高水溶性SJPt对Hela细胞杀伤作用的研究

高水溶性ＳＪＰｔ对Ｈｅｌａ细胞杀伤作用的研究侯晓明袁国卿（北京市临床药学研究所１０００３５）（中国科学院化学研究所北京１０００８０）自从具有强抗癌活性的顺式二氯二铵铂（ＤＤＰ）问世以来￣［１］，有关铂配合物的抗癌活性，构效关系及作用机理，受到人们的普...     

新型双氢青蒿素哌嗪-芳香酰胺类化合物的合成及其抗肿瘤活性的初步评价

以双氢青蒿素为起始原料,在草酰氯作用下无需分离,随后在"一锅煮"条件下与哌嗪作用得到胺类化合物,该类化合物与芳香酰氯作用,快速、高效地合成了6种新型芳酰胺类双氢青蒿素哌嗪衍生物。所有化合物通过IR、1H NMR、13C NMR和HR-MS得到结构确认。同时,以四甲基偶氮唑盐比色法(MTT法)研究了该类化合物对人肝癌细胞株SMMC-7721的抑制活性。初步研究结果表明,该类化合物具有明显抑制人肝癌细胞增殖、诱导其凋亡的细胞活性,给药72h,半抑制浓度IC50最优值仅为0.04μmol/L。在与青蒿素、双氢青蒿素的对比实验中发现,该类化合物的抗癌活性明显提高,表现出该类化合物具有潜在的开发和应用价值。     

溶血磷脂酰胆碱衍生物的合成及其对自然杀伤T细胞的激活作用

溶血磷脂酰乙醇胺（pLPE）是自然杀伤T细胞（NKT）的内源性抗原。溶血磷脂酰胆碱衍生物(LPC)与CD1d结合被NKT细胞识别并激活NKT细胞。本文以手性甘油醇缩丙酮为起始原料,合成了LPC衍生物（5和6）,其结构经¹H NMR, ¹³C NMR, ³¹P NMR和HR-MS(ESI)确证。研究了5和6〖STBZ〗的免疫活性。结果表明:100 ng·mL^-1 6负载mCD1d分子后可刺激2H4细胞释放IL-2因子（32.13±1.56 pg·mL^-1）,具有潜在的NKT激活性质。     

基于微流控芯片的乳腺癌细胞微环境酸化检测

建立了基于微流控芯片的乳腺癌微环境酸化模型和动态检测微环境酸化情况的分析方法。设计了一种多层复合式微流控芯片,将乳腺癌细胞悬液引入含有水凝胶前体的芯片培养室后,在硝酸纤维素薄膜上固化形成3D培养支架。芯片通道连续灌流模拟血流供应,并将非电化学的pH检测器引入芯片,通过图像分析得到实时的pH变化。通过观察癌细胞的存活率、增殖率、乳酸水平及pH值,分析微环境的酸化情况,同时与正常细胞进行比较。结果表明,连续灌流培养7 d,乳腺癌细胞的存活率保持在90%以上;随着培养天数的增加,芯片上癌细胞微环境的pH值逐渐降低,且灌流速度越低,pH值下降越明显,而正常细胞微环境的pH值无明显变化。基于微流控芯片的微环境酸化检测平台可实时动态检测微环境的pH值,有望成为相关肿瘤研究的有力工具。     

转铁蛋白偶联双氢青蒿素氧基苯甲酰肼的载药量分析

在含20%乙醇的B-R缓冲溶液(V/V,pH=7.2)中,利用电分析化学方法测得双氢青蒿素氧基苯甲酰肼(dihydroartemisininoxy benzoic acid hydrazide,DBAH)在银电极上于-0.66V(vs.SCE)处有一不可逆还原峰。DBAH与被高碘酸钠氧化后的转铁蛋白(holotransferrin,Tf)结合形成电活性复合物,其还原峰峰电位由-0.16V负移至-0.84V,峰电流随DBAH和Tf浓度的增加而增加。该法测得一个Tf分子可以与3个DBAH分子连接,其结合常数为5.1×1010。紫外可见吸收光谱法测定结果与电化学测定结果相吻合。实验结果表明,Tf与DBAH共价结合形成电活性复合物。     

