欧前胡素对顺铂的骨肉瘤细胞抗肿瘤作用的增效作用

目的 研究欧前胡素是否能提高人骨肉瘤细胞(human osteosarcoma cells,HOS细胞)对顺铂的敏感性。方法 HOS细胞用欧前胡素和顺铂处理,MTT法检测体外细胞活力,Western blot检测Bcl-2 蛋白家族成员(Mcl-1、Bcl-2、Bcl-xl、Bad和Bax)的表达水平,流式细胞术检测细胞凋亡水平和线粒体膜电位的变化情况。构建Mcl-1真核表达载体,MTT法检测Mcl-1表达载体转染对欧前胡素联合顺铂治疗骨肉瘤疗效的影响。结果 欧前胡素在体外可显著提高顺铂对骨肉瘤细胞系HOS的杀伤活性。欧前胡素可显著降低HOS细胞Mcl-1的表达,而顺铂对Mcl-1的表达水平无影响。相比于欧前胡素或顺铂单治疗组,两者联合可显著诱导HOS细胞发生凋亡并降低其线粒体膜电位。体外转染Mcl-1真核表达载体显著降低顺铂联合欧前胡素对HOS细胞的杀伤活性。结论 欧前胡素通过靶向于Mcl-1增强顺铂对骨肉瘤细胞的杀伤活性。     

Interaction of platinum agents,cisplatin, carboplatin and oxaliplatin against albumin in vivo rats and in vitro study using inductively coupled plasma‐mass spectrometory

The protein binding rates (PBR) of platinum‐containing agents cisplatin (CDDP), carboplatin (CBDCA) and oxaliplatin (L‐OHP) have been reported as 98%, 25–50% and 98%, respectively. To investigate the protein‐binding properties of albumin with cisplatin, carboplatin and oxaliplatin, inductively coupled plasma mass spectrometry (ICP‐MS) was used to measure their plasma concentration in rats over time. The study also examined the effects of cisplatin, carboplatin and oxaliplatin‐binding on albumin in vitro, using CD spectrometry and native‐polyacrylamide gel electrophoresis (native PAGE). The ratios of PBR to irreversible PBR, of cisplatin and oxaliplatin were 98%:98% and 90%:87%, respectively, indicating a higher affinity for irreversible binding with albumin. That of carboplatin was 25%:10%, indicating 60–70% reversible binding with albumin. The plasma protein binding rate concentrations of cisplatin, carboplatin and oxaliplatin after in vivo administration were 96%, 15% and 80%, respectively. The CD spectrometry of albumin was unaffected by cisplatin, carboplatin and oxaliplatin binding. Though similar protein binding rates were observed with oxaliplatin and cisplatin, oxaliplatin had a higher mobility rate during PAGE. It was confirmed that the binding of cisplatin and oxaliplatin with albumin affected its electric charge but not the structure. In conclusion, cisplatin and oxaliplatin bind irreversibly with albumin in plasma and may irreversibly interact with tissue protein and/or DNA. The difficulties involved with predicting the tissue concentrations of cisplatin and oxaliplatin from their plasma concentration inhibits their therapeutic drug monitoring. On the contrary, carboplatin, like some generic drugs, reversibly binds to plasma proteins. It is, therefore, possible to conduct therapeutic drug monitoring for carboplatin.     

Development and Application of a Simple Assay to Quantify Cellular Adducts of Platinum Complexes with DNA

Pharmaceutical Research -     

顺铂、樟脑胺氯乙酸铂及碳铂对染色体损伤的比较

以CHL细胞为材料,比较了顺铂(DDP)、碳铂(JM 8)和樟脑胺氯乙酸铂(CCP)的细胞毒作用和对CHL细胞的染色体损伤。实验表明,这三种铂络合物都有较强的细胞毒作用,其中顺铂最强而CCP最弱。DDP,JM-8和CCP的IC~(90)(±SD)分别为0.68±0.21,4.4±0.6,10.8±1.5μmol/L。在微核及染色体畸变实验中,观察到在药物浓度小于或等于IC_(90)时,这三种化合物诱发的微核及染色体损伤相当,但药物浓度增加时,CCP所诱发的微核及染色体畸变均大于DDP而JM-8最轻。     

