乳腺癌细胞株MCF-7/ADM中肿瘤干细胞的研究

目的： 通过对阿霉素耐药乳腺癌细胞系MCF-7/ADM中乳腺癌干细胞（breast cancer stem cells,BCSCs）成分分析，观察化疗耐药处理的MCF-7乳腺癌细胞株是否可高效富集乳腺癌干细胞，为研究乳腺癌的发病机制提供思路。方法: MTT法分别测定阿霉素耐药乳腺癌细胞系MCF-7/ADM及其亲本细胞株MCF-7对阿霉素的IC50，计算其耐药倍数。通过细胞侧群（side population,SP）分析、球囊培养、流式细胞仪检测MCF-7/ADM及MCF-7中CD+44CD-24细胞比例三方面鉴定MCF-7/ADM和MCF-7中乳腺癌干细胞比例。结果: 阿霉素耐药乳腺癌细胞系MCF-7/ADM相对于MCF-7对阿霉素的耐药倍数为37.1；MCF-7/ADM中SP细胞比例为（9.60±0.66）%，MCF-7细胞的SP细胞比例为（0.39±0.11）%；两者球囊形成率分别为 （10.27±0.64）%和（1.03±0.15）%；两者的CD+44CD-24细胞比例分别为（64.87±3.87）%和（3.70±0.53）%,差异显著（P<0.05）。结论: 阿霉素耐药乳腺癌细胞系MCF-7/ADM中乳腺癌干细胞比例明显高于MCF-7细胞。化疗耐药处理的MCF-7乳腺癌细胞株可高效富集乳腺癌干细胞，这对于乳腺癌发病机制的研究具有重要意义。     

Doxorubicin-loaded human serum albumin nanoparticles surface-modified with TNF-related apoptosis-inducing ligand and transferrin for targeting multiple tumor types

Human serum albumin (HSA) nanoparticles (NPs) surface modified with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and transferrin, and containing doxorubicin were designed and prepared. Surface amines of HSA were reversibly protected with dimethylmaleic anhydride (DMMA), and HSA-NPs were prepared using a desolvation technique. Furthermore, the surfaces of HSA-NPs were modified with thiolated TRAIL or transferrin using sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The prepared TRAIL/transferrin plus doxorubicin HSA-NPs were characterized by TEM, FE-SEM, and particle size analysis, and their cytotoxic and apoptotic activities were evaluated in several cancer cell lines, namely, HCT 116, doxorubicin-resistant MCF-7, and CAPAN-1. In addition, the tumor-targeting abilities of NPs were assessed using an infrared imaging system in HCT 116-xenografted nu/nu mice. Results showed that the TRAIL/transferrin/doxorubicin HSA-NPs had remarkable cytotoxic and apoptotic activities in all cancer cells examined with a general or a drug-resistant character, and that these NPs had obvious synergistic cytotoxic effects particularly on CAPAN-1 cells. Moreover, these HSA-NPs were effectively localized to tumors in a HCT 116-xenografted nu/nu mouse over 32 h. The findings of this study suggest that the described TRAIL/transferrin/doxorubicin HSA-NPs are a useful targeting agent capable of killing different types of tumor cells in various tissue organs.     

In vitro cytotoxic potential of friedelin in human MCF-7 breast cancer cell: Regulate early expression of Cdkn2a and pRb1, neutralize mdm2-p53 amalgamation and functional stabilization of p53

We aimed to explore the cytotoxic and apoptotic effect of friedelin on breast cancer MCF-7 cells. Cytotoxic effect of friedelin on MCF-7 cells was analyzed using MTT, cell and nuclear morphology. The apoptosis mechanism of friedelin on MCF-7 cells was analyzed using real-time PCR. Friedelin potentially inhibit 78% of MCF-7 cell’s growth, the IC₅₀ value was 1.8 μM in 24 h and 1.2 μM in 48 h. Friedelin increased ROS significantly and DNA damage was confirmed by tunel assay. We found characteristically 52% apoptotic cells and 6% necrotic cells in PI, AO/ErBr staining after 48 h treatment with 1.2 μM of friedelin. Apoptosis was confirmed by significantly (p ≤ 0.001) increased tumor suppressor gene Cdkn1a, pRb2, p53, Nrf2, caspase-3 and decreased Bcl-2, mdm2 & PCNA expression after 48 h. In conclusion, friedelin effectively inhibit breast cancer MCF-7 cell growth, it was associated with early expression of Cdkn1a, pRb2 and activation of p53 and caspases.     

