode of action of rapamycin asan anti-cancer agent.     

Cathepsins D,B, and L in transformed human breast epithelial cells

To investigate the regulation of lysosomal enzymes during carcinogenesis, we measured cathepsins (Cats) D, B, and L in MCF-10F, which is a human breast epithelial cell line, and cells evolved after treatment with carcinogen and transfected with c-Ha-ras oncogene. The clones used in this study, MCF-10FTras, D3, D3-1, and D3-1Tras, expressed no estrogen receptors and gradually increased invasive potential, while oncogenetransfected lines were also tumorigenic in SCID mice [16,19]. Cats D, B, and L were determined in the cells and in cell media using enzyme-linked immunosorbent assay (ELISA), specific enzyme activity measurements, and immunocytochemistry. The major intra- and extracellular lysosomal proteinase in these cells was Cat D (30–180 pm/mg), followed by Cat B (2–10 pm/mg) and Cat L (1–5 pm/mg). An inverse relationship between intracellular Cat D levels and invasive potential of carcinogen-treated and c-Ha-ras oncogene-transfected cell lines was observed. No significant changes in extracellular concentration of Cat D precursor in this series of cell lines was observed. Intracellular levels of Cats B and L were unchanged or slightly lower in carcinogentreated D3 and D3-1 cells, as well as in MCF-10FTras. On the other hand, in D3-1Tras cell line, evolving from c-Ha-ras transfected D3-1 line, 3.5 fold and 4.4 fold increases in Cat B and Cat L, respectively, but a 2 fold decrease in Cat D, were observed compared to the parental cell line. Immunocytochemical staining showed a granular, polarized perinuclear and cytoplasmic staining of cathepsins in all cell lines. Cysteine proteinases stained more frequently and more intensely in D3-1Tras compared to other lines, confirming the immunochemical assays. We hypothesize that several molecular events, caused by a carcinogen and an oncogene such as c-Ha-ras, are needed to increase Cat B and Cat L, but not Cat D, expression. Therefore, the cysteine and aspartic lysosomal proteinases are differentially expressed in the breast cell lines with more invasive phenotype.     

卡马西平对雌激素依赖性乳腺癌细胞增殖的抑制作用及其机制的研究

背景与目的：卡马西平是一种应用多年的抗癫痫药,最近研究证明其具有组蛋白脱乙酰化酶抑制剂（histone deacetylase inhibitor,HDI）的性质。而已知的HDI多具有抗肿瘤作用。本实验旨在探讨卡马西平对雌激素受体α（estrogenreceptorα,ERα）阳性乳腺癌细胞增殖的抑制作用及其机制。方法：利用磺酰罗丹明B色度法（sulforhodamine B,SRB）分析不同情况下卡马西平对细胞增殖的影响。Western blot法检测ERa、Cyclin D1等相关蛋白的表达水平：RT—PCR法检测相应mRNA水平的变化：免疫荧光法测定他莫昔芬耐药细胞MCF-7RT中HER-2的表达;免疫沉淀技术检测Hsp90的分子伴侣功能及乙酰化水平。结果：卡马西平处理ERα阳性细胞MCF-7及T47D时,显著抑制雌二醇刺激的细胞增殖效应（P〈0．01）;卡马西平与4羟基他莫昔芬（4-OHT）联合处理MCF-7细胞,对雌二醇引起的细胞增殖抑制呈相加作用（q=1．00）;在MCF-7RT细胞中,卡马西平逆转了4-OHT的刺激增殖作用（P〈0．01）;卡马西平处理可以降低ERα阳性细胞中ERα、Cyclin D1在蛋白及mRNA水平的表达,并降低MCF-7RT细胞中HER-2表达;ERα、Cyclin D1的表达降低可以被26s蛋白酶体抑制剂MG132抑制;卡马西平可以促进Hsp90乙酰化并破坏其分子伴侣功能。结论：卡马西平明显抑制ER阳性乳腺癌细胞增殖,该作用可能部分通过促进ERα、Cyclin D1的蛋白酶体降解途径实现。卡马西平可以通过降低HER-2表达逆转与HER-2相关的4-OHT耐药。     