血管内皮抑素与顺铂对Calu-6肺癌细胞联合作用的观察

目的:研究血管内皮抑素对人肺癌细胞的增殖抑制作用及机制,并观察其与顺铂(DDP)的联合作用。方法:经血管内皮抑素及DDP干预后,以MTT法检测其对肺癌细胞Calu-6的抑制、流式细胞术检测细胞凋亡、ELISA测定Bcl-2/Bax、sFas/sFasL的表达。结果:(1)血管内皮抑素具有抑制Calu-6细胞增殖的作用(P<0.05)并呈现时间依赖性、诱导Calu-6细胞凋亡,下调Bcl-2表达,但对Bax的表达无明显影响(P>0.05),未检测到sFas/sFasL的表达。(2)血管内皮抑素与DDP联合用药方案中,同时给药较单药DDP诱导凋亡及下调Bcl-2表达明显,序贯给药与DDP间无明显差异(P>0.05)。(3)2种血管内皮抑素(恩度、内皮抑素)的效应差异无统计学意义(P>0.05)。结论:(1)血管内皮抑素可抑制Calu-6细胞生长、诱导其凋亡,其机制与下调Bcl-2表达有关。(2)血管内皮抑素与DDP间有增效作用,联合用药方案中以同时给药效果最著。(3)各血管内皮抑素的作用机制相似。     

铂类抗肿瘤药物的研发进展及市场情况

近年来,铂类抗肿瘤药物的临床研究进展较快,其抗肿瘤谱广,抗肿瘤活性增强,不良反应降低,已成为目前有关抗肿瘤药物研发的重要领域。新的铂类抗肿瘤药物将从那些在临床研究中显示出低毒性、抗肿瘤谱广、与现有药物无交叉耐药性的化合物中产生。本文就其作用机制、国内外上市开发现状、国内外销售情况及国内研究开发进展等进行综述。     

Potentiation of Cytotoxicity of Mitoxantrone Toward CHO-K1 Cells in Vitro by Dipyridamole

Dipyridamole (DP), a clinically used vasodilator and an antiplatelet compound, augmented the activity of the anticancer drug mitoxantrone (MXN) toward Chinese hamster ovary (CHO-K1) cells in culture. Clonogenic assays indicated that DP (1.0, 2.5, and 5.0 µM) decreased the survival of cells treated with MXN (5 to 25 nM) in a dose-dependent manner. Further, DP (1 and 5 nM) decreased the MXN concentration required for 50% inhibition of cell growth from 3.2 to 1.8 and from 3.0 to 0.5 nM, respectively, over a period of 3 days. DP (10 µM) increased the accumulation of MXN by 1.8-fold in exponentially growing cells exposed to MXN. The enhanced levels of MXN in CHO-K1 cells in the presence of the chemosensitizer may account for the potentiation of MXN-cytotoxicity by DP.     

In Vitro Cytotoxic Effects of Dibromodulcitol in 9L Rat Brain Tumor Cells

Dibromodulcitol (DBD) is a halogenated hexitol active against animal and human brain tumors in vivo. In aqueous solution, DBD is transformed to products with different cytotoxicities. We studied the in vitro activation and inactivation of DBD with a bioassay in 9L rat brain tumor cells. We developed a mathematical model to calculate the rate constants of activation and inactivation. The kinetics of the activation and inactivation of DBD transformation products in cell culture medium were exponential, with rate constants of 0.139 and 0.0189 hr^–1, respectively. The maximum cell kill was caused by DBD that had been preincubated in medium for 13–16 hr. Thus cell kill is not caused by parent drug but by active transformation products.     