LyP-1-conjugated Fe3O4 nanoparticles suppress tumor growth by magnetic induction hyperthermia

To find a promising drug carrier to suppress tumor using magnetic induction hyperthermia (MIH) and targeted therapy, two superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylene glycol (PEG) and LyP-1, respectively, were prepared and compared. The particle size ranges of PEG-SPIONs and LyP-1-SPIONs were 10–15 nm, and 15–20 nm, respectively. In FTIR spectra, PEG-SPIONs and LyP-1-SPIONs had strong peaks between 575 and 1630 cm^?1. Specifically, the PEG-SPIONs mainly has peaks in 581 and 1630 cm^?1. The LyP-1-SPIONs mainly had peaks in 575, 1050 and 1625 cm^?1. The contents of Fe₃O₄ in the PEG-SPIONs and LyP-1-SPIONs were about 94.24 and 89.26%, respectively. The iron contents in the MCF-7 and CT-26 cells were 33.1 ± 1.8 and 27.9 ± 0.95 pg, respectively, after co-incubation with LyP-1-SPIONs for 8 h. The LyP-1-SPIONs accumulated in the nucleus of MCF-7 cells while PEG-SPIONs in cytoplasma. In vitro, after 30 days we can found the tumor almost stopped to grow in Group LyP-1-SPIONs. LyP-1-SPIONs are promising in treating cancer as they accumulated in the nucleus of MCF-7 cells which expressed p32 and almost stopped tumor growth by combined MIH and targeted therapy.     

Somatostatin receptor-mediated tumor-targeting drug delivery using octreotide-PEG-deoxycholic acid conjugate-modified N-deoxycholic acid-O, N-hydroxyethylation chitosan micelles

In this study, a ligand-PEG-lipid conjugate, octreotide-polyethene glycol-deoxycholic acid (OCT(Phe)-PEG-DOCA, or OPD) was successfully synthesized and used as a targeting molecule for N-deoxycholic acid-O, N-hydroxyethylation chitosan (DAHC) micelles for efficient cancer therapy. DAHC micelles exhibited good loading capacities for doxorubicin (DOX), a model anti-cancer drug, and the modification of OPD showed no significant effect on drug load while slightly increasing the particle size and partly shielding the positive charges on the surface of micelles. Accelerated release rate of DOX from micelles were also observed after OPD modification and the release profile exhibited pH-sensitive properties. Compared with DAHC-DOX micelles, OPD-DAHC-DOX micelles exhibited significantly stronger cytotoxicity to MCF-7 cells (SSTRs overexpression) but with hardly any difference from WI-38 cells (no SSTRs expression). The results of flow cytometry and confocal laser scanning microscopy further revealed that OPD-DAHC-DOX micelles could be selectively taken into tumor cells by SSTRs-mediated endocytosis. In vivo investigation of micelles on nude mice bearing MCF-7 cancer xenografts confirmed that OPD-DAHC micelles possessed much higher tumor-targeting capacity than the DAHC control and exhibited enhanced anti-tumor efficacy and decreased systemic toxicity. These results suggest that OPD-DAHC micelles might be a promising anti-cancer drug delivery carrier for targeted cancer therapy.     

Enhanced effect of pH-sensitive mixed copolymer micelles for overcoming multidrug resistance of doxorubicin

P-glycoprotein (P-gp) mediated drug efflux has been recognized as a key factor contributing to the multidrug resistance (MDR) in tumor cells. To address this issue, a new pH-sensitive mixed copolymer micelles system composed of hyaluronic acid-g-poly(l-histidine) (HA-PHis) and d-α-tocopheryl polyethylene glycol 2000 (TPGS2k) copolymers was developed to co-deliver doxorubicin (DOX) and TPGS2k into drug-resistant breast cancer MCF-7 cells (MCF-7/ADR). The DOX-loaded HA-PHis/TPGS2k mixed micelles (HPHM/TPGS2k) were characterized to have a unimodal size distribution, high DOX loading content and a pH dependent drug release profile due to the protonation of poly(l-histidine). As compared to HA-PHis micelles (HPHM), the HPHM/TPGS2k showed higher and comparable cytotoxicity against MCF-7/ADR cells and MCF-7 cells, respectively. The enhanced MDR reversal effect was attributed to the higher amount of cellular uptake of HPHM/TPGS2k in MCF-7/ADR cells than HPHM, arising from the inhibition of P-gp mediated drug efflux by TPGS2k. The measurements of P-gp expression level and mitochondrial membrane potential indicate that the blank HPHM/TPGS2k inhibited P-gp activity by reducing mitochondrial membrane potential and depletion of ATP but without inhibition of P-gp expression. In vivo study of micelles in tumor-bearing mice indicate that HPHM/TPGS2k could reach the tumor site more effectively than HPHM. The pH-sensitive mixed micelles system has been demonstrated to be a promising approach for overcoming the MDR.     