富勒烯衍生物对神经细胞的凋亡作用

机体代谢中产生的氧自由基可诱导生物细胞大分子发生破坏，导致细胞发生非程序性凋亡。特别是神经细胞，担负着接受且产生神经冲动从而支配全身活动的功能，而且有关研究已经表明一些神经退化及紊乱疾病与细胞产生氧自由基有关。富勒烯衍生物作为新型的具有高度吸收自由基活性的一种抗氧化剂，最有潜力用作神经细胞的保护剂。本文介绍了富勒烯衍生物作用的机理及近几年富勒烯衍生物阻断凋亡信号及阻止氧自由基对神经细胞破坏作用的研究进展。     

大黄素衍生物的合成及细胞毒性研究

以天然大黄素为母体, 经化学修饰得到一系列新的含氮衍生物6～14. 通过¹H NMR, IR, MS和元素分析确定了结构. 选择口腔底癌(KB)和乳腺癌(MFC-7)两种人癌细胞株, 采用标准MTT法测定了这类大黄素衍生物的细胞毒性. 研究表明大多数衍生物都有较强的抗癌活性, 其中位置异构体7a和7b的混合物表现出最强的活性, 与母体大黄素相比较, 活性分别提高了174倍(KB)和133倍(MFC-7).     

木香烃内酯诱导乳腺癌MCF-7细胞凋亡作用机制的研究

研究了木香烃内酯诱导人乳腺癌细胞MCF-7细胞凋亡的作用机制.采用流式细胞仪测定不同浓度木香烃内酯(0,2,4,8 μg/mL)作用于MCF-7细胞后细胞凋亡、活性氧(Reactive oxygen species,ROS)含量及线粒体跨膜电位(Mitochondrial transmembrane potential,MTP)的变化,气相色谱-质谱联用(GC-TOF/MS)技术分析加药组与未加药组的代谢差异物.结果表明,木香烃内酯能诱导MCF-7细胞凋亡,并具有浓度依赖性,能够促使ROS含量升高;MTP在2μg/mL木香烃内酯作用时升高,在4和8μg/mL时显著下降;基于GC-TOF/MS的细胞代谢组学研究,最终发现15种代谢差异物.基于上述结果,推测木香烃内酯通过引起ROS含量升高、MTP降低,扰乱线粒体的正常功能,进一步阻碍TCA循环,抑制ATP合成,扰乱了细胞内代谢物的平衡,并引起位于膜间隙的凋亡相关蛋白释放,最终导致MCF-7细胞的凋亡.     

硫酸钠对胰岛素淀粉样纤维化及其细胞毒性的作用

超过20种人类疾病与蛋白质或者多肽淀粉样纤维化密切相关,探究影响蛋白质的结构稳定性及其淀粉样纤维化的环境条件具有重要意义.本文采用牛胰岛素作为模型蛋白质,研究了Na2SO4对蛋白质淀粉样纤维化的作用.实验结果表明,不同浓度的Na2SO4对胰岛素淀粉样纤维化过程具有不同的影响,低浓度条件下可促进纤维化,较高浓度可明显抑制淀粉样纤维的形成,更高的浓度则使胰岛素形成非纤维状聚集体.ANS荧光分析结果表明,所有浓度的Na2SO4均可减小胰岛素聚集体的表面疏水性,并导致聚集体对细胞膜的损害作用降低.Na2SO4的上述作用可能与其改变蛋白质分子间的静电作用力及溶剂效应有关.     

熊果酸衍生物的合成、表征及其对癌细胞的抑制活性

以熊果酸为母体化合物,对其3-OH和17-COOH进行结构修饰,设计合成了9个熊果酸衍生物,其结构经IR、¹H NMR和MS测试技术确证;噻唑蓝(MTT)比色法考察了所合成衍生物对体外人肝癌细胞HepG2培养增殖的抑制作用,结果显示受试衍生物均对细胞增殖有一定抑制作用,且呈剂量依赖性,其中衍生物9的抑制作用最强;衍生物9与细胞作用24 h后,经Hoechst 33342/PI双染色,倒置荧光显微镜下观察,出现细胞凋亡所具有的高蓝色/低红色荧光图;流式细胞术检测细胞周期发现：细胞被阻滞于S期,且阻滞作用随药物浓度的增加而增强。     

Development of 1-(4-(Substituted)piperazin-1-yl)-2-((2-((4-methoxybenzyl)thio)pyrimidin-4-yl)oxy)ethanones That Target Poly (ADP-Ribose) Polymerase in Human Breast Cancer Cells