Cytotoxicity of Paclitaxel Incorporated in PLGA Nanoparticles on Hypoxic Human Tumor Cells

Purpose  The aim of this work was to prepare paclitaxel-loaded PLGA nanoparticles and determine cytotoxicity of released paclitaxel for two hypoxic human tumor cell lines: breast carcinoma (MCF-7) and carcinoma cervicis (HeLa). Methods  Poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing paclitaxel were prepared by o/w emulsification-solvent evaporation method. Physicochemical characteristics of nanoparticles were studied. Cellular uptake of nanoparticles was evaluated by transmission electronic microscopy and fluorescence microscopy. Flow cytometry quantified the number of cells held in G₂/M phase. Cell viability was determined by the ability of single cell to form colonies. Biodistribution of nanoparticles in mice was evaluated by fluorescence microscopy. Results  The nanoparticles were spherical with average diameter 318 ± 5.1 nm. The encapsulation efficiency was 88.52%. The drug release profile in vitro exhibited a biphasic pattern. Cellular uptake was observed. Co-culture of tumor cells with paclitaxel-loaded nanoparticles demonstrated that released paclitaxel retained its bioactivity to block cells in G₂/M phase. Paclitaxel-loaded nanoparticles exhibited cytotoxic effect on both hypoxic MCF-7 and HeLa cells and its cytotoxicity was more significant than that of free paclitaxel. Fluorescent nanoparticles were mainly distributed to liver and spleen of mice. Conclusions  Paclitaxel-loaded PLGA nanoparticles may be considered a promising drug delivery system to eradicate hypoxic tumor cells. Cheng Jin and Ling Bai contributed equally to this work.     

洛伐他汀联合顺铂对人肝癌HepG2细胞生物学特性的影响及其机制

目的 研究洛伐他汀单用或与化疗药物顺铂联用时对人肝癌HepG2细胞生物学特性的影响,初步探索其抗肿瘤作用机制。方法 不同浓度洛伐他汀、洛伐他汀联合顺铂处理细胞 48 h后,CCK-8法检测HepG2细胞的增殖抑制作用,金氏公式计算联合应用效果;平板克隆形成实验评价药物作用于肝癌细胞的远期效应;划痕实验检测药物对细胞迁移能力的影响;Transwell小室法检测药物对细胞侵袭能力的影响;流式细胞术检测药物处理后细胞周期和凋亡情况;蛋白印迹技术（Western-blot）检测凋亡相关蛋白Bcl-2、Bax、Caspase-3的表达水平变化。结果 洛伐他汀呈浓度依赖性抑制HepG2细胞的增殖,金氏公式计算结果显示洛伐他汀可协同增强顺铂的抗肿瘤作用;平板克隆形成实验结果表明洛伐他汀能显著抑制HepG2细胞克隆形成率;划痕实验提示洛伐他汀能显著降低肝癌细胞的迁移率;Transwell小室侵袭实验结果发现洛伐他汀能明显减少穿膜细胞数量;流式细胞检测发现洛伐他汀可引起G0/G1期细胞增加,S期细胞减少,细胞凋亡率增加;Western-blot检测结果显示洛伐他汀可下调Bcl-2蛋白表达,同时上调Bax和Caspase-3蛋白表达。结论 洛伐他汀可明显抑制HepG2细胞增殖、迁移和侵袭能力,与顺铂联用可增强顺铂体外抗肿瘤效果,通过线粒体途径诱导肿瘤细胞凋亡是其可能的抗肿瘤作用机制之一。     

Chemical Characterization and in Vitro Cytotoxicity on Squamous Cell Carcinoma Cells of Carica Papaya Leaf Extracts

In traditional medicine, Carica papaya leaf has been used for a wide range of therapeutic applications including skin diseases and cancer. In this study, we investigated the in vitro cytotoxicity of aqueous and ethanolic extracts of Carica papaya leaves on the human oral squamous cell carcinoma SCC25 cell line in parallel with non-cancerous human keratinocyte HaCaT cells. Two out of four extracts showed a significantly selective effect towards the cancer cells and were found to contain high levels of phenolic and flavonoid compounds. The chromatographic and mass spectrometric profiles of the extracts obtained with Ultra High Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry were used to tentatively identify the bioactive compounds using comparative analysis. The principal compounds identified were flavonoids or flavonoid glycosides, particularly compounds from the kaempferol and quercetin families, of which several have previously been reported to possess anticancer activities. These results confirm that papaya leaf is a potential source of anticancer compounds and warrant further scientific investigation to validate the traditional use of papaya leaf to treat cancer.     