Enhanced solubility and targeted delivery of curcumin by lipopeptide micelles

A lipopeptide (LP)-containing KKGRGDS as the hydrophilic heads and lauric acid (C₁₂) as the hydrophobic tails has been designed and prepared by standard solid-phase peptide synthesis technique. LP can self-assemble into spherical micelles with the size of ~30 nm in PBS (phosphate buffer saline) (pH 7.4). Curcumin-loaded LP micelles were prepared in order to increase the water solubility, sustain the releasing rate, and improve the tumor targeted delivery of curcumin. Water solubility, cytotoxicity, in vitro release behavior, and intracellular uptake of curcumin-loaded LP micelles were investigated. The results showed that LP micelles can increase the water solubility of curcumin 1.1 × 10³ times and sustain the release of curcumin in a low rate. Curcumin-loaded LP micelles showed much higher cell inhibition than free curcumin on human cervix carcinoma (HeLa) and HepG2 cells. When incubating these curcumin-loaded micelles with HeLa and COS7 cells, due to the over-expression of integrins on cancer cells, the micelles can efficiently use the tumor-targeting function of RGD (functionalized peptide sequences: Arg-Gly-Asp) sequence to deliver the drug into HeLa cells, and better efficiency of the self-assembled LP micelles for curcumin delivery than crude curcumin was also confirmed by LCSM (laser confocal scanning microscope) assays. Combined with the enhanced solubility and higher cell inhibition, LP micelles reported in this study may be promising in clinical application for targeted curcumin delivery.     

A novel cabazitaxel-loaded polymeric micelle system with superior in vitro stability and long blood circulation time

Cabazitaxel (CTX) is a second-generation semisynthetic taxane that demonstrates antitumor activity superior to docetaxel. However, the low aqueous solubility of CTX has hampered its use as a therapeutic agent. In this work, CTX-loaded N-t-butoxycarbonyl-L-phenylalanine end-capped monomethyl poly (ethylene glycol)-block-poly (D,L-lactide) (mPEG-PLA-Phe(Boc)/CTX) micelles were prepared to improve the solubility of CTX while retaining its superior stability before accessing the tumor site. The mPEG-PLA-Phe(Boc)/CTX micelles showed excellent stability in vitro compared with mPEG-PLA/CTX micelles. When stored at 25 °C, the mPEG-PLA/CTX micelles tended to aggregate within 1 h, whereas the mPEG-PLA-Phe(Boc)/CTX micelles were uniformly transparent even after three weeks. Dilution of mPEG-PLA/CTX micelles widened their size distribution and decreased the encapsulation efficiency, while significant change was not found in mPEG-PLA-Phe(Boc)/CTX micelles, even when diluted 1000-fold. Pharmacokinetic results in Sprague–Dawley rats indicated that, compared with Jevtana^®, intravenous administration of mPEG-PLA-Phe(Boc)/CTX micelles stably retained the CTX in plasma with 26.03-fold larger of the area under the time–concentration curve, 2.13-fold longer of the half-life, and 9.99-fold higher of the maximum concentration. In conclusion, mPEG-PLA-Phe(Boc) micelle may be a potential nanocarrier not only to improve the solubility of CTX but also to prolong the blood circulation time, which results in improved biological activity.     