A number of uracil amides cleave poly (ADP-ribose) polymerase and therefore novel thiouracil amide compounds were synthesized and screened for the loss of cell viability in a human-estrogen-receptor-positive breast cancer cell line. The synthesized compounds exhibited moderate to significant efficacy against human breast cancer cells, where the compound 5e IC₅₀ value was found to be 18 μM. Thouracil amide compounds 5a and 5e inhibited the catalytical activity of PARP1, enhanced cleavage of PARP1, enhanced phosphorylation of H2AX, and increased CASPASE 3/7 activity. Finally, in silico analysis demonstrated that compound 5e interacted with PARP1. Hence, specific thiouracil amides may serve as new drug-seeds for the development of PARP inhibitors for use in oncology.     

Regulating Effect of Cytochrome b5 Overexpression on Human Breast Cancer Cells

Imbalance in the cellular redox system is thought to be associated with the induction and progression of breast cancers, and heme proteins may regulate the redox balance. Cytochrome b₅ (Cyt b₅) is a small mitochondrial heme protein. Its function and regulating mechanism in breast cancer remain unknown. In this study, we elucidated the level of endogenous oxidative stress in breast cancer cells, MCF-7 cells (hormone receptor-positive cells) and MDA-MB-231 cells (triple-negative cells), and investigated the difference in Cyt b₅ content. Based on the low content of Cyt b₅ in MDA-MB-231 cells, the overexpression of Cyt b₅ was found to regulate the oxidative stress and apoptosis cascades, including ERK1/2 and Akt signaling pathways. The overexpressed Cyt b₅ MDA-MB-231 cells were shown to exhibit decreased oxidative stress, less phosphorylation of ERK1/2 and Akt, and less cleavage of caspases 3 and 9 upon treatment with H₂O₂, as compared to those of normal MDA-MB-231 cells. Moreover, the overexpressed Cyt b₅ most likely functioned by interacting with its protein partner, Cyt c, as suggested by co-immunoprecipitation studies. These results indicated that Cyt b₅ has different effects on breast cancer cells of different phenotypes, which provides useful information for understanding the multiple roles of Cyt b₅ and provides clues for clinical treatment.     

Modification of Branched Poly(ethylene imine) with d-Fructose for Selective Delivery of siRNA into Human Breast Cancer Cells

Branched poly(ethylene imine) (bPEI) is frequently used in RNA interference (RNAi) experiments as a cationic polymer for the delivery of small interfering RNA (siRNA) because of its ability to form stable polyplexes that facilitate siRNA uptake. However, the use of bPEI in gene delivery is limited by its cytotoxicity and a need for target specificity. In this work, bPEI is modified with d- fructose to improve biocompatibility and target breast cancer cells through the overexpressed GLUT5 transporter. Fructose-substituted bPEI (Fru−bPEI) is accessible in three steps starting from commercially available protected fructopyranosides and bPEI. Several polymers with varying molecular weights, degrees of substitution, and linker positions on d- fructose (C1 and C3) are synthesized and characterized with NMR spectroscopy, size exclusion chromatography, and elemental analysis. In vitro biological screenings show significantly reduced cytotoxicity of 10 kDa bPEI after fructose functionalization, specific uptake of siRNA polyplexes, and targeted knockdown of green fluorescent protein (GFP) in triple-negative breast cancer cells (MDA-MB-231) compared to noncancer cells (HEK293T).     

The Role of Serotonin in Breast Cancer Stem Cells

Breast tumors were the first tumors of epithelial origin shown to follow the cancer stem cell model. The model proposes that cancer stem cells are uniquely endowed with tumorigenic capacity and that their aberrant differentiation yields non-tumorigenic progeny, which constitute the bulk of the tumor cell population. Breast cancer stem cells resist therapies and seed metastases; thus, they account for breast cancer recurrence. Hence, targeting these cells is essential to achieve durable breast cancer remissions. We identified compounds including selective antagonists of multiple serotonergic system pathway components required for serotonin biosynthesis, transport, activity via multiple 5-HT receptors (5-HTRs), and catabolism that reduce the viability of breast cancer stem cells of both mouse and human origin using multiple orthologous assays. The molecular targets of the selective antagonists are expressed in breast tumors and breast cancer cell lines, which also produce serotonin, implying that it plays a required functional role in these cells. The selective antagonists act synergistically with chemotherapy to shrink mouse mammary tumors and human breast tumor xenografts primarily by inducing programmed tumor cell death. We hypothesize those serotonergic proteins of diverse activity function by common signaling pathways to maintain cancer stem cell viability. Here, we summarize our recent findings and the relevant literature regarding the role of serotonin in breast cancer.     