银杏黄酮对卡铂所致大鼠肾损害的防护作用

目的研究银杏黄酮(GBE)对卡铂(CBDCA)肾毒性的防护作用,并探讨其可能机制。方法灌胃给予大鼠GBE后腹腔内注射CBDCA,检测肾脏系数、血清尿素氮(BUN)及尿N-乙酰-β-D氨基葡萄糖苷酶(NAG)、血还原型谷胱甘肽(GSH)与肾皮质丙二醛(MDA)及线粒体谷胱甘肽过氧化酶(GSH-Px)及尿与肾皮质铂含量,观察GBE的防护作用的剂量依赖关系和经时过程。结果250、500和750mg/kgGBE预处理后第5天,CBDCA所致大鼠肾脏系数、NAG活性和BUN含量增高均不同程度减轻;500mg/kg组GBE预处理的效果最明显,该组肾脏系数、NAG活性和BUN含量分别为(0.79±0.12)g/100g、(15.86±3.28)U/gCre和(9.27±3.77)mmol/L,而CBDCA组这3项指标分别为(0.96±0.22)g/100g、(23.58±5.45)U/gCre和(31.08±15.00)mmol/L,上述各项指标两组间的差异有显著性(P<0.05或0.01)。GBE预处理可抑制CBDCA引起的MDA形成增高,GSH含量和GSH-Px活性下降,并能降低CBDCA所致大鼠肾皮质铂含量增高,促进铂经尿排泄。结论GBE有明显预防CBDCA的肾毒性,其部分机制为抗氧化作用,还可能与促进铂清除有关。     

HSV-1体外感染骨髓间充质干细胞的实验研究

目的:在体外原代培养大鼠骨髓间充质干细胞,观察单纯疱疹病毒1型(HSV-1)感染骨髓间充质干细胞情况。方法:分离骨髓间充质干细胞,并对其作鉴定。用HSV-1感染骨髓间充质干细胞,提取总DNA,PCR法扩增骨髓间充质干细胞内的HSV-1特异性片段。结果:骨髓间充质干细胞经14d诱导后,碱性磷酸酶含量增高,形成钙结节,表现出成骨细胞特性。HSV-1感染骨髓间充质干细胞,细胞出现典型的病变,PCR法成功扩增出骨髓间充质干细胞内的HSV-1特异性片段。结论:大鼠骨髓间充质干细胞在体外可以向成骨细胞方向分化,可作为组织工程学的种子细胞。HSV-1可以在体外感染骨髓间充质干细胞。     

MicroRNA -181a 对人宫颈癌细胞的铂类药物敏感性的调节作用

目的：观察不同铂类药物敏感性宫颈癌细胞中的 microRNA（miR）-181a 表达,提高宫颈癌的药物治疗效果。方法观察不同铂类药物敏感性人体标本与人宫颈鳞状癌细胞系中的 miR -181a 表达。通过聚合酶链式反应（PCR）与基因转染技术,在体内与体外试验中,对 miR -181a 表达与铂类药物的敏感性进行研究。结果在2株人宫颈鳞状癌细胞系与人体标本中对化学治疗（简称化疗）药物有耐受性的肿瘤细胞 miR -181a 高表达,在裸鼠移植瘤模型与经过转染的细胞系中 miR -181a 的过表达显著增强了化疗的耐药性,而转染了 miR -181a 抑制因子后,药物敏感性明显升高。结论 miR -181a 在调控肿瘤细胞化疗耐药性中有重要的协调作用,研究为预测人宫颈鳞状细胞癌的化疗耐药性提供了一种基因标志物。     

