Detection of Glycomic Alterations Induced by Overexpression of P-Glycoprotein on the Surfaces of L1210 Cells Using Sialic Acid Binding Lectins

P-glycoprotein (P-gp) overexpression is the most frequently observed cause of multidrug resistance in neoplastic cells. In our experiments, P-gp was expressed in L1210 mice leukemia cells (S cells) by selection with vincristine (R cells) or transfection with the gene encoding human P-gp (T cells). Remodeling of cell surface sugars is associated with P-gp expression in L1210 cells as a secondary cellular response. In this study, we monitored the alteration of cell surface saccharides by Sambucus nigra agglutinin (SNA), wheat germ agglutinin (WGA) and Maackia amurensis agglutinin (MAA). Sialic acid is predominantly linked to the surface of S, R and T cells via α-2,6 branched sugars that tightly bind SNA. The presence of sialic acid linked to the cell surface via α-2,3 branched sugars was negligible, and the binding of MAA (recognizing this branch) was much less pronounced than SNA. WGA induced greater cell death than SNA, which was bound to the cell surface and agglutinated all three L1210 cell-variants more effectively than WGA. Thus, the ability of lectins to induce cell death did not correlate with their binding efficiency and agglutination potency. Compared to S cells, P-gp positive R and T cells contain a higher amount of N-acetyl-glucosamine on their cell surface, which is associated with improved WGA binding. Both P-gp positive variants of L1210 cells are strongly resistant to vincristine as P-gp prototypical drug. This resistance could not be altered by liberalization of terminal sialyl residues from the cell surface by sialidase.     

长春新碱在β-环糊精修饰电极上的电化学行为

采用循环伏安法研究了长春新碱(VCR)在β-环糊精(β-CD)修饰金电极表面上的电化学行为,探讨了扫速、pH值和温度对表面包络反应的影响。实验结果表明:VCR的电极过程具有吸附性和不可逆性;在β-CD修饰电极上VCR的浓度与峰电流之间的关系符合Langmiur单分子层吸附等温方程;VCR与β-CD能有效包络,其包络常数K=4.6×103L/mol,速率常数k°=42.89 s-1。     

辅酶类物质对长春花细胞合成长春新碱的调控作用

添加5种不同浓度的辅酶类物质,根据单因素实验设计正交实验,研究其对抗肿瘤生物碱长春新碱合成的调控作用,并探讨其作用机理. 结果表明,单独添加每种辅酶类物质对长春新碱的合成均有促进作用,确定其最佳添加浓度为(mmol/L): DL-硫辛酸1.0, VB1 1.5, FMN 3.0, ATP 3.0,赖氨酸3.0,该条件下长春新碱合成量(细胞干重)为0.217 mg/g,为首次在长春花细胞培养中成功合成长春新碱.     

HPLC-MS/MS测定大鼠血浆中长春瑞滨的浓度及其药代动力学研究

An high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method for the determination of vinorelbine was presented, and the pharmacokinetics of the aqueous solution and lipid microspheres by intravenous were evaluated. Detection was performed using positive ion electrometry ionization followed by tandem mass spectrometry(ESI-MS/MS). The linear range of the standard curve of vinorelbine is from 1 to 1 000 μg•L^－1. The method shows that it is specificity, convenient and suitable for assaying the concentration of vinorelbine in rat plasma, and is used to support pharmacologic studies with vinorelbine and preparation technology for lipid microspheres.     

Dual Agent Loaded PLGA Nanoparticles Enhanced Antitumor Activity in a Multidrug-Resistant Breast Tumor Eenograft Model

Multidrug-resistant breast cancers have limited and ineffective clinical treatment options. This study aimed to develop PLGA nanoparticles containing a synergistic combination of vincristine and verapamil to achieve less toxicity and enhanced efficacy on multidrug-resistant breast cancers. The 1:250 molar ratio of VCR/VRP showed strong synergism with the reversal index of approximately 130 in the multidrug-resistant MCF-7/ADR cells compared to drug-sensitive MCF-7 cells. The lyophilized nanoparticles could get dispersed quickly with the similar size distribution, zeta potential and encapsulation efficiency to the pre-lyophilized nanoparticles suspension, and maintain the synergistic in vitro release ratio of drugs. The co-encapsulated nanoparticle formulation had lower toxicity than free vincristine/verapamil combinations according to the acute-toxicity test. Furthermore, the most effective tumor growth inhibition in the MCF-7/ADR human breast tumor xenograft was observed in the co-delivery nanoparticle formulation group in comparison with saline control, free vincristine, free vincristine/verapamil combinations and single-drug nanoparticle combinations. All the data demonstrated that PLGANPs simultaneously loaded with chemotherapeutic drug and chemosensitizer might be one of the most potential formulations in the treatment of multidrug-resistant breast cancer in clinic.     