磁性纳米颗粒载ABCG2-siRNA逆转乳腺癌细胞对阿霉素耐药性的研究

目的：研究抑制乳腺癌耐药蛋白（ABCG2）表达对乳腺癌细胞耐药性的逆转作用,为乳腺癌基因靶向治疗提供新思路。方法：通过透射电镜和纳米粒度电位分析仪对磁性纳米颗粒（polyMAG）的粒径、电位进行表征,利用凝胶电泳阻滞实验检测polyMAG 与siRNA的结合情况,并用polyMAG携载不同浓度（5、10、20、50、100 nmol/L）ABCG2-siRNA,将其转染入乳腺癌细胞耐药株（MCF-7/ADR）后,采用CCK-8检测siRNA转染后细胞的存活率,荧光定量PCR和Western blotting分别检测ABCG2 mRNA和蛋白表达水平,Calcein-AM/PI染色观察细胞凋亡。结果：polyMAG粒径约100 nm,呈正电荷,表面电位29.6 mV。当polyMAG与ABCG2-siRNA的比为1：1和1：2时ABCG2-siRNA可完全结合。当20~100 nmol/L polyMAG-siRNA干扰ABCG2表达后,细胞存活率较未转染组明显降低（P < 0.05）。20 nmol/L polyMAG-siRNA转染组ABCG2 mRNA表达显著下调,约为未转染组的1/8（P < 0.05）,且凋亡细胞较其他组明显增多;50 nmol/L polyMAG-siRNA转染后可明显干扰ABCG2蛋白表达（P < 0.05）。结论：polyMAG-siRNA能高效干扰ABCG2表达,增加MCF-7/ADR细胞对阿霉素敏感性,逆转细胞耐药。     

Pentacyclic triterpenes from Chrysobalanaceae species: cytotoxicity on multidrug resistant and sensitive leukemia cell lines

Plants are known as important source in the search for new anti-cancer agents. Cytotoxicity-guided fractionation of leaves and fruits from Licania tomentosa Bench and leaves from Chrysobalanus icaco L. resulted in the isolation of betulinic, oleanolic and pomolic acids. These triterpenoids inhibited the growth and induced apoptosis of K562, an erythroleukemia cell line. Most importantly, they also inhibited the proliferation of Lucena 1, a vincristine-resistant derivative of K562 that displays several multidrug resistance (MDR) characteristics. Taken together, our findings emphasize the anti-tumor activity of these triterpenes on leukemia cell lines and call attention to their potential as anti MDR agents.     

洛伐他汀对 MCF-7细胞增殖分化功能及间隙连接细胞通讯的影响

背景与目的:洛伐他汀( lovastatin)是细胞内源性胆固醇合成的抑制剂,临床已普遍用于治疗高脂血症.现有研究报道,洛伐他汀具有抗肿瘤作用,但分子机制尚不清楚.本文探讨洛伐他汀对人乳腺癌细胞 MCF-7增殖功能以及间隙连接细胞通讯( gap junctional intercellular communication,GJIC)的影响.方法:分别以 4、 8、 16 μ mol/L洛伐他汀处理 MCF-7细胞 24～ 72 h后,流式细胞仪检测细胞周期的时相分布及细胞凋亡率,硝基蓝四氮唑( NBT)还原实验鉴定细胞分化,并采用划痕标记染料示踪技术观察洛伐他汀对 MCF-7细胞 GJIC功能的影响.结果:不同浓度洛伐他汀处理细胞不同时间后, MCF-7细胞的增殖明显受抑,抑制率最高可达 75.8%,且同一处理时相点各组间比较差异均有显著性 (P< 0.05);细胞周期分析显示,各处理组内 G0/G1期细胞明显增多,处理 72 h后可高达 80%左右;同时洛伐他汀能明显促进 MCF-7细胞分化、各处理组间比较差异均有显著性 (P< 0.01),但洛伐他汀诱导该细胞凋亡的作用不明显.另外,洛伐他汀有上调或恢复 MCF-7细胞 GJIC的作用; 16 μ mol/L洛伐他汀处理细胞 72 h后,荧光染料传输的范围可达 4～ 5层细胞.以上作用均有浓度-效应和时间依赖关系.结论:洛伐他汀可抑制 MCF-7细胞增殖,使细胞生长阻滞于 G0/G1期,并能促进细胞分化,该作用可能与洛伐他汀上调或恢复 MCF-7细胞的 GJIC功能有关.