顺铂联合8-硝基白杨素对人卵巢癌细胞裸鼠移植瘤生长的影响

目的 研究顺铂联合8-硝基白杨素对人卵巢癌COC1细胞裸鼠移植瘤生长的影响。方法 建立人卵巢癌COC1细胞裸鼠皮下移植瘤模型,随机分成4组,每组5只：生理盐水组、8-硝基白杨素组、顺铂组、8-硝基白杨素+顺铂组。观察各组裸鼠移植瘤体积、重量及裸鼠体质量的变化;裸鼠血清乳酸脱氢酶、谷丙转氨酶、肌酐值和外周血白细胞计数的变化;FCM测定瘤组织细胞凋亡率;Western blot分析瘤组织细胞凋亡的可能分子生物学机制。结果 与顺铂组和8-硝基白杨素组相比,顺铂联合8-硝基白杨素联合作用COC1细胞裸鼠移植瘤后,移植瘤的体积、移植瘤的重量均明显降低(P<0.05);与顺铂组相比,顺铂联合8-硝基白杨素联合组裸鼠体质量无明显差异(P>0.05),裸鼠血清乳酸脱氢酶、谷丙转氨酶、肌酐值和外周血白细胞计数无明显差异(P>0.05);顺铂和8-硝基白杨素联合作用COC1细胞16 d后,COC1细胞发生凋亡,同时bcl-2蛋白表达降低,caspase-3蛋白表达增高。结论 顺铂联合8-硝基白杨素通过降低bcl-2蛋白表达,活化caspase-3蛋白表达来抑制人卵巢癌COC1细胞裸鼠移植瘤生长。     

顺铂对结直肠癌细胞增殖及侵袭能力的影响

【摘要】目的 探讨顺铂对人结直肠癌细胞SW480增殖及侵袭力的影响。方法 以SW480细胞为研究对象,设定未经处理的SW480细胞为对照组,给予不同浓度顺铂进行不同时间干预,应用MTT法、Transwell侵袭小室、定磷法检测SW480细胞的增殖、侵袭力及Na + -K + -ATP酶活性。结果 生理浓度顺铂（70μmol/L）在48h即可抑制SW480 细胞的增殖,与72和96h相比抑制率差异无统计学意义;70μmol/L顺铂处理48h时,即可降低穿越Matrigel膜基质的细胞数;分别采用17.5、35、70和140µmol/L4个梯度浓度的顺铂作用于SW480细胞48h后,35、70和140µmol/L组细胞中Na + -K + -ATP酶活性均显著升高,且顺铂浓度达70µmol/L时Na + -K + -ATP酶即可达到较高活性。结论 Na + - K + -ATP酶活性的降低可能导致对结直肠癌细胞增殖和侵袭能力调节作用减弱,可能与结直肠癌细胞SW480耐顺铂有关。     

非病毒载体携带Cbfa1诱导成人骨髓间充质干细胞向成骨细胞分化的研究

目的:观察核心结合因子a1(Cbfa1)对成人骨髓间充质干细胞(hBMSCs)向成骨细胞定向分化的诱导作用。方法:经密度梯度离心法和贴壁筛选法获得hBMSCs。非病毒载体FuGene HD携带Cbfa1转染hBMSCs后7、14d,经Real-time PCR分析hBMSCs成骨细胞标志基因表达,包括Cbfa1、碱性磷酸酶(ALP)、骨钙素(OC)、骨桥蛋白(OPN)和Ⅰ型胶原;培养1、2和3周,ALP染色、钙结节染色检测成骨分化。结果:培养的hBMSCs阳性表达CD73,阴性表达CD34。第3代细胞增殖曲线呈"S"形,符合对数生长曲线。Cbfa1转染hBMSCs表现出与成骨细胞相似的形态,成骨细胞标志基因ALP、OC、OPN和Ⅰ型胶原的表达均明显上调,ALP染色阳性,并且有明显的钙结节形成。结论:非病毒载体携带Cbfa1转染hBMSCs可诱导其向成骨细胞分化。     

Effect of Trans-resveratrol on Rotenone-induced Cytotoxicity in Human Breast Adenocarcinoma Cells