欧前胡素通过上调细胞表面死亡受体5的数量增强TRAIL对乳腺癌细胞的杀伤活性

目的:探讨中药活性成分欧前胡素对肿瘤坏死因子相关凋亡诱导配体(TRAIL)的协同抗乳腺癌效应及分子机制。方法:将人乳腺癌细胞T-47D和MCF-7按对照组、欧前胡素组、TRAIL组、欧前胡素+TRAIL组及欧前胡素+TRAIL+死亡受体5(DR5)siRNA组进行分组,MTT法检测T-47D和MCF-7细胞活力,流式细胞术检测T-47D细胞凋亡和线粒体膜电位,Western blot实验和流式细胞术检测T-47D细胞表面DR5的表达水平及caspase-8、caspase-3的活化水平。结果:MTT实验结果显示,欧前胡素联合用药能显著提高各浓度TRAIL对T-47D和MCF-7细胞的杀伤活性;流式细胞术和Western blot结果显示欧前胡素处理能显著提高T-47D细胞DR5的表达水平和活性氧簇产生水平(P0.05)。另外,流式细胞术和Western blot结果还显示,欧前胡素联合用药能显著增强TRAIL促进T-47D细胞线粒体膜电位损伤、caspase-8和caspase-3活化及凋亡的作用。结论:欧前胡素通过上调乳腺癌细胞DR5的表达水平发挥对TRAIL的协同抗乳腺癌效应。     

Synthesis and characterization of novel P(HEMA-LA-MADQUAT) micelles for co-delivery of methotrexate and Chrysin in combination cancer chemotherapy

A Novel poly [2-hydroxyethyl methacrylate-Lactide-dimethylaminoethyl methacrylate quaternary ammonium alkyl halide] [P(HEMA-LA-MADQUAT)] copolymer was synthesized through combination of ring opening polymerization (ROP) and ‘free’ radical initiated polymerization methods. This newly developed copolymer was fully characterized by FT-IR, ¹HNMR and ¹³CNMR spectroscopy. Micellization of the copolymer was performed by dialysis membrane method and obtained micelles were characterized by FESEM, dynamic light scattering (DLS), zeta potential (ξ), and critical micelle concentration (CMC) measurements. This copolymer was developed with the aim of co-delivering two different anticancer drugs: methotrexate (MTX) and chrysin. In vitro cytotoxicity effect of MTX@Chrysin-loaded P(HEMA-LA-MADQUAT) was also studied through assessing the survival rate of breast cancer cell line (MCF-7) and DAPI staining assays. Cationic micelle (and surface charge of + 7.6) with spherical morphology and an average diameter of 55 nm and CMC of 0.023 gL^?1 was successfully obtained. Micelles showed the drug loaded capacity around 87.6 and 86.5% for MTX and Chrysin, respectively. The cytotoxicity assay of a drug-free nanocarrier on MCF-7 cell lines indicated that this developed micelles were suitable nanocarriers for anticancer drugs. Furthermore, the MTX@Chrysin-loaded micelle had more efficient anticancer performance than free dual anticancer drugs (MTX @ chrysin), confirmed by MTT assay and DAPI stainingmethods. Therefore, we envision that this recently developed novel micelle can enhance the efficacy of chemotherapeutic agents, MTX and Chrysin, combination chemotherapy and has the potential to be used as an anticancer drug delivery system for in vivo studies. Therefore, this recently developed novel micelle can enhance the efficacy of chemotherapeutic agents, MTX and Chrysin, combination chemotherapy and has the potential to be used as an anticancer drug delivery system for in vivo studies.     

The use of α-conotoxin ImI to actualize the targeted delivery of paclitaxel micelles to α7 nAChR-overexpressing breast cancer

Alpha7 nicotinic acetylcholine receptor (α7 nAChR), a ligand-gated ion channel, is increasingly emerging as a new tumor target owing to its expression specificity and significancy for cancer. In an attempt to increase the targeted drug delivery to the α7 nAChR-overexpressing tumors, herein, α-conotoxin ImI, a disulfide-rich toxin with highly affinity for α7 nAChR, was modified on the PEG-DSPE micelles (ImI-PMs) for the first time. The DLS, TEM and HPLC detections showed the spherical nanoparticle morphology about 20 nm with negative charge and high drug encapsulation. The ligand modification did not induce significant differences. The immunofluorescence assay confirmed the expression level of α7 nAChR in MCF-7 cells. In vitro and in vivo experiments demonstrated that the α7 nAChR-targeted nanomedicines could deliver more specifically and faster into α7 nAChR-overexpressing MCF-7 cells. Furthermore, fluo-3/AM fluorescence imaging technique indicated that the increased specificity was attributed to the ligand-receptor interaction, and the inducitivity for intracellular Ca²⁺ transient by ImI was still remained after modification. Moreover, paclitaxel, a clinical frequently-used anti-tumor drug for breast cancer, was loaded in ImI-modified nanomedicines to evaluate the targeting efficacy. Besides of exhibiting greater cytotoxicity and inducing more cell apoptosis in vitro, paclitaxel-loaded ImI-PMs displayed stronger anti-tumor efficacy in MCF-7 tumor-bearing nu/nu mice. Finally, the active targeting system showed low systemic toxicity and myelosuppression evidenced by less changes in body weight, white blood cells, neutrophilic granulocyte and platelet counts. In conclusion, α7 nAChR is also a promising target for anti-tumor drug delivery and in this case, α-conotoxin ImI-modified nanocarrier is a potential delivery system for targeting α7 nAChR-overexpressing tumors.     