长春碱、长春新碱提取分离方法研究

研究了长春碱、长春新碱的提取及分离工艺,探讨了提取剂、提取方法、提取时间、料液比对长春碱、长春新碱提取率的影响,并用LSA-21型树脂进行分离纯化。实验表明,最佳工艺条件为:以苯为溶剂,超声强化15 min(功率1 000 W),按料液比1∶6.5提取1次,用LSA-21型大孔吸附树脂吸附,以80%乙醇为洗脱剂,洗脱速度3 mL/min,长春碱、长春新碱的提取率分别为78.4%,62.2%,纯度分别为88.2%,85.8%。     

Hyaluronic acid-coated cationic nanostructured lipid carriers for oral vincristine sulfate delivery

The goal of this research is to structure a hyaluronic acid modified nanostructured lipid carrier (HA-NLCs) for vincristine sulfate (VCR) delivery, and detect its efficiency to improve the oral bioavailability. Emulsion solvent evaporation method was used to prepare the HA-NLCs nanoparticles. The particle size, zeta potential and entrapment efficiency of VCR-NLCs and HA-NLCs were 187?±?3.52?nm, ?8.61?±?1.29?mV, 33.12?±?1.16% and 192?±?4.41?nm, ?32.82?±?2.64?mV, 34.41?±?2.21%, respectively. HA-NLCs could significantly improve the cellular uptake efficiency and cytotoxicity in MCF-7 cells than other VCR formulations. The expressions of apoptosis related protein Caspase-3, Caspase-9, Bax and Bcl-2 were estimated by western blot assay in MCF-7 cells, and HA-NLCs exhibited the strongest effect in promoting cell apoptosis. The pharmacokinetics of HA-NLCs was evaluated in Sprague–Dawley male rats and the relative oral bioavailability of HA-NLCs and VCR-NLCs was improved about 1.8-fold and two-fold compared with VCR solution, respectively. Therefore, these results indicated that HA-NLCs could significantly improve the oral bioavailability and was a promising vehicle for the oral delivery of VCR.     

长春新碱透皮给药系统筛选及其透皮机理研究

分别制备了含不同表面活性剂的载长春新碱传递体(VCR-T)和载长春新碱壳聚糖纳米粒(VCR-CS-NPs),通过体外透皮试验,比较了不同透皮给药系统的透皮效果,并用DSC扫描探索了透皮效果差异的原因.所制备的VCR-T包封率从50%至80%不等,粒径90nm左右;VCR-CS-NPs的包封率为50%,粒径200nm左右;透射电镜下观察VCR-T和VCR-CS-NPs均外形圆整光滑,不粘连.体外透皮结果显示含Brij78的VCR-T为最佳的VCR透皮给药系统,DSC扫描认为这与载体与Brij78的相互作用有关;VCR-CS-NPs不能很好地透过皮肤,这可能与其粒径较大有关.     

Combined Treatment with an Oncolytic Adenovirus and Antitumor Activity of Vincristine against Retinoblastoma Cells

Treatment trends of retinoblastoma (RB) have gradually evolved from eye enucleation and external radiation to local treatment. Combined treatment with an oncolytic virus and chemotherapy is currently a new method in RB treatment. To investigate the therapeutic effect of oncolytic adenovirus SG600 in combination with vincristine (VCR) on retinoblastoma in vitro, the cell viability, cell cycle effects and apoptotic activity of HXO-RB₄₄ cells treated with SG600, VCR or SG600 plus VCR were measured using a cell counting kit-8-based procedure and flow cytometry. Western blot analysis for Akt, p-Akt, p-p53 and p-Rb protein was performed to investigate the underlying mechanisms of combined therapy. The combination therapy exerted a synergistic antitumor effect via a type of G₂/M and S phase arrest rather than the induction of apoptosis. The combination of VCR and SG600 further reduced Akt phosphorylation compared with cells treated with VCR alone, suggesting that SG600 could overcome chemoresistance, perhaps by down-regulating Akt in RB cells. An increase in the expression of p-p53 and decrease in p-Rb expression in HXO-RB₄₄ after co-treatment might be associated with cell cycle block. Western blot examination revealed that VCR might enhance SG600 replication. These results suggest that viro-chemo combination therapy is a feasible and potentially promising approach for the treatment of retinoblastoma.