Rotenone, a botanical insecticide is known to cause apoptosis in various cell types. Trans-resveratrol, a natural phytophenol present in red grapes and wine, is also well documented for its antioxidant, anti-inflammatory, anti-mutagenic, and anticarcinogenic activities. Therefore, the present investigations were carried out to assess the protective effect of trans-resveratrol against rotenone-induced cell death in human breast adenocarcinoma (MCF-7) cells. MCF-7 cells were exposed with various concentrations of rotenone for 24 h, and the loss in percent cell viability was evaluated by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] and neutral red uptake (NRU) assays. A significant decrease in percent cell viability in MCF-7 cells was observed at 50 μM and above concentrations of rotenone, as compared to untreated control. Furthermore, various concentrations (5, 10, and 25 μM) of trans-resveratrol were used to see its protective role on cell viability in rotenone-induced cell death in MCF-7 cells. Pre- or post- treatment of trans-resveratrol for 24 h was given to the cells. The data exhibited a significant dose dependent increase in the percent cell viability under pre- and post-treatment conditions. However, post-treatment of trans-resveratrol for 24 h after rotenone exposure to the cells was relatively less effective. Overall, the results suggest that trans-resveratrol significantly protects MCF-7 cells from rotenone-induced cell death. This model can be used as an effective and economical alternative to animal models for screening the antioxidant activity of a variety of natural compounds/drugs.     

Effect of Low Dose Radiation on Differentiation of Bone Marrow Cells into Dendritic Cells

Low dose radiation has been shown to be beneficial to living organisms using several biological systems, including immune and hematopoietic systems. Chronic low dose radiation was shown to stimulate immune systems, resulting in controlling the proliferation of cancer cells, maintain immune balance and induce hematopoietic hormesis. Since dendritic cells are differentiated from bone marrow cells and are key players in maintaining the balance between immune activation and tolerance, it may be important to further characterize whether low dose radiation can influence the capacity of bone marrow cells to differentiate into dendritic cells. We have shown that bone marrow cells from low dose-irradiated (γ-radiation, 0.2Gy, 15.44mGy/h) mice can differentiate into dendritic cells that have several different characteristics, such as expression of surface molecules, cytokine secretion and antigen uptake capacity, when compared to dentritic cells differentiated from the control bone marrow cells. These differences observed in the low dose radiation group can be beneficial to living organisms either by activation of immune responses to foreign antigens or tumors, or maintenance of self-tolerance. To the best of our knowledge, this is the first report showing that total-body low dose radiation can modulate the capacity of bone marrow cells to differentiate into dendritic cells.     

Transfection Efficiency and Cytotoxicity of Nonviral Gene Transfer Reagents in Human Smooth Muscle and Endothelial Cells

PURPOSE: Evaluation of a nonviral transfection reagent with respect to efficient gene transfer into primary human vascular cells. METHODS: Complexes consisting of seven commercially available transfection reagents (DAC-30, DC-30, Lipofectin, LipofectAMINE PLUS, Effectene, FuGene 6 and Superfect) and EGFP encoding plasmid DNA were studied. The in vitro transfection efficiency and cytotoxicity in human aorta smooth muscle cells (HASMCs) and endothelial cells (HAECs) and rat smooth muscle cells (A-10 SMCs) were assayed in the presence of serum using flow cytometric analysis and ATP-quantitation assay, respectively. RESULTS: Human primary cells were transfected less efficiently compared to the rat smooth muscle cell line. Transfection efficiency depended on the type of reagent, the reagent/DNA ratio, and, most importantly, on the cell type used. Determination of cytotoxicity showed that the effects of transfection on cell viability did not significantly differ from one another depending on the cell type. The exception to this was Superfect, which obviously reduced cell viability in all cell types. CONCLUSIONS: Our experiments showed that DAC-30 is the preferred transfection reagent for HASMCs and HAECs, exhibiting an improved efficiency combined with an acceptable cytotoxicity. Therefore, it might offer a therapeutic option for the treatment of cardiovascular disease and prove suitable for further drug development.