蝎毒联合ADM对人乳腺癌MCF-7/细胞的凋亡诱导作用及其耐药逆转的研究

目的探讨蝎毒(BMK)联合阿霉素(ADM)对人乳腺癌MCF-7/ADM细胞的凋亡诱导及其耐药逆转作用。方法MTT法、荧光分光光度法、流式细胞术、紫外分光光度术和免疫细胞化学法。结果①非细胞毒性剂量(3.0μg/m l)蝎毒能显著降低MCF-7/ADM的IC50(P<0.01),增加MCF-7/ADM细胞内ADM的药物积累(P<0.01)。②蝎毒(3.0μg/m l)联合ADM后增强了对耐药细胞的凋亡诱导作用,凋亡率由8.9±0.01%上升为12.6±0.21%(P<0.01)。③蝎毒(3.0μg/m l)能够显著降低耐药细胞内谷胱甘肽S转移酶(GSTs)酶的活性(P<0.01)。结论蝎毒能够部分逆转人乳腺癌MCF-7/ADM细胞对阿霉素的耐药性,其逆转机制与抑制耐药细胞GSTs活性有关。     

Patupilone-loaded poly(L-glutamic acid)-graft-methoxy-poly(ethylene glycol) micelle for oncotherapy

Patupilone, an original natural anti-cancer agent, also known as epothilone B or Epo906, has shown promise for the treatment of a variety of cancers, however, the systematic side effects of patupilone significantly impaired its clinical translation. Herein, patupilone-loaded PLG-g-mPEG micelles were prepared. Patupilone was grafted to a poly(L-glutamic acid)-graft-methoxy-poly(ethylene glycol) (PLG-g-mPEG) by Steglich esterification reaction to give PLG-g-mPEG/Epo906 that could self-assemble to form patupilone-loaded micelles (Epo906-M). The Epo906-M was able to inhibit the proliferation of A549, MCF-7 cancer cells and BEAs-2B cells in vitro. For in vivo treatment of orthotopic xenograft tumor models (MCF-7), the Epo906-M exhibited higher tumor inhibition efficiency with lower side effects as compared with free Epo906. Seventeen percent of the body weight loss appeared in the group treated with free Epo906 of 0.25 mg kg^?1, while the group treated with Epo906-M of 10 mg kg^?1 showed less than ten percent of body weight loss and displayed stronger tumor inhibiting effect. Therefore, the polypeptide-patupilone conjugate has improved potential for oncotherapy.     

Synthesis,characterization, and in vitro evaluation of targeted gold nanoshelled poly(d,l-lactide-co-glycolide) nanoparticles carrying anti p53 antibody as a theranostic agent for ultrasound contrast imaging and photothermal therapy

Breast cancer is the leading cause of cancer-related deaths in women and earlier detection can substantially reduce deaths from breast cancer. Polymers with targeted ligands are widely used in the field of molecular ultrasound imaging and targeted tumor therapy. In our study, the nanotheranostic agent was fabricated through filling perfluoropropane (C₃F₈) into poly(d,l-lactic-co-glycolic acid) nanoparticles (PLGA NPs), followed by the formation of gold nanoshell on the surface, then conjugated with anti p53 antibody which has high specificity with the p53 protein overexpressing in breast cancer. The average diameter of the gold nanoshelled PLGA NPs carrying anti p53 antibody (p53-PLGA@Au NPs) was 247 ± 108.2 nm. The p53-PLGA@Au NPs had well-defined spherical morphology and hollow interiors observed by electron microscope, and had a good photothermal effect under the irradiation of an 808 nm laser. The results of laser scanning confocal microscope (LSCM) and flow cytometer (FCM) indicated the specific targeting of p53-PLGA@Au NPs conjugating with breast cancer MCF-7 cells overexpressing p53 protein in vitro. Also the ultrasound imaging experiments in vitro showed that p53-PLGA@Au NPs were suitable for ultrasound contrast imaging. In conclusion, the p53-PLGA@Au NPs are demonstrated to be novel targeted UCAs and may have potential applications in the early diagnosis and targeted near-infrared (NIR) photothermal therapy of breast cancer in the future.     