QCM Detection of Adhesion, Spreading and Proliferation of Human Breast Cancer Cells （MCF-7） on a Gold Surface

The quartz crystal microbalance (QCM) was used to monitor the one-day incubation of human breast cancer cells (MCF-7) on the gold electrode. In combination with an optical microscope simulation experiment, the cell-population pictures at various stages, the QCM responses to the cells' adhesion, spreading and proliferation on the electrode surface were discussed. The △f0 and △R1 responses were found mainly from mixed effects of viscodensity and surface stress, and in proportion to the cell coverage, rather than to the number of cells at the electrode. The significant fore-and-aft changes in cyclic voltammetry and electrochemical impedance spectroscopy of the ferri-ferrocyanide redox couple also proved that the cells were adhesion to the gold surface.     

Current Photoactive Molecules for Targeted Therapy of Triple-Negative Breast Cancer

Cancer is the second leading cause of death worldwide; therefore, there is an urgent need to find safe and effective therapies. Triple-negative breast cancer (TNBC) is diagnosed in ca. 15–20% of BC and is extremely aggressive resulting in reduced survival rate, which is mainly due to the low therapeutic efficacy of available treatments. Photodynamic therapy (PDT) is an interesting therapeutic approach in the treatment of cancer; the photosensitizers with good absorption in the therapeutic window, combined with their specific targeting of cancer cells, have received particular interest. This review aims to revisit the latest developments on chlorin-based photoactive molecules for targeted therapy in TNBC. Photodynamic therapy, alone or combined with other therapies (such as chemotherapy or photothermal therapy), has potential to be a safe and a promising approach against TNBC.     

Targeted Drug Delivery Biopolymers Effectively Inhibit Breast Tumor Growth and Prevent Doxorubicin-Induced Cardiotoxicity

The anticancer agent doxorubicin(dox) has been widely used in the treatment of a variety of hematological malignancies and solid tumors. Despite doxorubicin’s efficiency in killing tumor cells, severe damage to healthy tissues, along with cardiotoxicity, limits its clinical use. To overcome these adverse side effects, improve patient safety, and enhance therapeutic efficacy, we have designed a thermally responsive biopolymer doxorubicin carrier that can be specifically targeted to tumor tissue by locally applying mild hyperthermia (41 °C). The developed drug vehicle is composed of the following: a cell penetrating peptide (SynB1) to promote tumor and cellular uptake; thermally responsive Elastin-like polypeptide (ELP); and the (6-maleimidocaproyl) hydrazone derivative of doxorubicin (DOXO-EMCH) containing a pH-sensitive hydrazone linker that releases doxorubicin in the acidic tumor environment. We used the in vivo imaging system, IVIS, to determine biodistribution of doxorubicin-delivered ELP in MDA-MB-231 xenografts in nude mice. Tumor bearing mice were treated with a single IV injection of 10 mg/kg doxorubicin equivalent dose with free doxorubicin, thermally responsive SynB1 ELP 1-DOXO, and a thermally nonresponsive control biopolymer, SynB1 ELP 2-DOXO. Following a 2 h treatment with hyperthermia, tumors showed a 2-fold higher uptake when treated with SynB1 ELP 1-DOXO compared to free doxorubicin. Accumulation of the thermally non-responsive control SynB1 ELP2 –DOXO was comparable to free doxorubicin, indicating that an increase in dox accumulation with ELP is due to aggregation in response to thermal targeting. Higher levels of SynB1 ELP1–DOXO and SynB1 ELP2 –DOXO with respect to free doxorubicin were observed in kidneys. Fluorescence intensity from hearts of animals treated with SynB1 ELP1–DOXO show a 5-fold decrease in accumulation of doxorubicin than the same dose of free doxorubicin. SynB1-ELP1-DOXO biopolymers demonstrated a 6-fold increase in tumor/heart ratio in comparison to free doxorubicin, indicating preferential accumulation of the drug in tumors. These results demonstrate that thermally targeted polymers are a promising therapy to enhance tumor targeting and uptake of anticancer drugs and to minimize free drug toxicity in healthy tissues, representing a great potential for clinical application.     

Induction of Apoptosis and Regulation of MicroRNA Expression by (2E,6E)-2,6-bis-(4-hydroxy-3-methoxybenzylidene)-cyclohexanone (BHMC) Treatment on MCF-7 Breast Cancer Cells

(2E,6E)-2,6-bis-(4-hydroxy-3-methoxybenzylidene)-cyclohexanone (BHMC) is a synthetic curcumin analogue, which has been reported to possess anti-tumor, anti-metastatic, and anti-invasion properties on estrogen receptor (ER) negative breast cancer cells in vitro and in vivo. However, the cytotoxic effects of BHMC on ER positive breast cancer cells were not widely reported. This study was aimed to investigate the cytotoxic potential of BHMC on MCF-7 cells using cell viability, cell cycle, and apoptotic assays. Besides, microarray and quantitative polymerase chain reaction (qPCR) were performed to identify the list of miRNAs and genes, which could be dysregulated following BHMC treatment. The current study discovered that BHMC exhibits selective cytotoxic effects on ER positive MCF-7 cells as compared to ER negative MDA-MB-231 cells and normal breast cells, MCF-10A. BHMC was shown to promote G2/M cell cycle arrest and apoptosis in MCF-7 cells. Microarray and qPCR analysis demonstrated that BHMC treatment would upregulate several miRNAs like miR-3195 and miR-30a-3p and downregulate miRNAs such as miR-6813-5p and miR-6132 in MCF-7 cells. Besides, BHMC administration was also found to downregulate few tumor-promoting genes like VEGF and SNAIL in MCF-7. In conclusion, BHMC induced apoptosis in the MCF-7 cells by altering the expressions of apoptotic-regulating miRNAs and associated genes.     

Lysinated Multiwalled Carbon Nanotubes with Carbohydrate Ligands as an Effective Nanocarrier for Targeted Doxorubicin Delivery to Breast Cancer Cells

Multiwalled carbon nanotubes (MWCNTs) are elongated, hollow cylindrical nanotubes made of sp2 carbon. MWCNTs have attracted significant attention in the area of drug delivery due to their high drug-loading capacity and large surface area. Furthermore, they can be linked to bioactive ligands molecules via covalent and noncovalent bonds that allow for the targeted delivery of anticancer drugs such as doxorubicin. The majority of methodologies reported for the functionalization of MWCNTs for drug delivery are quite complex and use expensive linkers and ligands. In the present study, we report a simple, cost-effective approach for functionalizing MWCNTs with the carbohydrate ligands, galactose (GA), mannose (MA) and lactose (LA), using lysine as a linker. The doxorubicin (Dox)-loaded functionalized MWCNTs were characterized using FT-IR, NMR, Raman, XRD and FE-SEM. The drug–loaded MWCNTs were evaluated for drug loading, drug release and cell toxicity in vitro, in breast cancer cells. The results indicated that the carbohydrate-modified lysinated MWCNTs had greater Dox loading capacity, compared to carboxylated MWCNTs (COOHMWCNTs) and lysinated MWCNTs (LyMWCNTs). In vitro drug release experiments indicated that the carbohydrate functionalized LyMWCNTs had higher Dox release at pH 5.0, compared to the physiological pH of 7.4, over 120 h, indicating that they are suitable candidates for targeting the tumor microenvironment as a result of their sustained release profile of Dox. Doxorubicin-loaded galactosylated MWCNTs (Dox-GAMWCNTs) and doxorubicin loaded mannosylated MWCNTs (Dox-MAMWCNTs) had greater anticancer efficacy and cellular uptake, compared to doxorubicin–loaded lactosylated MWCNTs (Dox-LAMWCNTs) and pure Dox, in MDA-MB231 and MCF7 breast cancer cells. However, neither the ligand conjugated multiwall blank carbon nanotubes (GAMWCNTs, MAMWCNTs and LAMWCNTs) nor the lysinated multiwalled blank carbon nanotubes produced significant toxicity in the normal cells. Our results suggest that sugar-tethered multiwalled carbon nanotubes, especially the galactosylated (Dox-GAMWCNTs) and mannosylated (Dox-MAMWCNTs) formulations, may be used to improve the targeted delivery of anticancer drugs to breast cancer cells.