Exercise-induced elevation in plasma oxidative generating capability augments the temporal inflammatory response stimulated by lipopolysaccharide

Prolonged oxidative stress is detrimental to health; however, transient oxidative stress may improve immune capability. We examined whether exercise-induced increases in the plasma oxidative generating capability enhance immune responsiveness to potential pathogens. Twelve individuals underwent a 30-min row and pre and post-exercise bloods were collected for oxidative stress and immune assessment. We found that exercise induced a transient increase in plasma carbonyls (3.2–5.3 nmol/mg protein) and creatine kinase activity (0.5–1.2 absorbance/min/mg protein) and that lipopolysaccharide (LPS) stimulation (0.5–24 h) of pre- and post-exercise blood augmented temporal tumour necrosis factor-α (TNFα) secretion. Further characterisation of plasma using a modified dihydro-2′,7′-dichlorohydrofluorescein (DCF) assay revealed that addition of a sub-threshold of hydrogen peroxide to post-exercise (and not pre-exercise) plasma caused a sixfold increase in the radical oxygen species (ROS) generating capability after 15 min (555 ± 131 to 3607 ± 488 change in fluorescent intensity [ΔFI]), which was inhibited using 60 mM N-acetyl-l-cysteine (920 ± 154 ΔFI). Furthermore, cell experiments revealed that LPS stimulation of either THP-1 cells pre-incubated with post-exercise plasma or peripheral blood mononuclear cells pre-treated with pro-oxidants, modulated the temporal secretion of key cytokines that regulate the initiation, progression and resolution of an inflammatory response. These results indicate that exercise-induced changes in plasma parameters (e.g. oxidative generating capability—dependent or independent of inflammatory mediators) augment the temporal LPS response and support the notion that repeated transient oxidative stress (such as that induced by regular exercise) is important for a “healthy” immune system.     

Preparation, characterization, in-vitro drug release and cellular uptake of poly(caprolactone) grafted dextran copolymeric nanoparticles loaded with anticancer drug

Biodegradable and biocompatible polymers that are engineered to nanostructures play a key role in providing solution for sustained chemotherapy. This study is focused on preparation, drug encapsulation efficiency, in-vitro drug release, in-vitro cellular uptake and cell viability of poly(caprolactone) grafted dextran (PGD) nanoparticles (NPs) formulation containing vinblastine as the anticancer drug. Drug-loaded PGD NPs were prepared by a modified oil/water emulsion method and characterized by laser light scattering, atomic force microscopy (AFM), and zeta potential. The drug encapsulation efficiency was determined spectrophotometrically and in-vitro drug release was estimated using dialysis bag. Breast cancer cell line (MCF-7) was used to image and measure the cellular uptake of fluorescent PGD NPs. Cancer cell viability was assessed by treating MCF-7 cells with vinblastine-loaded PGD NPs by crystal violet staining method. Result showed that the vinblastine-loaded PGD NPs were superior in properties such as drug encapsulation efficiency, the cellular uptake and the cancer cell mortality.     

Restoration of chemosensitivity by multifunctional micelles mediated by P-gp siRNA to reverse MDR

One of the main obstacles in tumor therapy is multiple drug resistance (MDR) and an underlying mechanism of MDR is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins, especially P-glycoprotein (P-gp). In the synergistic treatment of siRNA and anti-cancer drug doxorubicin, it is crucial that both the siRNA and doxorubicin are simultaneously delivered to the tumor cells and the siRNA can fleetly down-regulate P-g before doxorubicin inactivates the P-gp and is pumped out. Herein, a type of micelles comprising a polycationic PEI-CyD shell to condense the siRNA and hydrophobic core to package doxorubicin is reported. The structure of the polymer is determined by ¹H NMR, FT-IR, DSC, and XRD and the micelles are characterized by DLS, 2D-NOESY NMR, and TEM to study the self-assembly of the micelles with siRNA and drugs. In vitro studies demonstrate controlled release and temporal enhancement of the therapeutic efficacy of P-gp siRNA and doxorubicin. Release of siRNA down-regulates the mRNA and protein levels of P-gp in the MCF-7/ADR cell lines effectively and the accumulated doxorubicin facilitates apoptosis of the cells to reverse MDR. Moreover, in vivo research reveals that the siRNA and doxorubicin loaded micelles induce tumor cell apoptosis and inhibit the growth of MDR tumor. The western blotting and RT-PCR results illustrate that the synergistic treatment of siRNA and doxorubicin leads to efficient reduction of the P-gp expression as well as cell apoptotic induction in MDR tumors at a small dosage of 0.5 mg/kg. The micelles have large clinical potential in drug/RNAi synergistic treatment via restoration of the chemosensitivity in MDR cancer therapy.     

IGFBP7对人乳腺癌MCF-7细胞增殖的影响及其机制

目的: 探究胰岛素样生长因子结合蛋白7(IGFBP7)对人乳腺癌MCF-7细胞增殖的影响及其分子生物学机制。方法: 将质粒pCMV6-IGFBP7转染MCF-7细胞,构建稳定表达IGFBP7的MCF-7细胞系;采用Western blotting检测IGFBP7在MCF-7细胞稳定转染子的表达;采用软琼脂培养克隆形成实验检测IGFBP7对MCF-7细胞克隆形成能力的影响;采用流式细胞术检测IGFBP7对MCF-7细胞周期的影响;采用Western blotting检测IGFBP7对MCF-7细胞细胞外信号调节激酶1/2(ERK1/2)、p-ERK1/2、细胞周期素D1(cyclin D1)、细胞周期素依赖性激酶4(CDK4)、cyclin E、CDK2、p21^CIP1/WAF1、p27^KIP1、p53、视网膜母细胞瘤蛋白(Rb)和p-Rb蛋白含量的影响。结果: (1)只有稳定转染质粒pCMV6-IGFBP7的MCF-7细胞表达IGFBP7。(2)IGFBP7能够显著降低MCF-7细胞的克隆形成率(P<0.01),阻止细胞从G₁期进入S 期,使其停滞于G₁期(P<0.01)。(3)IGFBP7能够显著抑制ERK1/2的磷酸化(P<0.01)。(4)IGFBP7能够下调cyclin D1和cyclin E蛋白表达(P<0.01),上调p27^KIP1、p21^CIP1/WAF1和p53蛋白表达(P<0.01),抑制Rb的磷酸化(P<0.01)。(5)MEK1/2阻断剂PD98059可部分模拟IGFBP7的肿瘤抑制效应。结论: (1) IGFBP7可通过下调cyclin D1和cyclin E蛋白表达,上调p27^KIP1、p21^CIP1/WAF1和p53蛋白表达,以及抑制Rb磷酸化发挥抗肿瘤作用;(2) IGFBP7对cyclin D1和p27^KIP1的调节可能与其抑制ERK1/2信号通路有关。     

Preparation,characterization, and in vitro drug release behavior of glutathione-sensitive long-circulation micelles based on polyethylene glycol prodrug

In this paper, a kind of glutathione-sensitive polymeric micelles was prepared through assembling in aqueous solution of an amphiphilic polymeric prodrug which was synthesized by linkage of 6-mercaptopurine (6-MP) and polyethylene glycol monomethyl ether using propiolic acid as a connecting arm. The glutathione (GSH)-sensitive strategy is based on a Michael addition–elimination reaction, that is the amphiphilic polymeric prodrug which contains α, β-unsaturated carbonyl group acts as a Michael acceptor to receive the attack of nucleophile – glutathione, and undergoes elimination reaction to release the original drug. Transmission electron microscope observation showed that the polymeric micelles (PMs) had a spherical-like morphology with a mean diameter of 28 ± 3.2 nm. The dynamic light scattering investigation data exhibited that the size and distribution changes of PMs are negligible after being placed for 15 days. In vitro drug release study indicated that only less than 13% of 6-MP was released from the micelles under GSH stimulation at micromolar level, while 34.5, 53.7, and 77.8% accumulative release rates were achieved under GSH stimulation at millimolar level (1, 2 and 10 mM), respectively. The cell inhibition rate of PM solution against HL-60 cells carried out by MTT method reached 85%. The cellular uptake and the intracellular drug release of PMs in HL-60 cells were observed through determining the intracellular 6-MP content by UV–vis spectrophotometer. In vitro macrophage uptake study showed a low phagocytosis rate, indicating the long-circulation ability of the PMs.